Production of the liver-protective compounds cynarin and silymarin from tissue cultures of Globe artichoke and Milk thistle plants

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Production of the liver-protective compounds cynarin and silymarin from tissue
cultures of Globe artichoke and Milk thistle plants
BEKHEET S.H.; H.S.Taha; M.K.El-Bahr and A.M.M.Gabr
Plant Biotechnology Dept., National Research Center, Dokki, 12622, Giza, Egypt.
Corresponding author: Shawky Bekheet. Email: shawky005@yahoo.com
Abstract
The production of the useful natural components form plants by the conventional
methods are met with several problems. The seasonal production, diseases, handling and
poor storage impede offering such demand compounds to pharmaceutical factories. The main
purpose of this work is to employ different biotechnology applications for production of
phenolic compounds, cynarin and silymarin from Globe artichoke and Milk thistle plants
respectively under in vitro conditions. Shoot tips of the two plant species were isolated from
seedlings grown in vitro and then cultured on Murashige and Skoog medium supplemented
with 2 mg/l Kinetin + 2 mg/l 6-benzyladenine + 0.1 mg/l Indole-3-acetic acid to get stock
tissue culture materials. Calli were obtained from leaf explants using Murashige and Skoog
medium + 5 mg/l 1-Naphthaleneacetic acid + 2 mg/l Kinetin + 0.1 mg/l Gibberellic acid.
Supplementation of culture medium with of picloram enhanced callus growth of both plants.
Addition of 3 mg/l picloram registered the best results of callus proliferation presented as
growth ratio. Otherwise, accumulation enhancement of cynarin and silymarin by addition of
chitosan, and methyl jasmonate was investigated. It was found that elicitation of culture
medium with chitosan and methyl jasmonate showed increasing of cynarin and silymarin
contents in callus cultures of Globe artichoke and Milk thistle respectively. Methyl
jasmonate had more positive effect on the contents of the interested compounds compared
with chitosan.
Key words: Cynarin, Globe artichoke, In vitro, Milk thistle, Silymarin.

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Introduction
Globe artichoke (Cynara scolymus L.) and Milk thistle (Silybum marianum L.) plants
(Asteraceae/Compositae family) are considered important medicinal herbs producing active
ingredients agents treat liver diseases (Morazzoni and Bombardelli, 1995; Orlovskaya et
al., 2007). Cynarin (1,5-di-O-caffeoylquinic acid) is the main biological active chemical in
artichoke plant (Gebhardt, 1997). It was originally thought to be the single active
component in artichoke leaf extract and was often used as a monosubstance. However, the
active ingredients of Milk thistle are chemicals called flavonoids. The flavonoids in Milk
thistle are silybin, silydianin, and silychristin. Together, they are called silymarin. Silymarin
protects the liver by acting as an antioxidant and by promoting the growth of new liver cells
(Barnes et al., 2002).
The production of the natural compounds form Globe artichoke and Milk thistle by the
conventional agricultural methods are met with the seasonal cultivation, diseases and storage
problems. The development of alternative methods to whole plant cultivation for the
production of pharmaceutically valuable compounds is an issue of considerable economic
importance. Plant tissue cultures are found to have potential as a supplement to traditional
agriculture in the industrial production of such compounds (Bekheet et al., 2014). Tissue and
cell cultures are used for the large scale culturing of plant cells from which biologically
active agents are extracted. The faster proliferation rates and shorter biosynthetic cycle of
cell and organ cultures leads to have a higher rate of metabolism when compared to field
grown plants (Rao and Ravishankar, 2002). The principal advantage of this technology is
that it ultimately provides a continuous, reliable source of active agent year-round.
Additional advantages of such processes include controlled production according to demand
and reduced requirements. On contrast, one of the obstacles to the use of tissue culture for
the pharmaceutical industry is the low yield of the metabolites of interest. For this reason,
several strategies have been adopted to improve the production of plant-derived secondary

