American Journal of Plant Sciences, 2011, 2, 847-850
doi :1 0.4236/ aj ps.2011 .26100 Publ i s hed Online December 2011 (http://www.SciRP.org/journal/ajps)
847
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
1Pharmaco gnos y Dep artment, Facu lty o f Phar macy, Helwan Univer sity, Cairo , Egypt ; 2Pharma cogn osy Dep artmen t, F acult y of P harma cy,
Cairo University, Cairo, Egypt; 3Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Braunschweig, Ger-
many.
Received S eptember 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 b y 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 an d 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
The genus Callis temon (Myrtaceae) contains 34 species
of beautiful evergreen shrubs and small trees. The majo-
rity of the Callistemon species is endemic to the more
temperate regions of Australia, four species are found in
New Caledonia and seven species have been introduced
to India as ornamental trees [1]. They are commonly kno-
wn as bottle brushes because of their cylindrical brush-
like flowers resembling the traditional bottle brush.
C. lanceolatus, also named C. citrinus, is a well-
known shrub. Leaves of this plant are used as a tea sub-
stitute and have a refreshing flavor. Many phenolic com-
pounds of this plant have been identified [2]. Due to the
over-exploitation for its volatile oil and secondary me-
tabolites, there is a great need to develop alternative stra-
tegies of conservation and industrial production of the
bioactive compounds from this plant [3]. No reports of
works were found concerning the other two species, C.
viridiflorou s and C. comboynensis.
In vitro cultures have the potential to form secondary
metabolites and to exhibit bioactivity comparable to the
original plant [4,5]. Cultured cells may serve industrial
purposes, e.g. by immobilization of cells in a matrix for
use in bioreactors. Besides the genetic potential of the
donor plant for callus induction and growth of this callus
in in vitro cultures, a medium containing sufficient nu-
trients, such as the preferred MS medium, is required [4].
Antioxidants play an important role in the prevention
of human diseases. Antioxidant compounds may function
as free radical scavengers, complexing agents for pro-
oxidant metals, as well as reducing agents and quenchers
of singlet oxygen formation [6-8]. Antioxidants are often
used in oils and fatty foods to retard their autoxidation.
Therefore, the importance of the search for natural anti-
oxidants has greatly increased in recent years [9]. A fo-
cus is on plant-derived polyphenols because of their po-
tential antioxidant and antimicrobial properties. Phenolic
compounds exhibit considerable free-radical scavenging
activity, which is determined by their reactivity as hy-
drogen- or electron- donating agents, their reactivity with
other antioxidants and their metal chelating properties, as
well as the stability of the resulting antioxidant-derived
radicals [10,11].
Our present work is a comparative study of leaves and

Total Phenolic Content and Antioxidant Activity of Standardized Extracts from Leaves and Cell Cultures of
848 Three Callistemon Species
cell cultures of three Callistemon species with respect to
their potential as antioxidant agents in relation to their
total content of phenolic compounds.
2. Materials and Methods
2.1. Plan t Ma teria l
Plants of three Callistemon species, C. lanceolatus (CL),
C. viridiflorus (CV), and C. comboynensis (CC), were
collected from a cultivated area in Cairo, Alexandria
Road, Egypt. They were kindly authenticated by Prof. Dr.
M. Gebali (Plant Taxonomy and Egyptian Flora De-
partment, National Research Center, Giza, Egypt). A
voucher specimen of each was deposited at the herbar-
ium of the Pharmacognosy Department, Faculty of Phar-
macy, Helwan University, Cairo, Egypt.
2.2. Calli and Cell Cultures
Callus of CL was induced by a combination of 0.9
mg·L–1 kinetin and 1.1 mg·L–1 NAA [12]. Calli of CV
and CC were similarly induced (the detailed methodo-
logy will be published later on). Calli material (0.3 g
each) were collected in the active growth phase (after the
15th day of subculture) and placed in 250 ml flasks con-
taining 100 ml liquid MS medium supplemented with 0.9
mg·L–1 kinetin in combination with 1.1 mg·L–1 NAA.
The resulting cell cultures of the three Callistemon spe-
cies were incubated in a horizontal shaker at 100 rpm
and 25˚C for 21 days.
