Journal of Photochemistry and Photobiology B: Biology 140 (2014) 223227

Contents lists available at ScienceDirect

Journal of Photochemistry and Photobiology B: Biology

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

Light-induced uctuations in biomass accumulation, secondary

metabolites production and antioxidant activity in cell suspension
cultures of Artemisia absinthium L.
Mohammad Ali, Bilal Haider Abbasi
Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan

a r t i c l e

i n f o

Article history:
Received 21 May 2014
Received in revised form 25 July 2014
Accepted 11 August 2014
Available online 20 August 2014
Keywords:
Artemisia
Light
Cell suspension
Phenolics
Flavonoids
Antioxidant

a b s t r a c t
Light is an important factor inuencing plant morphogenesis and biochemical pathways, including biosynthesis of primary and secondary metabolites. In the present study, we investigated the differential
effect of light on biomass accumulation and secondary metabolites production in cell suspension cultures
of Artemisia absinthium L. A prolonged log phase of 21 days was followed by light-grown cultures. Lightgrown cultures displayed 3.9-fold maximum increase (8.88 g/l) in dry biomass on day 30 of culture which
was comparable to 3.7-fold maximum increase (9.2 g/l) on day 27 in dark-grown cultures. Compared to
dark grown-cultures, enhanced levels of total phenolic content (5.32 mg/g DW), total phenolic production (42.96 mg/l) and total secondary metabolites (6.79 mg/g) were found in light-grown suspension cultures during the log phase of growth. Further, a positive correlation among maximum levels of
antioxidant activity (63.8%), total phenolic production (42.96 mg/l) and total secondary metabolites
(6.79 mg/g DW) was displayed by light-grown suspension cultures.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Artemisia absinthium L. (wormwood) is a well known medicinal
plant, mentioned in almost all books of herbal medicine in the
Western world [1]. It has been considered as a general remedy
for all diseases and was referred to as A herb of Mars for its
over-arching medical powers [2]. The plant has traditionally been
used as anti-helmintic, choleretic, antiseptic, balsamic, depurative,
digestive, diuretic, emmenagogue and in treating leukaemia and
sclerosis [3]. Phytochemical studies have shown the presence of
terpenes, antioxidant phenolics, avonoids, and other biologically
active compounds [4]. The dry leaves and stem contain, among
others, 0.251.32% essential oil, absinthin, anabsin, artemisinin,
anabsinthin, artabsin, and matricin [5].
Plant secondary products are of immense use as potential drugs,
nutraceuticals, and food additives [6]. Among different classes of
secondary metabolites, plant polyphenols constitute the largest
group of natural antioxidants [7]. These compounds posses biological properties like antioxidant, anti-aging, anti-carcinogen, protection from cardiovascular, immune/autoimmune diseases and brain
dysfunctions viz. Parkinsons, Alzheimers, Huntingtons diseases,
etc. [8,9]. Phenolics may have a direct contribution in the antioxi Corresponding author. Tel./fax: +92 51 90644121.
E-mail address: bhabbasi@qau.edu.pk (B.H. Abbasi).
http://dx.doi.org/10.1016/j.jphotobiol.2014.08.008
1011-1344/ 2014 Elsevier B.V. All rights reserved.

dant activity [10]. The antioxidant potential in various medicinal

plants has been shown mainly due to phenolic compounds [11
16]. Likewise, different species of the genus Artemisia including A.
absinthium L. have shown phenolics associated antioxidant activities [4,1719]. Furthermore, the importance of avonoids as antioxidants and their role in antimalarial and anticancer activities of
Artemisia annua has been reviewed [20].
Cell suspension cultures offer a simple system to study growth
and production kinetics that can help to evaluate and implement
optimal conditions for the production of a number of high value
medicinal compounds in good quantities [21]. Light plays an
important role in almost all plant developmental processes and
provides the fundamental building blocks for growth, development, primary and secondary metabolism [2224]. Secondary
metabolites production can be efciently stimulated by optimizing
in vitro conditions including light sources [25]. The stimulatory
effects of light on accumulation of secondary metabolites, including avonoids [26], anthocyanins [27], artemisinin [28] and caffeic
acid derivatives [22] have been shown. On the other hand, the
inhibitory effects of light on the accumulation of secondary metabolites such as nicotine and shikonin [29] were also reported. In
addition to its stimulatory and inhibitory effects on secondary
metabolites, light is also involved in regulating the secretion mechanism of secondary metabolites [30].

