Industrial Crops & Products 129 (2019) 525–535

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Industrial Crops & Products

journal homepage: www.elsevier.com/locate/indcrop

Effects of chitosan and salicylic acid on the production of pharmacologically T

attractive secondary metabolites in callus cultures of Fagonia indica
⁎
Taimoor Khana, Tariq Khana, Christophe Hanob, Bilal Haider Abbasia,b,c,
a
Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan
b
Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC EA1207), INRA USC1328, Plant Lignans Team, Université d’Orléans, F 28000 Chartres, France
c
EA2106 Biomolecules et Biotechnologies Vegetales, Universite de Tours, Tours, France

A R T I C LE I N FO A B S T R A C T

Keywords: Fagonia indica is valuable for its multiple uses such as anticancer, antimicrobial and antioxidant throughout the
Callus culture world. Various biotechnological approaches need to be employed for the sustainable production of plant biomass
Phenolics and its phytochemical content. Elicitation has been shown to be a very potent tool for enhanced production of
Flavonoids secondary metabolites in different in vitro cultures. The current study involves the application of various con-
Antioxidant
centrations of chitosan and salicylic acid as elicitors in callus cultures keeping Thiadizuron induced callus as a
Elicitation
control. The main aim was to enhance the accumulation of biomass and secondary metabolite contents. The
results show that maximum biomass; Fresh weight (FW: 38.0 g/100 mL) and secondary metabolites [Phenolic
content (16.9 μgGAE/mg; Flavonoid content (2.2 μgQE/mg)] were observed in CHT treated cultures. The SA
treated calli also showed better accumulation of biomass (FW: 37.5 g/100 mL) and phytochemicals [Phenolic
content (12.29 μgGAE/mg); Flavonoid content (1.73 μgQE/mg)] as compared with control. The antioxidant
potential was found in correlation with the production of PC/FC and found higher in CHT (94.3%) cultures than
SA (90.2%) and control. For instance, the antioxidative enzyme activities [peroxidase (POD: 4.41) and super-
oxide dismutase (SOD: 0.42 nM/min/mg FW)] were found optimum in CHT treated samples. HPLC analyses
revealed significant accumulation (5.5 ± 0.004 μg/mg DW) and (5.2 ± 0.001 μg/mg DW) of pharmacologi-
cally active components in CHT and SA treated samples, respectively. The results showed that both elicitors have
potential to enhance the biomass accumulation and polyphenols in callus cultures of Fagonia indica.

1. Introduction include flavonoids, phenolics, alkaloids, coumarins, terpenoids, sapo-

Fagonia indica commonly known as “Sacchi boti” belongs to the
family Zygophyllaceae. The species mostly grows on sandy warm lands
and found in various parts of the world like Pakistan, India,
Afghanistan, Egypt, UAE and Africa (Beier, 2005; Rizvi et al., 1996). It
is medicinally vital in traditional therapies that include digestive pro-
blems, fever, and urinary illnesses while liquid potion is famous remedy
to treat tumors mostly breast cancer in preliminary periods (Farheen
et al., 2015; Quattrocchi, 2012). Moreover, it is also employed as a
tonic in liver ailments, dropsy, and smallpox. The hotly oiled decoction
is prescribed to stimulate abortion (Pareek et al., 2013; Waheed et al.,
2012). Other main uses of F. indica are sedative, anti-malignant, anti-
microbial, antidiabetic, anti-inflammatory, antioxidant, antipyretic and
hepatoprotective (Alqasoumi et al., 2011; Lam et al., 2012; Rasool
et al., 2014; Saleem et al., 2014; Waheed et al., 2012).
These activities are due to medicinally important compounds that
nins, glycosides, sterols, ß-carboline alkaloids, coumarins and rare
elements (Ebrahimi and Payan, 2013; Eman, 2011; Qureshi et al.,
2016). Shehab et al. (2015) stated that F. indica has reasonably rich
amounts of flavonoids and other polyphenols to other vascular plants.
The bioactive role of phenolics and flavonoids are linked with their
antioxidant potency (Ghasemzadeh et al., 2010). The antioxidants play
a vital protective role in stress conditions as part of the defense system.
The enzymatic and non-enzymatic defense systems have been evolved
in plants for scavenging of reactive oxygen species (ROS) that includes
superoxide dismutase (SOD), and peroxidase (POD) (Lee and Lee, 2000;
Zhang et al., 1995). While the non-enzymatic antioxidants are low
molecular weight quenchers such as ascorbic acid (Noctor and Foyer,
1998; Wingsle and Hallgren, 1993).
Plant in vitro culture is an attractive alternative technology for
enhancing secondary metabolites that are either difficult to synthesize
chemically or produced in limited quantities in wild plants (Kolewe

⁎
Corresponding author at: Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan.
E-mail address: bhabbasi@qau.edu.pk (B.H. Abbasi).