2.3. Preparation of the Extracts
The three cell suspension cultures were aseptically fil-
tered and the cells dried in a vacuum oven at 40˚C, to-
gether with the leaves of the three species. They were
then macerated in 80% ethanol for two days, filtered and
macerated for another two days. After filtration, they
were concentrated under vacuum at 50˚C.
2.4. Evaluation of the Antioxidant Activity
Determination of the free radical scavenging activity of
the different extracts was carried out using a modified
quantitative DPPH (1,1-diphenyl-2-picrylhydrazyl; Sigma-
Aldrich, St. Louis, MO, USA) assay [13]. Various con-
centrations of sample extracts in methanol were prepared
(1000, 500, 250, and 100 µg·ml–1). Gallic acid was used
as a positive control at concentrations of 100, 50, 25, and
10 µg·ml–1. Blan k sample s were r un using 1 ml metha nol
in place of the test extract. One ml of 0.2 mM DPPH in
methanol was added to 1 ml of the test solution, or stan-
dard, plus 1 ml of methanol for dilution and allowed to
stand at room temperature in a dark chamber for 30 min.
The change in colour from deep violet to light yellow
was then measured at 517 nm. Inhibition of free radical
in percent (I%) was calculated according to the following
equation:


I%A0A1 A0100 
 
, with A0 being
the absorbance of the control reaction (containing all re-
agents except for the extract) and A1 the absorbance of
the extract. Measurements were carried out in triplicates.
2.5. Determination of Total Phenolic Content
A spectrophotometric method after MacDonald [14] was
adopted for the determination of total polyphenols in the
prepared extracts. Folin-Ciocalteu reagent from Merck
(Darmstadt, Germany) was used and a standard calibra-
tion curve was prepared using different concentrations of
gallic acid in methanol (0.025 - 0.400 mg·ml–1). Cell cul-
ture and leaf extracts were prepared in methanol at a
concentration of 0.06 g/3 ml and 0.06 g/20 ml, respec-
tively. Absorbance was measured at 765 nm. For each
sample, three replicate assays were performed. The total
phenolic content was calculated as gallic acid equivalent
(GAE) by the following equation: TCVM . T is
the total phenolic content in mg·g–1 of the extracts as
GAE, C is the concentration of gallic acid established
from the calibration curve in mg·ml–1, V is the volume of
the extract solution in ml and M is the weight of the ex-
tract in g.
3. Results and Discussion
3.1. Phenolic Content of the Extracts
Ethanolic (80%) extracts from the leaves and cell cul-
tures of the three Callistemon species were standardized
for their contents of phenolic compounds. The calibra-
tion curve showed linearity for gallic acid in the range of
25 - 400 µg·ml–1, with a correlation coefficient (R2) of
0.999 (Figure 1). Leaves of CL contained the highest
content 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 were standardized to
contain more phenolics (14.9 ± 0.6) than the cell suspen-
sions of the other two species, CL and CC, which con-
tained 12.2 ± 0.16 and 9.12 ± 0.16 mg·g–1, respectively
(Figure 2).
3.2. Antioxidant Activity of the Extracts
It is well known that there is a strong relationship be-
tween total phenol content and antioxidant activity, as
phenols possess strong scavenging ability for free radi-
cals due to their hydroxyl groups. Therefore, the pheno-
lic content of plants may directly contribute to their an-
tioxidant action [11,15,16].
The standardized Callistemon extracts were assessed
for their capacity to scavenge DDPH free radical along
with gallic acid as a positive control. The antioxidant
activity data are presented as percent of free radical inhi-

Total Phenolic Content and Antioxidant Activity of Standardized Extracts from Leaves and Cell Cultures of
Three Callistemon Species
849
Figure 2. Total phenolic content of leaf and cell culture ex-
tracts from three Callistemon species determined by the
Folin-Ciocalteu assay and calculated as GAE in mg·g–1 ex-
tract base d on dry we ight. Results are t he aver age of t ripl i-
cates ± SD.