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M. Ali, B.H. Abbasi / Journal of Photochemistry and Photobiology B: Biology 140 (2014) 223227

The exploitation of in vitro cultures for secondary metabolites

enhancement is a promising approach to overcome various limitations posed by wild plants. The present study was aimed to investigate the effect of light on total phenolic and total avonoid
accumulation and to nd out a correlation of these secondary
metabolites with antioxidant activities in cell suspension cultures
of A. absinthium L. under continuous light conditions.

Absorbance of the mixtures was recorded at 517 nm by spectrophotometer. For background correction, a methanolic solution of
DPPH that had decayed with no resultant 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 pure
methanol. The radical scavenging activity was calculated by the
following formula and expressed as %age of DPPH discoloration:

% scavenging DPPH free radical 100  1-AE=AD

2.1. Cell suspension cultures establishment

Cell suspension cultures were established as has been described
in our previous study [31]. Briey, leaf explants from 28-day old
seed derived plantlets were cultured on Murashige and Skoog
(MS) [32] medium supplemented with TDZ 1.0 mg/l and NAA
1.0 mg/l to obtain friable calli. To prepare inoculum culture, 35day old yellowish friable calli were cultured in liquid MS media
with the same composition of plant growth regulators. The cultures were placed in gyratory shaker (25 C, 120 rpm) in dark for
the development of stock cell suspension cultures. Fine cell suspension cultures were collected after a period of 14 days. Subsequent experiments were carried out in 250 ml Erlenmeyer asks
containing 50 ml MS media with 30 g/l sucrose, 1.0 mg/l TDZ and
1.0 mg/l NAA in combination and 1.5 g fresh cell suspension was
inoculated in each ask. The same protocol was repeated for the
establishment of inoculum and cell suspension cultures in continuous light. Observations and data recording of the growth kinetics
were performed with an interval of 3 days for 39 days period. Triplicate asks were used in all experiments.
The pH of all media was adjusted to 5.8 (Eutech Instruments pH
510, Singapore) before autoclaving (121 C, 20 min, Systec VX 100,
Germany). To establish suspension cultures in light, cultures were
placed in continuous light with intensity of 40 lmol m2 s1 and
temperature in the shaker was maintained at 25 1 C. Murashige
and Skoog basal medium (MS0) was used as control.
2.2. Analytical methods
To investigate dry biomass accumulation (DBM), each cell suspension culture obtained with an interval of 3 days in response
to continuous light and dark was oven dried (60 C, 24 h) over a
39 days period.
Dried cell suspension culture samples were extracted as has
been described previously [31]. Briey, each nely ground dried
sample (100 mg) was mixed with 80% (v/v) methanol (10 ml).
The mixtures were sonicated (10 min; Toshiba, Japan) 3 times with
a resting period of 30 min in between and centrifuged (8000 rpm,
10 min). The supernatants were collected and either immediately
used for analysis or stored at 4 C.
Total phenolic content was determined using FolinCiocalteu
reagent according to the protocol of Velioglu et al. [33]. Absorbance
was measured at 725 nm using UV/VIS-DAD spectrophotometer
(Halo DR-20, UVVIS spectrophotometer, Dynamica Ltd., Victoria,
Australia). The calibration curve (050 lg/ml, R2 = 0.968) was plotted using gallic acid as standard and the TPC was expressed as gallic acid equivalents (GAE)/g of dry weight.
Total avonoid content was determined using aluminum chloride colorimetric method as described by Chang et al. [34]. Absorbance of the reaction mixtures was measured at 415 nm using UV/
VIS-DAD spectrophotometer. The calibration curve (040 lg/ml,
R2 = 0.998) was plotted using quercetin as standard. The TFC was
expressed as quercetin equivalents (QE)/g of dry weight.
For antioxidant activity determination, the DPPH free radical
scavenging assay (RSA) as described by Abbasi et al. [35] was used.

