International Journal of Medicine and

Pharmaceutical Sciences (IJMPS)

ISSN(P): 2250-0049; ISSN(E): 2321-0095
Vol. 4, Issue 6, Dec 2014, 55-62
TJPRC Pvt. Ltd.

ANTIBACTERIAL, ANTIOXIDANT AND CYTOTOXIC ACTIVITIES

OF CINNAMOMUM TAMALA NEES. LEAVES
MUNMI BORKATAKY & KAUSHAL SOOD
Department of Life Sciences, Dibrugarh University, Dibrugarh, Assam, India

ABSTRACT
Cinnamomum tamala Nees. (Lauraceae) is an important medicinal plant used as a spice in traditional cuisine.
The Ayurvedic Pharmacopoeia of India indicates the use of dried mature leaves of Cinnamomum tamala in sinusitis and
the plant is reported to be hypoglycaemic, stimulant, carminative. It is and also used in cough, diarrhoea, gonorrhoea,
rheumatism, irritations, boils conjunctivitis and itching. The objective of the present study was to determine the
antibacterial activity, antioxidant activity and cytotoxicity of C. tamala leaves. The in vitro antimicrobial activity assay was
carried out by agar well diffusion method andin vitro antioxidant activity was quantified by DPPH radical scavenging
assay. Brine shrimp lethality assay was used to evaluate the cytotoxicity of the ethanol extract of the leaves. The
experiments were performed in triplicate and data was analyzed statistically. The ethanol extract of C. tamala leaves
exhibited antimicrobial activity, antioxidant activity and brine shrimp lethality. The extract was found to exhibit the
antimicrobial activity against S. epidermidis followed by S. aureus. The extract exhibited the scavenging of DPPH free
radicals and recorded an IC50 of 26.02 0.61 g/mL. The extract also exhibited brine shrimp lethality in a dose-dependent
manner and LD50= 4.86 0.08 g/ mL was recorded. The study indicated that C. tamala leaves have potent antibacterial,
antioxidant and cytotoxic activities and justified the use of the plant in traditional medicine and cuisine.

KEYWORDS:Cinnamomum tamala, Antibacterial, Antioxidant, Cytotoxic, IC50, LD50

INTRODUCTION
Cinnamomum tamala Nees.(Lauraceae) also known as the Indian cassia in English and tejpat or tezpaata in
folk; is an evergreen aromatic tree occurring in tropical and subtropical Himalayas including Khasi and Jaintia Hills of
Northeast India. The Ayurvedic Pharmacopoeia of India indicates the use of dried mature leaves of Cinnamomum tamala
in sinusitis. It is popular as a flavouring agent and is inevitable in the preparation of traditional cuisines, especially in the
North-eastern region. Although C. tamala leaves are mainly used as spices, the plant has many medicinal properties. It is
reported to be hypoglycaemic, stimulant, carminative and also used in cough, diarrhoea, gonorrhoea, rheumatism,
irritations, boils, conjunctivitis and itching [1]. Studies have also been conducted for its hypolipidemic effects [2],
anti-diabetic and antioxidant [3], anti-ulcer [4], anti-inflammatory [5], anti-diarrhoeal [6], immunosuppressive [7]
activities. C. tamala is listed as threatened species as the plant population is declining day by day due to over exploitation
and habitat destruction in India [8].

MATERIALS AND METHODS

Chemicals and Equipment

All the chemicals used in the study were of analytical grade. Dimethyl Sulfoxide (DMSO), Ciprofloxacin and

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Munmi Borkataky & Kaushal Sood

2, 2-Diphenyl-1- picrylhydrazyl (DPPH), and other general purpose laboratory chemicals and reagents were procured from
Merck Specialities Pvt. Ltd, Mumbai, India. Microbiological media were obtained from HiMedia, Mumbai, India.

Preparation of Plant Extracts

The leaves were collected locally and processed. The cleaned and shade dried material was ground into fine

powder using electric blender. Plant extract was prepared by cold maceration method. Dried powder was extracted by
soaking in ethanol (1: 2 w/v) for 48 hours with intermittent shaking. The extract was filtered through Whatman No. 1 filter
paper into pre-weighed beakers and dried in a rotatory vacuum evaporator (IKA RV 10 Digital) until a constant dry weight
of each extract was obtained. The residues were stored aseptically at 5C for further use.

Qualitative Phytochemical Analysis

Qualitative phytochemical analysis was performed for alkaloids, saponins, flavonoids, phenols and tannins,

sterols, cardiac glycosides and anthraquinone glycosides by following the standard methods [9-11].

In vitro Antibacterial Activity Assay

Test organisms

The ethanol extract of the plant was screened against 5 bacterial strains, two of which were Gram positive and
three Gram negative. The test organisms were Staphylococcus aureus MTCC 96, Proteus mirabilis MTCC 1429,
Escherichia coli MTCC 739, Pseudomonas aeruginosa MTCC 1688, Staphylococcus epidermidis MTCC 435. The test
strains were obtained from the IMTECH, Chandigarh, India.

