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Recent Advances Toward Development of Plant Cell Culture Process for Sustainable Production of Lignans and Their Health Benefits

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Cancer has been recognized as a global health burden in rates of morbidity and mortality. Much effort has been devoted to discover promising cancer therapeutic agents from natural sources, and there has been some progress in introducing new anticancer drugs into the pharmaceutical market. Plant-derived natural products have played a very important role as cancer chemotherapeutic agents, either in their unmodified (naturally occurring) or synthetically modified forms. In this respect, lignans which are presented in a wide range of plants such as flaxseed, sesame, and other seeds, as well as vegetables, fruits, and beverages such as coffee and tea, show diverse spectrum of health-promoting effects, such as anticancer, antioxidant, antiviral, antidiabetic, and protective against cardiovascular diseases. A number of lignans (arctigenin, matairesinol and its glycosides pinoresinol and phillygenin) have come to the fore in research. The production of lignans from plant by the conventional methods is met with several problems. The seasonal production, agricultural practices, handling, and poor storage of plant materials impede offering such demand compounds to pharmaceutical factories. The biotechnological methods, particularly plant cell cultures, are an attractive alternative source to whole plant for the production of such compounds with significant amounts which are not always easily available. The aim of this review is to focus on lignans: biosynthesis in plant, sources, health benefit, and production of flaxseed lignans by plant biotechnological methods.
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249© Springer Nature Switzerland AG 2021
S. Malik (ed.), Exploring Plant Cells for the Production of Compounds of
Interest, https://doi.org/10.1007/978-3-030-58271-5_10
Recent Advances Toward Development
ofPlant Cell Culture Process
forSustainable Production ofLignans
andTheir Health Benets
AhmedM.M.Gabr, HodaB.Mabrok, OksanaSytar, andIrynaSmetanska
Abstract Cancer has been recognized as a global health burden in rates of morbid-
ity and mortality. Much effort has been devoted to discover promising cancer thera-
peutic agents from natural sources, and there has been some progress in introducing
new anticancer drugs into the pharmaceutical market. Plant-derived natural prod-
ucts have played a very important role as cancer chemotherapeutic agents, either in
their unmodied (naturally occurring) or synthetically modied forms. In this
respect, lignans which are presented in a wide range of plants such as axseed,
sesame, and other seeds, as well as vegetables, fruits, and beverages such as coffee
and tea, show diverse spectrum of health-promoting effects, such as anticancer, anti-
oxidant, antiviral, antidiabetic, and protective against cardiovascular diseases. A
number of lignans (arctigenin, matairesinol and its glycosides pinoresinol and
phillygenin) have come to the fore in research. The production of lignans from plant
by the conventional methods is met with several problems. The seasonal production,
agricultural practices, handling, and poor storage of plant materials impede offering
such demand compounds to pharmaceutical factories. The biotechnological
methods, particularly plant cell cultures, are an attractive alternative source to whole
plant for the production of such compounds with signicant amounts which are not
A. M. M. Gabr (*)
Department of Plant Biotechnology, Genetic Engineering and Biotechnology Research
Division, National Research Centre (NRC), Cairo, Egypt
H. B. Mabrok
Department of Nutrition and Food Sciences, Food Industry and Nutrition Division, National
Research Centre (NRC), Cairo, Egypt
O. Sytar (*)
Plant Biology Department, Educational and Scientic Center “Institute of Biology and
Medicine”, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine
Department of Plant Physiology, Slovak Agricultural University in Nitra,
Nitra, Slovak Republic
I. Smetanska
Department of Plant Food Processing, Agricultural Faculty, University of Applied Science
Weihenstephan-Triesdorf, Weidenbach, Germany
250
always easily available. The aim of this review is to focus on lignans: biosynthesis
in plant, sources, health benet, and production of axseed lignans by plant
biotechnological methods.
Keywords Lignans · Anticancer · Flax and biotechnology production
Abbreviations
AC Aberrant crypts
ACF Aberrant crypt foci
BA Benzyladenine
CHD Coronary heart disease
CVD Cardiovascular disease
DP Dirigent protein
DW Dry weight
ED Enterodiol
EL Enterolactone
ER Estrogen receptor
FW Fresh weight
HDL High-density lipoprotein
IAA Indole acetic acid
LAR Lariciresinol
LDL Low-density lipoprotein
LS Linsmaier & Skoog medium
MAT Matairesinol
MeJA Methyl jasmonate
MS Murashige and Skoog medium
NAA Naphthaleneacetic acid
NO synthase Nitric oxide synthases
PAL Phenylalanine ammonia lyase
PINO Pinoresinol
SDG Secoisolariciresinol diglucoside
SECO Secoisolariciresinol
TAG Triacylglycerol
1 Introduction
Plants have been an important source of medicine for thousands of years. Since the
early days of mankind, plants and their secondary metabolites have been used by
humans to treat infections, health disorders, and illness (van Wyk and Wink 2004).
A. M. M. Gabr et al.
251
Plants are also the source of many modern medicines. According to recent estimates,
25% of all prescribed medicines in the developed world contain ingredients derived
from plants, and roughly 80% of the world’s population living in the developing
world relies on herbal remedies for their primary healthcare needs (Tripathi and
Tripathi 2003). Some of the most effective cancer treatments to date are natural
products or compounds derived from plant products. Isolation of anticancer
pharmaceuticals from plants is difcult due to their extremely low concentrations.
The industry currently lacks enough methods for producing all the desired plant-
derived pharmaceutical molecules. Some substances can only be isolated from
extremely rare plants. Plant cell cultures are an attractive alternative source to whole
plant to produce high-value secondary metabolites. The biotechnological method
offers a quick and efcient method for producing these high-value medical
compounds in cultivated cells.
As a folk medicine, lignans from phenolic compounds have been used owing to
anticancer and laxative effect for centuries. Lignans are naturally occurring
phenylpropanoid dimers (C6-C3 unit; e.g., coniferyl alcohol), in which the
phenylpropane units are linked by the central carbons of the side chains (Ayres and
Loike 1990; Hearon and MacGregor 1995). They are derived from the phenylpro-
panoid pathway (Imai etal. 2006). Numerous structurally different forms of lignans
exist, even if their molecular backbone consists only of two phenylpropane units
(Mazur and Adlercreutz1998; Imai etal. 2006).
Lignans are commonly included in the human diet, as they are widespread in the
plant kingdom (Umezawa 2003; Milder et al. 2005a, b; Penalvo et al. 2008).
Nowadays, with the growing interest toward nutraceuticals and pharmaceuticals,
plant lignans are becoming important therapeutically active class of compounds
because of their putative benecial health effects, such as antioxidant, antiviral,
anticancer (Hano etal. 2017; Garros etal. 2018; Ayres and Loike 1990; Saarinen
et al. 2007; Willfor et al. 2003), antidiabetic, and antiobesity (Bhathena and
Velasquez 2002) effects, and they may protect against cardiovascular diseases
(Vanharanta etal. 1999). Secoisolariciresinol and matairesinol were among the rst
lignan precursors identied in the human diet and are therefore the most extensively
studied, especially as anticancer agents (Fuss 2003).
The highest lignan concentrations have been found in axseeds and sesame
seeds (Milder etal. 2005a; Smeds etal. 2007, 2012; Thompson etal. 2006), and
lower concentrations are present in, e.g., Brassica vegetables (Milder etal. 2005a),
nuts, and cereals (Mazur and Adlercreutz1998; Milder etal. 2005a; Penalvo etal.
2008; Smeds etal. 2007). Flaxseed is the richest source of plant lignans due to its
high content of secoisolariciresinol diglucoside (SDG) and secoisolariciresinol
(SECO) and relatively high matairesinol (MAT). SDG and MAT are metabolized by
the intestinal microora into their biologically active forms enterodiol (ED) and
enterolactone (EL), respectively (Adlercreutz and Mazur 1997). Lignans (SDG,
SECO, and MAT) are conceded as natural cancer chemopreventive substances,
effective against the onset of breast, prostate, and colon cancer, and display a wide
range of health-promoting activities for humans in indirect manner (Laine etal.
2009). The benecial effects of these compounds on human health are well
Recent Advances Toward Development of Plant Cell Culture Process for Sustainable…
252
recognized (Westcott and Muir 2003; McCann etal. 2005), and the invivo studies
conrm the observed invitro effect (Bylund etal. 2005).
In an effort to study the biosynthesis and the accumulation of secondary metabo-
lites, cell cultures have been established as a very useful tool because this system
allows uniformity, accessibility, and reduced complexity (Facchini 2001; Mesnard
etal. 2002; Verpoorte etal. 2002). Many studies were aimed to accumulate the dif-
ferent lignans (podophyllotoxin, justicidin B, SDG, SECO, or MAT) in cell culture
of different Linum species (Mohagheghzadeh et al. 2002; Schmidt et al. 2006;
Hemmati etal. 2007; Hano etal. 2006; Attoumbre etal. 2006; Renouard etal. 2012;
Ionkova etal. 2013). Additionally, Agrobacterium rhizogenes-mediated transforma-
tion system was found to be very useful for hairy root induction and production of
phytochemicals (Choi etal. 2000; Veena and Taylor 2007). Successful induction of
hairy root has been reported in some Linum spp. such as L. avum (Oostdam etal.
1993; Lin et al. 2003), L. austriacum (Mohagheghzadeh et al. 2002), L. leonii
(Vasilev etal. 2006), L. tauricum (Ionkova and Fuss 2009), L. usitatissimum (Gabr
etal. 2016, 2018), and L. album (Tashackori etal. 2016), and main metabolites
(lignans) from resulted hairy roots have been shown to have biological activity.