https://doi.org/10.1016/j.indcrop.2018.12.048
Received 1 April 2018; Received in revised form 11 December 2018; Accepted 13 December 2018
Available online 19 December 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
T. Khan et al.
et al., 2008). The chemical synthesis of these compounds is difficult or
expensive. Plants have biologically active phytochemicals which are
responsible for their medicinal values. Phytochemicals in plants are
produced in response against some stress (Ahmad et al., 1998; Dubey
et al., 2004; Duraipandiyan et al., 2006; Joshi et al., 2009). Naturally,
plants produce phytochemicals in response to internal and external
stimuli, but the biosynthesis of these chemicals is low or confined to
specific parts (Kolewe et al., 2008). Plant cell and tissue culture is an
efficient technology for sustainable production but still, the issue of
production exists. For instance, the issues can be resolved by employing
agents i.e. elicitors is a feasible solution to enhance the yield of these
compounds (Teoh, 2016). Elicitation is an efficient strategy to promote
plants defense mechanism for more production of phytochemicals from
in vitro cultures. It is also adapted to determine the role of various
agents on plants by using in vitro cell culture technology (Yang and
Stöckigt, 2010; Yue et al., 2016; Zhao et al., 2005). Research from our
group has explored different strategies to elicit the production of dif-
ferent secondary metabolites in various in vitro cultures of F. indica. For
instance (Khan et al., 2017) explored the used of Methyl Jasmonate and
phenyl acetic acid as elicitors for the enhanced production of phenolic
compounds in adventitious root cultures of F. indica. Similarly, in a
recent study, (Khan et al., 2018) reported the use of various types of
carbohydrates for the enhancement of phenolic compounds in callus
cultures of F. indica.
There is a broad range of agents used as elicitors, generally cate-
gorized into two groups; biotic and abiotic elicitors. Abiotic elicitors are
of non-living or non-biological nature or origin and further grouped
into physical, hormonal and chemical factors (light, osmotic stress,
salinity, drought stress, thermal stress, salicylic acid, Jasmonate, and
gibberellic acid) (Jochum et al., 2007; Liang et al., 2013; Liu et al.,
2011; Munns and Tester, 2008; Vasconsuelo and Boland, 2007; Zhu
et al., 2009) while the biotic elicitors are biological substances or
agents, compounds from cell wall (microbes or plants) that includes
CHT, Chitin, Polysaccharides, yeast extracts, etc (Naik and Al-Khayri,
2016). Elicitor specificity, elicitor concentration, nutrient composition,
elicitor exposure duration and age of culture are some factors which can
influence elicitation (Awad et al., 2014; Chodisetti et al., 2015).
Due to overexploitation from natural habitats, the inconsistency of
chemical constituents and adulteration with other species, F. indica
needs proper attention and continuous platforms for its production
(Khan et al., 2015). In the current work, we have applied elicitors in
callus culture with the aim to establish a strategy for enhancement in
the accumulation of growth and commercially vital metabolites in
callus cultures of F. indica.
2. Materials and methods
2.1. Seed germination
The F. indica seeds were collected from the wild habitat of Khyber
Pakhtunkhwa and properly identified at Quaid-i-Azam University
Islamabad, Pakistan. The slightly modified protocol of Abbasi et al.,
2010 was followed to sterilized seeds. Briefly, the seeds were rinsed
with distilled water followed by drenched in mercuric chloride (0.1%)
for 40 s and ethanol (70%) for 2 min. The seeds were washed 3–4 times
with distilled water to remove ethanol traces and moved to the ster-
ilized filter paper.
The seeds were then inoculated on MS0 (Murashige and Skoog basal
medium; Phytotechnology Labs, USA; Murashige and Skoog, 1962)
supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar
(Phytotechnology Labs, USA), pH: 5.6-5.7. Later, the flasks were shifted
to a growth chamber having temperature 25 ± 1̊C and light intensity
of ∼45 to 50 μmol m − 2 s−1 for 16/8 (L/D) photoperiod.
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2.2. Callus establishment
To induce callogenesis and obtain sufficient amount of callus, the
stem explant (0.1 cm) from 28 days old seed germinated plantlets were
inoculated on Murashige and Skoog (MS) (1962) media having 3%
sucrose and 0.8% agar, supplemented with TDZ (4.4 μM) as optimized
previously by Tariq et al., (2015). Prior to autoclave media (121 °C,
20 min, Systec VX 100, Germany), the pH of the medium was adjusted
to 5.6 (Eutech Instruments pH 510, Singapore) using NaOH (0.1 N) and
HCl (1.0 N). The cultures were shifted and maintained at temperature
25 ± 1 °C under 16/8 h (Light/Dark) photoperiod with light intensity
of 40–50 μmol m−2 sec-1 (Philips TLD 35 Florescent lamps).
2.3. Callus establishment with elicitors
2.3.1. Preparation of elicitors
CHT was prepared by dissolving in 0.1 M acetic acid. The reaction
mixture was stirred continuously at 60ᴼC for 5 h to dissolve properly.
The salicylic acid (1 mM) was prepared by dissolving in distilled water,
followed by continuous stirring for 1 h for proper mixing of SA.
2.3.2. Callus inoculation
The calli (0.5 g FW) were cultured on MS (Murashige and Skoog
1962) media containing 3% sucrose, 0.8% agar, fortified with various
concentrations of CHT (6.5, 13.10, 32.75, 65.50, 131.20 μM) and
Salicylic acid (10, 20, 40, 80, 160 μM). The cultures were shifted to
growth room and maintained properly at temperature 25 ± 1 °C under
16/8 h (Light/Dark) photoperiod with light intensity of 40–50 μmol
m−2 sec-1 (Philips TLD 35 Florescent lamps). The calli samples of each
elicitor treatment were harvested after 7 days for total 7 weeks. The
calli morphology and other dimensions were recorded. Furthermore,
the calli were oven dried overnight after fresh weight and samples were
stored for further analysis.
2.4. Sample extraction
The protocol of Ahmad et al., (2010) was used to prepare metha-
nolic extracts of calli. Briefly, finely ground samples (100 mg) were
mixed in 10 mL methanol (80%), followed by sonication (10 min;
Toshiba, Japan) and vortexing (20 min) for proper mixing and extrac-
tion. The procedure was repeated three times and then the mixtures
were centrifuged at 13,000 rpm for 15 min. The supernatants were
taken and stored at 4ᴼC for further analysis.
2.4.1. Total phenolic contents
To determine the total phenolic contents, Folin-Ciocalteu reagent
was used according to the protocol of Slinkard and Singleton (1977)
with slight modifications. Briefly, the Folin-Ciocalteu reagent (90 μL)
was added each well containing 20 μL of the samples in 96 well plates.
Then sodium carbonate (90 μL) was added from the 6% stock solution
to the wells and kept plates at room temperature for 90 min under
normal room light. Gallic acid (1 mg/mL) and methanol (20 μL) were
used as a positive and negative control, respectively. The absorbance
was measured at 725 nm by using UV/VIS-DAD spectrophotometer
(Halo DR-20, UV-VIS spectrophotometer, Dynamica Ltd, Victoria,
Australia). The calibration curve (0–50 μg/mL, R2 = 0.968) was plotted
by using gallic acid as standard and the TPC was expressed as gallic acid
equivalents (GAE)/g of dry weight. Total phenolic production (TPP)
was measured by using the following formula and estimated in mg
GAE/L.
Total phenolic production mg/L = Dry weight (g/L)×Total phe-
nolic contents (mg/g).
2.4.2. Total flavonoid contents
For determination of total flavonoid contents, the aluminium
chloride the following method was used as described by Chang et al.
T. Khan et al. Industrial Crops & Products 129 (2019) 525–535