where AE is 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.
2.3. 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 were subjected
to analysis of variance (ANOVA) and signicant difference was separated using Duncans Multiple Range Test (DMRT). SPSS (Windows version 7.5.1, SPSS Inc., Chicago) was used to determine the
signicance at P < 0.05. Pearson correlation coefcients were
determined using GraphPad Prism 5.01. Figures were generated
using Origin 8.5.
3. Results and discussion
3.1. Effect of light on biomass accumulation
Cell suspension cultures of A. absinthium L. established under
continuous light followed relatively longer lag and log phases of
9 and 21 days, respectively, over a 39 days culture period, compared to cultures established under continuous dark. Maximum
dry biomass accumulation of 8.88 g/l was observed on day 30
which was comparative to dry biomass of 9.03 g/l on day 30 under
continuous dark (Fig. 1).
Starting with the initial values of 2.27 g/l and 2.47 g/l on day 0,
almost doubling in dry biomass with the values 4.7 g/l and 5.8 g/l
was observed on day 15 under light and dark, respectively. However, log phase under continuous light displayed relatively lower
values for dry biomass until the start of stationary phase. Stationary phases of both cultures showed signicantly similar dry bio-

11

Light

10

Dark

9
8

Dry Biomass (g/l)

2. Materials and methods

7
6
5
4
3
2
1
0
0

12

15

18

21

24

27

30

33

36

39

Time (days)
Fig. 1. Biomass accumulation of cell suspension cultures of Artemisia absinthium L.
on MS medium supplemented with 1.0 mg/l TDZ + 1.0 mg/l NAA under continuous
light and dark. Values are mean standard error of three replicates.

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M. Ali, B.H. Abbasi / Journal of Photochemistry and Photobiology B: Biology 140 (2014) 223227

3.3. Effects of light on total avonoid accumulation

In the present study, a decrease in total avonoid accumulation
in light-grown suspension cultures was found, unlike total phenolic accumulation. Maximum corresponding levels for total avonoid content and total avonoid production were found to be
1.57 mg/g DW and 12.92 mg/l on day 27 (log phase) in response
to light and 1.89 mg/l and 16.12 mg/l on day 33 (stationary phase)
in response to dark (Fig. 4). The reason for lesser accumulation
under continuous light might be the increase in transformation
efcacy of some secondary metabolites which may lead to diversion in some chain reaction of biosynthetic pathways. Poutaraud
et al. [41] have observed that the transformation efciency of
hypericin increases under continuous light conditions, resulting
in less accumulation of pigments in Hypericum perforatum. Similarly, enhanced hypericin content in cell suspension cultures of
H. perforatum under continuous dark was reported [42]. It is likely
that secondary metabolites can constitute a photo-block, resulting
in hindered photo-conversion under continuous light conditions
[43].
3.4. Antioxidant activity and its dependence on secondary metabolites
The radical scavenging activity found in cell suspension cultures
in response to continuous light and dark varied considerably in
terms of its percent potential to scavenge the free radical DPPH

Total Phenolic Content (mg/g DW)

45

TPC in Dark
TPC in Light
TPP in Dark
TPP in Light

7
6

40
35
30

25
4
20
3
15
2

10

5
0

0
0

12

15

18 21

24

27

30

33

36

39

Time (days)
Fig. 3. Total phenolic content (mg Gallic Acid/g dry weight) and total phenolic
production (mg Gallic Acid/l) in cell suspension cultures of Artemisia absinthium L.
under continuous light and dark. Values are mean standard error of three
replicates. TPC vs TPP in dark: Pearson Correlation Coefcient (r) = 0.987
(P < 0.0001). TPC vs TPP in light: Pearson Correlation Coefcient (r) = 0.923
(P < 0.0001)

3.0

18

TFC in Dark
TFC in Light
TFP in Dark
TFP in Light

2.7
2.4

16
14

2.1

12

1.8
10
1.5
8
1.2
6

0.9
0.6

0.3

Total Flavonoid Production (mg/l)

Total phenolic accumulation in cell suspension cultures of A.

absinthium L. under continuous light and dark was found to adopt
different growth phases (Fig. 3). Maximum values observed for
total phenolic content and total phenolic production in cultures
under continuous light were found to be 5.32 mg/g DW and
42.96 mg/l on day 24 and day 27 (Log phase), respectively. On contrary, suspension cultures under dark displayed total phenolic content and total phenolic production with its respective values of
3.57 mg/g DW and 32.47 mg/l on day 30 (stationary phase) and
day 37 (late log phase). Overall, light induced higher level of total
phenolic accumulation in terms of its content and production,
compared to dark. Continuous light may turn the cell suspension
cultures to stress condition and act as a triggering factor for
enhanced accumulation of phenolic metabolites. Recently, we have
reported enhanced levels of total phenolic content in callus cultures of A. absinthium L. in response to light which is in agreement
with our current results [36]. In another study [22], increased level
of anthocyanins accumulation in light-grown hairy root cultures of
Echinacea purpurea was reported. Additionally, light stress induced
secondary metabolites production is reported for vegetative tissues
and cell cultures of other plants as well [27,3740].

