Differential Effects of Thidiazuron on Production of Anticancer Phenolic Compounds in Callus Cultures of Fagonia indica

7/24/2019 Differential Effects of Thidiazuron on Production of Anticancer Phenolic Compounds in Callus Cultures of Fagonia in

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Differential Effects of Thidiazuron on Production

of Anticancer Phenolic Compounds in Callus CulturesofFagonia indica

Tariq Khan1 &Bilal Haider Abbasi1,3

&

Mubarak Ali Khan2 &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 gquercetin 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 chroma-

tography (HPLC) data indicated higher amounts of commercially important anticancer sec-

ondary metabolites such as gallic acid (125.10 5.01g/mL), myricetin (32.5 2.05g/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) radical-

scavenging 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 multipotenthormone as elicitor in callus cultures ofF. indica.

Keywords Callus . TDZ .Fagonia . Phenolic acids . Anticancer. HPLC

Appl Biochem Biotechnol

DOI 10.1007/s12010-016-1978-y

* Bilal Haider Abbasi

[email protected]

1Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan

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

Technology (CIIT), Abbottabad, Pakistan

3Pakistan Academy of Sciences, Islamabad, Pakistan
http://crossmark.crossref.org/dialog/?doi=10.1007/s12010-016-1978-y&domain=pdf


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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 thereported 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], anti-

inflammatory [8], antimicrobial, analgesic [9], and hepato-protective activities [10]. Theanticancer 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 consistentFagonia 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 commer-

cially important anticancerous secondary metabolites inF. 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.



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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) wasdissolved 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 evapo-

rator (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 toeach 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 appropriatereaction 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 chroma-

tography (HPLC) by adopting the method described by Shah et al. [19] with minor modifi-

cations. 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 (acetoni-

trile-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 wave-

lengths. 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 micro-

grams 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



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(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. IC50values were calculated for micrograms of ascorbic acid (used as apositive 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



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Table1

EffectofdifferentPGRsongrowthparametersincallusculturesofFagoniaindica

PGR

Initiationday

Percentinduction

Areaof

callus(cm

2)

Biom

ass(g/flask)

Morphology/

Characteristics

FW

DW

Type

Concen

tration

(mg/L)