The aim of the present chapter is to highlight on lignans: plant biosynthesis,
sources, health benets, and the effort for lignan production in plant cell cultures.
2 Lignans
Lignans are widely distributed in the plant kingdom. They can be already found in
mosses and ferns (Stafford 2000). Most lignans are dimers of phenylpropanoid units
which are linked via their β-carbon atoms (Fig.1). This general structure leads to a
broad variety of derivatives (Fig.1). Also, most lignans contain chiral C-atoms.
Other natural compounds with chiral centers usually occur only in one enantiomeric
form. In contrast, lignans can be found in both forms in different plant species or
organs of the same species (Umezawa 2003).
3 Lignan Biosynthesis inPlants
Lignans are di-phenolic compounds derived from the oxidative dimerization of two
phenylpropanoid (C6-C3) units (Moss 2000). They are dened by the link of the C3
side chains between their two central carbon atoms (Harmatha 2005). The
deamination of phenylalanine acid by phenylalanine ammonia lyase is the rst step
in phenylpropanoid pathway to form cinnamic acid. After the oxidation steps, the
hydroxycinnamic acids are formed. These reactions are activated by coenzyme A
(CoA) to their aldehydic forms and into alcohols including the coniferyl alcohols
(Davin and Lewis 1992; Boerjan etal. 2003; Costa etal. 2003).
A. M. M. Gabr et al.
253
The formation of lignans starts via stereoselective coupling of two coniferyl
alcohols. The dirigent protein (DP), which catalyzes the dimerization of trans-
coniferyl alcohol, entraps the coniferyl alcohol radicals such that the C6-C3 moi-
eties are aligned to produce only one stereoisomer (pinoresinol, PINO) as shown in
Fig.2 (Umezawa etal. 1990; Davin etal. 1997; Halls etal. 2004).
The lignans can be further converted into tetrahydrofurans (lariciresinol, LAR)
and dibenzylbutanes (secoisolariciresinol, SECO), via the action of pinoresinol-
lariciresinol reductase (PLR; so-called LuPLR1 LuPLR1), and to
dibenzylbutyrolactones (matairesinol, MAT) by secoisolariciresinol dehydrogenase
(SDH) or to secoisolariciresinol diglucoside (SDG) by secoisolariciresinol
diglucosyl transferase (SDT) as shown in Fig.3 (Ayres and Loike 1990; Lewis and
Davin 1999; Ford etal. 2001).
SDG is hardly present in an unbound form (Hano etal. 2006), but is linked in a
macromolecular structure, called the lignan macromolecule or lignan complex
(Kamal-Eldin etal. 2001), as represented in Fig.4. The SDG moieties are esteried
to each other via the linker molecule 3-hydroxy-3-methyl glutaric acid (HMGA)
(Klosterman and Smith 1954). CoA-activated HMGA links to the C-6 of the
glucosyl moieties of SDG resulting in dimer and higher oligomer formation (Ford
etal. 2001).
An average chain length of 5 SDG units has been suggested based on NMR
analysis of the lignan macromolecule, resulting in an average molecular mass of
Fig. 1 Basic chemical structure of lignans and their derivatives
Recent Advances Toward Development of Plant Cell Culture Process for Sustainable…
254
Fig. 2 Dirigent protein-mediated stereoselective radical coupling of coniferyl radicals, DP: diri-
gent protein. (Modied and adapted from Davin and Lewis 2005)
PINO LAR SECO MAT
SDG
PLRPLR SDH
SDT
Fig. 3 Biosynthetic pathways of lignans, where PLR, pinoresinol-lariciresinol reductase; SDH,
secoisolariciresinol dehydrogenase; and SDT, secoisolariciresinol diglucosyl transferase (modied
and adapted from Gabr etal. 2016)
A. M. M. Gabr et al.
255
4000 (Kamal-Eldin etal. 2001). Besides SDG and HMGA, coumaric acid glucoside
(CouAG) and ferulic acid glucoside (FeAG) have been identied as constituents of
the lignan macromolecule (Ford etal. 2001; Johnsson etal. 2002).
The conversion from coniferyl alcohol to matairesinol is believed to be the gen-
eral biosynthetic pathway (Umezawa 2003; Suzuki and Umezawa 2007; Davin and
Lewis 2003). Matairesinol is believed to be a central intermediate leading to all
diverse lignan structures (Fig.5), i.e., podophyllotoxin, justicidin B, savinin, and
()–hinokinin.
Podophyllotoxin: During the last decades, most work was performed to under-
stand the biosynthesis of podophyllotoxin and the related 6-methoxypodophyllo-
toxin. The steps from matairesinol to deoxypodophyllotoxin are most hypothetical
since they were gured out by feeding of possible intermediates to Anthriscus syl-
vestris which accumulates mainly yatein (Sakakibara et al. 2003). In addition,
Marques etal. (2013) reported that the podophyllum biosynthetic pathway though
remains largely unknown, with the last unequivocally demonstrated intermediate
Fig. 4 Chemical structure of the lignan macromolecule (modied and adapted from Kamal-Eldin
etal. 2001)
O
O
H
H
O
CH
3
OH
O
CH
3
HO
(-)matairesinol
O
O
O
O
O
O
Savinin
O
O
O
O
MeO
MeO
H
Justicidin B
O
O
O
O
O
O
(-)-hinokinin
O
HO
O
OMe
OMe
MeO
H
O
O
podophyllotoxi
n
Fig. 5 Possibly from matairesinol-derived lignans (modied and adapted from Fuss 2007)
Recent Advances Toward Development of Plant Cell Culture Process for Sustainable…
256
being ()–matairesinol. Justicidin B: The biosynthesis of justicidin B is not investi-
gated to the present (Hemmati etal. 2007). But, it can be assumed that the rst steps
are similar to the lignan biosynthesis in Forsythia intermedia shown by Davin and
Lewis (2003). But, one can presume that the biosynthesis starts with the dimeriza-
tion of coniferyl alcohol to pinoresinol which is further converted to matairesinol.
Several unknown steps can lead to the biosynthesis of justicidin B. ()–Hinokinin:
()–Hinokinin biosynthesis has not been investigated to date (Bayindir etal. 2008).
Takaku etal. (2001) suggested two possible hypothetical pathways for the further
biosynthesis of ()–hinokinin according to the lignan composition found in
Chamaecyparis obtusa. In the rst pathway, (+)-pinoresinol is reduced via (+)-lar-
iciresinol to ()–secoisolariciresinol by a pinoresinol- lariciresinol reductase (PLR).
Subsequently, ()–matairesinol is formed by secoisolariciresinol dehydrogenase. In
the last two steps, ()–hinokinin may be synthesized by the formation of two meth-
ylenedioxy bridges, either via ()–pluviatolide or via ()–haplomyrfolin, depend-
ing on the benzene ring on which the rst methylenedioxy bridge is formed. In the
second hypothetical pathway, the two methylenedioxy bridges are formed directly
on (+)–pinoresinol by piperitol-sesamin synthase to give (+)–sesamin (Jiao etal.
1998; Kato etal. 1998). (+)–sesamin may be converted to ()–dihydrocubebin by a
predicted sesamin-dihydrosesamin reductase (SDR), with a two-step reduction
reaction similar to the reductions catalyzed by PLR.Finally, ()–hinokinin could be
formed by a secoisolariciresinol dehydrogenase-like enzyme.
Cytotoxic lignans derived from podophyllotoxin are currently used in cancer
chemotherapy. Podophyllotoxin for semisynthetic derivatization is isolated from the
rhizomes of Podophyllum plants growing wild, some of which are counted as
endangered species (Petersen and Alfermann 2001). An alternative source for
podophyllotoxin or related lignans may in future be cell cultures derived from
different plant species, such as Podophyllum spp. or Linum spp. These cell cultures
were shown to accumulate considerable amounts of podophyllotoxin or
5-methoxypodophyllotoxin. Etoposide, teniposide, and etopophosare semisynthetic
derivatives of podophyllotoxin and are used in the treatment of cancer.
Biotechnological approaches, including the use of cell cultures, biotransformation,
or metabolic engineering techniques to manipulate the biosynthetic pathway,
represent an alternative to produce podophyllotoxin. As chemical synthesis of
podophyllotoxin is not yet economic, it still must be isolated from wild growing
Podophyllum species, some of which are endangered species. Therefore plant
invitro cultures may serve as alternative sources for podophyllotoxin and for other
types of lignans as well (Fuss 2003). Hairy root system with growth medium D
(HRGM-D) containing hormone-free MS basal medium with an extra 1-day
preincubation period at 35 °C can be a suitable platform for optimization and
production of satisfactory level of aryltetralin lignans like podophyllotoxin and its
derivatives from L. mucronatum (Samadi etal. 2012).
A. M. M. Gabr et al.
257
4 Lignan Sources
When comparing results of the lignan content of foods obtained in different studies,
it is important to consider that there are several factors affecting results. The lignan
content and composition is greatly dependent on natural variations (both genetic
and environmental); on the sample pre-treatment applied, including sampling,
storage, drying, and extraction methods; and on the analytical method used (Smeds
etal. 2012; Durazzo etal. 2018).