Total Flavonoid Content (mg/g DW)

3.2. Effects of light on total phenolic accumulation

Total Phenolic Production (mg/l)

mass accumulation. Light-induced suspension cultures were found

to be milky white in color during log and stationary phase and
brownish during decline phase (Fig. 2).

0.0
3

12

15

18

21

24

27

30

33

36

39

Time (days)
Fig. 4. Total avonoid content (mg Quercetin/g dry weight) and total avonoid
production (mg Quercetin/l) in cell suspension cultures of Artemisia absinthium L.
under continuous light and dark. Values are mean standard error of three
replicates. TFC vs TFP in dark: Pearson Correlation Coefcient (r) = 0.995
(P < 0.0001). TFC vs TFP in light: Pearson Correlation Coefcient (r) = 0.931
(P < 0.0001).

and its dependence on secondary metabolites. Light induced cultures showed maximum activity of 63.8% while dark grown cultures showed 82.7% activity. Furthermore, the activity showed by
light-grown cultures was found to be biomass-accumulation
dependent but total phenolic content and total avonoid content
independent while that of dark-grown cultures was found to be
total phenolic content dependent but biomass accumulation independent (Fig. 5). The correlation difference among antioxidant
activity and phenolic and avonoid metabolites suggest the
involvement of antioxidant secondary metabolites other than

Fig. 2. Light-grown suspension cultures during lag phase (a) log phase, (b) stationary phase and (c) decline phase (d).

TFC in dark
TFC in Light
TPC in Dark
TPC in Light

2.0
1.6

5
4

1.2

0.8

0.4

0.0
90
80
70
60
50
40
30
20

0
RSA in Dark
RSA in Light

12

15

18

21

24

27

30

33

36

39

Radical Scavenging
Total Secondary
Activity (%)
Metabolites (mg/g DW)

M. Ali, B.H. Abbasi / Journal of Photochemistry and Photobiology B: Biology 140 (2014) 223227

Total Phenolic
Content (mg/g DW)

Radical Scavenging Total Flavonoid

Activity (%)
Content (mg/g DW)

226

8
7
6
5
4
3
2
1
0

80
70
60
50
40
30
0

Fig. 5. DPPH radical scavenging activity (%) with respect to total phenolic content
(mg Gallic Acid/g dry weight) and total avonoid content (mg Quercetin/g dry
weight) in cell suspension cultures of Artemisia absinthium L. under continuous light
and dark. Values are mean standard error of three replicates.

Total Flavonoid
Production (mg/l)

90
80
70
60
50
40
30
20

50
40
30
20
10
0

Total Phenolic
Production (mg/l)

Radical Scavenging
Activity (%)

phenolics and avonoids in light-grown suspension cultures. Light

might be a key factor to induce some secondary metabolites with
antioxidant activities that are not produced in the dark. However,
when compared with total phenolic and total avonoid production,
suspension cultures showed a signicantly positive correlation of
antioxidant activity and total phenolic production in response to
light and of total avonoid production and antioxidant activity in
response to dark (Fig. 6).
Furthermore, enhanced level of maximum total secondary
metabolites (combination of total phenolic content and total avonoid content) with the value 6.79 mg/g DW was observed on day
27 (late log phase) for light-grown cultures which was in a significantly positive correlation with its corresponding radical scavenging activity (Fig. 7). Recently, we have reported a direct
relationship of total phenolic content and antioxidant activity in
callus cultures of A. absinthium L. [44]. Additionally, several reports
are available on phenolics related antioxidant activity in the
in vitro cultures of different plants [4548].
In conclusion, light-grown suspension cultures of A. absinthium
L. are potential entities for enhanced accumulation of phenolics.
Total secondary metabolites, representing total phenolic content
and total avonoid content, were found to be in positive correlation with antioxidant activities in cell suspension cultures. Furthermore, depending on the demand for secondary metabolites,

TFP in Dark
TFP in Light
TPP in Dark
TPP in Light

RSA in Dark
RSA in Light

RSA in Dark
RSA in Light

90

Time (days)

18
16
14
12
10
8
6
4
2
0

TSM in Dark
TSM in Light

12 15 18 21 24 27 30 33 36 39

Time (days)
Fig. 6. DPPH radical scavenging activity (%) with respect to total phenolic
production (mg Gallic Acid/l) and total avonoid production (mg Quercetin/l) in
cell suspension cultures of Artemisia absinthium L. under continuous light and dark.
Values are mean standard error of three replicates.