Fig. 1. Variations in callus color and morphology under different concentrations of chitosan and salicylic acid after 28 days of cultures.

Table 1
The effects of elicitors (Chitosan and Salicylic acid) with varying levels (4.5–160 μM) on certain growth parameters and phytochemicals accumulation in F. indica
callus cultures. Values represent mean of three replicates ± standard error (SE).
Elicitor Initiation day Callus dimensions Maximum biomass Optimum values Color/
(g/100 mL) Texture

Type Concentration Max.Width (cm) Max.Height (cm) FW DW TPC TFC DPPH

(%) (g/100 mL) (g/100 mL) (μgGAE/g DW) (μgQE/g DW) (%)

TDZ (Cont) 4.54 μM 3rd 48 ± 0.01 18 ± 0.41 17.84 ± 0.06 0.81 ± 0.01 7.66 ± 0.02 1.16 ± 0.04 83.5 ± 0.01 GB/C
Chitosan 6.5 μM 3rd 34 ± 0.03 10 ± 0.33 15.18 ± 0.32 0.71 ± 0.02 12.84 ± 0.04 1.15 ± 0.02 83.2 ± 0.02 LG/C
13.10 μM 5th 30 ± 0.40 12 ± 0.23 13.91 ± 0.29 0.66 ± 0.02 10.83 ± 0.02 1.19 ± 0.06 83 ± 0.03 LG/C
32.75 μM 4th 35 ± 0.02 14 ± 0.22 18.46 ± 0.33 0.83 ± 0.03 11.22 ± 0.02 1.16 ± 0.05 88.4 ± 0.02 G/C
65.50 μM 4th 51 ± 0.12 20 ± 0.19 28.74 ± 0.48 1. 49 ± 0.04 13.87 ± 0.09 1.83 ± 0.06 92.9 ± 0.04 GB/C
131.02 μM 3rd 49 ± 0.05 18 ± 0.05 15.82 ± 0.23 0.74 ± 0.012 11.61 ± 0.31 1.15 ± 0.02 87.6 ± 0.01 GB/C
Salicylic Acid 10 μM 4th 48 ± 0.01 20 ± 0.30 24.58 ± 0.19 1.25 ± 0.04 8.93 ± 032 1.51 ± 0.01 82.1 ± 0.01 DG/C
20 μM 4th 51 ± 0.01 22 ± 0.04 25.16 ± 0.29 1.33 ± 0.018 9.04 ± 0.1 1.62 ± 0.01 90.8 ± 0.04 G/C
40 μM 5th 35 ± 0.03 16 ± 0.02 17.76 ± 0.58 0.95 ± 0.02 7.26 ± 0.03 1.19 ± 0.02 85.1 ± 0.05 GB/C
80 μM 5th 28 ± 0.04 10 ± 0.01 9.78 ± 0.27 0.67 ± 0.03 7.27 ± 0.4 1.21 ± 0.02 80 ± 0.02 GB/C
160 μM 6th 25 ± 0.03 10 ± 0.03 9.08 ± 0.23 0.59 ± 0.023 7.07 ± 02 1.28 ± 0.03 82 ± 0.03 GB/C

