Production of commercially of important secondary metabolites and antioxidant activity in cell suspension cultures of Arthemisia absinthium L.

  

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Industrial Crops and Products

  j o u r n a l h o m e p a g e :

  Production of commercially important secondary metabolites and antioxidant activity in cell suspension cultures of Artemisia absinthium L. _,

  Mohammad Ali , Bilal Haider Abbasi , Ihsan-ul-haq a

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

  Department of Pharmacy, Quaid-i-Azam University, Islamabad 45320, Pakistan a r t i c l e i n f o a b s t r a c t

  Article history:

  in vitro Artemisia absinthium An culture of L. was established for production of commercially impor-

  Received

  15 February 2013

  tant secondary metabolites. Callus cultures were obtained by inoculating leaf explants on Murashige

  Received in revised form

  22 May 2013 −1

  and Skoog (MS) medium supplemented with Thidiazuron (TDZ; 0.5–5.0 mg l ) alone or in combination

  Accepted

  27 May 2013 −1 −1

  with either ␣-naphthalene acetic acid (NAA; 1.0 mg l ) or Indole acetic acid (IAA; 1.0 mg l ). The cal-

  −1 −1

  lus obtained in response to 1.0 mg l TDZ and 1.0 mg l NAA was subcultured on the same medium

  Keywords:

  to investigate its biomass accumulation and secondary metabolites production on weekly basis for

  7 Callus weeks. For submerged cultivation, 35 day old calli were cultured on MS basal media supplemented

  Cell suspension −1 −1

  with 1.0 mg l TDZ and 1.0 mg l NAA. Growth kinetics and secondary metabolites production were

  Phenolics

  investigated in 3 day old suspension cultures for 42 days. Additionally, high performance liquid chro-

  Flavonoids

Antioxidant matography (HPLC) based quantification of gallic acid, caffeic acid and catechin was carried out in cell

Artemisia

  suspension cultures. Seed germinated plantlets were used as control. Maximum levels of total phenolic content 3.57 mg GAE/g DW (control: 2.75 mg GAE/g DW), total flavonoid content 1.89 mg QE/g DW (con- trol:

  1.20 mg QE/g DW), and antioxidant activity 82.7% (control: 72.3%) were displayed by suspension

  −1 −1

  cultures. Among the phenolic compounds, maximum level of gallic acid 104 ␮g g (control: 21.3 ␮g g ),

  −1 −1 −1 −1

  caffeic acid 27.40 g (control: 28.5 g ) and catechin 92.0 g (control: 68.10 g ) were ␮g ␮g ␮g ␮g

  A. absinthium detected in suspension cultures. The results indicate that cell suspension cultures of L. have the potential for enhanced production of phenolics and, hence, highest antioxidant activity than callus culture and seed derived plantlets.

  © 2013 Elsevier B.V. All rights reserved.

1. Introduction

   These compounds are considered to play a protective role

  against a wide range of diseases such as coronary heart disease and Secondary metabolites produced in plants are low molecular certain types of cancers Plant cell cul- relationship exists between the phenolic compounds produced by ture is an attractive alternative technology for enhancing secondary the cultures of different plants and their antioxidant activ- in vitro metabolites that are either difficult to synthesize chemically or ities plant cell suspension cultures containing undifferentiated cells Artemisia absinthium L., commonly known as “Wormwood” are attractive industrially, especially compared to differentiated has been used as herbal medicine throughout Europe, Middle cultures, due to their relative similarity to microbial cell culture East, North Africa, and Asia popular drink absinthe, prepared from

  A. absinthium L., is con- Among different classes of secondary metabolites, plant sidered to stimulate creativity and excitement The plant has traditionally been used as anti-

   diuretic, emmenagogue and in treating leukaemia and sclerosis ( Recently, the aerial part of A. absinthium L. has shown to possess anti-snake venom activity ( Antimalarial and anticancer activities

  ∗ Corresponding author. Tel.: +92 51 90644121; fax: +92 51 90644121.

  are among the prominent biological effects reported for different

  E-mail addresses: (B.H. Abbasi).

  2013 Elsevier B.V. All rights reserved.

• – 0926-6690/$ see front matter ©

  M. Ali et al. / Industrial Crops and Products 49 (2013) 400– 406 401

2. Materials and methods

  TDZ and 1.0 mg l

  NAA. For this purpose, 1.0 g of the callus sections were subcultured on MS media containing 1.0 mgl

  −1

  TDZ and 1.0 mg l

  −1

  NAA. Investigations of the biomass accumulation were performed with an interval of 7 days for 49 days period. Triplicate flasks were used in all experiments.

