Appl Biochem Biotechnol

DOI 10.1007/s12010-016-1978-y

Differential Effects of Thidiazuron on Production

of Anticancer Phenolic Compounds in Callus Cultures
of Fagonia indica
Tariq Khan 1 & Bilal Haider Abbasi 1,3 &
Mubarak Ali Khan 2 & Zabta Khan Shinwari 1,3

Received: 15 October 2015 / Accepted: 3 January 2016

# Springer Science+Business Media New York 2016

Abstract Fagonia indica, a very important anticancer plant, has been less explored for its in
vitro potential. This is the first report on thidiazuron (TDZ)-mediated callogenesis and
elicitation of commercially important phenolic compounds. Among the five different plant
growth regulators tested, TDZ induced comparatively higher fresh biomass, 51.0 g/100 mL
and 40.50 g/100 mL for stem and leaf explants, respectively, after 6 weeks of culture time.
Maximum total phenolic content (202.8 g gallic acid equivalent [GAE]/mL for stem-derived
callus and 161.3 g GAE/mL for leaf-derived callus) and total flavonoid content (191.03 g
quercetin equivalent [QE]/mL for stem-derived callus and 164.83 g QE/mL for leaf-derived
callus) were observed in the optimized callus cultures. The high-performance liquid chromatography (HPLC) data indicated higher amounts of commercially important anticancer secondary metabolites such as gallic acid (125.10 5.01 g/mL), myricetin (32.5 2.05 g/mL),
caffeic acid (12.5 0.52 g/mL), catechin (9.4 1.2 g/mL), and apigenin (3.8 0.45 g/mL).
Owing to the greater phenolic content, a better 2-2-diphenyl-1-picrylhydrazyl (DPPH) radicalscavenging activity (69.45 % for stem explant and 63.68 % for leaf explant) was observed in
optimized calluses. The unusually higher biomass and the enhanced amount of phenolic
compounds as a result of lower amounts of TDZ highlight the importance of this multipotent
hormone as elicitor in callus cultures of F. indica.
Keywords Callus . TDZ . Fagonia . Phenolic acids . Anticancer . HPLC

* Bilal Haider Abbasi

bhabbasi@qau.edu.pk

Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan

Biotechnology Program, Department of Environmental Sciences, COMSATS Institute of Information

Technology (CIIT), Abbottabad, Pakistan

Pakistan Academy of Sciences, Islamabad, Pakistan

Appl Biochem Biotechnol

Introduction
Cancer has caused 7.9 million deaths (around 13 % of total deaths) in 2007, and this number is
increasing every day with expected 12 million deaths in 2030 [1]. Among the different types of
cancer including lung, stomach, liver, and colon, breast cancer accounts for 10.9 % of the
reported cases and is considered as the second major diagnosed type [2]. Recently, natural
products have gained a tremendous attention due to their wider health-promoting effects. The
exploration of plant-derived natural products, specifically against breast cancer, is under focus
by many research groups worldwide [3, 4]. Fagonia indica belongs to the family
Zygophyllaceae and has been recently indicated for having potential against breast cancer
[3]. It is commonly known as sacchi boti, meaning Btrue herb,^ and is widely distributed in
Pakistan, Afghanistan, India, and Egypt [5]. The plant possesses distinct compounds, having
multiple therapeutic properties such as antioxidant [6], anticancer [4], antidiabetic [7], antiinflammatory [8], antimicrobial, analgesic [9], and hepato-protective activities [10]. The
anticancer activities of F. indica can be attributed to the important phenolic acids such as
apigenin, myricetin, and gallic acid as well as to the saponins and various triterpenoids
ubiquitously found in its different parts [11, 12]. Due to its high medicinal importance,
Fagonia products, especially the virgins mantle tea, are marketed against breast cancer and
mainly exported from the Indian subcontinent. However, the lesser phytochemical content,
extreme variability, and lack of procedures for sustainable harvest from wild-grown plants are
the major bottlenecks for formulation of phytochemically consistent Fagonia products [13].
These issues can be circumvented through the application of in vitro cultures, specifically cell
culture systems [14]. The advantage of cell cultures lies in their potential for continuous,
uniform, and enhanced production of important phytochemicals followed by easier extraction
methods, irrespective of geography and season [15]. The present study was, therefore, aimed at
the establishment of an in vitro callus culture system for the enhanced production of commercially important anticancerous secondary metabolites in F. indica.

