American Journal of Plant Sciences, 2011, 2, 847-850 847

doi:10.4236/ajps.2011.26100 Published Online December 2011 (http://www.SciRP.org/journal/ajps)

Total Phenolic Content and Antioxidant Activity of

Standardized Extracts from Leaves and Cell
Cultures of Three Callistemon Species
Mohamed I. S. Abdelhady1,3*, Amira Abdel Motaal2, Ludger Beerhues3
1
Pharmacognosy Department, Faculty of Pharmacy, Helwan University, Cairo, Egypt; 2Pharmacognosy Department, Faculty of Pharmacy,
Cairo University, Cairo, Egypt; 3Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Braunschweig, Ger-
many.
Email: *[email protected]

Received September 26th, 2010; revised October 24th, 2011; accepted November 5th, 2011.

ABSTRACT
A comparative study was carried out with ethanolic (80%) extracts from leaves and cell cultures of three Callistemon
species, namely C. lanceolatus (CL), C. viridiflorous (CV), and C. comboynensis (CC). Cell suspensions of the three
species were grown in liquid Murashige and Skoog (MS) medium (100 ml) supplemented with 0.9 mg·g–1 kinetin in
combination with 1.1 mg·g–1 NAA. The CL leaf extract was standardized to contain the highest amount of phenolics
(104 ± 2.0 mg·g–1), followed by CC (95.8 ± 1.2 mg·g–1) and CV (79.8 ± 4.6 mg·g–1). On the other hand, cell cultures of
CV contained more phenolics (14.9 ± 0.6 mg·g–1) than those of the other two species, CL and CC, which contained 12.2
± 0.16 and 9.12 ± 0.16 mg·g–1, respectively. Nevertheless, CV leaf extract exhibited the highest antioxidant activity
(91.4% ± 0.4%) at a concentration of 1000 µg·ml–1, comparable to 100 µg·ml–1 gallic acid (90.8% ± 1.5%).

Keywords: Callistemon, Phenolic Content, Antioxidant Activity, Callus, Cell Cultures

1. Introduction purposes, e.g. by immobilization of cells in a matrix for

use in bioreactors. Besides the genetic potential of the
The genus Callistemon (Myrtaceae) contains 34 species
donor plant for callus induction and growth of this callus
of beautiful evergreen shrubs and small trees. The majo-
in in vitro cultures, a medium containing sufficient nu-
rity of the Callistemon species is endemic to the more
trients, such as the preferred MS medium, is required [4].
temperate regions of Australia, four species are found in
Antioxidants play an important role in the prevention
New Caledonia and seven species have been introduced
to India as ornamental trees [1]. They are commonly kno- of human diseases. Antioxidant compounds may function
wn as bottle brushes because of their cylindrical brush- as free radical scavengers, complexing agents for pro-
like flowers resembling the traditional bottle brush. oxidant metals, as well as reducing agents and quenchers
C. lanceolatus, also named C. citrinus, is a well- of singlet oxygen formation [6-8]. Antioxidants are often
known shrub. Leaves of this plant are used as a tea sub- used in oils and fatty foods to retard their autoxidation.
stitute and have a refreshing flavor. Many phenolic com- Therefore, the importance of the search for natural anti-
pounds of this plant have been identified [2]. Due to the oxidants has greatly increased in recent years [9]. A fo-
over-exploitation for its volatile oil and secondary me- cus is on plant-derived polyphenols because of their po-
tabolites, there is a great need to develop alternative stra- tential antioxidant and antimicrobial properties. Phenolic
tegies of conservation and industrial production of the compounds exhibit considerable free-radical scavenging
bioactive compounds from this plant [3]. No reports of activity, which is determined by their reactivity as hy-
works were found concerning the other two species, C. drogen- or electron- donating agents, their reactivity with
viridiflorous and C. comboynensis. other antioxidants and their metal chelating properties, as
In vitro cultures have the potential to form secondary well as the stability of the resulting antioxidant-derived
metabolites and to exhibit bioactivity comparable to the radicals [10,11].
original plant [4,5]. Cultured cells may serve industrial Our present work is a comparative study of leaves and