FW = Fresh Weight, DW = Dry Weight; GB = Green Brown, DG = Dark Green; LG = Light Green, LB = Light Brown, G = Greenish, TPC = Total Phenolic contents,
TFC = Total Flavonoid contents.

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Fig. 2. Variations in color and morphology of weekly harvested callus cultures of F. indica grown in chitosan and salicylic acid (Comparative cultures shown in rows
and weekly in columns).

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T. Khan et al.
Fig. 3. Fresh weight growth kinetics of F. indica callus cultures elicited with
chitosan 65.5 μM and salicylic acid 20 μM Values represent means ± standard
errors from triplicates.
Fig. 4. Dry weight growth kinetics of F. indica callus cultures elicited with
chitosan 65.5 μM and salicylic acid 20 μM. Values represent means ± standard
errors from triplicates.
(2002). Briefly, the 10% aluminium trichloride solution (10 μL) and
potassium acetate (10 μL) were added to the reaction well containing
20 μL of the samples. The final volume was adjusted to 200 μL by ad-
dition of 160 μL dH₂O and kept for 30 min at room temperature. The
absorbance of the reaction mixtures was measured at 415 nm by using
UV/VIS-DAD spectrophotometer. The calibration curve (0–40 μg/mL,
R2 = 0.998) was plotted by using quercetin as standard. The TFC was
expressed as mg Quercitin Equivalent (QE)/g of dry weight. Total fla-
vonoid production (TFP) was calculated by using the following formula
and expressed in mg QE/L.
Total flavonoid production mg/L = Dry weight (g/L)×Total
Flavonoid Contents (mg/g)
2.4.3. DPPH activity
The 2, 2-diphenyl-1-picrylhydrazyl (DPPH), Free Radicals
Scavening Activity (FRSA) was employed to determine the antioxidants
activity of cultures by using the modified method of Amarowicz et al.
(2004). Briefly, the 180 μl DPPH reagent was diluted with 20 μl of ex-
tract sample and placed in the dark for an incubation time of 1 h at
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Industrial Crops & Products 129 (2019) 525–535
room temperature. The ascorbic acid concentrations (5, 10, 20 and
40 μg/mL) and DMSO (20 μl) with DPPH (180 μl) were used as positive/
negative controls, respectively. The absorbance of the reaction mixture
was measured at 517 nm with a spectrophotometer.
Finally, the FRSA activity was calculated and expressed as percent
DPPH discoloration by using formula;
% scavenging DPPH free radical = 100 × (1-AE/AD)
Where AE denotes absorbance of the mixture at 517 nm with sample
addition and AD shows the absorbance of the DPPH solution without
adding anything.
2.5. Peroxidase (POD) activity
2.5.1. Extraction of samples
The Nayyar and Gupta (2006) described protocol was employed for
sample extraction with little modifications. Briefly, 100 mg of the fresh
sample was homogenized with 1 mL potassium phosphate buffer
(50 mM, pH 7) containing 1% PVP. The resultant mixture was cen-
trifuged at 15,000 rpm for 30 min; the supernatant was taken for further
analysis.
The POD activity was performed by employing the method
Lagrimini (1980) with minor modifications. The reaction mixture was
prepared by taking 40 μl potassium phosphate buffer (50 mM; pH 7),
20 μl guaiacol (100 mM; 10x), 20 μl fresh sample extract, 100 μl dH2O
and 20 μl H2O2 (27.5 mM; 10x). The control was taken by applying
equal amounts of all the reagents except sample extract. After 20 s of
the incubation period, the absorbance was recorded at 470 nm through
(Thermo Scientific Multiskan GO) and the enzymatic activity was
measured using the formula given below:
A = ELC
A denotes Absorbance, E = Extinction coefficient (6.39
mM−1 cm−1), L = Length of wall (0.25 cm), C = enzyme concentration
(value calculated in nM/min/mg FW) and FW represent fresh weight of
sample.
2.5.2. Superoxide dismutase (SOD) activity
An updated protocol of Ullah and Alamri (2013) was employed to
perform the SOD assay. Briefly, the activity was done by mixing 20 μl
EDTA (1 mM), 20 μl methionine (130 mM), 20 μl NBT (0.75 mM), 78 μl
phosphate buffer (50 mM, pH 7), 2 μl riboflavin (0.02 mM) and 60 μl
fresh sample extract. The same mixture excluding extract samples was
run as a control. After 7 min of incubation period under fluorescent
light, the absorbance was measured at 660 nm with a microplate reader
(Thermo Scientific Multiskan GO). In order to measure the enzymatic
activity, the same formula was used as for POD.
A = ELC (given above)
2.6. HPLC quantification
Important phenolic and flavonoids compounds were quantified
through Varian HPLC system equipped with an online degasser
(Metachem Degasit), an autosampler (Prostar 410) and a photodiode
array detector (PDA, Prostar 335) by adopting the method described
previously by other authors (Bourgeois et al., 2016; Wang et al., 2015)
with minor modifications. The comparative standards: apigenin, ca-
techin, kaempferol, isorhamnetin, and myricetin were purchased from
PlantMetaChem, Germany; caffeic acid, gallic acid, hederagenin, ur-
solic acid and betulinic acid were purchased from Sigma-Aldrich
Company, France.
The separation was performed at 35 °C on an RP-18 column
(250 x 4.0 mm id, 5 μm; Purospher Merck). The mobile phase was
composed of acetonitrile (solvent A) and 0.1% (v/v) formic acid acid-
ified ultrapure water (solvent B). The composition of the mobile phase
varied during a 1-hour run according to a linear gradient ranging from a
T. Khan et al. Industrial Crops & Products 129 (2019) 525–535