  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 for- mula and expressed as %age of DPPH discoloration: % scavenging DPPH free radical

  species of the genus Artemisia

  For antioxidant activity determination, the DPPH free radical scavenging assay (FRSA) as described by used. Absorbance of the mixtures was recorded at 517 nm by spec- trophotometer. For background correction, a methanolic solution of DPPH that had decayed with no resultant purple colour (2 mg of butylated hydroxyanisole (BHA) dissolved in

  1.0 ml/min. Gallic acid was analyzed at 257 nm, catechin at 279 nm and caffeic acid at 325 nm. Every time column was reconditioned for 10 min before the next analysis.

  20 ␮l and flow rate was

  B. A gradient of time 0–20 min for 0–50% B, 20–25 min for 50–100% B and then isocratic 100% B till 30 min was used. Injection volume was

  Quantitative analysis of phenolic compounds was carried out by using HPLC–DAD attached with Discovery C-18 analytical column, by the method described by minor modifi- cation. Methanol–acetonitrile–water–aectic acid (10:5:85:1) were used as mobile phase A and methanol-acetonitrile-acetic acid (60:40:1) were used as mobile phase

  = 0.998) was plotted by using quercetin as standard. The TFC was expressed as quercetin equivalents (QE)/g of dry weight.

  2

  For total flavonoid content determination, the aluminium chlo- ride colorimetric method as described by was used. Absorbance of the reaction mixtures was measured at 415 nm by using UV/VIS–DAD spectrophotometer. The calibration curve (0–40 ␮g/ml, R

  = 0.968) was plotted by using gallic acid as standard and the TPC was expressed as gallic acid equivalents (GAE)/g of dry weight.

  2

  Absorbance was measured at 725 nm by using UV/VIS–DAD spec- trophotometer (Halo DR-20, UV–VIS spectrophotometer, Dynam- ica Ltd., Victoria, Australia). The calibration curve (0–50 ␮g/ml, R

  4 C. For total phenolic content determination, Folin-Ciocalteu reagent was used according to the protocol of

  2.4. Analytical methods For fresh weight (FW) determination, calli were harvested from the media and weighed while cell suspensions were filtered through

  0.45 ␮m stainless steel sieve (Sigma), washed with dis- tilled water, pressed gently on filter paper to remove excess water and weighed. Subsequently, calli and cell suspension cultures were oven dried (60

  ◦

  C,

C. Murashige and Skoog basal medium (MS0) was used as control.

  −1

  −1

  10 min). The supernatants were col- lected and either immediately used for analysis or stored at

  −1

  

  

  In vitro studies of the genus Artemisia have mainly been focused on enhanced production of artemisinin; strategies should be adopted to enhance medicinally important phenolics and flavonoids in the species of this commercially important genus. This study aimed to establish cell suspension culture and to inves- tigate phenolics and flavonoids and antioxidant potential in the suspension cultures of A. absinthium L.

  2.1. Seed germination and explant inoculation Seeds of

  A. absinthium L. were obtained from National Agri- culture Research Centre (NARC) Islamabad, Pakistan. Following a single wash with running tap water seeds were immersed in ethanol (3 min), followed by treatment with mercuric chloride (2 min) and then washed with autoclaved water and dried on ster- ilized filter papers. MS0 (Murashige and Skoog basal medium; Phytotechnology Labs, USA; contain- ing 3% sucrose and 0.8% (w/v) agar (Phytotechnology Labs, USA) was used for seed germination.

  2.2. Callus induction and biomass yield To study the effects of plant growth regulators on callus induc- tion, approximately

  1.5 cm of the leaf sections from 28-days old seed germinated plantlets were incubated on Murashige and Skoog (MS) (1962) media supplemented with TDZ (0.5, 1.0, 2.0, 3.0, 4.0, 5.0 mg l

  −1

  ) alone and in combination with 1.0 mg l

  −1

  of NAA or 1.0 mg l

  IAA. Before autoclaving (121

  1.0 mg l

  ◦

  C, 20 min, Systec

  VX 100, Germany), pH of all media was adjusted to 5.8 (Eutech Instruments pH 510, Singapore). All cultures were placed in

  16 h photoperiod with light intensity of 40 ␮mol m

  −2

  s

  −1

  and temperature of the growth room was maintained at

  25 ±

  1

  ◦

  Differential callus formation frequency and changes in callus morphology were recorded on weekly basis. Furthermore, growth curve was established for the biomass accumulation of the rapidly growing friable callus, obtained in response to

  24 h) for dry weight (DW) determination. Extraction of calli, cell suspension culture samples and in vitro seed derived plantlets (control) was performed according to the protocol described by minor modification. Briefly, each finely 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,

2.3. Cell suspension culture and growth kinetics

  42 days period. Triplicate flasks were used in all experiments.