Lignans are detected at relatively high concentrations in coniferous trees, espe-
cially in knotwood of Norway spruce (Willfor etal. 2004). Also, lignans occur in
plants included in our food, while the concentrations are signicantly lower than in
coniferous trees. The highest concentrations have been found in axseeds and ses-
ame seeds (Milder etal. 2005a; Smeds etal. 2007, 2012; Thompson etal. 2006),
and lower concentrations are present in, e.g., Brassica vegetables (Milder etal.
2005a), nuts, and cereals (Mazur and Adlercreutz 1998; Milder etal. 2005a; Smeds
Table 1 Lignans in selected food
Food and foods group SDG content (μg/100g)
Flaxseed 369,900
Seeds Pumpkin 21,370
Sunower 610
Soybean 273
Caraway 221
Legumes Peanut 333
Urad dal bean 240
Pigeon pea 50
Nuts Blackberry 3710
Lingberry 1510
Walnut 163
Almond 107
Berries Cranberry 1510
Strawberry 1210
Red currant 160
Oat bran 24
Oatmeal 10
Cereals Rye bran 132
Rye meal, whole grain 50
Vegetables Broccoli 414
Garlic 379
Carrots 192
Coffee Green tea 2460
Tea Black tea 1590
Arabica coffee (instant) 716
Modied and adapted from Patel etal. (2012)
Recent Advances Toward Development of Plant Cell Culture Process for Sustainable…
258
etal. 2007; Penalvo etal. 2008). Plant SDG is known to be part of a macromolecule
in which they are connected through the linker-molecule HMGA.Hence, SDG has
a different extraction method including saponication step to be degraded to its
monomeric constituents. The food contents of SDG were presented in Table 1 as
analyzed by Adlercreutz and Mazur (1997) and Mazur (1998) and reviewed by Patel
etal. (2012)
Thompson etal. (2006) developed a lignan database and reported in the follow-
ing order: nuts and oilseeds (25–379,012μg per 100g by serving weight (g)), cere-
als and breads (2.0–7239.3), legumes (1.8–979.4), fruits (0.3–61.8), vegetables
(1.2–583.2), soy products (2.2–269.2), meat products and other processed foods
(0.2–415.1), alcoholic beverages (1.1–37.3), and nonalcoholic beverages (0.9–12).
Matairesinol was the least-concentrated lignan in most studied foods, whereas
secoisolariciresinol reached the highest concentration in 63 foods, lariciresinol in
44 foods, and pinoresinol in 14 foods among 120 investigated foods (Thompson
etal. 2006).
4.1 Seeds andNuts
Flaxseed is the richest source of plant lignans (Mazur and Adlercreutz 1998; Smeds
etal. 2012). Defatted axseed is containing high amounts of SDG (9–30mg/g dry
weight, DW) which is considered 800 times higher than any other food (Dobbins
and Wiley 2004). SECO concentration in axseed (294,210μg/g fresh weight, FW)
is higher than in other food as well. PINO, LARI, and MAT are also present in
substantial amounts (Milder etal. 2005a). Other authors have also analyzed lignan
concentrations in axseed (Johnsson et al. 2000; Kraushofer and Sontag 2002;
Mazur and Adlercreutz 1998; Obermeyer etal. 1995; Thompson etal. 2006; Smeds
etal. 2012). The second highest lignan concentration was detected in sesame seeds,
with PINO and LARI as the main constituents, 29,331 μg/100 g FW and
9470μg/100g FW, respectively (Milder etal. 2005a). However, the major lignans
in sesame seeds are the oil-soluble sesamin and sesamolin which was not included
in these analyses. After lignan-rich food sources (axseeds and whole sesame
seeds), cloudberry seeds are considered the third richest lignan food source, with
total lignan content 43,876μg/100g DW and LARI and medioresinol (MED) as the
main constituents, 11,581 and 13,595μg/100g DW, respectively, and total lignan
content 43,876μg/100g DW (Smeds etal. 2012). Also, as a rst report, Smeds etal.
(2012) found that camelina seeds (whole) seem to have a relatively high lignan
content, with LAR and SECO dominating. Several other members of the oilseeds,
i.e., cashew nuts and peanut, are also found to be relatively high in their content of
total lignans (629 and 94μg/100g FW), respectively (Milder etal. 2005a). Lignan
contents in several food sources are presented in Table2.
A. M. M. Gabr et al.
259
Table 2 Lignan contents in different food sources (μg/100g)
Food SECO PINO LARI MAT References
Seeds
Flaxseed 294,210 3324 3041 553 Milder etal. (2005a)
369,900 1087 Mazur and
Adlercreutz (1998)
385,000–
670,000
Johnsson etal.
(2000)
1,261,700 Liggins etal. (2000)
495,700–
1,006,200
700–
2850
Kraushofer and
Sontag (2002)
81,700 Obermeyer etal.
(1995)
379012.3 729.6 2807.5 153.3 Thompson etal.
(2006)
Sesame seed 66 29,331 9470 481 Milder etal. (2005a)
7.3 6814.5 1052.4 123.1 Thompson etal.
(2006)
Sunower seeds 53 167 671 0 Milder etal. (2005a)
26.2 33.9 149.7 0.5 Thompson etal.
(2006)
127.8 0 Horn-Ross etal.
(2000)
Nuts
Cashew 133 0 496 0 Milder etal. (2005a)
Peanut 53 0 41 0 Milder etal. (2005a)
Cereals
Rye 462 1547 1505 729 Smeds etal. (2007)
Wheat 868 138 672 410 Smeds etal. (2007)
Oat 90 567 766 440 Smeds etal. (2007)
Corn 125 33 69 21 Smeds etal. (2007)
Vegetables
Asparagus 743 122 92 14 Penalvo etal. (2008)
68.4 Horn-Ross etal.
(2000)
Brassica vegetables
(Brassica oleracea L.)
19 1691 599 12 Milder etal. (2005a)
Broccoli 5.8 6.1 82.0 0.1 Thompson etal.
(2006)
Cabbage 2.6 44.2 32.3 0.1 Thompson etal.
(2006)
Carrot 93 19 60 0 Milder etal. (2005a)
Courgette 18 37 64 0 Milder etal. (2005a)
Corn 7 14 Penalvo etal. (2008)
French beans 29 24 220 0 Milder etal. (2005a,
b)
(continued)
Recent Advances Toward Development of Plant Cell Culture Process for Sustainable…
260
Table 2 (continued)
Food SECO PINO LARI MAT References
Garlic 42.0 481.9 54.4 4.8 Thompson etal.
(2006)
Glover sprouts 0 0 Horn-Ross etal.
(2000)
Mushroom 0 0 0 0 Penalvo etal. (2008)
Myoga spike 7 7 1 Penalvo etal. (2008)
Salad green, rucola 78.3 3.4 Valsta etal. (2003)
Seri 449 491 746 13 Penalvo etal. (2008)
Spinach 0.2 0.3 3.1 0.1 Thompson etal.
(2006)
Cucumber 8 1 59 0 Milder etal. (2005a)
Sweet peppers (green) 7 1 164 0 Milder etal. (2005a)
Sweet peppers (red) 7 1 106 Milder etal. (2005a)
Tomatoes 1.2 1.9 6.0 0.0 Thompson etal.
(2006)
Tomatoes (cherry) 16 19 38 Penalvo etal. (2008)
Wheat sprout juice 1.6 0 Valsta etal. (2003)
White potatoes 0.4 0.2 0.6 0 Thompson etal.
(2006)
Fruits
Apricot 328.3 Tr Horn-Ross etal.
(2000)
31 105 314 0 Milder etal. (2005a)
Brussels sprouts 21 0.4 Valsta etal. (2003)
Dried apricots 147.6 190.1 62.1 0.6 Thompson etal.
(2006)
328.3 Tr Horn-Ross etal.
(2000)
Dried dates 106.2 100.2 116.9 0.3 Thompson etal.
(2006)
Dried prunes 1.8 103.8 71.5 2.1 Thompson etal.
(2006)
75.8 Tr Horn-Ross etal.
(2000)
Dried raisins 9.2 0.8 11.5 0.2 Thompson etal.
(2006)
Tr 52.2 Horn-Ross etal.
(2000)
Grape, green 0.2 1.3 Valsta etal. (2003)
Grapefruit 26.3 0 Valsta etal. (2003)
0Tr Horn-Ross etal.
(2000)
Kiwi 174.6 1.2 Valsta etal. (2003)
(continued)
A. M. M. Gabr et al.
261
Table 2 (continued)
Food SECO PINO LARI MAT References
Nashi (Japanese pear) 7 0 21 Penalvo etal.
(2008).
Olive 55.9 2.7 Valsta etal. (2003)
Pear 9.9 0.7 Valsta etal. (2003)
Raspberries 11.6 17.7 8.2 0.3 Thompson etal.
(2006)
Rosehip 78.8 2.2 Valsta etal. (2003)
Strawberries 5.1 20.8 22.9 0.1 Thompson etal.
(2006)
5 212 117 0 Milder etal. (2005a)
Valencia orange 56 51 193 Penalvo etal. (2008)
Yuzu 26 654 192 Penalvo etal. (2008)
Nonalcoholic beverage
Coffee Tr 0 Horn-Ross etal.
(2000)
9.4–16.1 0.4–1.5 9.0–
13.1
0.0–0.7 Milder etal. (2005a)
4.7 0.2 0.9 0.1 Thompson etal.
(2006)
Orange juice 8.0 0.1 0.2 0.0 Thompson etal.
(2006)
Pomegranate juice 0 2.1 0 0 Bonzanini etal.