Table 2
Kinetics of various growth parameters in callus cultures of F. indica. Values represent mean of three replicates ± standard error (SE).
Weeks Elicitor used Callus Dimensions Characteristics/ Morphology

Type Concentrations Max.Width Max.Height Color Texture

Used (mm) (mm)

W1 (TDZ) 4.54 μM 12 04 DG C
Chitosan 65.50 μM 15 05 GB C
Salicylic Acid 20 μM 12 05 GB C
W2 TDZ 4.54 μM 21 08 LG C
Chitosan 65.50 μM 45 12 LG C
Salicylic Acid 20 μM 15 12 DG C
W3 TDZ 4.54 μM 45 09 LG C
Chitosan 65.50 μM 50 15 DG C
Salicylic Acid 20 μM 52 15 DG C
W4 TDZ 4.54 μM 49 14 DG C
Chitosan 65.50 μM 52 18 G C
Salicylic Acid 20 μM 48 19 G C
W5 TDZ 4.54 μM 52 20 G C
Chitosan 65.50 μM 51 20 DG C
Salicylic Acid 20 μM 51 22 DG C
W6 TDZ 4.54 μM 56 22 LG C
Chitosan 65.50 μM 55 25 DG C
Salicylic Acid 20 μM 51 24 DG (p) C
W7 TDZ 4.54 μM 55 22 GB C
Chitosan 65.50 μM 58 25 GB C
Salicylic Acid 20 μM 51 22 DG (p) C
W8 TDZ 4.54 μM 45 20 GB F
Chitosan 65.50 μM 50 25 GB C
Salicylic Acid 20 μM 51 22 LG (p) C

FW = Fresh Weight, DW = Dry Weight; GB = Brown Green, DG = Dark Green; LG = Light Green, LB = Light Brown, G = Greenish, C = Compact, F = Friable.

Fig. 5. Accumulation of total phenolic contents under elicitation with chitosan Fig. 6. Effects of chitosan 65.5 μM and salicylic acid 20 μM on total phenolic
65.5 μM and salicylic acid 20 μM in callus cultures of F. indica on distinct production in callus cultures of F. indica. Values represent means ± standard
growth periods. Values represent means ± standard errors from triplicates. errors from triplicates.

5:95 (v/v) to 100:0 (v/v) mixture of solvents A and B, respectively, at a
flow rate of 0.6 mL/min. Detection was performed at 210 and 254 nm.
Compounds were identified by comparison with authentic standards
and standard additions. All the compounds were quantified against 5-
point calibration curves (R2 > 0.999). Nahagenin was quantified as
hederagenin equivalent. All the samples analysis was carried out in
triplicates and the results were expressed as μg/mg DW of the sample.
2.7. Experimental design and data analysis
All the experiments were conducted in triplicates and were repeated
twice. In each experiment, the mean value was found and standard
error was calculated to formalize statistical analysis (Microsoft Excel
Program). Data were shown as mean and ± SEM.
3. Results and discussion
3.1. Preliminary optimization
Sustainable production of phenolics and flavonoids in response to
elicitors may depend on a variety of factors including cultures’ age, a
dose of elicitor and contact time (Murthy et al., 2014; Namdeo, 2007).
The optimum biomass (FW = 28 g/100 mL; DW = 1.492 g /100 mL)
was recorded on MS media fortified with 65.50 μM CHT while lower
biomass (FW = 13.9 g/100 mL, DW = 0.660 g/100 mL) was found on
13.10 μM CHT treatment with green and green brownish compact
features (Fig. 1). As compared to the callus cultures grown under the