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metabolites such as genetic transformation, bioreactor engineering and elicitors application.
In this respect, enhancement of phenolic compounds accumulation in tissue cultures of Globe
artichoke (Bekheet et al., 2014; Menin et al., 2013; Pandino et al., 2017) and Milk thistle
(Sánchez-Sampedro et al., 2005; Hasanloo et al., 2008; Rahimi et al., 2011; Gabr et al.,
2016) have been investigated by many researchers. This work aims to optimize in vitro
system for production of cynarin and silymarin from Globe artichoke and Milk thistle using
different biotechnological techniques.
Materials and Methods
1- Establishment of in vitro shootlet cultures
Seeds of Globe artichoke and Milk thistle (Egyptian varieties) were washed with
distilled water and then immersed in 70 % ethanol for 1 min followed by 50 % commercial
Clorox (containing 5.25 % sodium hypochlorite) for 20 min and finally washed three times
with distilled sterilized water. The steps of disinfestation were took place under aseptic
conditions in a laminar air-flow cabinet. The disinfected seeds were placed in jars containing
50 ml of MS-basal medium (Murashige and Skoog, 1962). Shoot tips were isolated from
the aseptic grown seedlings and re-cultured on fresh medium contained 2 mg/l Kinetin (kin)
+ 2 mg/l benzyladenine (BA) + 0.1 mg/l indole-3-acetic acid (IAA) to get stock in vitro
shootlet cultures.
2- Callus induction
For callus induction, leaf explants were excided from in vitro grown seedlings and
cultured on MS medium + 5 mg/l 1-Naphthaleneacetic acid (NAA) + 2 mg/l kin + 0.1 mg/l
Gibberellic acid (GA3). The cultures were incubated in the dark for two weeks before
transferring on normal light conditions for four weeks to initiate callus cultures.

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3- Effect of picloram on callus growth
To assess the effect of picloram (4-amino-3,5,6-trichloropicolinic acid) on callus
growth of Globe artichoke and Milk thistle, about 250 mg of callus tissue were sub-cultured
on callus growth medium (MS + 5 mg/l NAA + 2 mg/l Kin + 0.1 mg/l GA3) supplemented
with 1, 2, 3 and 4 mg/l of picloram.
Growth ratio were determined after five weeks of sub-culturing.
Harvested fresh weight - Inoculated fresh weight
*Growth ratio = ________________________________________
Inoculated fresh weight
4- Incubation conditions and experimental design
Cultures were normally maintained at 25˚C and 16 hr photoperiod provided by white
fluorescent tubes (3000 Lux) except light experiment. Each experiment was set up as a
separate completely randomized design.
5- Effect of chitosan and methyl jasmonate on cynarin and silymarin accumulation
Callus cultures of Globe artichoke and Milk thistle were re-cultured on free B5
medium supplemented with different concentrations (200, 400 and 800 mg/l) of chitosan
and methyl jasmonat (20 mg/l, 40 mg/l and 80 mg/l) and maintained at 25 ± 2°C in the dark.
Two weeks of elucidated samples were harvested, weighed and immersed in liquid nitrogen
to avoid any possible enzyme degradation then followed with freeze-drying. The
lyophilized samples were grounded by flint mill (Retsch, Germany) (20000 rpm, 2 min) to a
fine powder.
6- Determination of cynarin
Cynarin content was determined in the different treatments using High Performance
Liquid Chromatography (HPLC) according to the method described by Menin et al. (2013).

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Briefly, 50 mg grounded samples were extracted for 20 min using 1 ml 80% aqueous ethanol
(v/v) in an ultrasonic bath. Samples were centrifuged for 10 min at 6000 rpm. The
supernatants were collected and the pellets were re-extracted twice with 500 µl 80% ethanol.
After centrifugation (10 min at 10,000 rpm), the supernatant was filtered through a 0.45 μm
Anotop 10 filter (Whatman, Maidstone, UK) into a 2 ml glass vial, and 10 μl of the filtrate
was injected into an HPLC instrument (Dionex Summit P680A HPLC-System), equipped
with P680 pump, ASI-100 automated sample injector, a Narrow-Bore AcclaimPA C16-
column (3 μm, 2.1 × 150 mm, Dionex) and PSA-100 photodiode array detector (Dionex) and
software Chromeleon 6.8 (Dionex, USA). The mobile phases consisted of a 1:1,000 (v/v)
mix of degassed glacial acetic acid: ultrapure water (Eluant A) and a 1:1,000 (v/v) mix of
glacial acetic acid: acetonitrile (Eluant B). The elution gradient started at 5% (v/v) B: 95%
(v/v) A, and increased linearly to 35% (v/v) B: 65% (v/v) A over 28 min. The column was
equilibrated with 100% (v/v) A between injections. The flow rate was 0.5 ml min–1 and the
absorbance of the output was monitored at 300 nm and at 330 nm. Cynarin reference
standard was purchased from Sigma-Aldrich.
7- Determination of silymarin
Flavonolignans were extracted from the lyophilized with 10 ml of methanol at 40 °C
for 8 h. The methanol solution was evaporated and concentrated to a dry residue. The extract
was dissolved in 1 ml of methanol and kept at 4 °C in darkness. The content of silymarin
compounds was determined by HPLC on a UNICAM CRYSTAL 200 Liquid
Chromatograph. The mobile phase consisted of methanol and water (both acidified with
0.3% orthophosphoric acid p.a. - w/v). The flow-rate was 1.4 ml/min. Substances were
detected by absorption at k = 288 nm and their identification were carried out by the
comparison of retention times and absorption spectra with standards complex of silymarins
(Sigma-Aldrich). The silymarin content was expressed as mg/g dry weight and derived using
a known concentration of standard and sample peak areas.