12

15

18

21

24

27

30

33

36

39

Time (days)
Fig. 7. DPPH radical scavenging activity (%) with respect to total secondary
metabolites (mg Gallic Acid and Quercetin/g dry weight) in cell suspension cultures
of Artemisia absinthium L. under continuous light and dark. Values are mean standard error of three replicates.

strategies should be adopted for biomass dependent production

of total phenolic content and total avonoid content, for enhanced
antioxidant activities in cell suspension cultures grown under continuous dark and light, respectively.
Acknowledgement
Financial support of Higher Education Commission (HEC) of
Pakistan is appreciated.
References
[1] S. Krebs, T.N. Omer, B. Omer, Wormwood (Artemisia absinthium) suppresses
tumour necrosis factor alpha and accelerates healing in patients with Crohns
disease a controlled clinical trial, Phytomedicine 17 (2010) 305309.
[2] P. Baker, The Book of Absinthe: A Cultural History, Grove Press, 2007.
[3] J.M. Canadanovic-Brunet, S.M. Djilas, G.S. Cetkovic, V.T. Tumbas, Free-radical
scavenging activity of wormwood (Artemisia absinthium L.) extracts, J. Sci. Food
Agric. 85 (2005) 265272.
[4] R. Singh, P.K. Verma, G. Singh, Total phenolic, avonoids and tannin contents in
different extracts of Artemisia absinthium, J. Intercult. Ethnopharmacol. 1
(2012) 101104.
[5] S. Kordali, A. Cakir, A. Mavi, H. Kilic, A. Yildirim, Screening of chemical
composition and antifungal and antioxidant activities of the essential oils from
three Turkish Artemisia species, J. Agric. Food Chem. 53 (2005) 14081416.
[6] G.K. Devi, K. Manivannan, P. Anantharaman, Evaluation of antibacterial
potential of seaweeds occurring along the coast of Mandapam, India against
human pathogenic bacteria, J. Coast. Life Med. 2 (2014) 196202.
[7] . Ciesla, I. Kowalska, W. Oleszek, A. Stochmal, Free radical scavenging
activities of polyphenolic compounds isolated from Medicago sativa and
Medicago truncatula assessed by means of thin-layer chromatography DPPH
rapid test, Phytochem. Anal. 24 (2013) 4752.
[8] H. Lai, N.P. Singh, Oral artemisinin prevents and delays the development of 7,
12-dimethylbenz [a] anthracene (DMBA)-induced breast cancer in the rat,
Cancer Lett. 231 (2006) 4348.
[9] J. Sun, Y.-F. Chu, X. Wu, R.H. Liu, Antioxidant and antiproliferative activities of
common fruits, J. Agric. Food Chem. 50 (2002) 74497454.
[10] A.M. Bidchol, A. Wilfred, P. Abhijna, R. Harish, Free radical scavenging activity
of aqueous and ethanolic extract of Brassica oleracea L. var. italica, Food
Bioprocess Technol. 4 (2011) 11371143.
[11] C. Jayasinghe, N. Gotoh, T. Aoki, S. Wada, Phenolics composition and
antioxidant activity of sweet basil (Ocimum basilicum L.), J. Agric. Food
Chem. 51 (2003) 44424449.
[12] M.B. Ali, S. Khatun, E.-J. Hahn, K.-Y. Paek, Enhancement of phenylpropanoid
enzymes and lignin in Phalaenopsis orchid and their inuence on plant
acclimatisation at different levels of photosynthetic photon ux, Plant Growth
Regul. 49 (2006) 137146.
[13] H.-J. Kim, F. Chen, X. Wang, J.-H. Choi, Effect of methyl jasmonate on phenolics,
isothiocyanate, and metabolic enzymes in radish sprout (Raphanus sativus L.),
J. Agric. Food Chem. 54 (2006) 72637269.
[14] M.B. Ali, E.-J. Hahn, K.-Y. Paek, Methyl jasmonate and salicylic acid induced
oxidative stress and accumulation of phenolics in Panax ginseng bioreactor
root suspension cultures, Molecules 12 (2007) 607621.