Figure 1. Standard calibration curve of gallic acid at con-
centrations of 25, 50, 100, 200, 300 and 400 µg·ml–1. Spec-
trophotometric detection was at 765 nm.
Table 1. Antioxidant activity of Callistemon leaf and cell culture extracts assayed by the DPPH assay.
Conc. of ex tr a ct
µg/ml CL leaves CV leaves CC leaves CL cultures CV cultures CC cultures Conc . of s tandard µg/ml Gallic acid
1000 73.5 ± 3.2 91.4 ± 0.4 74.4 ± 0.350.7 ± 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 ± 0.941.7 ± 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 ± 0.738.3 ± 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 ± 0.335.9 ± 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.
4. Acknowledgements
bition in Table 1. The ethanolic (80%) extracts of the
leaves of CV exhibited pronounced antioxidant activity
(91.4% ± 0.4%) at a concentration of 1000 µg·ml–1, com-
parable to 100 µg·ml–1 gallic acid (90.8% ± 1.5%), al-
though its phenolic content was less than that of CL and
CC (Figure 2). Furthermore, extracts of CV cell cultures
showed antioxidant activity (71.1% ± 0.4%) comparable
to that of leaf extracts of CL and CC at 1000 µg·ml–1,
even though their phenolic contents were approximately
7-fold that of CV cell cultures (Figure 2). It was previ-
ously reported that non-phenolic antioxidants might also
contribute to the antioxidant activity of plant extracts
[17,18]. Thus, compounds other than phenolics might be
responsible for the pronounced antioxidant activity ob-
served with CV extracts, which requires further investi-
gation. Polyphenolic compounds are also believed to
have chemopreventive and suppressive activities against
cancer cells by inhibition of metabo lic enzymes involved
in the activation of potential carcinogens or arresting the
cell cycle [19]. Nevertheless, a compound with strong
antioxidant potential can also contribute to DNA protec-
tion and prevent apoptosis [20]. Further studies are there-
fore required to detect potential anticancer activities of
the extracts reported here.
Part of this research work was funded by the DFG (De ut-
sche Forschungs G emeins chaft).
REFERENCES
[1] P. C. Kanjilal and A. Das, “Flora of Assam,” Omsons
Publications, New Delhi, 1992.
[2] I. I. Mahmoud, F. A. Moharram, M. S. Marzouk, M. W.
Linscheid and M. I. Salch, “Polyphenolic Constituents of
Callistemon Lanceolatus Leaves,” Pharmazie, Vol. 57,
No. 7, 2002, pp. 494-496.
[3] R. K. Sharma, R. Kotoky and P . R. Bhattach arya, “Vola-
tile Oil from the Leaves of Callistemon lanceolatus D.C.
Grown in Northeastern India,” Flavonoid and Fragrance
Journal, Vol. 21, No. 2, 2006, pp. 239 -240.
doi.org/10.1002/ffj.1564
[4] M. R. Ahuja, D. A. Evens, W. R. Sharp and P. J. Am-
mirato, “Handbook of Plant Cell Culture,” Macmillan,
New York, 1986, pp. 626-651.
[5] A. Parsaeimehr, E. Sargsyan and K. Javidnia, “A Com-
parative Study of the Antibacterial, Antifungal and Anti-
oxidant Activity and Total Content of Phenolic Com-
pounds of Cell Cultures and Wild Plants of Three En-
demic Species of Ephedra,” Molecules, Vol. 15, No. 3,
2010, pp . 1668-167 8 .
doi.org/10.3390/molecules15031668

Total Phenolic Content and Antioxidant Activity of Standardized Extracts from Leaves and Cell Cultures of
850 Three Callistemon Species
[6] E. A. Bell, “The Possible Significance of Secondary
Compounds in Plant,” In: E. A. Bell and B. V. Charlwood,
Eds., Secondary Plant Products, Springer-Verlag, New
York, 1980, pp.11-21.
[7] F. Constable, O. L. Gamborg, W. G. W. Kurz and W.