Copyright © 2011 SciRes. AJPS

848 Total Phenolic Content and Antioxidant Activity of Standardized Extracts from Leaves and Cell Cultures of
Three Callistemon Species

cell cultures of three Callistemon species with respect to in percent (I%) was calculated according to the following
their potential as antioxidant agents in relation to their equation: I%   A0  A1 A0  100 , with A0 being
total content of phenolic compounds. the absorbance of the control reaction (containing all re-
agents except for the extract) and A1 the absorbance of
2. Materials and Methods the extract. Measurements were carried out in triplicates.
2.1. Plant Material
2.5. Determination of Total Phenolic Content
Plants of three Callistemon species, C. lanceolatus (CL),
A spectrophotometric method after MacDonald [14] was
C. viridiflorus (CV), and C. comboynensis (CC), were
adopted for the determination of total polyphenols in the
collected from a cultivated area in Cairo, Alexandria
prepared extracts. Folin-Ciocalteu reagent from Merck
Road, Egypt. They were kindly authenticated by Prof. Dr.
(Darmstadt, Germany) was used and a standard calibra-
M. Gebali (Plant Taxonomy and Egyptian Flora De-
tion curve was prepared using different concentrations of
partment, National Research Center, Giza, Egypt). A
gallic acid in methanol (0.025 - 0.400 mg·ml–1). Cell cul-
voucher specimen of each was deposited at the herbar-
ture and leaf extracts were prepared in methanol at a
ium of the Pharmacognosy Department, Faculty of Phar-
concentration of 0.06 g/3 ml and 0.06 g/20 ml, respec-
macy, Helwan University, Cairo, Egypt.
tively. Absorbance was measured at 765 nm. For each
2.2. Calli and Cell Cultures sample, three replicate assays were performed. The total
phenolic content was calculated as gallic acid equivalent
Callus of CL was induced by a combination of 0.9
(GAE) by the following equation: T  C  V M . T is
mg·L–1 kinetin and 1.1 mg·L–1 NAA [12]. Calli of CV
the total phenolic content in mg·g–1 of the extracts as
and CC were similarly induced (the detailed methodo-
GAE, C is the concentration of gallic acid established
logy will be published later on). Calli material (0.3 g
from the calibration curve in mg·ml–1, V is the volume of
each) were collected in the active growth phase (after the
the extract solution in ml and M is the weight of the ex-
15th day of subculture) and placed in 250 ml flasks con-
tract in g.
taining 100 ml liquid MS medium supplemented with 0.9
mg·L–1 kinetin in combination with 1.1 mg·L–1 NAA. 3. Results and Discussion
The resulting cell cultures of the three Callistemon spe-
3.1. Phenolic Content of the Extracts
cies were incubated in a horizontal shaker at 100 rpm
and 25˚C for 21 days. Ethanolic (80%) extracts from the leaves and cell cul-
tures of the three Callistemon species were standardized
2.3. Preparation of the Extracts
for their contents of phenolic compounds. The calibra-
The three cell suspension cultures were aseptically fil- tion curve showed linearity for gallic acid in the range of
tered and the cells dried in a vacuum oven at 40˚C, to- 25 - 400 µg·ml–1, with a correlation coefficient (R2) of
gether with the leaves of the three species. They were 0.999 (Figure 1). Leaves of CL contained the highest
then macerated in 80% ethanol for two days, filtered and content of phenolics (104 ± 2.0 mg·g–1), followed by CC
macerated for another two days. After filtration, they (95.8 ± 1.2 mg·g–1) and CV (79.8 ± 4.6 mg·g–1). On the
were concentrated under vacuum at 50˚C. other hand, cell cultures of CV were standardized to
contain more phenolics (14.9 ± 0.6) than the cell suspen-
2.4. Evaluation of the Antioxidant Activity
sions of the other two species, CL and CC, which con-
Determination of the free radical scavenging activity of tained 12.2 ± 0.16 and 9.12 ± 0.16 mg·g–1, respectively
the different extracts was carried out using a modified (Figure 2).
quantitative DPPH (1,1-diphenyl-2-picrylhydrazyl; Sigma-
3.2. Antioxidant Activity of the Extracts
Aldrich, St. Louis, MO, USA) assay [13]. Various con-
centrations of sample extracts in methanol were prepared It is well known that there is a strong relationship be-
(1000, 500, 250, and 100 µg·ml–1). Gallic acid was used tween total phenol content and antioxidant activity, as
as a positive control at concentrations of 100, 50, 25, and phenols possess strong scavenging ability for free radi-
10 µg·ml–1. Blank samples were run using 1 ml methanol cals due to their hydroxyl groups. Therefore, the pheno-
in place of the test extract. One ml of 0.2 mM DPPH in lic content of plants may directly contribute to their an-
methanol was added to 1 ml of the test solution, or stan- tioxidant action [11,15,16].
dard, plus 1 ml of methanol for dilution and allowed to The standardized Callistemon extracts were assessed
stand at room temperature in a dark chamber for 30 min. for their capacity to scavenge DDPH free radical along
The change in colour from deep violet to light yellow with gallic acid as a positive control. The antioxidant
was then measured at 517 nm. Inhibition of free radical activity data are presented as percent of free radical inhi-