(2009)
Milk, cow 0.4 0.3 0.3 0.1 Thompson etal.
(2006)
Milk, soya 1.1 30.0 6.6 0.0 Milder etal. (2005a)
Tea, black 5.0–6.2 27.0–
40.6
28.9–
30.8
1.1–1.5 Milder etal. (2005a)
Tea, green 12.9 5.7 18.7 2.0 Milder etal. (2005a)
Alcoholic beverage
Beer 0.0–1.0 12.6–
22.2
5.9–9.2 0.0 Milder etal.
(2005a,)
Wine, red 29.4 0.4 7.4 0.0 Thompson etal.
(2006)
41.7–61.3 6.3–
11.9
8.6–
15.9
5.9–7.8 Milder etal. (2005a)
Wine, white 5.9 0.1 0.4 1.6 Thompson etal.
(2006)
5.2–7.6 1.7–3.0 4.6–
11.9
2.7–3.1 Milder etal. (2005a)
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4.2 Cereals
In grains, the plant lignans are localized in the outer ber-containing layers of the
kernel, with high concentrations in the aleurone layer. Mazur etal. (1998a, b)
reported that lignan concentrations in whole grain were 48–112μg/100g while in
grain brans the concentration is higher than in whole grain (63–299μg/100g DW).
On the other hand, lignan concentrations in the our (8–32μg/100g DW) are low.
Determination of plant lignans in different cereal species were listed them from the
highest total lignans concentration to the lowest as follow: rye, wheat, triticale, oat,
spelt wheat, Japanese rice, wild rice, buckwheat, barley, amaranth, corn, millet,
quinoa, red rice, brown rice, dhurra were conrmed by Smeds etal. (2007) and
Durazzo etal. (2013).
4.3 Vegetables andFruits
It is worth noting that vegetables contain higher concentration of lignans than fruits.
The total lignan concentrations in vegetables are 16–3874μg/100g DW while in
fruits are 5.0–1510μg/100g DW (Mazur etal. 1998a, b). Brassica vegetables (cab-
bages, brussels sprouts, kale), asparagus, seri, and garlic contain high levels of total
lignans in the selected vegetables for analysis by Horn-Ross etal. (2000), Milder
etal. (2005a), Thompson etal. (2006), and Penalvo etal. (2008) with values 2321,
1034, 1724, and 583.2μg/100g FW, respectively. French beans (273μg/100g FW),
sweet peppers (green, 172μg/ 100g; red, 113μg/100g FW), carrots (171μg/100g
FW), and courgettes (119 μg/100 g FW) also have relatively high lignan
concentrations.
The richest source among fruits analyzed by Penalvo etal. (2008) was yuzu, a
citrus fruit originating in East Asia. Mazur etal. (2000) found relatively high levels
of SECO in berries. Horn-Ross etal. (2000) were able to detect SECO and MAT in
a few dried fruits (apricots, prunes, raisins) and in peaches, while a higher
concentration of MAT was reported in raisins (52 μg/100g FW) and only trace
amounts of SECO.
Moreover, Penalvo etal. (2008) showed for avocado a prole of decreasing con-
centration of lignans, syringaresinol > pinoresinol > medioresinol > secoisolarici-
resinol > lariciresinol > matairesinol, and for pineapple, syringaresinol > lariciresinol
> matairesinol > secoisolariciresinol > pinoresinol > medioresinol, whereas the
most representative lignan for navel orange was lariciresinol and secoisolarici-
resinol for kiwifruit. In berries, as reported by Smeds etal. (2009), the most repre-
sentative lignans among those studied were lariciresinol for cloudberries
(5008μg/100g dry weight) and secoisolariciresinol for blackberries (2902μg/100g
dry weight) (Smeds etal. 2009).
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263
4.4 Beverage
According to Mazur etal. (1998a, b), lignans are also found in beverages including
tea, coffee, and wine. The lignan concentration in tea (770–3050μg/100g dry tea
leaves) is higher than that in coffee powder (393–716μg/100g DW). Total lignan
concentrations in red wine are 69.1–91.3μg/100ml with SECO being the superior
one (Milder etal. 2005a). In 2008, Peñalvo etal. reported that plant lignan concen-
tration in Japanese food varies from 0 to 1724μg/100g wet weight. Within the
coffee samples from different countries, secoisolariciresinol varies from 27.9 to
52.0 μg L1 and lariciresinol from 5.3 to 27.8 μg L1, respectively, contrary to
matairesinol that was not possible to detect in each type of coffee (Angeloni
etal. 2018).
5 Daily Intake
Daily intake of SECO and MAT ranges from 0.18 to 0.65mg/d in studied popula-
tions in the Netherlands, the United States, and Finland (Horn-Ross etal. 2000; de
Kleijn etal. 2001; Keinan-Boker etal. 2002; McCann etal. 2003; Valsta etal. 2003).
Milder etal. (2005b) reported that the intake of the total plant lignan MAT, SECO,
PINO, and LARI was 1.2mg/day in Dutch adults. Similar results were obtained by
Nurmi etal. (2010) in a study population of 100 Finnish men. In 2010, Pellegrini
etal. conducted a cross-sectional study on 242 men and postmenopausal women in
Northern Italy in which the daily intake of individual lignans including the daily
intake of MAT, SECO, PINO, and LARI were 20.9, 335.3, 96.7, and 175.7μg/d,
respectively, and the total lignans were 665.5μg/d. The major dietary sources for
lignan intake in the Dutch cohorts were coffee and tea (Nurmi etal. 2010). In
contrast, in an Italy cohort, red wine accounts for about one-third of total lignan
intake (Pellegrini etal. 2010).
6 Lignans forHuman Health
Since lignans can show, e.g., antiviral, fungicidal, antibacterial, and cytotoxic activ-
ities, they are thought to be involved in the plant defense against pathogens and are
important for human health benets against diseases such as breast cancer, prostate
cancer, colon cancer, cardiovascular disease, obesity, and diabetes (Landete 2012).
Experimental evidence in animals has shown clear anticarcinogenic effects of ax-
seed or pure lignans in many types of cancer. Most of the plant lignans in human
foods are converted by the intestinal microora to enterolactone (EL) and enterodiol
(ED), called mammalian lignans or enterolignans (Adlercreutz 2007). Intestinal
bacteria catalyze the transformation of plant lignans to enterodiol and enterolactone
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by a series of metabolic steps. ED and EL are thought to be responsible for the
health benets for lignan (Mabrok etal. 2012).
6.1 Effect ofLignans onBreast Cancer
Breast cancer is one of the most common cancers in the world. It accounts for ~30%
of all diagnosed cancer cases in women each year (Bange et al. 2001) and is
considered a major public health issue with over 1 million newly diagnosed cases
per year. More than 400,000 annual cases of death and 4.4 million women living
with breast cancer are reported (Malik etal. 2010). Plant lignans (SDG, SECO, and
MAT) are metabolized by human intestinal bacteria to the enterolignans, ED and EL
(Borriello etal. 1985), as shown in Fig.6. ED and EL exhibit structural similarity to
estradiol and have therefore been hypothesized to modulate hormone-related
cancers such as breast cancer (Adlercreutz etal. 1982). Studies revealed that the
enterolignans are mostly associated with the reduction of breast cancer (Serraino
and Thompson 1992; Thompson etal. 1996; Wang etal. 2005; Fabian etal. 2010).
The chemopreventive effect of lignans is well established in human cancer cell lines
and animal cancer models (Boccardo etal. 2006; Saarinen etal. 2007, 2008). In
Mousavi and Adlercreutz (1992) reported that the combination of EL (0.5–2.0μM)
and estradiol (1.0 nM) resulted in a lower cell proliferation of human estrogen
receptor-positive (ER+) breast cancer cell (MCF-7 cell). This anti-proliferative
effect of lignan in MCF-7 cells depends on the dose and the estradiol status. Flaxseed
consumption reduces the incidence, number, and growth of tumors in carcinogen-
treated rats at the pre-initiation, promotion, or progression stages of carcinogenesis
(Serraino and Thompson 1992; Thompson etal. 1996). Moreover, consumption of
rye decreased tumor number and size in breast cancer model (Davies etal. 1999).
Tumor growth and incidence of metastases are also reduced by dietary axseed in
athymic mice injected with human ER-negative (Dabrosin etal. 2002; Chen etal.
2002) or ER-positive (Chen etal. 2004) breast cancer cells. The tumor size and
number of breast tumors as well as breast cancer biomarkers were reduced in breast
cancer rats fed with SDG and/or axseed diet (Saggar etal. 2010a, b; Mabrok etal.
2012). Dietary axseed and its equivalent amount of pure SDG reduced tumor size
and cell proliferation in ovariectomized-athymic mice with an increase in apoptosis
in this model. This effect may be mediated through the inhibition of ER- and growth
factor-mediated signaling pathways (Chen etal. 2002, 2009a; Dabrosin etal. 2002;
Saggar etal. 2010a, b). One of the main components of axseed is the lignans, of
which 95% are made of the predominant secoisolariciresinol diglucoside (SDG).
SDG is converted into enterolactone and enterodiol, both with antiestrogen activity
and structurally similar to estrogen; they can bind to cell receptors with farther
decreasing cell growth. Some clinical trials showed that axseed can have an impor-
tant role in decreasing breast cancer risk, mainly in postmenopausal women
(Velentzis etal. 2009; Calado etal. 2018).