Stem

derived

Leaf

derived

Stem

derived

Leaf

derived

Stem

derived

Leaf

derived

Stem

derived

Leafderived

Stemd

erived

Leafderived

Stemderive

d

Leafderived

MS0

0

5th

5th

42%c,d

35%d

0.9

0d,e

0.3

5d

1.83

0.3

1e,f

1.0

40.0

9f

0.1

00.0

5c,d

0.0

90.0

2e

YG,compa

ctYG,compact

TDZ

1

3rd

3rd

96%a

90%a

11.2

4b

6.3

4b

13.991.2

2a,b

10.2

11.7

0c

1.2

10.3

6a

1.1

40.0

9a,b

LG,

friable

DG,compact

2

4th

4th

95%a

88%a,b

12.5

3b

9.5

3a

14.571.9

9a,b

11.5

21.3

1b,c

1.3

20.2

2a

1.2

10.5

2a

LG,

friable

DG,compact

3

4th

5th

95%a

87%a,b

15.3

6a

10.2

4a

17.501.0

7a

13.0

41.7

1b

1.4

20.9

3a

1.2

30.3

5a

LG,granular

DG,compact

4

4th

5th

93%a

89%a,b

14.0

8a

9.0

1a

17.071.8

5a

13.4

01.5

2b

1.3

30.7

2a

1.2

40.4

1a

LG,granular

DG,compact

5

4th

5th

91%a

82%a,b

9.6

9b,c

7.3

2a,b

13.012.0

5b

10.3

42.0

4c

0.9

20.1

0a,b

0.8

20.0

9b

LG,

friable

DG,compact

BAP

1

4th

6th

78%b

70%b

8.4

5b,c

7.0

4a,b

9.20

1.5

2c

6.2

20.8

1d

0.5

30.1

4b

0.3

10.1

0c,d

LG,granular

DG,granular

2

5th

6th

76%b

68%b,c

9.5

3b,c

8.3

2a,b

9.57

1.9

1c

6.5

11.0

1d

0.5

50.0

9b

0.3

90.1

2c,d

LG,

friable

DG,

friable

3

5th

6th

85%a,b

83%a,b

10.6

3b

8.0

4a,b

10.612.0

7b,c

7.8

21.2

2c,d

0.5

90.0

9b

0.4

10.0

6c

LG,

friable

DG,compact

4

5th

7th

78%b

75%b

11.0

1b

8.5

4a,b

10.671.5

2b,c

7.9

00.8

5c,d

0.6

30.1

1a,b

0.4

30.9

3c

LG,

friable

DG,compact

5

6th

7th

68%b,c

65%bc

7.0

3c

6.5

1b

8.01

1.0

5cd

6.5

41.4

1d

0.4

20.2

0bc

0.3

40.0

7cd

LG,

friable

DG,compact

2,4-D

1

5th

6th

71%b

70%b

4.8

1c,d

4.7

0b,c

4.50

1.0

2d,e

4.5

10.4

6d,e

0.3

00.1

1c

0.2

40.0

9d

YG,

friable

YG,

friable

2

5th

6th

77%b

74%b

8.5

3b,c

6.9

8b

6.57

1.9

9d

6.2

11.7

2d

0.3

50.1

0c

0.3

40.0

9c,d

LB,compact

DB,compact

3

6th

6th

57%c

40%c,d

4.8

9c,d

2.4

9c,d

4.04

1.0

7e

2.7

20.9

3e,f

0.2

80.0

9d

0.1

00.0

7d,e

YB,

friable

YG,

friable

4

7th

38%d

4.0

3c,d

3.67

1.0

9e

0.2

20.0

7d

YG,

friable

NAA

1

4th

5th

75%b

70%b

5.3

0c,d

4.5

0b,c

5.20

1.0

2d,e

4.9

21.0

2d,e

0.5

10.1

0b

0.4

20.1

4c

W,

friable

W,compact

2

4th

5th

70%b

66%b,c

5.2

1c,d

3.9

1c

5.09

0.9

9d,e

4.0

21.0

1e

0.4

20.1

0b,c

0.3

20.1

1c,d

W,

friable

W,compact

3

4th

5th

68%b,c

60%b,c

3.8

1d

3.0

8c

4.04

0.9

7e

3.5

01.0

0e

0.3

10.0

9c

0.3

00.1

2c,d

W,

friable

W,compact

IAA

1

6th

7th

53%c

50%c

1.4

1d,e

1.0

4c,d

2.50

0.6

2e,f

1.8

10.7

9e,f

0.0

90.0

3d

0.0

60.0

2d,e

B,

friable

LG,compact

2

5th

6th

55%c

32%d

2.0

3d

0.9

3d

3.57

0.9

2e

1.8

20.7

3e,f

0.1

00.0

8c,d

0.0

70.0

2d,e

LB,

friable

LG,compact

3

5th

6th

63%b,c

58%c

2.5

1d

0.5

8d

4.50

1.9

1d,e

2.4

00.6

1e,f

0.1

30.0

3c,d

0.0

90.0

4e

LG,

friable

DG,compact



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Table1

(continued)

PGR

Initiationday

Percentinduction

Areaof

callus(cm

2)

Biom

ass(g/flask)

Morphology/

Characteristics

FW

DW

Type

Concen

tration

(mg/L)

Stem

derived

Leaf

derived

Stem

derived

Leaf

derived

Stem

derived

Leaf

derived

Stem

derived

Leafderived

Stemd

erived

Leafderived

Stemderive

d

Leafderived

4

6th

68%b,c

3.2

3d

5.67

1.2

1d,e

0.2

10.0

9c

LG,

friable

5

6th

63%b,c

2.3

1

4.05

1.0

1e

0.1

20.0

6c,d

YG,

friable

TDZ+NAA

1:1

4th

4th

92%a

85%a,b

6.9

4c

5.0

4b,c

4.57

2.3

0d,e

3.9

21.0

9e

1.0

10.2

2a,b

0.9

20.8

1a,b

LG,

friable

LG,compact

Valuesrepresentmean

standarderror(SE).Meansfollowedbythesameletterswithineach

columnarenotsignificantlydifferent(P=0.0

5)usingDuncanscomparisonmeantest

FWfreshweight,DW

dryweight,YGyellowishgreen,D

Gdarkgreen,

LGlightgreen,

LBlightbrown,

DBdarkbrown,

Wwhitish,

Bbrownish



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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 Fagoniaspp. [24,25], no previous reports on TDZ-

induced tissue culture responses in Fagonia spp. are available. Palmer and Keller [26],

however, reported TDZ-induced callus in Tribulus terrestris, another member ofZygophyllaceae. Interestingly, MS mediums devoid of any PGRs also favored callogenesis

inF. 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 (Table1). 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), whilefor leaf-derived callus, more TPC (161.3g GAE/mL) and TFC (164.8g 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-



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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 com-

pounds, 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

Fig. 1 aColorimetric estimation

of total phenolic content in stem-

and leaf-derived calluses at differ-

ent concentrations of thidiazuron.

bColorimetric estimation of total

flavonoid content in stem- andleaf-derived calluses at different

concentrations of thidiazuron



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an important anticancer role. Recent studies suggest that gallic acid, myricetin, caffeic acid,

catechin, and apigenin exhibit anticancer activities through suppression of oncogenes, reduc-

tion 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-

Fig. 2 Phenylalanine ammonium

lyase activity expressed as units/

gram of fresh weight of stem- and

leaf-derived calluses at different

TDZ concentrations

Table 2 Quantification of phenolic acids in callus cultures ofFagonia indica

Phenolic compounds Retention

time (min)

Quantity (g/mL of sample)

Stem-derived callus extract Leaf-derived

callus extract

Apigenin 21.937 3.8 0.45g 3.2 0.2g

Caffeic acid 9.412 12.5 0.52e 11.4 0.65e,f

Catechin 7.314 9.4 1.2f 8.2 0.43f,g

Gallic acid 4.039 125.10 5.01a 105.1 2.76b

Myricetin 15.672 32.5 2.05c 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



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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 ofL-phenylalanine totrans-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 accumu-

lation 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 ofF. 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 inF. indica. It can produce a high amount of

callus containing elevated levels of commercially important phenolic compounds. The

Fig. 3 Percentage of free radical-

scavenging activity in different

stem- and leaf-derived callus

samples



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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 forF. indicais 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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