26 Thidiazuron-Induced Changes in Biomass Parameters, Total Phenolic Content, and Antioxidant...

Thidiazuron-Induced Changes in Biomass Parameters,Total Phenolic Content, and Antioxidant Activity in CallusCultures of Artemisia absinthium L.

Mohammad Ali & Bilal Haider Abbasi

Received: 22 September 2013 /Accepted: 28 November 2013 /Published online: 28 December 2013# Springer Science+Business Media New York 2013

Abstract Callus culture of Artemisia absinthium L. was established for enhanced productionof phenolics and higher antioxidant activity. Callus was induced from seed-derived leafexplants, incubated on to MS media supplemented with thidiazuron (TDZ; 0.55.0 mg/l)either alone or in combination with -naphthalene acetic acid (NAA; 1.0 mg/l). These calluscultures were investigated for their growth kinetics, total phenolic content, and antioxidantactivity on weekly basis for a period of 49 days. Maximum dry biomass accumulation of8.73 g/l was observed on day 42 in response to 1.0 mg/l TDZ and 1.0 mg/l NAA. Furthermore,maximum level of total phenolic content of 8.53 mg GAE/g DW and highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of 72.6 % were observed in calliformed in response to 1.0 mg/l TDZ on day 42. The results showed a positive correlationof total phenolic content and DPPH radical scavenging activity in most of the callus cultures ofA. absinthium L.

Keywords Artemisia . Callus . Phenolics . Antioxidant activity . Thidiazuron

Introduction

Artemisia absinthium L. (Wormwood) is a well-known traditional herb, mentioned in almostall books of herbal medicine in the Western world [1]. This plant is a rich source of terpenes,antioxidant phenolics, flavonoids, and other biologically active compounds [2]. The dry leavesand stems contain, among others, 0.251.32 % essential oil, absinthin, anabsin, artemisinin,anabsinthin, artabsin, and matricin [3]. The plant has traditionally been used as anti-helmintic,choleretic, antiseptic, balsamic, depurative, digestive, diuretic, emmenagogue, and in treatingleukaemia and sclerosis [4].

Plant secondary metabolites are unique sources for pharmaceuticals, food additives, flavors,and other industrial materials either as a part of final product or as a raw material [5]. Amongdifferent classes of secondary metabolites, plant polyphenols constitute the largest

Appl Biochem Biotechnol (2014) 172:23632376DOI 10.1007/s12010-013-0663-7

M. Ali : B. H. Abbasi (*)Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistane-mail: [email protected]

group of natural antioxidants [6]. Phenolics and flavonoids posses biological proper-ties like antioxidant, anti-aging, anti-carcinogen, and protection from cardiovascular,immune/autoimmune diseases, and brain dysfunctions, viz., Parkinsons, Alzheimers,Huntingtons diseases, etc. [7, 8]. Phenolics are considered more potent antioxidants thanvitamin C, E and carotenoids [9].

There are various limitations in using wild plants as the sole source of secondary metab-olites. The use of plant in vitro cultures is an alternative for obtaining secondary metabolitesthat are either difficult to obtain by conventional techniques or whose production is econom-ically not feasible [10]. The genus Artemisia has been exploited for enhanced production ofartemisinin; however, strategies should be adopted to enhance medicinally important phenoliccompounds by exploiting in vitro cultures of this genus. The aim of present study was toinvestigate the interrelationship of biomass accumulation, total phenolic content, andantioxidant activity in response to application of thidiazuron (TDZ), in callus cultures ofA. absinthium L.

Materials and Methods

Seed Germination

Seeds ofA. absinthiumL.were obtained fromNational Agriculture Research Centre, Islamabad,Pakistan. Following a single wash with running tap water, seeds were immersed in ethanol(3min), followed by treatment with mercuric chloride (2min), and then washed with autoclavedwater and dried on sterilized filter papers. Murashige and Skoog basal medium (MS0, 1962;Phytotechnology Labs, USA) containing 3 % sucrose and 0.8 % (w/v) agar (PhytotechnologyLabs, USA) was used for seed germination.