  NAA. The cultures were placed in gyratory shaker (25

  To establish cell suspension culture, 35-day old proliferated calli were transferred to 500 ml Erlenmeyer flasks containing MS basal media supplemented with combination of 1.0 mg l

  −1

  2.5. 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 sub- jected to analysis of variance (ANOVA) and significant difference was separated using Duncan’s Multiple Range Test (DMRT). SPSS

  AE AD 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.

  1 −

  ×

  = 100

  TDZ and 1.0 mg l

  ◦

  NAA in combi- nation and 1.5 g fresh cell suspension was inoculated in each flask. Observations and data recording of the growth kinetics were per- formed with an interval of three days for

  C, 120 rpm) in dark for the development of stock cell suspension cultures as inoculum cultures. Fine cell suspension cultures were collected after a period of

  14 days. Subsequent experiments were carried out in 250 ml Erlenmeyer flasks containing 50 ml MS media with

  30 g l

  −1

  sucrose, 1.0 mg l

  −1

  TDZ and 1.0 mg l

  −1

  −1

  402 M. Ali et al. / Industrial Crops and Products 49 (2013) 400– 406 Table

  35

  and 9.20 g l

  −1

  , respectively. Fur- thermore, cell suspension cultures were found to be milky white, green and brownish in colour during log, stationary and decline phases, respectively (

  

  7

  14

  21

  28

  42

  ) were recorded on day 9 of cul- ture. However, maximum fresh weight and dry weight displayed by culture at day 27 were 171 g l

  49

  20

  40

  60

  80 100 120 140 160 180 200 _{FW DW} Cultur e time ( days) F resh we ight ( g/ l)

  1

  2

  3

  4

  −1

  −1

  6

  ) in dry biomass was observed on day 42 of culture. Decline phase was observed after

  IAA in combination with TDZ. Furthermore, combination of TDZ and NAA was more efficient for callus formation compared to TDZ alone. Previously, best callogenic responses have been observed on BA in combination with NAA which, in accordance with our results, show that the combination of cytokinins and auxins is superior in inducing friable calli in leaf explants of

  A. absinthium L. Biomass formation of the callus culture showed a 7-day lag phase with relatively slow growth

   Almost threefold

  increase in dry biomass (DW: 5.67 g l

  −1

  ) was achieved on day

  14 and, as a whole, four and half-fold increase (DW: 8.73 g l

  −1

  42 days of culture, characterized by decrease in dry biomass (DW: 7.77 g l

  ) and dry weight (4.07 g l

  −1

  ) on day 49.

  3.2. Cell suspension culture development and growth kinetics Biomass formation of the cell suspension culture of

  A. absinthium L. displayed a relatively quick growth curve and was characterized by a lag phase of

  6 days for fresh and dry biomass, followed by a long log phase of 21 days, and a subsequent stationary phase dur- ing

  42 day period of study (

   Almost doubling in fresh weight

  (63 g l

  −1

  5

  7

  TDZ. The callus was green and friable. However, green compact or brownish compact calli were produced in response to combination of TDZ and

  6

  60

  80 100 120 140 160 180 200 FW DW

  Culture time ( days) F resh we ight (g/l )

  1

  2

  3

  4

  5

  7

  20

  8

  9

  10

  11

  12

  13

  14 Dr y we ight ( g/l ) Fig.

  2. Growth kinetics of cell suspension culture of Artemisia absinthium L. on MS medium supplemented with 1.0 mg l

  −1 TDZ + 1.0 mg l

  40

  42

  8

  6

  9

  10

  11

  12 Dr y we ight (g/l ) Fig.

  1. Growth kinetics of callus culture of Artemisia absinthium L. on MS medium supplemented with

  1.0 mg l −1

  TDZ + 1.0 mg l −1

  NAA. Values are mean ± standard error of three replicates.