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Results and Discussion
1- Establishment of in vitro shootlet cultures
In vitro grown seedlings were used to obtain stock materials of Globe artichoke and
Milk thistle shootlet cultures. Based on primary experiments, MS medium supplemented
with 2 mg/l kin + 2 mg/l BA + 0.1 mg/l IAA was used for in vitro shootlets proliferation.
Healthy and vigorous shoot cultures of both Globe artichoke and Milk thistle plants were
obtained after five weeks of the third re-culturing (Fig. 1). Adventitious shootlets carrying
normal leaves and without callus or root formation were obtained on this medium. The
proliferated shootlets of Milk thistle were normal and enough to obtain stock materials
within 2-3 sub-culturing. However, we faced some difficult with verification phenomenon
which appeared in Globe artichoke cultures. We increased the level of agar used for
solidifying culture medium from 7 to 8 g/l to overcome this problem.
Fig. (1). In vitro proliferation of shootlet cultures of Globe artichoke
(A) and Milk thistle (B)
grown on MS medium supplemented with 2 mg/l kin + 2 mg/l BA + 0.1 mg/l IAA.
2- Callus induction
Callus cultures of Globe artichoke and Milk thistle were obtained
from leaf explants
using MS medium + 5 mg/l NAA + 2 mg/l Kin + 0.1 mg/l GA3
(based on the primary
experiments). Our observations reveal that callogenesis of Milk thistle was more
responsive
to the used medium compared with
callus of Globe artichoke. Little calli were obtained after

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five weeks and callusing was increasing as sub-
culturing increased. The calli were brownish
to creamish in color, hard and compact (Fig. 2). T
he callus turned dark brown after six
weeks, therefore regular sub-culturing of callus was done every four weeks.
Fig. (2). Callus derived from leaf explants of Globe artichoke (A) and
Milk thistle (B)
grown for five weeks on MS medium supplemented with
5 mg/l kin + 0.5 mg/l
IAA + 0.1 mg/l GA3.
3- Effect of picloram on callus growth
Data of Table (1) show the effect supplementation of culture medium with
picloram on
growth of callus proliferated from leaf segments of Globe artichoke and
Milk thistle. Results
indicated that supplementation of culture medium with picloram generally enhanced callus
growth of callus of both Globe artichoke and Milk thistle plants
. Addition of 3 mg/l picloram
to culture medium registered the best results of
callus growth presented as growth value. At
this medium, significant differences in callus
growth within the two plant species have been
observed. It was noticed that growth dynamic of Milk thistle
callus was higher than Globe
artichoke. The highest growth ratio of Milk thistle callus was 7.0, however, it was 6.7
in
Globe artichoke (Table 1).
Callus induction is the unique technique in tissue culture for production of active
constituents, many factors play important role in callus formation such as
type of explants,
composition of MS medium and environment (Renu and Bansal., 2011). Picloram
is used as
an auxin source in plant tissue cultures. The data indicate its potential uses
in routine callus

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cultures, in regeneration of plants from calli, and in research concerning the physiological
development of plant tissues. In our study, we found that supplementation of culture medium
with picloram generally enhanced callus growth of both Milk thistle and Globe artichoke
callus cultures. It gave rise to the best responses towards callus growth. These results are
accordance with those reported by Bekheet et al. (2014). They mentioned that addition of 3
mg/l picloram to culture medium registered the highest results of growth value of Globe artichoke and
Milk thistle calli. In this respect, Genady (2017) found that supplementation of culture
medium with 2 mg/L of picloram was the best concentration for degree of callus formation
and callus induction percent of Verbena bipinnatifida Nutt. However, Tanur Erkoyuncu et
al. (2017) found that After four weeks of culture, the highest callus induction rate (100%) of
Burak and Şafak maize cultivars was obtained in the Şafak, medium containing 8 mg/L of
Picloram.
Table (1). Growth ratio of callus derived from leaf explants of Globe artichoke and Milk
thistle grown for five weeks o various concentrations of picloram.
Milk thistleGlobe artichoke
Picloram
(mg/l)
4.
2
3.
5
0.0
5.
4
5.
5
0.1
6.
7
6.
3
0.2
7.
0
6.
7
0.3
6.
5
5.
6
0.4
4- Effect of chitosan and methyl jasmonate on cynarin accumulation
Cynarin contents of callus cultures Globe artichoke affected by chitosan and methyl
jasmonate are presented in Table (2). Generally, cynarin accumulation increased by
elicitation with the two elicitors. Moreover, the levels of cynarin in callus cultures treated
with methyl jasmonate were higher than those treated with chitosan. The highest value of