M. Ali, B.H. Abbasi / Journal of Photochemistry and Photobiology B: Biology 140 (2014) 223227
[15] R. Bahri-Sahloul, R. Ben Fredj, N. Boughalleb, J. Shriaa, S. Saguem, J.-L. Hilbert,
F. Trotin, S. Ammar, S. Bouzid, F. Harzallah-Skhiri, Phenolic composition and
antioxidant and antimicrobial activities of extracts obtained from Crataegus
azarolus L. var. aronia (Wild.) Batt. ovaries calli, J. Bot. 2014 (2014).
[16] M.H. Abdille, R. Singh, G. Jayaprakasha, B. Jena, Antioxidant activity of the
extracts from Dillenia indica fruits, Food Chem. 90 (2005) 891896.
[17] F.H. Afshar, A. Delazar, H. Nazemiyeh, S. Esnaashari, S.B. Moghadam,
Comparison of the Total Phenol, Flavonoid Contents and Antioxidant Activity
of Methanolic Extracts of Artemisia spicigera and A. splendens Growing in Iran,
2012.
[18] A.D. Ruikar, E. Khatiwora, N. Ghayal, A. Misar, A. Mujumdar, V. Puranik, N.
Deshpande, Studies on aerial parts of Artemisia pallens wall for phenol,
avonoid and evaluation of antioxidant activity, J. Pharm. Bioallied Sci. 3
(2011) 302.
[19] Y.-J. Lee, M. Thiruvengadam, I.-M. Chung, P. Nagella, Polyphenol composition
and antioxidant activity from the vegetable plant Artemisia absinthium L., AJCS
7 (2013) 19211926.
[20] J.F. Ferreira, D.L. Luthria, T. Sasaki, A. Heyerick, Flavonoids from Artemisia
annua L. as antioxidants and their potential synergism with artemisinin
against malaria and cancer, Molecules 15 (2010) 31353170.
[21] P. Srivastava, V. Sisodia, R. Chaturvedi, Effect of culture conditions on synthesis
of triterpenoids in suspension cultures of Lantana camara L., Bioprocess
Biosyst. Eng. 34 (2011) 7580.
[22] B.H. Abbasi, C.-L. Tian, S.J. Murch, P.K. Saxena, C.-Z. Liu, Light-enhanced caffeic
acid derivatives biosynthesis in hairy root cultures of Echinacea purpurea, Plant
Cell Rep. 26 (2007) 13671372.
[23] A. Shohael, M. Ali, K. Yu, E. Hahn, R. Islam, K. Paek, Effect of light on oxidative
stress, secondary metabolites and induction of antioxidant enzymes in
Eleutherococcus senticosus somatic embryos in bioreactor, Process Biochem.
41 (2006) 11791185.
[24] C. Liu, Y. Zhao, Y. Wang, Artemisinin: current state and perspectives for
biotechnological production of an antimalarial drug, Appl. Microbiol.
Biotechnol. 72 (2006) 1120.
[25] H. Senger, Blue light responses: phenomena and occurrence in plants and
microorganisms, 1987.
[26] F. Kreuzaler, K. Hahlbrock, Flavonoid glycosides from illuminated cell
suspension cultures of Petroselinum hortense, Phytochemistry 12 (1973)
11491152.
[27] J.J. Zhong, T. Seki, S.I. Kinoshita, T. Yoshida, Effect of light irradiation on
anthocyanin production by suspended culture of Perilla frutescens, Biotechnol.
Bioeng. 38 (1991) 653658.
[28] C.-Z. Liu, C. Guo, Y.-C. Wang, F. Ouyang, Effect of light irradiation on hairy root
growth and artemisinin biosynthesis of Artemisia annua L., Process Biochem.
38 (2002) 581585.
[29] M. Tabata, H. Mizukami, N. Hiraoka, M. Konoshima, Pigment formation in
callus cultures of Lithospermum erythrorhizon, Phytochemistry 13 (1974) 927
932.
[30] D. Kim, H. Pendersen, C. Chin, Effects of light on berberine production in cell
suspension cultures of Thalictrum rugosum, Biotechnol. Lett. (United Kingdom)
(1988).
[31] M. Ali, B.H. Abbasi, Ihsan-ul-haq, Production of commercially important
secondary metabolites and antioxidant activity in cell suspension cultures of
Artemisia absinthium L., Ind. Crops Prod. 49 (2013) 400406.