Copyright © 2011 SciRes. AJPS

 Total Phenolic Content and Antioxidant Activity of Standardized Extracts from Leaves and Cell Cultures of 849
Three Callistemon Species

Figure 2. Total phenolic content of leaf and cell culture ex-

tracts from three Callistemon species determined by the
Figure 1. Standard calibration curve of gallic acid at con- Folin-Ciocalteu assay and calculated as GAE in mg·g–1 ex-
centrations of 25, 50, 100, 200, 300 and 400 µg·ml–1. Spec- tract based on dry weight. Results are the average of tripli-
trophotometric detection was at 765 nm. cates ± SD.

Table 1. Antioxidant activity of Callistemon leaf and cell culture extracts assayed by the DPPH assay.

Conc. of extract CL CV CC CL CV CC Conc. of standard

Gallic acid
µg/ml leaves leaves leaves cultures cultures cultures µg/ml

1000 73.5 ± 3.2 91.4 ± 0.4 74.4 ± 0.3 50.7 ± 0.2 71.1 ± 0.4 47.3 ± 1.9 100 90.8 ± 1.5

500 67.3 ± 0.2 78.4 ± 0.2 66.9 ± 0.9 41.7 ± 1.4 68.4 ± 0.2 44.4 ± 0.3 50 83.7 ± 0.6

250 60.3 ± 2.0 75.4 ± 0.4 57.8 ± 0.7 38.3 ± 0.4 53.5 ± 0.2 35.3 ± 0.2 25 76.3 ± 0.2

100 48.9 ± 3.7 57.4 ± 0.4 56.3 ± 0.3 35.9 ± 0.8 46.7 ± 0.2 34.1 ± 0.1 10 65.4 ± 0.1

Activity is expressed as inhibition of free radical in percent, I% ± SD (n = 3). Leaf and cell culture extracts were tested at 1000, 500, 250 and 100 µg·ml–1 and
the positive control (gallic acid) at 100, 50, 25 and 10 µg·ml–1.

bition in Table 1. The ethanolic (80%) extracts of the 4. Acknowledgements

leaves of CV exhibited pronounced antioxidant activity Part of this research work was funded by the DFG (Deut-
(91.4% ± 0.4%) at a concentration of 1000 µg·ml–1, com- sche Forschungs Gemeinschaft).
parable to 100 µg·ml–1 gallic acid (90.8% ± 1.5%), al-
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Three Callistemon Species

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