  3

  9

  39

  12

  15

  18

  21

  24

  27

  30

  33

  36

  IAA. NAA in combination with TDZ was more responsive for callogensis than

  −1

  1 Effects of different concentrations of TDZ alone and in combination with

  4.53 ± 0.203

  2.31 Brownish, compact 90.7 ±

  2.33 5.03 ± 0.260

  6 TDZ

  5.0

  53.3 ±

  2.40 Brownish, compact

  67.3 ±

  2.91

  7 TDZ 0.5 + NAA

  5 TDZ

  1.0 77.3 ±

  2.03 Yellowish, friable 118 ±

  2.60 6.17 ± 0.120

  8 TDZ 1.0 + NAA

  1.0 83.3 ±

  1.20 Yellowish, friable 132 ±

  3.46 6.50 ± 0.115

  9 TDZ 2.0 + NAA

  1.0 77.3 ±

  4.0 60.0 ±

  3.79 5.13 ± 0.240

  3.18 6.03 ± 0.145

  2.31 6.40 ± 0.265

  IAA and NAA on callus formation frequency, callus morphology and callus growth (fresh weight and dry weight). Values are mean ± standard error of three replicates.

  S. no. Growth regulator (mg l −1

  ) Callus (%) Callus morphology FW (g l −1

  ) DW (g l −1

  )

  1 TDZ

  0.5 71.7 ±

  2.03 Green, friable 112 ±

  2 TDZ

  1.73 Green, compact 103 ±

  1.0 80.3 ±

  1.45 Green, friable 129 ±

  3.21 6.50 ± 0.289

  3 TDZ

  2.0 75.3 ±

  2.40 Green, compact 119 ±

  2.40 6.47 ± 0.240

  4 TDZ

  3.0 66.0 ±

  2.33 Yellowish, friable 120 ±

  10 TDZ 3.0 + NAA

  frequency of 80.3% was observed on 1.0 mg l

  1.0 42.7 ±

  1.86 5.10 ± 0.153

  17 TDZ 4.0 +

  IAA

  1.0 53.3 ±

  3.48 Brownish, compact 80 ±

  1.15 5.00 ± 0.346

  18 TDZ 5.0 +

  IAA

  1.45 Brownish, compact 59.3 ±

  1.0 61.0 ±

  1.76 3.53 ± 0.203 (Windows version 7.5.1, SPSS Inc., Chicago) was used to determine the significance at P <

  0.05.

  3. Results and discussion

  3.1. Callus formation and growth kinetics The leaf explants of A. absinthium L. treated with 1.0 mg l

  −1 TDZ in combination with

  1.0 mg l −1

  NAA resulted into highest callus formation frequency (83.3%) and maximum callus biomass (FW: 132 g l

  −1 ) with yellowish friable features after 5 weeks of culture

   When TDZ was used alone, maximum callus formation

  1.53 Brownish, compact 83.7 ±

  IAA

  1.0 72.7 ±

  1.45 5.43 ± 0.296

  2.91 Yellowish, friable 111 ±

  1.53 5.43 ± 0.176

  11 TDZ 4.0 + NAA

  1.0 66.0 ±

  1.15 Brownish, compact 98 ±

  3.46 5.60 ± 0.153

  12 TDZ 5.0 + NAA

  1.0 58.7 ±

  2.33 Brownish, compact 76.3 ±

  13 TDZ 0.5 +

  16 TDZ 3.0 +

  IAA

  1.0 67.7 ±

  2.03 Green, compact 101 ±

  2.40 6.00 ± 0.173

  15 TDZ 2.0 +

  IAA

  1.0 63.7 ±

  2.60 Green, compact 89 ±

  3.79 5.57 ± 0.120

  −1 NAA. Values are mean ± standard error of three replicates. M. Ali et al. / Industrial Crops and Products 49 (2013) 400– 406 403

3.3. Total phenolic content (TPC) and total flavonoid content

  metabolites including phenolics and flavonoids in the in vitro cultures of A. annua has been reviewed (

  5. Total phenolic content (mg gallic acid/g dry weight) and total flavonoid con- tent (mg quercetin/g dry weight) in cell suspension culture of Artemisia absinthium L. with respect to growth curve (DW). Values are mean ± standard error of three

  g) Tot a l f lavonoi d c o n te nt ( m g QE/ g ) Fig.