227

[32] T. Murashige, F. Skoog, A revised medium for rapid growth and bio assays with
tobacco tissue cultures, Physiol. Plant. 15 (1962) 473497.
[33] Y. Velioglu, G. Mazza, L. Gao, B. Oomah, Antioxidant activity and total
phenolics in selected fruits, vegetables, and grain products, J. Agric. Food
Chem. 46 (1998) 41134117.
[34] C.-C. Chang, M.H. Yang, H.M. Wen, J.C. Chern, Estimation of total avonoid
content in propolis by two complementary colorimetric methods, J. Food Drug
Anal. 10 (2002) 178182.
[35] B.H. Abbasi, M.A. Khan, T. Mahmood, M. Ahmad, M.F. Chaudhary, M.A. Khan,
Shoot regeneration and free-radical scavenging activity in Silybum marianum
L., Plant Cell, Tissue Organ Culture (PCTOC) 101 (2010) 371376.
[36] U. Tariq, M. Ali, B.H. Abbasi, Morphogenic and biochemical variations under
different spectral lights in callus cultures of Artemisia absinthium L., J.
Photochem. Photobiol. B: Biol. 130 (2014) 264271.
[37] A.G. Beckwith, Y. Zhang, N.P. Seeram, A.C. Cameron, M.G. Nair, Relationship of
light quantity and anthocyanin production in Pennisetum setaceum cvs.
Rubrum and Red Riding Hood, J. Agric. Food Chem. 52 (2004) 456461.
[38] T.S. Feild, D.W. Lee, N.M. Holbrook, Why leaves turn red in autumn. The role of
anthocyanins in senescing leaves of red-osier dogwood, Plant Physiol. 127
(2001) 566574.
[39] T. Nagata, S. Todoriki, T. Masumizu, I. Suda, S. Furuta, Z. Du, S. Kikuchi, Levels of
active oxygen species are controlled by ascorbic acid and anthocyanin in
Arabidopsis, J. Agric. Food Chem. 51 (2003) 29922999.
[40] W. Zhang, C. Curtin, M. Kikuchi, C. Franco, Integration of jasmonic acid and
light irradiation for enhancement of anthocyanin biosynthesis in Vitis vinifera
suspension cultures, Plant Sci. 162 (2002) 459468.
[41] A. Poutaraud, F. Di Gregorio, V.C.F. Tin, P. Girardin, Effect of light on hypericins
contents in fresh owering top parts and in an extract of St. Johns wort
(Hypericum perforatum), Planta Med. 67 (2001) 254259.
[42] T.S. Walker, H. Pal Bais, J.M. Vivanco, Jasmonic acid-induced hypericin
production in cell suspension cultures of Hypericum perforatum L. (St. Johns
wort), Phytochemistry 60 (2002) 289293.
[43] K. Hahlbrock, D. Scheel, Physiology and molecular biology of phenylpropanoid
metabolism, Ann. Rev. Plant Biol. 40 (1989) 347369.
[44] M. Ali, B.H. Abbasi, Thidiazuron-induced changes in biomass parameters, total
phenolic content, and antioxidant activity in callus cultures of Artemisia
absinthium L., Appl. Biochem. Biotechnol. (2013) 114.
[45] A. Amid, N.N. Johan, P. Jamal, W.N.W.M. Zain, Observation of antioxidant
activity of leaves, callus and suspension culture of Justicia gendarusa, Afr. J.
Biotechnol. 10 (2013) 1865318656.
[46] L. Giri, P. Dhyani, S. Rawat, I.D. Bhatt, S.K. Nandi, R.S. Rawal, V. Pande, In vitro
production of phenolic compounds and antioxidant activity in callus
suspension cultures of Habenaria edgeworthii: a rare Himalayan medicinal
orchid, Ind. Crops Prod. 39 (2012) 16.
[47] W. Al Khateeb, E. Hussein, L. Qouta, M. Aludatt, B. Al-Shara, A. Abu-Zaiton, In
vitro propagation and characterization of phenolic content along with
antioxidant and antimicrobial activities of Cichorium pumilum Jacq, Plant
Cell, Tissue Organ Culture (PCTOC) 110 (2012) 103110.
[48] R. Diwan, A. Shinde, N. Malpathak, Phytochemical composition and
antioxidant potential of Ruta graveolens L. in vitro culture lines, J. Bot. 2012
(2012).