Callus Induction and Biomass Accumulation

For callus induction, approximately 1.5 cm of the leaf sections from 28-day-old seed-derived plantlets were inoculated on MS [11] media supplemented with varyingconcentration of TDZ (0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mg/l) either alone or incombination with -naphthalene acetic acid (NAA; 1.0 mg/l). Callus formation wasobserved on weekly basis through visual observations. All calli were subcultured onthe same media after 20 days. To investigate biomass accumulation, 1 g of each40 days old callus, formed in response to the above mentioned plant growth regula-tors (PGRs) types and concentrations, was subcultured on fresh media with samecomposition of PGRs. Each experiment was carried out in 20 flasks, containing 20 gof inoculated calli as starting culture/treatment. Fresh weight (FW) and dry weight(DW) accumulation were recorded with an interval of 7 days for a period of 49 days.Triplicate flasks were used in all experiments.

Analytical Methods

To investigate biomass accumulation, calli were harvested from the media and weighed forFW determination and then oven dried (60 C, 24 h) for DW determination.

The callus samples were extracted as described by Ali et al. [12]. Briefly, each finelyground dried callus sample (100 mg) was mixed with 80 % (v/v) methanol (10 ml). Themixtures were sonicated (10 min; Toshiba, Japan) three times with a resting period of 30 min

2364 Appl Biochem Biotechnol (2014) 172:23632376

in between and centrifuged (8,000 rpm, 10 min). The supernatant was either collected andstored at 4 C or immediately used for analysis.

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Phenolics were quantified by using FolinCiocalteu reagent according to the protocolof Velioglu et al. [13]. Absorbance was measured at 725 nm by using UV/VisDAD

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Fig. 2 a Fresh callus biomass and b dry callus biomass of A. absinthium L. on MS medium supplemented withTDZ (0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mg/l) and NAA (1.0 mg/l). Values are meanSE of three replicates


spectrophotometer (HaloDR-20, UVVis spectrophotometer, Dynamica Ltd., Victoria, Australia).The calibration curve (050 g/ml, R2=0.968) was plotted by using gallic acid as standard,

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Fig. 3 a Total phenolic content (milligrams GAE per gram DW) in callus cultures of A. absinthium L. on MSmedium supplemented with TDZ and b TDZ+NAA. Values are meanSE of three replicates


and the total phenolic content (TPC) was expressed as gallic acid equivalents (GAE) pergram of DW.

For antioxidant activity determination, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) freeradical scavenging assay (FRSA) was exploited [14]. Briefly, absorbance of the mixture wasrecorded at 517 nm by spectrophotometer. For background correction, a methanolic solution ofDPPH that had decayed and showed no purple color (2 mg of butylated hydroxyanisole (BHA)dissolved in 4 ml of methanol with 0.5 ml of DPPH solution added) was used instead of puremethanol. The radical scavenging activity was calculated by the following formula andexpressed as percent DPPH discoloration:

% scavenging DPPH free radical 100 1AE=AD where AE is absorbance of the solution when an extract was added at a particular concentrationand AD is the absorbance of the DPPH solution with nothing added.

Experimental Design and Data Analysis

All experiments were conducted in a completely randomized design and were repeated twice.Each treatment was consisted of three replicates. Mean values of various treatments weresubjected to analysis of variance, and significant difference was separated using Duncansmultiple range test. SPSS (Windows version 7.5.1, SPSS Inc., Chicago) was used to determinethe significance at P

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Starting with the inoculum culture of 20 g/l, more than doubling in FCB with the values55.7, 44.3, and 51.3 g/l was observed on day 14 while maximum FCB with the values 112,130, and 118 g/l was recorded on day 42 of culture in response to 0.5, 1.0, and 2.0 mg/l ofTDZ, respectively. On the other hand, more than twofold increase in FCB was found to be 56,60.7 and 43.3 g/l on day 21 of culture and maximum accumulation recorded was 103, 90, and67.7 g/l on day 35 in response to 3.0, 4.0, and 5.0 mg/l of TDZ, respectively (Fig. 1a).Furthermore, twofold increase in DCB accumulation was not found strictly linked to particularday/days and maximum DCB with the values 7.63, 8.1, and 6.8 g/l was observed on day 42 inresponse to 0.5, 1.0, and 2.0 mg/l of TDZ, respectively, followed by 5.04, 5.0, and 4.5 g/l inresponse to 3.0, 4.0, and 5.0 mg/l, respectively (Fig. 1b).