  0.0 ^{0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0} Tot a l phe n o li c co n te nt ( m g GAE/

  3 ^{6 9 12 15 18 21 24 27 30 33 36 39 42 2 4 6 8 10 12 DW TPC TFC} Culture time (days ) Dr y we ight ( g/l )

  (3.57 mg GAE/g DW) in conjunction with highest antioxidant activ- ity was recorded in 30-day old suspension cultures in stationary phase. Decline in activity was recorded after 39 days and 59% of

   Maximum total phenolic content

  ) was observed on day 27 while highest antioxidant activity (82.7%) was found in 30-day old suspension cultures, in stationary phase. How- ever, antioxidant activity was found to be associated with total phenolics production

  −1

  On the other hand, the DPPH radical scavenging activity in cell suspension cultures was not found to be strictly culture growth associated. Maximum biomass accumulation (DW: 9.03 g l

  35 days of culture. These results show a positive correlation of phenolic compounds and antioxidant activity in callus cultures of A. absinthium L.

   Decline in antioxidant activity was recorded after

  35 day old calli

  3.4. Antioxidant activity The DPPH free radical scavenging activity in callus cultures was found to be independent of callus biomass accumulation. However, it was found to be dependent on secondary metabolites produc- tion during the culture growth. Highest antioxidant activity (63.3%) and maximum accumulation of total phenolics (1.48 mg GAE/g DW) and total flavonoids (0.48 mg QE/g DW) were recorded in

  (TFC) in callus and cell suspension cultures Phenolics and flavonoids induction in callus culture of A. absinthium L. was not found to be strictly growth dependent

  

  0.0 ^{0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0} T ota l ph e noli c c ont e nt (m g GAE /g ) T ota l fl av o no id co nt e nt (m g Q E/g ) Fig.

  7 ^{14 21 28 35 42 49 1 2 3 4 5 6 7 8 9 10} DW TP TF Culture time (days ) Dr y we ight ( g/l )

   Furthermore, the presence of different secondary

  Silybum marianum were reported to be more produc- tive for phenolic acids among nine different plant species

  1.89 mg QE/g DW (day 33) were observed in the early stationary phase of the growth curve. Previously, the cell cultures of Artemisia fragida and

  On contrary to callus profile, peak values of total phenolics with 3.57 mg GAE/g DW (day 30) and total flavonoids with

  

  A considerable increase with more than doubling in TPC and TFC was observed in 6-day old suspension cultures (

  

  highest levels of flavonoids production in callus cultures of differ- ent medicinal species during the log phase of the culture

   Our results were consistent with the previous reports for

  0.48 mg QE/g DW (4.1-fold increase) on day 35. Earlier studies have been undertaken on the investigations of total phenolic content in callus culture of various medicinal plants

  Initial increase in TPC and TFC was observed in the log phase (day 21), with respective peak values of 1.48 mg GAE/g DW (4.9- fold increase) and

  4. Total phenolic content (mg gallic acid/g dry weight) and total flavonoid con- tent (mg quercetin/g dry weight) in callus culture with respect to growth curve

  404 M. Ali et al. / Industrial Crops and Products 49 (2013) 400– 406

  92.0 ␮g g

  erties ( Similarly, Caffeic acid has exhibited pharmacological antioxidant, anticancer and antimu- tagenic activities

   and anticarcinogenic prop-

  The phenolic compounds detected in the present study are medicinally important phytochemicals. Gallic acid is a low molec- ular weight antioxidant (LMWA) having antiapoptotic

  −1 DW) on day-36 and day-42, respectively.

  DW (control: 68.1 ␮g g

  −1

  DW and

  3.6. Relationship between total phenolic content, total flavonoid content, phenolic compounds and antioxidant activity in cell suspension culture

  −1

  75.3 ␮g g

  with the values

  42 43.7 ± 0.291 ND ND a SDP, seed derived plantlets. b ND, not detected.