Preparation of Inoculums

Stock cultures were maintained at 4C on slants of nutrient agar. Active cultures for experiments were prepared
by transferring a loopful of culture from the stock to test tubes of nutrient broth and incubating for 24 hours at 37C.
The cultures were diluted with fresh nutrient broth to achieve optical densities corresponding to 0.5 McFarland
standardwhich corresponded to a cell density of 106 CFU mL-1.

Antibacterial Susceptibility Test

The agar well diffusion method [12, 13] was used to determine the antimicrobial activity of the extract. In vitro
antibacterial activity was screened by using Nutrient Agar obtained from Himedia (Mumbai). The plates were prepared by
pouring 25 ml of molten media into sterile petri-plates (diameter 100 mm). The plates were allowed to solidify at room
temperature and 100 L inoculum suspensions was spread uniformly with the help of a sterile glass spreader after which
four 6 mm diameter wells were bored into the medium with the help of a sterile glass well borer. The plant extract was
dissolved in DMSO to obtain a final concentration of 200 mg/mL and 100 L of it was loaded into one of the wells.
The extract was allowed to diffuse for 45 minutes at room temperature, after which the plates were transferred for
incubation at 35oC. After 24 hours of incubation, the inhibition zones around the well were measured. The experiment was
performed in triplicate and mean and standard deviation were calculated. Ciprofloxacin (10g/ml) was used the standard
drug while DMSO and distilled water were used as negative control.

In Vitro Antioxidant Activity Assay

The free radical scavenging activity of the extract was measured in vitro by 1, 1- diphenyl-2-picrylhydrazyl

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Antibacterial, Antioxidantand Cytotoxic Activities of Cinnamomum tamala Nees.Leaves

(DPPH) assay [14]. 0.30 mM solution of DPPH was prepared in DMSO and 1 mL of this solution was added to 3.0 mL of
the different concentrations of the plant extract dissolved in DMSO. The mixture was incubated at room temperature for 30
min and the absorbance was measured at 517 nm against blank (SHIMADZU UV-1800 Spectrophotometer, Japan).
A reaction mixture without test sample served as control. The percentage scavenging was calculated by the following
equation:
% inhibition =

Absorbance Control Absorbance Test 100

Absorbance Control

IC50 was defined as the concentration of the extract required to achieve 50% inhibition of DPPH radicals.
The experiment was performed in triplicate and IC50 was expressed as mean standard deviation. Ascorbic acid was used
as the standard.

Determination of Cytotoxicity
Cytotoxic potential of the plant extract was measured by in vitro brine shrimp lethality assay [15]. The eggs of

brine shrimp (Artemia salina) were collected and hatched in artificial sea water prepared by dissolving 28 g of sodium
chloride in 1L of distilled water under constant aeration and illumination at 28oC for 36 hours. After hatching, the active
nauplii were separated from the shells and unhatched eggs by siphoning with a plastic tube. For the purpose of toxicity
testing, 10 brine shrimps were transferred to test tubes containing 4.5 mL of artificial sea water. Each test consisted of
exposing groups of 10 Artemia larvae to various concentrations of the plant extract. 0.5 mL of the diluted plant extract
(1000 g/mL to 0.001 g/mL) was added to the test tube and the number of survivors in each test tube was counted after
24 hours. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of
observation. A control tube was maintained and the percent of nauplii that survived for control and for the experimental
were calculated. DMSO was used as negative control. Mortality was calculated as follows:
Mortality (%) =Sc - St
Where, Sc = percent of nauplii that survived in the control tube
and St = percent of nauplii that survived in the experimental tube
LD50 was defined as the dose that corresponded to 50 % mortality during the test. Mortality (%) was plotted
against the logarithm of dose and LD50 was determined by four parameter logistic curve.

Statistical Analysis
Statistical analysis was performed using SigmaStat 3.5 and the results were expressed as the mean of the three

replicates standard deviation of the replicates.

RESULTS AND DISCUSSIONS

Qualitative Phytochemical Analysis

The ethanol extract of C. tamala leaves was tested positive for important secondary metabolites viz. alkaloids,

flavonoids, phenolics and tannins, steroids, cardiac glycosides and anthraquinone glycosides except saponins (Table 1).