Materials and Methods

Establishment of Callus Cultures
Stem explants (~1.0 cm) and leaf explants (~0.5 cm2) were excised from 50-day-old in
vitro-germinated seedlings and were cultured on a Murashige and Skoog (MS) basal
medium (MS0, 1962; PhytoTechnology Laboratories, USA) containing 3 % sucrose and
0.8 % (w/v) agar (PhytoTechnology Laboratories, USA) supplemented with various plant
growth regulators (PGRs). The different PGRs used included -naphthalene acetic acid
(NAA), benzylaminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D), indoleacetic
acid (IAA), and thidiazuron (TDZ) at concentrations of 1.05.0 mg/L each or in the
combination TDZ + NAA (1:1). An MS medium devoid of PGRs (MS0) was used as a
control treatment. The cultures were maintained at 25 2 C under a 16/8 (light/dark)
photoperiod (40 mol m2 s1; Philips TLD 35 fluorescent lamps). All experiments were
performed in triplicate culture flasks and were repeated twice. The data on callus
formation were recorded as (1) callus induction frequency, (2) callus diameter, and (3)
callus biomass. The calluses were harvested after 42 days of culture period and were
oven dried after fresh weight (FW) determination.

Appl Biochem Biotechnol

Analytical Scheme
For the determination of the total phenolic content (TPC) and the total flavonoid content (TFC)
through colorimetric assays, the samples were subject to extraction according to the modified
protocol of Bahri-Sahloul et al. [16]. Briefly, a powdered callus sample (300 mg) was
dissolved in 10 mL of 50 % aqueous methanol, kept on a shaker (24 rpm; 24 h; room
temperature), and sonicated for (30 min). The mixture was then centrifuged (6500 rpm;
10 min), the supernatant was collected and syringe filtered, and the filtrate was transferred
to already weighed 1.5-mL Eppendorf tubes. The solvent was placed in a centrifugal evaporator (Eppendorf 5301 Concentrator) for an hour, and the final weight of the Eppendorf tube
with the crude extract was recorded. In this way, the final weight of the crude extract was
measured and a final dilution of 10 mg/mL was prepared by the addition of methanol to the
crude extract. The TPC was determined according to the Folin-Ciocalteu method [17]. Briefly,
90 L of the Folin-Ciocalteu reagent (10 diluted in deionized distilled water) was added to
each well containing 20 L of the samples followed by the addition of 90 L of sodium
carbonate (6 g/100 mL distilled water) and was kept at room temperature for 90 min. Gallic
acid (1 mg/mL) and methanol (20 L) were used as a positive and a negative control,
respectively. The TFC was determined through the aluminum trichloride (AlCl3) method
[18]. Briefly, 10 L of aluminum trichloride solution (10 g/L of distilled water) and 10 L
of potassium acetate (98.15 g/L of distilled water) were added to the reaction well containing
20 L of the samples. The final reaction volume was adjusted to 200 L by adding 160 L
distilled water and kept for 30 min at room temperature. Quercetin (1 mg/mL) and methanol
(20 L) were used as a positive and a negative control, respectively. After an appropriate
reaction time, the absorbance of samples was recorded at 630 nm for TPC and at 450 nm for
TFC, respectively, with a microplate reader (ELx800 Absorbance Reader, BioTek Inc., USA).
The results are expressed as micrograms of gallic acid equivalent (GAE) per milliliter and
micrograms of quercetin equivalent (QE) per milliliter, respectively.
Important phenolic compounds were quantified through high-performance liquid chromatography (HPLC) by adopting the method described by Shah et al. [19] with minor modifications. The reference standards used were apigenin, caffeic acid, catechin, gallic acid,
myricetin, and rutin (Sigma Company, USA). Standards and plant extract stock solutions were
prepared in methanol, at concentrations of 200 g/mL and 10 mg/mL, respectively. The
samples were filtered through a 0.45-m membrane filter and then separated in an RP-C8
column (4.6 mm 250 mm internal diameter [i.d.], 5 m; Purospher, Merck) using mobile
phase A (acetonitrile-methanol-water-acetic acid; 5:10:85:1 v/v) and mobile phase B (acetonitrile-methanol-acetic acid; 40:60:1 v/v) having a flow rate of 1 mL/min in an isocratic mode. A
gradient of time 020 min for 050 % B, 2025 min for 50100 % B, and then isocratic 100 %
B until 40 min were used. All the samples were analyzed at 257, 279, and 368 nm wavelengths. The identification of phenolic compounds was carried out based on the retention time
of corresponding reference standards. All the samples were assayed in triplicate, the mean
value of content (standard error) was calculated, and the results were expressed as micrograms per milliliter of sample.
The activity of phenylalanine ammonia lyase (PAL) was determined according to the
protocol followed by Khan et al. [20]. Briefly, freeze-dried calluses (100 mg FW) were
homogenized with ice-cold 100 mM potassium borate buffer (pH 8.8) plus 2 mM
mercaptoethanol and then subjected to centrifugation (12,000 rpm; 10 min; 4 C). The reaction
mixture (2 mL) had 0.5 mL of 4 mM phenylalanine, 1 mL of 100 mM potassium borate buffer