  1.18 2.97 ± 0.384 92.0 ± 0.240

  36 70.2 ±

  1.18 27.40 ± 0.441 ND 30 104 ± 1.13 11.0 ± 0.321 75.3 ± 0.173

  

  Many reports are available on the protective effects of natural antioxidants against oxidative stress related disorders like ageing, degenerative diseases and cancer ( phenolic compounds may have a direct contribution in the antioxidant activity. The antioxidant potential in various medicinal plants has been shown to be mainly due to phenolic compounds

  18 72.7 ± 0.769 10.60 ± 0.203 ND

  nolics and antioxidant activity in wild plant of

   Furthermore, cell suspension culture was found to be more

  Ruta graveolens

  DW) was recorded in the seed derived plantlets. Recently, phenolics associated enhanced antiox- idant activities over wild plants have been reported for the callus culture of Habenaria edgeworthii cell suspen- sion and in vitro shoot cultures of

  −1

  A. absinthium L. showed increase in TPC, TFC, gallic acid, catechin and, hence, antioxidant activity as compared to that in seed derived plantlets. However, enhancement in caffeic acid con- centration was not observed in cell suspension culture and its peak value (Control: 28.4 ␮g g

  A. absinthium L. It shows that polyphenols may be the major antioxidants present in this species. Additionally, in the present study cell suspension cul- ture of

   also reported a positive correlation between total phe-

   Although artemisinin has been reported

  A. absinthium L. was observed

  30 day old suspension cultures. Our results are in agreement with a previous report where a positive correlation between high TPC and TFC and antiradical and antioxidant activities in

  ) and antioxidant activity (82.7%) in

  −1

  relation was observed among TPC (3.57 mg GAE/g DW), gallic acid accumulation (104 ␮g g

   In the present study, a positive cor-

  to be the major compound against malaria in Artemisia species, reports are available on the synergistic effects of dietary flavonoids alone and in combinations against this disease

  24 99.8 ±

  8.70 ± 0.265 ND

  7 ^{14 21 28 35 42 49 10 20 30 40 50 60 70 80} d cd cd bcd bcd abc

  7. DPPH radical scavenging activity (%) with respect to total phenolic content and total flavonoid content in cell suspension cultures of Artemisia absinthium L. Values are mean ± standard error of three replicates. Columns with similar alphabets are not significantly different at P <

  −1

  from 43.3 ␮g g

   Overall accumulation of gallic acid ranged

  3.5. HPLC based quantification of phenolic compounds in cell suspension culture Gallic acid was found to be the major phenolic compound which accumulated in conjunction with biomass accumulation in cell culture

  radical scavenging activity was observed in 42 day old suspension cultures.

  0.05.

  0.0 ^{0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0} T ota l ph e noli c c ont e nt (m g GAE /g ) T ota l fl av o no id co nt e nt (m g Q E/g ) Fig.

  −1

  RSA TPC TFC Culture time (days ) Ra d ic a l s c av e ng ing activity (% )

  3 ^{6 9 12 15 18 21 24 27 30 33 36 39 42 10 20 30 40 50 60 70 80 90 100} de fg ef fg g g cd cd c cd cd b b b a

  0.05.

  P <

  6. DPPH radical scavenging activity (%) with respect to total phenolic content and total flavonoid content in callus cultures of Artemisia absinthium L. Values are mean ± standard error of three replicates. Columns with similar alphabets are not significantly different at

  0.00 ^{0.32 0.64 0.96 1.28 1.60 1.92 2.24 2.56} T ota l ph e noli c c ont e nt (m g GAE /g ) T ota l fl av o no id co nt e nt (m g Q E/g ) Fig.

  ^RSA ab ^{TPC TFC} Culture time (days ) R a dical sc av e ng ing activity (% ) a

  to 104 ␮g g

  DW (control: 21.3 ␮g g

  71.4 ± 0.433

  DW). However, its maximum accumulation was recorded in the mid log phase (day 24). Catechin was accu- mulated only in the stationary phase (day 36 and 42) of culture

  12

  6 53.8 ± 0.612 3.37 ± 0.233 ND

   21.3 ± 0.252 28.5 ± 0.176 68.10 ± 0.0882 43.3 ± 0.551 1.03 ± 0.0882

  ) Day Gallic acid Caffeic acid Catechin Control (SDP

  Phenolic compounds (␮g g −1

  2 Quantification of gallic acid, caffeic acid and catechin in cell suspension cultures of Artemisia absinthium L. Values are mean ± standard error of three replicates.

  Table

  −1

  −1

  28.4 ␮g g

  DW (control:

  −1

  DW to 27.40 ␮g g

  −1

  1.03 ␮g g

  DW) and maximum accumulation was observed during the onset of stationary phase (day-30) of culture which is significantly higher than the content found in control. These results are in correlation with our findings of maximum TPC accumulation in 30 day old cell suspension cultures. Caffeic acid was the second major phenolic compound observed in terms of its accumulation in the log phase of culture and ranged from

  productive for phenolics and flavonoids and showed enhanced

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