A. M. M. Gabr et al.
265
Bacteria in colon
Bacteria in colon
Plant
Lignans
Mammalian
Lignans
Bacteria in colon
SECO
SDG
MAT
Enterodiol
Enterolactone
Fig. 6 Metabolism of plant lignans
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The urinary excretion of EL is lower in breast cancer patients than those in con-
trols (Adlercreutz etal. 1982). In Stuedal etal. (2005) investigated possible associa-
tions between circulating EL and normal breast tissue morphology. They reported
that EL concentration in plasma is negatively correlated with breast mammographic
density in postmenopausal Norwegian women. In pilot study, the observed modula-
tion of breast cancer biomarkers in premenopausal women showed a signicant
decrease in Ki-67 index (80% of the studied subjects) with 9- and 16-fold increase
in plasma of EL and total lignans, respectively (Fabian etal. 2010). A very recent
study showed that lignan intakes are associated with lower risks of breast cancer in
premenopausal women with more positive prognostic characteristics (McCann
etal. 2012).
6.2 Effect ofLignans onProstate Cancer
Prostate cancer is the second most frequently diagnosed cancer of men and the sixth
leading cause of cancer death among men. About 899,000 new cases of prostate
cancer are diagnosed every year worldwide and cause of over 200,000 deaths. The
incidence of the disease is increasing with a rise of 1.7% over 15years (Ferlay etal.
2008). There are numerous reports on the potential tumor-suppressive inuence of
lignans. Previous studies have shown that Asian men have much lower incidences
of prostate cancer and possibly of benign prostatic hyperplasia (BPH) than their
Western counterparts. Vegetarian men also have a lower incidence of prostate cancer
than omnivorous males (Serraino and Thompson 1992). Lignans and their derived
metabolites (ED and EL) are believed to be partly responsible for growth inhibition
of human prostate cancer cell lines (Lin etal. 2001). In prostate cancer cell lines
(PC-3, DU-145, and LNCaP), 10–100μM ED and EL signicantly inhibit growth
of all cell lines (Lin etal. 2001). The density of LNCaP human prostate cancer cells
was reduced by 57.5%, the metabolic activity of the tumor cells by 55%, and the cut
secretion of prostate specic antigen by 48.50% after treatment of prostate cancer
cells with a subcytotoxic concentration of EL (60 μM for 72 h) compared to
untreated cells (McCann et al. 2008). Additionally, several key genes such as
MCMs, survivin, and CDKs, which produce important effects on programmed cell
death of prostate cancer cells, were benecially regulated by EL treatment (McCann
etal. 2008). Activation of insulin-like growth factor-1 (IGF-1) receptor signaling is
critical for prostate cancer cell growth and progression (Chen etal. 2009b). EL
suppresses proliferation and migration of prostate cancer cells, at least partially,
through inhibition of IGF-1/IGF-1R signaling (Chen etal. 2009b). High local levels
of estrogen can initiate prostate cancer, and then aromatase activity must be involved,
as aromatase converts testosterone to estrogen (Friedman 2009). The ability of
lignans to inhibit aromatase can decrease excess estrogen while simultaneously
increasing benecial free testosterone (Grifths etal. 1998), which might protect
against prostate cancer.
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267
In a human pilot study, rye whole grain and bran intake has shown benecial
effects on prostate cancer progression as assessed by prostate-specic antigen
concentrations (Landberg et al. 2010). Morton and others (Morton et al. 1997)
reported that higher EL levels in prostatic uid were associated with populations
(from Portugal, Hong Kong, and the United Kingdom) with a low risk of prostate
cancer, indicating a potential prostate cancer protective effect of lignans. A case-
control study conducted in Scotland on 433 prostate cancer cases found that higher
serum EL concentrations were associated with a lower risk of prostate cancer (Heald
etal. 2007). In a small clinical study, prostate cancer cell proliferation decreased
and apoptosis increased in men fed 30g of axseed per day (Demark-Wahnefried
etal. 2001). A signicant factor which may have inuenced their study was that the
subjects were on a low-fat diet. A subsequent study by those authors further sup-
ported the role of axseed in combination with a low-fat diet to control prostate
growth (Demark-Wahnefried etal. 2004). In Azrad et al. (2013) investigated possi-
ble associations between urinary ED and EL with Ki-67 (cell proliferation bio-
marker) among 147 patients with prostate cancer who participated in a presurgical
trial of axseed supplementation (30g/day) for ~30days. They reported that total
urinary enterolignans and EL were inversely correlated with Ki67in the tumor tissue.
6.3 Effect ofLignans onColon Cancer
Colorectal cancer is the third most common cancer (9.4%) worldwide after lung and
breast cancers. It ranks fourth in mortality (7.9%), after lung, stomach, and liver
cancers (Stewart and Kleihues 2003). ED and EL at 10–100μM levels have been
shown to signicantly decrease the cell proliferation of four cancer cell lines
(LS174T, T84, Caco-2, and HCT-15) (Sung etal. 1998; Cos etal. 2003). These cell
lines are not estrogen-dependent, because estradiol at 0.5, 1, and 10μM did not
enhance their cell proliferation. Hence the reduction in colon cancer cell proliferation
by ED and EL may be due to non-hormone-related mechanisms (Sung etal. 1998).
Ayella and Wang (2007) assessed the effect of different doses of commercially
available plant lignans (SDG) on human colon cancer cell growth. It was found that
cancer cell growth was signicantly inhibited by SDG treatment. Increasing SDG
dosage (0–40μM) did not kill the cancer cells as cell viability was not affected but
instead resulted in S-phase cell cycle arrest as measured by DNA ow cytometry
analysis (Ayella and Wang 2007). ED and EL both alone and in combination at
different concentrations caused a signicant increase in apoptotic cells and decrease
in cell proliferation of human colon adenocarcinoma Caco-2 cell line (Bommareddy
etal. 2010). EL and ED signicantly inhibited cell proliferation of Caco-2 cells
starting at 50μM and 150μM, respectively. Both EL and ED at the concentration of
100μM signicantly induced apoptosis in Caco-2 cells. However, EL-treated cells
showed higher increase of apoptotic cells when compared to ED (Bommareddy
etal. 2010). A new tetrahydrofuran lignan called (+)-camerarialdehyde extract of
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268
the stems of Cameraria latifolia was found to show cytotoxicity toward HT-29
human colon cancer cell line (Ren etal. 2013).
The impact of lignans on colon carcinogenesis has been studied mainly with
carcinogen-treated rats (Serraino and Thompson 1992; Jenab and Thompson 1996;
Davies etal. 1999). Flaxseed and defatted axseed meal (at a level of 5% or 10%)
decreased the number of aberrant crypts (AC) and aberrant crypt foci (ACF) in the
descending colon of carcinogen-treated rats by 41–53% and 48–57%, respectively
(Serraino and Thompson 1992). In a subsequent study, where carcinogen-treated
rats were fed either a control diet or a diet supplemented with axseed and defatted
axseed meal (at level of 2.5% or 5%) or pure SDG, the number of AC per focus
(AC multiplicity) was signicantly reduced in the distal colon of axseed and SDG
groups (Jenab and Thompson 1996). Feeding 30% rye bran diet, which contains
lignans, to AOM-treated rats for 31weeks lowered the number of large ACF (>6AC
per ACF) and the number of tumors compared with the control (Davies etal. 1999).
In a case-cohort study of Danish middle-aged men and women, Kuijsten etal.
(2006) investigated the association between plasma enterolignans and the incidence
of colon cancer. The authors observed a substantial reduction in colorectal adenoma
risk among subjects with high plasma concentrations of enterolignans, in particular
ED (Kuijsten etal. 2006). In an Ontario-based colon cancer population case-control
study involving 1095 cases and 1890 control subjects, the dietary lignan intake is
associated with a signicant reduction in colorectal cancer risk (Thompson etal.
2006). Recently, Johnsen etal. (2010) examined the association between plasma EL
concentration and incidence of colon and rectal cancer in 57,053 participants aged
50–64. They concluded that higher EL levels are associated with lower risk of colon
cancer among women and higher risk of rectal cancer among men.
6.4 Effect ofLignans onCardiovascular Disease (CVD)
Oxidative stress, inammation, obesity, diabetes, dyslipidemia, and hypertension
are interrelated and contribute to an atherogenic environment that promotes the
development of CVD (O’Keefe etal. 2009; Mathieu et al. 2009). Psychosocial
stress may increase cardiovascular risk by activating the sympathetic nervous
system and increasing cortisol, blood glucose, and lipid levels as well as elevating
blood pressure (O’Keefe et al. 2009). Lignan intake may have an important
protective effect against vascular disease. Dupasquier etal. (2007) demonstrated
how dietary axseed can inhibit the atherogenic effects of a high-cholesterol diet in
the LDLrKO mouse through a reduction of circulating cholesterol levels and, at a
cellular level, via anti-proliferative and anti-inammatory actions. A series of
studies from the same laboratory investigated the effects of ax lignan complex and
pure SDG on atherosclerosis in rabbits (Prasad et al. 1998; Prasad 1999, 2005,
2007, 2008, 2009). They used similar methods for each study; however, they
included important variations such as the level of cholesterol in the atherogenic diet
(0.25–1.0%), the dosage, and duration of the treatment (2–4months). Despite these
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269
differences, all of these studies found that consumption of ax lignan complex and
SDG resulted in improvements in the lipid prole and was also shown to be effective
in reducing the development of aortic atherosclerosis. Hypercholesterolemia
induced endothelial cell dysfunction, and decreased endothelial nitric oxide
formation results in impaired angiogenesis and subsequent cardiovascular disorders.