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T. Khan et al.
Fig. 7. Accumulation of total flavonoids contents under elicitation with chit-
osan 65.5 μM and salicylic acid 20 μM in callus cultures of F. indica on distinct
growth periods Values represent means ± standard errors from triplicates.
Fig. 8. Effects of chitosan 65.5 μM and salicylic acid 20 μM on total flavonoids
production in callus cultures of F. indica. Values represent means ± standard
errors from triplicates.
effect of TDZ, the callus grown under the effect of different con-
centrations of SA and CHT shows variety both in color and yield of
callus. SA also stimulated biomass accumulation (FW = 25 g/100 mL;
DW = 1.325 g /100 mL) at concentration of 20 μM. The higher con-
centrations of SA were found to be inhibitory for callus proliferation.
This could be due to stress induced by higher concentrations of SA
(Abbasi et al., 2011; Fazal et al., 2016). The characteristic parameters
(callus height and width) were also investigated. Both CHT and SA
enhanced these parameters as compared with control. Furthermore, the
optimum TPC (13.87 μg/mg) and TFC (1. 826 μg/mg) were recorded in
callus grown on CHT (65.50 μM).
The results showed that better DPPH activity was observed for CHT
(92.9% at 65.50 μM) treated cultures than SA (90.8% at 20 μM) treated
cultures and control (83.5%). Overall, the CHT and salicylic acid
treatments (CHT: 65.50 μM, SA: 20 μM) produced better results than
controls (Table 1) (Fig. 2).
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Fig. 9. Comparison between chitosan (65.5 μM) and salicylic acid (20 μM)
impacts over fresh biomass accumulation and Total phenolics production in
callus cultures of F. indica, Values represent means ± standard errors from
triplicates.
Fig. 10. Comparison between chitosan (65.5 μM) and salicylic acid (20 μM)
impacts over Dry biomass accumulation and Total phenolics production in
callus cultures of F. indica. Values represent means ± standard errors from
triplicates.
3.2. Biomass production and growth kinetics
In present study, the optimum biomass accumulation was recorded
for 7th week of cultures grown on CHT (FW: 38.0 g/100 mL; DW: 1.8 g/
100 mL) or SA supplemented media (FW: 37.5 g/100 mL, DW: 1.6 g/
100 mL) than control (FW: 34.7 gm/100 mL; DW: 1.3 gm/100 mL;
Figs. 3 and 4). Contrarily, SA reported inhibiting biomass accumulation
in some other plant species (Chaichana and Dheeranupattana, 2012).
Bulgakov et al. (2002) revealed that higher concentrations of SA put
significant stress on growth parameters of cells which results in lower
biomass accumulation in Rauwolfia cordifolia callus culture (Bulgakov
et al., 2002). Nevertheless, negligible effects of SA were observed on
cell growth and biomass accumulation in grape and Rosa hybrida callus
cultures (Ali et al., 2012; Obinata et al., 2003; Ram et al., 2013).
Other parameters were also found the optimum in CHT cultures
(H = 51.20 mm; W = 20 mm) than salicylic acid (H = 51 mm,
W = 22 mm) on week 7th (Table 2). Mostly, compact calli with green,
green brown texture were observed in CHT and controls. However, SA-
mediated calli showed dark reddish patches (Fig. 2). The appearance of
dark reddish colors on cultures in SA mediated cultures could be due to
T. Khan et al.
Fig. 11. Free radical scavenging activity (FRSA) in callus cultures of F. indica
treated with chitosan (65.50 μM) and salicylic acid (20 μM). Values represent
means ± standard errors from triplicates.
Fig. 12. Peroxidase activity in callus cultures of F. indica treated with chitosan
(65.50 μM) and salicylic acid (20 μM). Values represent means ± standard
errors from triplicates.
the possible biosynthesis of anthocyanins under stress. Anthocyanins
are glycosylated, water dissolving coloring pigments far apportioned in
plants that give colors from orange to blue covering red, pink, purple
(Appelhagen et al., 2018). In plants under stress, the accumulation of
secondary metabolites raises as of increase photosynthesis and pre-
dominant carbon fixation towards secondary metabolism (Seigler,
1998). This stress exerts markable impacts over phytochemicals levels
e.g. phosphate and nitrogen deficiencies enhance the lignification and
phenylpropanoids accumulation(Chalker-Scott, 1989; Dixon et al.,
2002; Dixon and Paiva, 1995). SA plays a significant role throughout
the plant’s life cycle in the regulation of vital plant biochemical and
physiological processes (Rivas-San Vicente and Plasencia, 2011). The
current study is in correlation with previous reports (Sudha and
Ravishankar, 2003; Xing et al., 2011; Xu et al., 2012). Furthermore, SA