Appl Biochem Biotechnol

(pH 8.8), and 0.5 mL of extract. The absorbance of reaction mixture was recorded before
incubation (BioTek microplate reader). The mixture was incubated at 30 C, and the reaction
was terminated by the addition of 0.2 mL of 6 M HCl. The absorbance was recorded at 290 nm
after 30 min of the reaction. The increase in absorbance was treated as a function of the amount
of product formed.
The ability of the callus extract to scavenge free radical (free radical-scavenging activity
[FRSA]) was determined according to the protocol described by Amarowicz et al. [21].
Briefly, 190, 195, 197.5, and 199 L of 2-2-diphenyl-1-picrylhydrazyl (DPPH) solution
(4.8 mg/50 mL of methanol) were added to 10, 5, 2.5, and 1 L of the sample, taking ascorbic
acid as a positive control. The final concentrations of the samples were adjusted to 1000, 750,
500, and 250 g/mL. The absorbance was recorded at 515 nm, 1 h after the reaction (ELx800
Absorbance Reader, BioTek Inc., USA). The DPPH results are expressed as half-maximal
inhibitory concentration (IC50), which is a measure of the effectiveness of the sample in
inhibiting the reaction. IC50 values were calculated for micrograms of ascorbic acid (used as a
positive control) equivalent per milliliter of extract. The radical-scavenging activity was
calculated by the following formula and expressed as percent DPPH discoloration:
% scavenging DPPH free radical 100  1AE=AD
where AE is the absorbance of the solution when an extract was added at a particular
concentration and AD is the absorbance of the DPPH solution with nothing added.

Statistical Analysis
All experiments were conducted in a completely randomized design at least three times. Each
treatment consisted of three replicates. Statistical analysis was carried out using SPSS 22.0 and
Statistix 8.1. The relationship between different parameters was assessed using Pearsons
correlation coefficient (r). One-way ANOVA was used to check the significant mean difference
with Tukeys HSD for post hoc analysis. A P < 0.05 was used to define significant results. All
the figures were made using Origin 8.1.