Biomass accumulation in response to combination of different concentrations of TDZand 1.0 mg/l NAA was also investigated. Maximum values of FCB 153, 163, and 144 g/land DCB 8.17, 8.73, and 7.52 g/l accumulation were recorded on day 42 of culture inresponse to 0.5, 1.0, and 2.0 mg/l of TDZ (in combination with 1.0 mg/l), respectively.On the other hand, maximum levels of FCB 111, 98.3, and 75.8 g/l and DCB 5.44, 5.30and 4.83 g/l in response to the respective concentrations 3.0, 4.0, and 5.0 mg/l of TDZ(in combination with 1.0 mg/l) were recorded on day 35 of culture (Fig. 2a, b). Theseresults suggest that higher concentrations of TDZ alone and in combination with NAAdecrease the callus formation frequency as well as reduce the log phase for biomassaccumulation in callus cultures of A. absinthium L.

Total Phenolic Content and Its Dependence on Biomass Accumulation

Differential TPC profiles displayed by callus cultures of A. absinthium L. were found to bedependent upon PGR concentration and type and age of the callus. Maximum levels of TPC inresponse to different concentrations of TDZ alone were found on day 42 of culture, except forTDZ 2.0 mg/l where it was observed on day 35 (Fig. 3a). In response to combinations of TDZand NAA, maximum TPC levels were observed on day 35 of culture except for 1.0 mg/l TDZand 1.0 mg/l NAAwhere it was observed on day 42 (Fig. 3b).

When correlated with dry biomass accumulation, maximum TPC was found to beeither growth dependent or independent in response to TDZ lower concentrations (0.5 and1.0 mg/l) and higher concentrations (3.0, 4.0, and 5.0 mg/l), respectively, while maximumTPC in response to 2.0 mg/l was observed during the log phase of growth. On the otherhand, maximum total phenolic content in response to lower concentrations of TDZ(0.5 and 3.0 mg/l) and higher concentrations (3.0, 4.0, and 5.0 mg/l) were observed in logphase and stationary phase, respectively. Exceptionally, maximum TPC in response to2.0 mg/l was also found in stationary phase of growth. These results suggest that a mixedpattern of phenolics accumulation is followed by calli of A. absinthium L. in response todifferent concentration of TDZ either alone or in combination with 1.0 mg/l NAA, inrelation to DBM.

The importance of TDZ as an effective plant growth regulator for plant morphogenesis hasbeen reviewed several times [2427]. Additionally, TDZ had been reported for the productionof economically important secondary metabolites in some plant species [28].

Antioxidant Activity and Its Relationship with Biomass Accumulation and Total PhenolicContent

The DPPH radical scavenging activity in callus cultures of A. absinthium L. wasdetermined with an interval of 7 days for a period of 49 days. In response to lower