mediated accumulation of anthocyanins is previously reported
(Rajendran et al., 1992; Sudha and Ravishankar, 2003).
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Industrial Crops & Products 129 (2019) 525–535
Fig. 13. Superoxide dismutase activity in callus cultures of F. indica treated
with chitosan (65.50 μM) and salicylic acid (20 μM). Values represent means
± standard errors from triplicates.
3.3. Total phenolic and flavonoid content
Plant phenolic compounds are one of the most studied classes of
compounds because of their enormous potential in providing defense
against pathogenic attack, as signaling molecules and regulating vital
biochemical processes including antioxidant activity (Karabourniotis
et al., 2014, 2001). The phenolic and flavonoid contents were analyzed
in all cultures harvested weekly for a total period of 8 weeks. In case of
CHT (65.50 μM) the optimum level (PC: 16.9, PP: 28.6 μg/mg) was
observed at 6th/7th week while in SA (20 μM) derived calli the pro-
duction (PC: 12.29, PP: 19.9 μg/mg) was optimum on 7th week, as
compared with control (Figs. 5 and 6). The reason for the higher re-
sponse in case of CHT compared with SA might be the fact that SA is a
plant growth regulator and exogenous application of SA promotes pri-
mary growth. While CHT elicits plant defense response and thus trigger
secondary metabolism that ultimately produces higher quantities of
phenolic compounds compared to SA. The flavonoids content and fla-
vonoids production were also investigated and the results showed that
the highest production of FC and FP (2.2, 3.8 μg/mg) was recorded in
CHT (65.50 μM) cultures at 6th week, followed by FC and FP (1.7,
2.9 μg/mg) in cultures derived at SA (20 μM) as compared with control
(Fig. 7 and 8). A positive correlation was found in biomass accumula-
tion, phenolic production and flavonoid production in both types of
cultures (Figs. 9 and 10).
Induction of plant defense mechanism by CHT could be the cause of
the enhanced production of phenolic and flavonoids by mimicking
naturally occurring fungal pathogenicity (Iriti and Faoro, 2009). CHT
also affect plant signal transduction pathways including nitric oxide
signals and phenylalanine ammonia lyase and its direct interaction with
chromatin may help in gene expression control. CHT and SA play a vital
role in the enhanced production of phenolic and flavonoid compounds
and plant endogenous enzymes such as SOD which helps in deactiva-
tion of the harmful effects of ROS (Fazal et al., 2016; Valko et al., 2007).
3.4. DPPH activity
Plants balance their production by quenching free radicals with
phytochemicals that work as antioxidants (Chen et al., 2009; Pinto
et al., 2005; Zheng et al., 2014). One of the leading class of these
compounds acting as free radical scavenging molecules is phenolic
compounds (Sánchez‐Moreno et al., 1998). To measure the antioxidant
activity, the DPPH, as a free radical, is typically employed, followed by
examining sample for its percentage of scavenging free radicals (Abbasi
T. Khan et al.
Table 3
HPLC-quantification of various medicinally vital phenolic and flavonoid compounds in F. indica callus culture. Values are the mean of three triplicates
± standard error (SE).
Quantity
(μg/mg DW of Sample)
TDZ (4.54 μM)
Gallic Acid 0.067 ± 0.002
Caffeic Acid 0.089 ± 0.0008
Myricetin 0.471 ± 0.001
Catechin 0.471 ± 0.001
Kaempherol 0.489 ± 0.0006
Isorhamnetin 0.489 ± 0.0006
Apigenin 0.525 ± 0.001
Nahagenin 0.198 ± 0.001
Hederagenin 0.029 ± 0.0006
Ursolic Acid 0.094 ± 0.001
Betulinic Acid 0.151 ± 0.002
Total Concentration 3.077 ± 0.009
et al., 2010). The optimum level of DPPH activity was detected in CHT
cultures (94.3%), SA (92.0%) treated cultures and controls (87.0%)
(Fig. 11). This is because phenolics and flavonoids are mostly re-
sponsible for antioxidant potential of plants (Fazal et al., 2016);
(Abraham et al., 2011). It was reported that SA stimulated some genes
of antioxidant enzymes such as SODs, catalases and ascorbate perox-
idases to protect cells in response to ROS (El-Esawi et al., 2017).
Moreover, high antioxidant activities of CHT and SA have been shown
in earlier reports (Abraham et al., 2011; Popova et al., 2009).
3.5. Peroxidase and superoxide dismutase activities
The SOD and POD are iso-enzymes, differentiated by various phy-
sical and chemical features and amino acid sequence but have the same
reaction to catalyze. The effects of CHT and SA were also analyzed on
enzymatic activities (SOD and POD) in callus cultures of F. indica. These
activities were found increasing from the 4th-7th week. The optimum