Results and Discussion

Callus Induction
TDZ is considered as one of the most potent bioregulators for callogenesis in many plant
species [22]. In the present study, callus formation was initiated in both explants by all PGR
treatments. The highest callus induction frequency (96 %) was observed in stem explants,
incubated on MS medium supplemented with 1.0 mg/L TDZ (Table 1). No significant
differences in callus formation were observed among explants in response to all levels of
TDZ; however, 1.0 mg/L of TDZ was the most effective for callus organogenesis. TDZ alone
(1.0 mg/L) produced maximum callus biomass (FW). Furthermore, a higher callus biomass
(17.50 g FW/35 mL) was recorded at the optimal range (1.05.0 mg/mL) of TDZ treatment.
Culture characteristics showed that the calluses formed were light green in color (Table 1).
Within the optimal range, the TDZ-stimulated growth parameters in callus cultures may be
ascribed to the ability of the hormone to trigger the production of purine cytokinins for

4th

4th

4th

IAA

NAA

2,4-D

BAP

4th

5th

4th

5th

4th

7th
4th

4
1

6th

6th

5th

6th

5th

5th

5th

4th
5th

1
2

6th

6th

7th

5th

5th

5th

6th

6th

6th

7th

7th

6th

6th
6th

5th

5th

5th

4th

3rd

60 %b,c 3.81d
50 %c
32 %d
58 % c

53%c

55 % c

63 %b,c

2.51d

2.03d

1.41d,e

66 %b,c 5.21c,d

4.03
5.30c,d

c,d

4.89

68%b,c

70 %b

40 %

c,d

8.53b,c

70 % b

38 %
75 %b

57 %

0.58d

0.93d

1.04c,d

3.08c

3.91c

4.50b,c

2.49

c,d

6.98b

74 %b

77 %b
c,d

4.70b,c

4.81c,d

70 % b

8.54

71 %b

11.01
6.51 b

75 %

68 % b,c 65 % bc 7.03 c

78 %

a,b

8.04a,b

7.32

85 % a,b 83 %a,b 10.63b

9.69
7.04a,b
8.32a,b

82 %

a,b

9.01a

10.24a

9.53a

6.34

0.35d

70 %b 8.45b,c
68 %b,c 9.53b,c

78 % b
76 %b

91 %

b,c

89 %a,b 14.08a

93 %a
a,b

87 %a,b 15.36a

95 %a

11.24

0.90d,e

88 %a,b 12.53b

FW

10.21 1.70

1.04 0.09f
1.21 0.36a

6.22 0.81d
6.51 1.01d

10.34 2.04

13.40 1.52b

13.04 1.71b

4.50 1.91d,e

3.57 0.92e

2.50 0.62e,f

4.04 0.97e

5.09 0.99d,e

3.67 1.09
5.20 1.02d,e

4.04 1.07

6.57 1.99d

4.50 1.02d,e

8.01 1.05cd

10.67 1.52

b,c

2.40 0.61e,f

1.82 0.73e,f

1.81 0.79e,f

3.50 1.00e

4.02 1.01e

4.92 1.02d,e

2.72 0.93

e,f

6.21 1.72d

4.51 0.46d,e

6.54 1.41d

7.90 0.85

c,d

10.61 2.07b,c 7.82 1.22c,d

9.20 1.52c
9.57 1.91c

13.01 2.05

17.07 1.85a

17.50 1.07a

LG, friable

0.43 0.93c

0.41 0.06c

LG, friable

LG, friable

YG, friable

YG, friable

DG, compact

DG, compact

DG, compact

0.42 0.14c

YG, friable
W, friable

0.10 0.07d,e YB, friable

0.06 0.02d,e B, friable

0.30 0.12c,d W, friable

0.13 0.03c,d 0.09 0.04e

LG, friable

DG, compact

LG, compact

LG, compact

W, compact

W, compact

W, compact

YG, friable

0.34 0.09c,d LB, compact DB, compact

0.24 0.09d

0.10 0.08c,d 0.07 0.02d,e LB, friable

0.09 0.03d

0.31 0.09c

DG, compact

LG, granular DG, compact

0.42 0.10b,c 0.32 0.11c,d W, friable

0.22 0.07
0.51 0.10b

0.28 0.09

0.35 0.10c

0.30 0.11c

DG, compact
LG, granular DG, compact

0.31 0.10c,d LG, granular DG, granular

0.39 0.12c,d LG, friable
DG, friable

0.82 0.09b

1.24 0.41a

1.23 0.35a

LG, friable