concentrations of TDZ (0.5 and 1.0 mg/l), a positive correlation of antioxidant activity,total phenolic content, and dry biomass accumulation was observed. The calli devel-oped on MS medium supplemented with 0.5 and 1.0 mg/l of TDZ displayed maxi-mum corresponding values for antioxidant activity (72.3 and 72.6 %), total phenoliccontent (8.45 and 8.53 mg GAE/g DW), and dry biomass accumulation (7.63 and8.10 g/l) on day 42 of culture (onset of stationary phase) (Fig. 4a, b). While biomassdependent and total phenolic content independent antioxidant potential was observedat higher concentrations of TDZ (4.0 and 5.0 mg/l) (Fig. 4e, f). Contrarily, totalphenolic content dependent and biomass independent antioxidant activity was ob-served in calli obtained in response to TDZ and 1.0 mg/l NAA, except for thebiomass accumulation at higher concentrations of TDZ (3.0, 4.0, and 5.0 mg/l). Theseresults suggested the involvement of phenolics in antioxidant activity of calli formedin response to lower concentrations of TDZ. However, a mixed pattern of antioxidantactivity, total phenolic content, and biomass was observed in response to higherconcentrations of TDZ. Maximum antioxidant activity (61.3 %) and maximum drybiomass (7.54 g/l) were observed on day 42 (Onset of stationary phase), whilemaximum TPC (5.04 mg GAE/g DW) was recorded on day 35 (log phase) of culturein callus formed in response to 3.0 mg/l TDZ (Fig. 4d), which could be linked withthe assumption that antioxidants other than phenolics are produced in response tosome PGRs. On the other hand, the calli developed in response to 4.0 and 5.0 mg/l ofTDZ showed maximum corresponding values of antioxidant activity (61.2 and60.8 %) and dry biomass (5.0 and 4.5 g/l) on day 35 (onset of stationary phase)while maximum TPC (7.14 and 6.81 mg GAE/g DW) on day 42 of culture (stationary phase)(Fig. 4e, f).

Antioxidant activity was found to be TPC dependent in almost all cultures inresponse to combination of TDZ and NAA (Fig. 5). Callus cultures developed inresponse to lower concentrations of TDZ (0.5, 2.0, and 3.0 mg/l) and 1.0 mg/l NAAdisplayed maximum corresponding values for antioxidant potential (61.4, 60.7, and58.8 %) and total phenolic content (7.35, 7.14, and 6.88 mg GAE/g DW) on day 35of culture. The remaining cultures showed a mixed pattern regarding the correlation ofantioxidant activity with TPC and biomass accumulation. With increase in TDZ con-centration to 5.0 mg/l, the maximum antioxidant activity (49 %) was found to be TPCand biomass accumulation independent, suggesting the role of antioxidants other thanphenolics in the log phase of culture. Previously, we have reported phenolics dependentantioxidant activity in callus and cell suspension cultures of A. absinthium L [12].Several reports are available on the positive correlation of phenolics and antioxidantactivities in different plants [2933]. Earlier studies have been undertaken on theinvestigations of total phenolic content in callus culture of various medicinal plants[3436]. Furthermore, a strong relationship has been observed between the phenoliccompounds produced by the in vitro cultures of different plants and their antioxidant activities[3739]. These studies suggested the involvement of phenolics as major antioxidants insome plants.

Fig. 5 Antioxidant activity (percent), total phenolic content (milligrams GAE per gram DW) and dry biomass(grams per liter) in calli formed in response to a TDZ 0.5 mg/l+NAA 1.0 mg/l, b TDZ 1.0 mg/l+NAA 1.0 mg/l,c TDZ 2.0 mg/l+NAA 1.0 mg/l, d TDZ 3.0 mg/l+NAA 1.0 mg/l, e TDZ 4.0 mg/l+NAA 1.0 mg/l, and f TDZ5.0 mg/l+NAA 1.0 mg/l. Values are meanSE of three replicates. Columns with similar alphabets are notsignificantly different at P

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Conclusions

The present study showed that phenolics production was positively regulated in callus culturesof A. absinthium L. in response to TDZ. A linear relationship of total phenolic content andanti-oxidative potential was observed in most of the calli formed. The results suggest theexploitation of callus cultures of A. absinthium L. by treatment with other PGRs and elicitorsto enhance total phenolic content and antioxidant activity and subsequently to be used for theestablishment of suspension cultures.

Acknowledgments Financial support from Higher Education Commission (HEC) is gratefully acknowledged.

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Thidiazuron-Induced...AbstractIntroductionMaterials and MethodsSeed GerminationCallus Induction and Biomass AccumulationAnalytical MethodsExperimental Design and Data Analysis

Results and DiscussionEffects of Plant Growth Regulators on Biomass Accumulation and Growth KineticsTotal Phenolic Content and Its Dependence on Biomass AccumulationAntioxidant Activity and Its Relationship with Biomass Accumulation and Total Phenolic Content

ConclusionsReferences

26 Thidiazuron-Induced Changes in Biomass Parameters, Total Phenolic Content, and Antioxidant...

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