enzyme activities (POD: 4.41; ± 0.05; SOD: 0.42 ± 0.004 nM/min/mg
FW) were found in CHT derived cultures and SA (POD: 4.23 ± 0.03;
SOD: 0.37 ± 0.003 nM/min/mg FW) as compared with control (POD:
3.9 ± 0.025; SOD: 0.31 ± 0.002 nM/min/mg FW) (Figs. 12 and 13).
Previous studies reported that SA and CHT possibly enhanced SOD and
POD activities (Chong et al., 2005; Dang et al., 2010; Liu et al., 2007;
Yu et al., 1999). It shows the involvement of POD and SOD to mediate
CHT and SA-induced stress in callus cultures of F. indica (du Jardin,
2015; Zheng and Zhu, 2003). The findings of this study provide the
distinct role of CHT and SA with ROS for systemic signaling in plants to
cope with biotic and abiotic stress (Wasternack and Parthier, 1997). For
instance, both types of elicitors showed peak activity on 7th week that
shows a positive correlation with biomass and PC and FC.
3.6. HPLC analyses of callus culture of F. indica
CHT has been reported to stimulate the accumulation of alkaloids,
trans-resveratrol, and viniferins in Ruta graveolens and Vitis vinifera in
vitro cultures (Orlita et al., 2008; Singh et al., 2017; Taurino et al.,
2015). SA also has positive effects on plants metabolism in inducing
accumulation of the triterpenoids ginsenosides in ginseng and glycyr-
rhizin in licorice (Ali et al., 2012; Shabani et al., 2009). In the current
study, total 11 metabolites were quantified in callus cultures of F. in-
dica, fed with elicitors (Table 3). HPLC analysis showed that total
production of these metabolites was raised both by CHT
(5.507 ± 0.004 μg/mg) and salicylic acid (5.296 ± 0.001 μg/mg)
than controls (3.077 ± 0.009 μg/mg) (Table 3). The current data
shows enhancement of metabolites and is comparable with previous
reports. Optimum catechins concentrations were found
533
Industrial Crops & Products 129 (2019) 525–535
Chitosan (65.5 μM) Salicylic Acid (20 μM)
0.205 ± 0.001 0.065 ± 0.0003
0.189 ± 0.003 0.087 ± 0.0021
0.649 ± 0.007 1.697 ± 0.0008
1.129 ± 0.003 1.572 ± 0.0008
0.862 ± 0.002 0.525 ± 0.001
0.524 ± 0.005 0.418 ± 0.0003
0.987 ± 0.009 0.720 ± 0.003
0.114 ± 0.0034 0.085 ± 0.0017
0.104 ± 0.004 0.029 ± 0.0003
0.311 ± 0.003 0.076 ± 0.001
0.433 ± 0.004 0.021 ± 0.001
5.507 ± 0.004 5.296 ± 0.001
(1.129 ± 0.003 μg/mg) in CHT cultures than salicylic acid. Catechins
have adequate anti-oxidative and anti-cancerous activities (Lee and Lee,
2000; Li et al., 2000; Nanjo et al., 1996; Rice-Evans et al., 1997). The
current data is also in relation with recent studies reported on catechins
production in vitro, the dominant compounds were epicatechin and
epicatechin-3-O-gallate in calli of Uncaria elliptica and Fagopyrum es-
culentum, respectively (Moumou et al., 1992; Trotin et al., 1993). The
mechanistic approaches reported previously are, catechins induced
scavenging activities with dilated R-tocopherol and carotenes demo-
tions as well as lipid oxidation (Lotito and Frei, 2006). As for salicylic
acid derived cultures, myricetin (1.697 ± 0.0008 μg/mg) was the most
accumulated compound as compared with CHT and control. Myricetin
is an important flavonoid with strong anti-tumor, antioxidant and anti-
inflammatory properties and acts as antioxidants via affecting tran-
scriptional and enzymatic motions (Higdon and Frei, 2003). Moreover,
these phenolics and flavonoids are originated via phenylpropanoid
pathway and proclaimed with functions in photophosphorylation, oxi-
dative phosphorylation and provoking RNA construction (Khan et al.,
2015; Zaletok et al., 2015). The findings provide a strong strategy for
enhanced production of phenolics in callus cultures and elicitors
treatments.
4. Conclusion
Conclusively, the F. indica callus cultures showed enhanced pro-
duction of biomass and bioactive compounds with elicitors’ application.
The CHT derived callus cultures exhibited higher levels of phenolics
and flavonoids and antioxidants activity. The SA acid was also found to
stimulate biomass accumulation and phytochemicals biosynthesis. The
optimal production of metabolites detected via HPLC was in CHT eli-
cited cultures than salicylic acid and control. The findings of the current
study suggest that elicitors, especially CHT possess strong potential in
biomass production and enhancement of polyphenolic compounds in
callus culture.
Conflict of interest
Authors declare no conflict of interest
Contribution of authors
TK performed experiments, compile data and prepared manuscript,
BHA conceived the idea, supervised research and reviewed MS, TK
established and maintained these cultures, CH performed HPLC and
interpreted HPLC data
T. Khan et al.
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