0.42 0.20bc 0.34 0.07cd LG, friable

0.63 0.11

a,b

0.59 0.09b

0.53 0.14b
0.55 0.09b

0.92 0.10

a,b

1.33 0.72a

1.42 0.93a

1.21 0.52a

DG, compact

YG, compact YG, compact

Stem derived Leaf derived

Morphology/
Characteristics

1.14 0.09a,b LG, friable

0.10 0.05c,d 0.09 0.02e

Stem derived Leaf derived

DW

14.57 1.99a,b 11.52 1.31b,c 1.32 0.22a

13.99 1.22

a,b

1.83 0.31e,f

Leaf derived

Biomass (g/flask)

Stem
Leaf
Stem derived
derived derived

Area of
callus (cm2)

95 %a

96 %

90 %

3rd

TDZ

35 %d

42 %c,d

5th

MS0

5th

Leaf
derived

Percent induction

Concentration Stem
Leaf
Stem
(mg/L)
derived derived derived

Initiation day

Type

PGR

Table 1 Effect of different PGRs on growth parameters in callus cultures of Fagonia indica

Appl Biochem Biotechnol

Initiation day

6th

4th

2.31

b,c

92 %a

85 %a,b 6.94 c

3.23d

68 %b,c

63 %

FW

5.04b,c

4.57 2.30d,e

4.05 1.01

5.67 1.21d,e
3.92 1.09e

Leaf derived

Biomass (g/flask)

Stem
Leaf
Stem derived
derived derived

Area of
callus (cm2)

YG, friable

LG, friable

LG, compact

Stem derived Leaf derived

1.01 0.22a,b 0.92 0.81a,b LG, friable

0.12 0.06c,d

0.21 0.09c

Stem derived Leaf derived

DW

Morphology/
Characteristics

FW fresh weight, DW dry weight, YG yellowish green, DG dark green, LG light green, LB light brown, DB dark brown, W whitish, B brownish

Values represent mean standard error (SE). Means followed by the same letters within each column are not significantly different (P = 0.05) using Duncans comparison mean test

4th

6th

Leaf
derived

Percent induction

Concentration Stem
Leaf
Stem
(mg/L)
derived derived derived

TDZ + NAA 1:1

Type

PGR

Table 1 (continued)

Appl Biochem Biotechnol

Appl Biochem Biotechnol

enhanced cellular growth [23]. In our study, the combination of TDZ with an auxin (NAA)
showed lower callogenic response in explants tested. Although there are a few studies on auxin
(2,4-D and NAA)-induced callogenesis in Fagonia spp. [24, 25], no previous reports on TDZinduced tissue culture responses in Fagonia spp. are available. Palmer and Keller [26],
however, reported TDZ-induced callus in Tribulus terrestris, another member of
Zygophyllaceae. Interestingly, MS mediums devoid of any PGRs also favored callogenesis
in F. indica. However, the response was as low as negligible. Overall, stem explants showed a
higher response (96 %) than leaf explants (90 %) as shown in Table 1. The differential
response of different explants of the same species to the same hormonal treatment may be
due to the selective physiological and biochemical potential of different tissues. Callus
formation usually depends on the optimal concentration of PGRs, plant genotype, explant
type, PGR type, and in vitro growth conditions [27]. TDZ at higher and lower concentrations
beyond the optimal level significantly decreased the callus formation frequency and resulted in
marked reduction in biomass (Table 1). This is in agreement with the results of Ali and Abbasi
[28]. Similarly, Nikam et al. [29] found that increasing the cytokinin concentration decreases
the proliferation of callus in T. terrestris.