Steek, “Production of Secondary Metabolites in Plant Cell
Cultures,” Planta Me dica, Vol. 25, 1974, pp. 158-165.
do i.org/10. 1055/s-0028-1097926
[8] C. A. Rice-Evans, N. J. Miller and G. Paganaga, “Anti-
oxidant Properties of Phenolic Compounds,” Trends in
Plant Science, V ol. 2, No. 4, 1997, pp. 152-159.
doi.org/10.1016/S1360-1385(97)01018-2
[9] C. Zollman and A. Vickers, “Complementary Medicine
and the Patient,” British Medical Journal, Vol. 319, 1999,
pp. 1486 -1494.
[10] M. K. Ang-Lee, S . J. Moss and C. S. Yu an, “P.P . Herbal
Medicines and Preoperative Care,” The Journal of the
American Medical Association, Vol. 286, No. 2, 2001, pp.
208-216. doi.org/10.1001/jama.286.2.208
[11] A. Wojdylo, J. Oszmianski and R. Czemerys, “Antioxi-
dant Activity and Phenolic Compounds in 32 Selected
Herbs,” Food Chemistry, Vol. 105, No. 3, 2007, pp. 940-
949. doi.org/10.1016/j.foodchem.2007.04.038
[12] R. S. D. Paul Raj, S. M. Morais and K. Gopalakrishnan,
“In Vitro Propagation of Callistemon citrinus. L.,” Indian
Journal of Science and Technology, Vol. 3, No. 1, 2010,
p. 67.
[13] L. L. Mensor, F. S. Menezes, G. G. Leitão, A. S. Reis, T.
C. dos Santos, C. S. Coube and S. G. Leitão, “Screening
of Brazilian Plant Extracts for Antioxidant Activity by
the Use of DPPH Free Radical Method,” Phytotherapy
Research, Vol. 15, No. 2, 2001, pp. 127-130.
doi.org/10.1002/ptr.687
[14] S. Mcdonald, P. D. Prenzler, M. Autolovich and K. Ro-
bards, “Phenolic Content and Antioxidant Activity of Olive
Oil Extracts,” Food Chemistry, Vol. 73, No. 1, 2001, pp.
73-84. doi.org/10.1016/S0308-8146(00)00288-0
[15] A. Bendini, L. Cerretani, L. Pizzolante, T. Gallina-Toschi,
F. Guzzo, S. Ceoldo, A. M. Marconi, F. Andreetta and M.
Levi, “Phenol Content Related to Antioxidant and An-
timicrobial Activity of Passiflora Spp. Extracts,” Euro-
pean Food Research and Technology, Vol. 223, No. 1,
2006, pp . 102-109.
doi.org/10.1007/s00217-005-0150-7
[16] A. Dlugosz, J. Lembas-Bogaczyk and E. Lamer-Zaraw-
ska, “Antoxid Increases Ferric Reducing Antioxidant Po-
wer (FRAP) even Stron ger than Vitamin C,” Acta Poloniae
Pharmaceutica, Vol. 63, 2006, pp. 446- 4 48.
[17] R. Harish and T. Shivanandappa, “Antioxidant Activity
and Hepatoprotective Potential of Phyllanthus niruri,”
Food Chemistry, Vol. 95, No. 2, 2006, pp . 18 0-185.
doi.org/10.1016/j.foodchem.2004.11.049
[18] N. Hassimotto, M. Genovese and F. Lajolo, “Antioxidant
Activity of Dietary Fruits, Vegetables, and Commercial
Frozen Fruit Pulps,” Journal of Agricultural and Food
Chemistry, Vol. 53 , No. 8 , 20 0 5 , pp. 29 28-2935.
doi.org/10.1021/jf047894h
[19] D. J. Newman, G. M. Gragg, S. Holbeck and E. A. Saus-
ville, “Natural Products as Leads to Cell Cycle Pathway
Targets in Cancer Ch emotherapy,” Current Cancer Drug
Targets, V ol. 2, N o. 4, 20 02, pp. 279-308.
doi.org/10.2174/1568009023333791
[20] V. Rajkumar, H. Guha and R. A. Kumar, “Antioxidant
and Anticancer Potentials of Rheum Emodi Rhizome Ex-
tracts,” Food and Chemical Toxicology, Vol. 49, No. 2,
2011, pp . 363-369 .