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cynarin content (60.4 µg/mg) was registered with 40 mg\l methyl jasmonate containing
medium.
Table (2). Effect of different concentrations of chitosan and methyl jasmonate on cynarin
accumulation in callus cultures Globe artichoke.
Treatment Cynarin (µg/mg)
Control
12.7
200 mg
\
l Chitosan
22.3
400 mg
\
l Chitosan
33.3
800 mg
\
l
Chitosan
29.8
20 mg
\
l Methyl jasmonat
e
49.6
40 mg
\
l Methyl jasmonat
e
60.4
80 mg
\
l Methyl jasmonat
e
52.9
5- Effect of chitosan and methyl jasmonate on silymarin accumulation
To improve the phenolic compounds production particularly silymarin in Milk thistle
tissue cultures, calli were treated with different concentration of chitosan, and methyl
jasmonate. The cultures showed increasing of silymarin compositions as well as the total
silymarin by treating with different concentrations of the two elicitors. It was noticed that
methyl jasmonate was more effective on silymarin accumulation compared with chitosan.
The highest value (23.41 mg/g dry weight)) of total silymarin was registered with 20 mg\l
methyl jasmonat (Table 3). However, 200 mg\l chitosan containing medium gave highest
silybine B (1.08 mg/g dry weight)) contents.

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Table (3). Effect of chitosan and methyl jasmonat on accumulation of silymarin (mg/g
dry weight) in callus cultures of Milk thistle.
Treatment
Silychrisrin Silydianin
Silybine A Silybine B
Total
Silymarin
Control
0
0
0
0
0
200 mg
\
l Chitosan
6.88
8.18
0.21
1.08
16.34
400 mg
\
l Chitosan
7.39
8.38
0.04
0.04
15.85
800 mg
\
l Chitosan
3.88
2.33
0.06
0
6.27
20 mg
\
l Methyl jasmonat
e
11.31
3.59
8.49
0.01
23.41
40 mg
\
l Methyl jasmonat
e
11.04
3.54
0.01
0.71
15.30
80 mg
\
l Methyl jasmonat
e
5.72
0.52
0.02
0.23
6.48
The use of biotic and abiotic elicitors substances that can induce plant defense
responses, is one of the methods used to increase the yields of plant secondary metabolites
by in vitro cultures (Eilert, 1987; Bohlmann and Eilert, 1994). Jasmonates (jasmonic
acid and its related compounds) are generally considered to modulate many
physiological event s in higher plants, e.g. flowering and senescence, and are regarded as
a class of phytohormones and it is also a signal molecule in elicitation process (Gundlach et
al., 1992). Otherwise, chitosan elicitation proved to be one of the most effective strategies to
enhance the production of bioactive compounds in plant tissue cultures (Tocci et al., 2011;
Yin et al., 2012).
Our results of chitosan, and methyl jasmonate as elicitors used in callus cultures of
Globe artichoke and Milk thistle showed increasing in cynarin and silymarin production.
Methyl jasmonate had more positive effect on the interested compounds compared with
chitosan. From the results presented, it is concluded that feeding a medium with chitosan,
and methyl jasmonate offers the possibility to enhance the content of some components of

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cynarin and silymarin extracted from Globe artichoke and Milk thistle respectively. Various
additives have so far been reported to favor the growth of S. marianum L. cells toward
silymarin production. It was found that the use of elicitor such as methyl jasmonate, salicylic
acid, and yeast extract alone or in combination and manipulation of culture medium could
improve the production of silymarin dramatically (Cacho and Moran, 1999; Hasanloo et
al., 2009). Sanchez-Sampedro et al. (2005) mentioned that one of the jasmonic acid
derivatives, methyl jasmonate (MeJA), strongly promoted the accumulation of silymarin.
Methyl jasmonate acted in a number of steps of the metabolic pathway of flavonolignans and
its stimulating effect was totally dependent of “de novo” protein synthesis. Otherwise,
Chitosan was found to enhance secondary metabolite production in cell suspensions and calli
of various species (Dörnenburg and Knoor 1994; Tumová and Backovská 1999; Putalun
et al. 2007).
Acknowledgement
This research was supported by National Research Center (NRC), Dokki, 12622, Giza, Egypt, through in
house projects of NRC (The Ninth Research Plan, 2010-2013). I am thankful to all project team who
provided expertise that greatly assisted the research.

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