Accumulation of Secondary Metabolites in Callus Cultures

Phenolic compounds are low molecular weight, antioxidative secondary metabolites found in
different plant species having a magnitude of effects against many ailments [30]. The highest
TPC (202.8 g GAE/mL) and TFC (191.03 g QE/mL) were recorded for stem-derived callus,
raised in vitro at 1.0 mg/L TDZ, as compared to the control treatment (92.7 g GAE/mL), while
for leaf-derived callus, more TPC (161.3 g GAE/mL) and TFC (164.8 g QE/mL) were detected
at TDZ (1.0 mg/L). The impact of TDZ on the profound production of TPC and TFC in in vitro
cultures is well documented [31]. In our study, the TPC and TFC in callus cultures were found
dependent on the concentration of TDZ and explants tested (Fig. 1a, b). The higher amounts of
TPC and TFC detected in calluses as compared to those in the control paralleled the involvement
of TDZ in the organogenesis of callus. It is extrapolated from our data that TDZ might have
triggered stress on the plant cells during the growth of callus; as a result, the phenylpropanoid
pathway might have switched on to produce a sufficient amount of phenolic acids and other
antioxidants, to cope with the stress condition [32]. There are instances where TDZ has been
employed for the production of commercially important secondary metabolites in some medicinal
plants [20, 33]. Although, TDZ at a lower concentration (1.0 mg/L) enhanced the accumulation
of TPC and TFC in callus cultures, increasing the concentration decreased the production of
phenolic compounds. The anticipated reason for this trend is that TDZ at higher concentrations
produce excessive ethylene that suppresses the production of secondary metabolites [34] (Fig. 2).

HPLC-DAD-Based Quantification of Phenolic Acids

HPLC is a powerful analytical tool for the quantification of phenolic compounds with
sufficient precision and selectivity in less time. In collaboration with the data from colorimetric
tests, the quantification of the callus cultures raised in vitro on an MS medium fortified with
1.0 mg/L TDZ accumulated higher amounts of important phenolic acids (Table 2).
Furthermore, the stem-derived callus produced higher amounts of these compounds such as
gallic acid (125.10 5.01 g/mL) followed by myricetin (32.5 2.05 g/mL), caffeic acid
(12.5 0.52 g/mL), catechin (9.4 1.2 g/mL), and apigenin (3.8 0.45 g/mL). The leaf-

Appl Biochem Biotechnol

Fig. 1 a Colorimetric estimation
of total phenolic content in stemand leaf-derived calluses at different concentrations of thidiazuron.
b Colorimetric estimation of total
flavonoid content in stem- and
leaf-derived calluses at different
concentrations of thidiazuron

derived callus, which showed comparatively lower quantities of these polyphenols, had gallic
acid (105.1 2.76 g/mL), myricetin (28.3 1.8 g/mL), caffeic acid (11.4 0.65 g/mL),
catechin (8.2 0.43 g/mL), and apigenin (3.2 0.2 g/mL). Among these reference compounds, rutin was detected (96.4 1.5 g/mL) only in callus samples supplemented with
higher doses of TDZ (>1.0 mg/L). More gallic acid were produced in the callus cultures,
suggesting its elicitation by TDZ as a potent bioregulator. The wider taxonomic distribution,
higher structural diversity, and maximum accumulation of gallic acid in dry weight make it a
precious metabolite of plants [35]. Gallic acid has a stimulatory role in activating the plant
antioxidant system against reactive oxygen species (ROS) via antioxidative enzymes such as
superoxide dismutase (SOD) and peroxidase (POD). Besides being very important in
protecting from other diseases, the phenolic compounds detected in the present study play

Appl Biochem Biotechnol

Fig. 2 Phenylalanine ammonium
lyase activity expressed as units/
gram of fresh weight of stem- and
leaf-derived calluses at different
TDZ concentrations

an important anticancer role. Recent studies suggest that gallic acid, myricetin, caffeic acid,
catechin, and apigenin exhibit anticancer activities through suppression of oncogenes, reduction of antioxidative stress, and induction of apoptosis and cell cycle arrest in different cancer
cell lines [3640].