SDG (20mg SDG/kg per d) increased expression of vascular endothelial function,
endothelial NO synthase, and heme oxygenase-1-mediated myocardial angiogenesis
in a hypercholesterolemic myocardial infarction model indicating its cardioprotective
effect (Penumathsa etal. 2008).
The intake of lariciresinol was linked by 30% to the reduced odds for hypercho-
lesterolemia. The data on lignan content in food, i.e., lariciresinol, matairesinol,
pinoresinol, and secoisolariciresinol, were collected from the available lignan data-
bases. It was shown the existing concept that dietary total lignans are linked to CVD
risk factors such as hypertension, hypercholesterolemia, and central obesity
(Witkowska etal. 2018).
Similar to most animal studies, several human studies have shown cardiovascular
benets from lignans. A study consisted of the prospective follow-up for an average
of 12·2years of 1889 men free of CVD at baseline showed a signicant association
between elevated serum EL concentrations and reduced risk of CHD-related and
CVD-related mortality (Vanharanta et al. 2003). Since axseed is rich sources of
plant lignan precursors for EL, the results of these studies suggest that axseed
might provide cardiovascular benets. Hypercholesterolemic participants receiving
600mg SDG/d had decreased plasma total cholesterol and LDL-cholesterol. Plasma
concentrations of SECO, ED, and EL were also measured, and the observed
cholesterol-lowering values were correlated with SECO and ED concentrations
(Zhang et al. 2008). In 6-week-long, randomized, double-blinded, placebo-
controlled study, the effects of axseed lignans on cardiovascular risk factors were
investigated. High-lignan axseed (0.41g lignans) decreased total cholesterol by
12%, LDL-cholesterol by 15%, and oxidized-LDL by 25% (Almario and
Karakas 2013).
6.5 Effect ofLignans onDiabetes
Diabetes and the metabolic syndrome are risk factors for CVD.Thus, dietary inter-
ventions that lower the risk of diabetes and the metabolic syndrome (increases in
central adiposity, serum TAG, serum glucose, blood pressure, and inammation;
decreases in HDL-cholesterol) will help to decrease the incidence of CVD (Cornish
etal. 2009). SDG from axseed has been shown effective in preventing/delaying the
development of type 2 diabetes through suppressing the expression of
phosphoenolpyruvate carboxykinase (PEPCK) gene, a rate-limited enzyme in the
gluconeogenesis in the liver (Prasad 2002). Lignan-rich fraction improves glucose
homeostasis by increasing glucose disposal rates and enhancing hepatic insulin
sensitivity by working as a peroxisome proliferator-activated receptor (PPAR)-γ
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270
agonist in type 2 diabetic rats (Kwon etal. 2011). Dietary axseed supplementation
in diabetes mellitus may have benecial effects on diabetic nephropathy evolution
by reducing the levels of oxidative stress and increasing the antioxidant defense
systems (Haliga etal. 2009).
SDG is effective in retarding the development of diabetic complications. In a
multidose 14-day study, lower doses of SDG (5 and 10 mg/kg B.W.) exhibited
moderate reduction in glucose levels and lipid prole, restoration of antioxidant
enzymes, and improvement of the insulin and c-peptide levels which shows the
regeneration of β-cell which secretes insulin. The synthetic SDG exerts anti-
hyperglycemic effect by preventing the liver from peroxidation damage through
inhibition of ROS level-mediated increased level of enzymatic and nonenzymatic
antioxidants (Moree etal. 2013).
Lignans have been shown to provide benets among type 2 diabetic patients.
Supplementation of lignan capsules (360mg/d) reduced glycosylated hemoglobin
(HbA1C) compared with placebo in participants with type 2 diabetes, and there was
no effect on fasting glucose and insulin concentrations, homeostasis model
assessment of insulin resistance (HOMA-IR), and blood lipid proles (Pan etal.
2007). Blood glucose and cholesterol, particularly low-density lipoprotein
cholesterol of type 2 diabetes patients (n=60), were decreased after consumption
of wheat our chapatis containing axseed gum (5g) (Thakur etal. 2009). In a
randomized, crossover study of overweight or obese men and postmenopausal
women (n=25) with pre-diabetes, axseed intake decreased glucose and insulin
and improved insulin sensitivity as part of a habitual diet in overweight or obese
individuals with pre-diabetes (Hutchins etal. 2013).
7 Biotechnological Approaches forLignan Production
7.1 Plant Cell Cultures
Today, the potential of plant cells to produce secondary metabolites in dedifferenti-
ated cultures is used extensively to produce plant-derived drugs. Screening by selec-
tion of high-yielding cell lines is the most common method to enhance
productivity.
Biotechnological approaches for producing aryltetralin lignans from Linum spe-
cies have been studied (Malik etal. 2014). The Linum sp. contains lignans such as
podophyllotoxin (PTOX), deoxypodophyllotoxin (a precursor of both PTOX and
6-methoxypodophyllotoxin, the latter via β-peltatin and β-peltatin-A-methyl ether),
and various derivatives. Lignans are natural compounds derived from two 8,8-linked
C6C3 (propylbenzene) units. PTOX is an aryltetralin lignan with strong cytotoxic
and antiviral activities. Thus, it is used as a starting material for producing various
semisynthetic derivatives that are widely used in chemotherapy, such as etoposide,
teniposide, and etopophos. It is currently produced largely from Podophyllum
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hexandrum and P. peltatum, slow-growing endangered species of the Berberidaceae
(Malik etal. 2014).
Biotechnological production of PTOX by invitro plant cell and hairy root cul-
tures from genera such as Podophyllum, Linum, Callistris, or Juniperus has been
reported but far from an industrial level (Ionkova 2008). Tissue cultures of
Podophyllum peltatum to produce podophyllotoxin were initiated from various
explants such as rhizomes, roots, and leaf segments of eld-grown plants. The
efciency of various factors such as the source of explant, growth regulators, and
light conditions on callus growth and podophyllotoxin production was studied
(Kadkade 1981, 1982). Callus was also induced from P. hexandrum invitro-grown
seedlings (van Uden etal. 1989). When the callus derived from P. hexandrum was
incubated in B5 medium containing 2,4-D, gibberellic acid (GA3), and
6-benzylaminopurine (BA), podophyllotoxin, 4-demethyl-podophyllotoxin, and
podophyllotoxin-4-O-glucoside were produced, and the levels of podophyllotoxin
and its derivatives were similar to those in the parent plant (0.3% DW) (Heyenga
et al. 1990). Different plant parts such as roots, shoots, and embryo have been
cultured and produce compounds similar to that from the whole plant. Embryogenic
roots from a P. peltatum callus were induced in liquid MS medium supplemented
with NAA, kinetin, and casein hydrolyzate. The roots were then transferred to the
medium without growth regulators, whereupon 1.6% of podophyllotoxin was
detected in the dried tissues, which was sixfold higher than in the mother plant
(Sakata etal. 1990). Rapidly producing and high-yielding P. peltatum plants were
developed from invitro propagation protocol by growing rhizome tips on the basal
MS medium containing sucrose, supplemented with benzyladenine and activated
carbon. The podophyllotoxin contents of invitro-rooted bud and plantlet cultures
were similar to the contents found in the wild (Moraes-Cerdeira etal. 1998). As the
levels found in these cultures were quite low, the interest in podophyllotoxins was
revived when root cultures of L. avum were found to contain high levels (1% DW)
of 5-methylpodophyllotoxin and its glucosides (Berlin et al. 1988). Other
podophyllotoxin-producing callus cultures are those from sterile leaves in Juniperus
chinensis, needles of Callitris drummondii, and the stem and leaves of young
seedlings of L. album on different media (van Uden etal. 1990a; Smollny etal.
1992; Wichers etal. 1991; Muranaka etal. 1998). One of the cell lines produced
0.3% podophyllotoxin of dried cells, together with small amounts of 5-methyl
podophyllotoxin, lariciresinol, and pinoresinol after 3weeks of cultivation (Smollny
etal. 1992). Uden etal. (1990a) studied the accumulation of the phenylpropanoid-
derived lignan of 5-methoxyl podophyllotoxin in cell suspension cultures of
L. avum; cultures that were routinely grown on a NAA-containing medium
accumulated low levels of 5-methylpodophyllotoxin. In cell culture of L. album,
L. avum, L. nodiorum, L. mucronatum, and L. tauricum, 6-methyl podophyllotoxin
is the main lignan accumulated in cell lines (Fuss 2003; Mohagheghzadeha
etal. 2007).
Mohagheghzadeh etal. (2002) established cell cultures of L. austriacum, which
accumulates the arylnaphthalene lignan justicidin B.The identication of justicidin
B or other arylnaphthalene lignans in L. glaucum invitro cultures agrees with the
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272
morphological data. L. glaucum has many morphological similarities with
L. austriacum (Mohagheghzadeh et al. 2003). However, arylnaphthalene-type
lignans were not found in L. bienne and L. tenuifolium in vitro cultures
(Mohagheghzadeh etal. 2009). In this respect, Hemmati etal. (2007) accumulated
justicidin B as the main lignan in Linum species before in cell suspension culture of
L. perenne Himmelszelt. The accumulation of justicidin B was with 23mg/g DW
2–3 times higher than that observed in cell cultures of L. austriacum. Cell cultures
of L. austriacum accumulate 6.7 mg/g DW justicidin B (Mohagheghzadeh etal.