Antioxidant Activity in Callus Cultures

The abundance of ROS or other free radicals causes oxidative stress in vivo that can damage
the body directly or indirectly. To scavenge these free radicals and thus protect the body from
their damaging effect, plant secondary metabolites are employed as antioxidants that act as an
antidote to many disorders of the body [41]. To confirm the presence of antioxidants in a
specific sample, the DPPH, as a free radical, is usually used and then the sample is analyzed for
its percentage of FRSA [42]. In our study, a high FRSA was observed in callus cultures
compared to the control (Fig. 3). It is evident that in the cases of both stem- and leaf-derived
calluses, the DPPH FRSA is dose dependent, the dose being 0.251.0 mg/mL. The stem-

Table 2 Quantification of phenolic acids in callus cultures of Fagonia indica

Phenolic compounds

Retention
time (min)

Quantity (g/mL of sample)

Stem-derived callus extract

Apigenin
Caffeic acid
Catechin
Gallic acid
Myricetin

21.937

3.8 0.45g

9.412

7.314
4.039
15.672

12.5 0.52
f

Leaf-derived
callus extract
3.2 0.2g
11.4 0.65e,f
8.2 0.43f,g

9.4 1.2

125.10 5.01
32.5 2.05c

105.1 2.76b
28.3 1.8d

Values represent mean standard error (SE). Means followed by the same letters within each column are not
significantly different (P = 0.05) using Duncans comparison mean test. In any column if the difference is not
significant, it is shown by the same letters

Appl Biochem Biotechnol

Fig. 3 Percentage of free radicalscavenging activity in different
stem- and leaf-derived callus
samples

derived callus, obtained at 1.0 mg/L of TDZ, showed a higher activity (69.45 0.75 %) at
1.0 mg/mL as compared to the control (40.89 1.09 %) (Fig. 3). Similarly, the leaf-derived
callus obtained at 1.0 mg/L TDZ showed higher inhibition (63.68 1.73 %) as compared to the
control (35.22 1.15 %) (Fig. 3). Furthermore, a higher DPPH FRSA was recorded for the
stem-derived callus (IC50 = 709 g/mL) compared to the leaf-derived callus (IC50 = 801 g/
mL).

The Correlation of PAL Activity with Metabolic Content and Antioxidant Activity
PAL plays an important role in the biosynthesis of many important phenolic compounds. PAL
is the strategic enzyme that starts the phenylpropanoid biosynthesis pathway in plants, with
conversion of L-phenylalanine to trans-cinnamic acid, which acts as the precursor for synthesis
of phenylpropanoids such as lignins, flavonoids, and coumarins [43, 44]. In this study, a high
PAL activity (4.2 U/g of FW) in the stem-derived callus was observed in response to TDZ
(1.0 mg/L) as compared to the control sample (1.0 U/g of FW). Similarly, the callus derived
from leaf explants in response to the same concentration of TDZ produced PAL activity
(3.8 U/g of FW) compared to that in the control samples (0.7 mg/L). The enhanced accumulation of these important secondary metabolites can be correlated with the higher levels of PAL
and FRSA. A positive correlation was observed between PAL activity and the accumulation of
metabolites in callus cultures of F. indica. Furthermore, just as for TPC and TFC, an increase
in the concentration of TDZ caused a decrease in the activity of PAL (Fig. 2). PAL activity has
been shown to enhance at transcriptional level in response to application of exogenous
cytokinins [45].

Conclusions and Future Prospects

TDZ is a potent bioregulator for callus induction in F. indica. It can produce a high amount of
callus containing elevated levels of commercially important phenolic compounds. The

Appl Biochem Biotechnol

production of a high amount of fresh, friable, and viable callus in response to a low level of this
PGR and simple basal medium can result in the production of commercially important
secondary metabolites very easily and cost efficiently. The establishment of feasible callus
cultures for F. indica is a step toward its cell suspension culture and the ultimate scale-up for
the mass production of commercially important secondary metabolites.
Acknowledgments Tariq Khan acknowledges the indigenous PhD fellowship program of the Higher Education
Commission (HEC), Pakistan. Bilal Haider Abbasi acknowledges the financial support from the Pakistan
Academy of Sciences (PAS), Pakistan.
Authors Contributions TK did the research work and wrote the manuscript. BHA conceived the idea and
supervised the work. MAK and BHA analyzed the data. BHA and ZKS critically reviewed the manuscript and
added to its technical part.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no competing interest.

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