2002). The diarylbutyrolactone hinokinin was detected for the rst time in the genus
Linum in in vitro cultures of L. strictum ssp. corymbulosum (Mohagheghzadeh
etal. 2006).
Flax lignans (SECO and SDG) were succeeded to accumulate in plant cell cul-
ture as reported by Attoumbre etal. (2006) accumulated lignans in L. usitatissimum
cell suspension on LS medium supplemented with NAA alone or in combination
with BA.However, SDG was barely detected, and MAT was not detected. Anjum
etal. (2017) developed a protocol for SDG production in invitro callus derived
from leaf and stem explants of L. usitatissimum inoculated with different concentra-
tion of plant growth regulators (α-naphthalene acetic acid (NAA), thidiazuron
(TDZ), and 6-benzyl adenine (BA)). They reported that the stem-derived calli
(1.0mg/l NAA) accumulated optimum concentrations of SDG (2.7±0.021mg/g
DW) compared with other growth regulators. Nadeem etal. (2018) found that
L. usitatissimum were grown in invitro culture on 200mg/l yeast extract enhanced
the accumulation of SDG 3.36-fold or 10.1mg/g DW than untreated culture.
7.2 Optimization ofCulture Media Composition
andCulture Conditions
Medium and culture condition optimization can increase the productivity in suspen-
sion culture by a factor of 20–30 and is very important in order to obtain an efcient
system for the production of high levels of secondary metabolites from plant cells.
Podophyllotoxin accumulation was strongly affected by light, with red light
stimulating its production in P. peltatum cell cultures (Kadkade 1982). Illumination
stimulated the endogenous production of podophyllotoxin-β-D-glucoside, to a
concentration of 0.11% (DW), in L. avum cultures (Uden 1993). Dark-grown
suspension cultures of P. hexandrum accumulated 0.1% podophyllotoxin, which
was three- to fourfold that in light-grown cultures (Uden 1993). Similar results were
also observed in the authors’ laboratory (Chattopadhyay etal. 2002a). P. hexandrum
cells growing in shake asks were found to be sensitive to hydrodynamic stresses
generated by changing rotational speeds. A rotational speed of 150rpm showed
more than 80% viability of P. hexandrum cells. A medium pH of 6.0 was favorable
for high biomass production and podophyllotoxin accumulation in P. hexandrum
cell cultures (Chattopadhyay et al. 2002b). MS medium with 60 mM nitrogen
A. M. M. Gabr et al.
273
having an ammonium to nitrate salt ratio of 1:2, 60 g/l glucose, and 1.25 mM
phosphate was found to be optimum for podophyllotoxin production (Chattopadhyay
etal. 2003a). Statistical optimization methodology, such as Plackett-Burman design
and response surface methodology, has been employed to optimize the media and
culture conditions for growth and podophyllotoxin production in P. hexandrum
cultures. The optimized values of important nutritional parameters were found to be
medium pH 6.0, 1.25mg/l IAA concentration, 72g/l glucose concentration, and an
inoculum level of 8 g/l. When P. hexandrum was cultivated under statistically
optimized culture conditions, a maximum of 20.2g/l (DW) of biomass and 48.8mg/l
podophyllotoxin were obtained (Chattopadhyay etal. 2002b).
Optimization of cell/tissue culture of Linum persicum for production of lignan
derivatives including podophyllotoxin was established. The farther evaluation for
anticarcinogenic activity on MCF7 cell line found that shoot extract of the invitro
plantlets has greater cytotoxic effect on breast cancer cells and leads to signicant
raise in ratio of Bax/Bcl-2 value=1.42 and Rbl1 gene expression that are well-
known as apoptosis-inducing agents (Esfandiari etal. 2018).
7.3 Plant Transformation by Agrobacterium rhizogenes
Various species of bacteria are capable of transferring genes to higher plant species
(Chung et al. 2006). Among them, Agrobacterium tumefaciens is most widely
studied. The soil bacterium A. rhizogenes infects the plant tissues and leads to the
formation of adventitious roots called “hairy roots.” Fast and hormone-independent
growth and high genetic stability make hairy root cultures superior in comparison to
cell cultures. Hairy roots are known to produce the same or even higher amounts of
the metabolites found in normal roots (Christensen and Muller 2009; Mukundan
etal. 1998). Integration of new genes into hairy roots has opened another way for
metabolic engineering (Guillon etal. 2006) which is based on the ability to transform
plant cells with the root-inducing (Ri) plasmid of A. rhizogenes. Two important
regions in the Ri plasmid are essential for transformation by transferred DNA
(T-DNA). These are the T-DNA itself as a mobile element and the virulence region
(vir) (Klee etal. 1987). The T-DNA is anked by 25bp directly repeated sequences.
Any DNA between these sequences will be transferred to the plant cell. The Ri
plasmids are grouped into two main classes according to the opines synthesized by
hairy roots: agropine-type (e.g., A4, 15834, LBA9402, 1855) and mannopine-type
(e.g., 8196, TR7, TR101) strains (Sevón and Oksman-Caldentey 2002). The T-DNA
of the agropine-type Ri plasmid consists of two separate T-DNA regions: the
TL-DNA and TR-DNA. The genes encoding auxin (tms1 and tms2) have been
localized on the TR-DNA of the agropine-type Ri plasmid. The mannopine-type Ri
plasmids contain only one T-DNA that shares considerable DNA sequence homology
with the TL-DNA of the agropine-type plasmids. The TL-DNA contains four
genetic loci, rolA, rolB, rolC, and rolD, the rst three of which mainly contribute to
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the root initiation and maintenance. A plant cell becomes susceptible to
Agrobacterium when it is wounded.
A threefold increase in podophyllotoxin content in comparison to controls was
obtained in transformed calli of P. hexandrum developed by transformation of
embryo using different strains of A. rhizogenes, viz., A4, 15834, and K599 (Giri
etal. 2001). Hairy root cultures of L. avum were also reported to produce up to 1%
of 5-methoxypodophyllotoxin and its glucoside derivative on a dry cell weight
basis. 5-Methoxypodophyllotoxin, isolated from the root cultures of L. avum
grown on vitamin-free MS basal medium supplemented with sucrose, appeared to
have about the same cytotoxic potency as podophyllotoxins and its semisynthetic
derivatives (Uden etal. 1992). The levels of podophyllotoxins varied from 0.05% to
0.3% DW, depending on the culture conditions and the tendency to differentiate.
Thus, hairy root cultures of L. avum, producing 1.5–3.5% podophyllotoxins in
DW, seem to be most suitable for future research efforts (Oostdam et al. 1993).
Hairy root cultures were induced from leaf explants of L. tauricum by infection with
A. rhizogenes strains TR 105 and ATCC 15834. The cultures produced 2.6% of the
lignan 4-demethyl-6-methoxypodophylotoxin and 3.5% of the lignan
6-methoxypodophyllotoxin on a DW basis, which was 10–12 times higher than in
L. tauricum cell suspensions (Ionkova and Fuss 2009). Whereas cell cultures of
L. austriacum accumulate 6.7mg/g DW justicidin B, hairy roots of the same species
can accumulate 16.9mg/g DW justicidin B (Mohagheghzadeh etal. 2002). More
recently, Ionkova et al. (2013) reported that the content of justicidin B in the
L. narbonense intact roots is about 0.5mg/g DW and in callus cultures is 1.57mg/g
DW.Suspensions of L. narbonense contain much smaller amounts of justicidin B,
0.09mg/g DW.Hairy roots produced vefold higher justicidin B (7.78mg/g DW)
compared to callus. As a rst report for lignans SECO, SDG and MAT accumulation
in L. usitatissimum transformed with A. rhizogenes A4 strain with fourfold lignan
higher than in untransformed root culture (Gabr etal. 2018).
The study with determination of lignans, phenolic acids, and antioxidant capac-
ity in transformed hairy root culture of Linum usitatissimum showed that total lignin
content (secoisolariciresinol diglucoside, secoisolariciresinol, and matairesinol)
was 55.5% higher in transformed root cultures than in the non-transformed root
culture. In the transformed root culture were detected secoisolariciresinol
diglucoside and matairesinol, but they were not found in the non-transformed root
Fig. 7 Schematic model of hairy root application
A. M. M. Gabr et al.
275
culture. Secoisolariciresinol content and overall production of phenolic acids in
transformed roots were approximately 3.5 times higher than that of the correspond-
ing non-transformed culture (Gabr etal. 2018).
The hairy root application can be used in the different directions (Fig. 7).
Different aspects and applications of hairy root cultures include recombinant protein
production, phytochemicals, introduction of desirable foreign genes,
phytoremediation, molecular breeding and crop improvement, rhizosphere
physiology and biochemistry, metabolic engineering, bioreactor design, and general
overviews of the system (Ono and Tian 2011). Elicitation, bioreactor, and scale-up
studies plus overextending accumulation of secondary metabolites of lignans have
been developed during last years. The hairy root culture gets more attention as
biological matrices for producing valuable metabolites as they have several attractive
features, including high genetic stability and relatively fast growth rates (Panda
etal. 2017).
7.4 Feeding ofPrecursors
The addition of precursors to the media in order to direct metabolic ux toward
enhanced production of the desired products represents an interesting approach to
exploit the biosynthetic potential of the enzymes present in plant cell cultures.
The addition of coniferyl alcohol complexed with β-cyclodextrin to P. hexan-
drum cell suspension cultures increased the concentration of podophyllotoxin four-
fold, to 0.013% on a DW basis. Noncomplexed coniferyl alcohol, suspended in the
medium, also enhanced podophyllotoxin production, albeit to a lower degree
(Woerdenbag etal. 1990). Coniferin, the β-d-glucoside of coniferyl alcohol, was
found to be a more potent precursor in terms of yield of the anticancer compound
(0.055%) but is not available commercially (Woerdenbag etal. 1990). Various phen-
ylpropanoid precursors (phenylalanine, tyrosine, cinnamic acid, caffeic acid, cou-
maric acid, ferulic acid, coniferyl alcohol, coniferin, etc.) were utilized in cell
cultivation for the improvement of podophyllotoxin levels in P. hexandrum cell cul-
tures. Of these, only coniferin at a concentration of 2.1mM was able signicantly
to increase podophyllotoxin accumulation on the tenth day of cultivation, by a fac-
tor of 12.8. However, most of the coniferin was transformed into unknown products
(Woerdenbag etal. 1990; van Uden etal. 1990b). Feeding cultures with the precur-
sor L-phenylalanine resulted in a three- to vefold increase in 5-methoxyl podo-
phyllotoxin levels but caused the levels of phenylalanine ammonia lyase (PAL)
activity to fall. Treatment of the cultures with the elicitor nigeran, either alone or in
combination with phenylalanine, caused the 5-methoxyl podophyllotoxin production
to cease, even though PAL activity was rapidly enhanced by these treatments.
Transfer of the cultures to NAA-free medium resulted in a 40–50-fold higher level
of 5-methoxyl podophyllotoxin accumulation, the PAL activity levels being lowered
compared to the routinely grown cells. With these more differentiated cultures,
phenylalanine feeding and elicitor treatment, both on its own and in combination
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276
with the precursor, had no effect on 5-methoxyl podophyllotoxin production, even
though the PAL activity levels were higher than in the untreated cells (van Uden
etal. 1990c). Co-cultured hairy roots of L. avum and P. hexandrum suspensions
resulted in increased podophyllotoxin concentrations by 240% in shake asks and
by 72% in a dual bioreactor, as compared to P. hexandrum suspension cultured
alone. The coniferin provided by L. avum hairy roots acted as a precursor for the
production of podophyllotoxin by P. hexandrum suspension cultures (Lin etal. 2003).
7.5 Elicitors
Elicitation is the induction of secondary metabolite production by molecules or
treatments known as “elicitors.” Elicitation is used to induce the expression of genes
often associated with the enzymes responsible for synthesis of secondary metabolites
by mimicking the pathogen defense or wound response in plants (Zhao etal. 2005).
Therefore, the treatment of plant cells with biotic and/or abiotic elicitors has been a
useful strategy to enhance secondary metabolite production in cell cultures. The
most frequently used elicitors in previous studies were fungal carbohydrates, yeast
extract, methyl jasmonate (MeJA), and chitosan (Ionkova 2009).
Attempts have been made to increase the accumulation of aryltetralin lignans in
Linum spp. cultures by inducing a hypersensitive defense reaction with a variety of
elicitors. However, nigeran, Phytophthora megasperma cell wall fractions, methyl
jasmonate, hydrogen peroxide, and salicylic acid were not found signicantly to
increase lignan accumulation in Linum spp. (Aapro 1998). Bhattacharyya et al.
(2012) showed approximately seven- to eightfold change in accumulation of
podophyllotoxin on P. hexandrum, after 9days of elicitation, elicited with 100μM
methyl jasmonate as compared to the control. The simulative effect of addition of
autoclaved and lter-sterilized culture ltrate of Piriformospora indica (a root
endophytic fungus) to the growing Linum album hairy root cultures on growth and
lignan production was observed as well. The podophyllotoxin and
6-methoxypodophyllotoxin (the lignans) concentrations were maximally improved
by 3.8 times and 4.4 times in comparison to control cultures together with increasing
of phenylalanine ammonia lyase activity at 3.1 times (Kumar etal. 2012).
The addition of methyl jasmonate to cell cultures of Forsythia intermedia resulted
in three- and sevenfold accumulations of the lignans, pinoresinol and matairesinol,
predominantly as glucosides (Schmitt and Petersen 2002). The effect of adding
methyl jasmonate to various cell lines in suspension culture of L. album has also
been studied, with a twofold increase in podophyllotoxin (7.69±1.45mg/g DW)
and 6-methoxypodophyllotoxin (1.11±0.09mg/g DW) being achieved as maximum
in one of the cell lines (Furden etal. 2005). The effect of yeast extract and abiotic
elicitors (Ag2+, Pb2+, and Cd2+) was also studied on podophyllotoxin production
in L. album suspension cultures, though only Ag2+ at 1mM concentration was
found to enhance production up to 0.24 mg/g cell DW (Shams-Ardakani etal.
2005). Ionkova (2009) concentrated on the induction of production of ariltetralin
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277
lignans in hairy root cultures of L. tauricum, transformed by Agrobacterium
rhizogenes, ATCC 15834, by exposing them to different concentrations of methyl
jasmonate during the culture period. The content of 4-demethyl-6-methoxypodo-
phylotoxin and 6-methoxypodophyllotoxin, the main constituents in hairy roots of
L. tauricum, increased about 1.2-folds by elicitation of MeJA; however, the fresh
weight, DW, and growth ratio were inhibited by increasing methyl jasmonate
concentrations. The highest total lignan yield was obtained with 150μM methyl
jasmonate treatment. Hano etal. (2006) studied the effects of fungal elicitors on
lignan metabolism in L. usitatissimum cell suspensions. They found that ax
(L. usitatissimum) cell cultures accumulated SECO and no MAT could be detected
either in control or elicited cells.
7.6 Bioreactor andScale-Up Studies
The environment in which plant cells grow usually changes when cultures are scaled
up from shake asks to bioreactors, and this may result in reduced productivity.
With the ultimate aim of implementing an industrial-scale process, the behavior of
cell cultures in bioreactors has received much attention.
Submerged batch and fed-batch cultivation of P. hexandrum cells for podophyl-
lotoxin production was studied in a 3L stirred-tank bioreactor. A 36% increase in
volumetric productivity of podophyllotoxin was achieved in fed-batch cultivation of
P. hexandrum cells over batch cultivation in a stirred-tank bioreactor (Chattopadhyay
etal. 2002b, 2003b, c). Continuous cultivation of P. hexandrum with cell retention
was also carried out in a 3L bioreactor equipped with a spin lter mounted on the
agitator shaft of the bioreactor. The result was an accumulation of 53g/l biomass
and 48.8mg/l podophyllotoxin, with volumetric productivity of 0.8mg/l per day)
(Chattopadhyay etal. 2003b). A maximal product yield of podophyllotoxin, up to
0.2% DW, was achieved when Linum album cell suspensions were cultured in a
20L bioreactor (Arroo etal. 2002). Ionkova etal. (2013) found that on L. narbo-
nense maximum biomass of 22.5g/l DW was harvested from the bioreactor culture
vessel (recording about eight times increase over initial inoculum), with 1.42%±0.12
justicidin B, greater than contents obtained from asks.
8 Conclusion
Some of the most effective cancer treatments to date are natural products or com-
pounds derived from plant products. Recently, the interest of international pharma-
ceutical industries has been directed more and more to plant-based anticancer
compounds. Isolation of anticancer pharmaceuticals from plants is difcult due to
their extremely low concentrations. The industry currently lacks sufcient methods
for producing all of the desired plant-derived pharmaceutical molecules. Some
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278
substances can only be isolated from extremely rare plants. Plant cell cultures are an
attractive alternative source to whole plant for the production of high-value
secondary metabolites. From the previous data in this review, it’s very clear the
importance of lignans as anticancer agents, and lignan production through hairy
root transformation is the optimum biotechnological method for high productivity
than cell suspension culture or elicited culture. Bioreactor studies represent the nal
step leading to commercial production of economically important lignans from
plant cell cultures.
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Lignans are widely occurring plant compounds and are closely related to lignin, which forms the woody component of trees and other plants. The lignans are characterized by their dimeric composition from cinnamic acids, and they are attracting increasing attention as a result of their pharmacological properties. The volume surveys the chemical, biological and clinical properties of lignans as well as providing information on their isolation, purification, identification and chemical synthesis. The volume also explores fully the potential use of these compounds as antiviral and antitumour agents, and thus provides a wide-ranging survey of their pharmacology and chemistry. The text is fully documented and referenced and provides the only up-to-date compilation on this subject. The volume is suitable for research scientists in the fields of organic chemistry, biochemistry, oncology, pharmacology, toxicology and botany.
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Hairy root culture is a promising alternative method for the production of secondary metabolites. In this study, transformed root of Linum usitatissimum was established using Agrobacterium rhizogenes A4 strain from root cultures for lignans, phenolic acids and antioxidant capacity determination. Total lignin content (secoisolariciresinol diglucoside, secoisolariciresinol and matairesinol) was 55.5% higher in transformed root cultures than in the non-transformed root culture. Secoisolariciresinol was detected in higher concentration (2.107 μmol/g DM) in the transformed root culture than non-transformed culture (1.099 μmol/g DM). Secoisolariciresinol diglucoside and matairesinol were exclusively detected in the transformed root culture, but were not found in the non-transformed root culture. The overall production of phenolic acids in transformed roots was approximately 3.5 times higher than that of the corresponding non-transformed culture. Free radical scavenging DPPH˙ and ABTS˙⁺ assays showed 2.9-fold and 1.76-fold higher anti-oxidant activity in transformed root culture as compared to non-transformed.