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Munmi Borkataky & Kaushal Sood

Table 1: Qualitative Phytochemical Analysis of Extracts of C. tamala leaves

Extract

Alkaloids

Saponins

Flavonoids

Phenols
and Tannins

Steroids

Cardiac
glycosides

Anthraquinone
glycosides

Ethanol

In Vitro Antibacterial Activity

The extract exhibited antibacterial activity against all the tested microorganisms (Table 2). The zone of inhibition

ranged from 15 1 mm (P. aeruginosa) to 22 0 mm (S. epidermidis). Maximum activity was observed against
S. epidermidis followed by S. aureus. The negative control did not inhibit the test strains.
Table 2: Antibacterial Activity of the Ethanol Extract of C. tamala Leaves
Diameter of Zone of Inhibition (in mm)
Ethanol
Ciprofloxacin
Staphylococcus aureus
20 1
28 1
Proteus mirabilis
18 1
26 1
Escherichia coli
18 0
27 1
Pseudomonas aeruginosa
15 1
22 1
Staphylococcus epidermidis
22 0
28 1
Results expressed as mean standard deviation of three replicates
Strains

In Vitro Antioxidant Activity

The extract was observed to scavenge DPPH free radicals in a concentration dependent manner (Figure 1).

IC50 for the ethanol extract of C. tamala leaves was obtained to be 26.02 0.61g/mL while that for ascorbic acid was
observed to be 4.19 0.01 g/ mL.

Cytotoxicity Assay
The extract was observed to cause the mortality of brine shrimp nauplii in a dose- dependent manner (Figure 2).

The extract recorded a LD50 value of 4.86 0.08 g/ mL. The negative control did not cause mortality of the brine shrimps
during the experiment.

Figure 1: DPPH Free Radical Scavenging Activity of C. tamala Leaves

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Antibacterial, Antioxidantand Cytotoxic Activities of Cinnamomum tamala Nees.Leaves

59

Figure 2: Cytotoxicity of C. tamala Leaves

The leaf extracts of C. tamala inhibited both Gram positive and Gram negative strains and thus showed a broad
spectrum antibacterial activity. The extract exhibited highest activity against S. epidermidis and S. aureus. S. epidermidis is
a common flora of human body and is reported to cause infections in immune-compromised patients while S. aureus is a
gram positive coccus which is a common human commensal. It is commonly responsible for the skin diseases, respiratory
infections and food poisoning. Various workers have already shown that Gram positive bacteria are more susceptible
towards plants extracts as compared to Gram negative bacteria [16, 17]. These differences may be attributed to the fact that
the cell wall in Gram positive bacteria is single layered whereas in the Gram negative bacteria, it is multi-layered [18, 19].
The observed antibacterial activity may be due to the presence of phytochemicals in the leaf extract. Although saponins
were not detected, other secondary metabolites- alkaloids, flavonoids, phenolics and tannins, steroids and glycosides were
observed in the leaf extract. These have been reported to exhibit antibacterial activity by various workers [20-26]. Free
radical scavenging activity is considered as an important indicator of the total antioxidant activity of plant extracts. DPPH
assay has been used successfully by various workers for quantifying the free radical scavenging of plant extracts.
The concentration of the plant extract required to cause 50 % inhibition of DPPH free radicals, also referred to as the IC50,
is determined from the inhibition of DPPH radicals caused by the different concentrations of the extract. A lower IC50 value
implies higher antioxidant activity. C. tamala leaves exhibited promising antioxidant activity which was approximately six
times that of Ascorbic acid. Ascorbic acid is a well-known and important antioxidant which is widely used in food
industry. The DPPH radical scavenging activity of C. tamala leaves may be attributed to the presence of various
phytochemicals. The phenolic constituents found in vegetables and spices have received considerable attention due to their
antioxidant activity. The antioxidant activity of phenolic constituents has been attributed to their oxide-reduction
properties, which play an important role in the adsorption and neutralization of free radicals [27]. Antioxidant-based drug
formulations are used for the prevention and treatment of complex diseases like atherosclerosis, stroke, diabetes,
Alzheimers disease and cancer [28]. Brine shrimp lethality assay is considered as a useful tool for the preliminary
assessment of cytotoxicity [29]. The technique is rapid, simple, easily mastered and inexpensive and uses small amounts of
test material (220 mg or less). It has been reported that brine shrimp toxicity is predictive of cytotoxicity, fungicidal
activity and pesticidal activity [30]. In particular, a positive correlation is reported between brine shrimp lethality and
cytotoxicity towards the 9 KB cell line (human nasopharyngeal carcinoma) and other solid tumors, as well as for the P388
cell line (in vivo murine leukemia) [31]. Brine shrimp (Artemia salina) larvae have been used for over three decades in

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Munmi Borkataky & Kaushal Sood

toxicological studies [29]. LD50 values less than 250 g/mL are considered significant and are regarded to have potential
for further investigation as cytotoxic and antitumor agents [32].

CONCLUSIONS
From the findings of the present study, it may be concluded that C. tamala leaves have potent antibacterial and
antioxidant activities. Furthermore, the preliminary results suggest that the leaves of C. tamala are cytotoxic and thus,
studies may be pursued for antitumor and anticancer activities using cell lines. C. tamala leaves have traditionally been
used in tea and in cuisine. The current work also justifies the use of C. tamala in traditional medicine and in cuisine and the
use of the same is encouraged for reaping the potential health. Further works may be pursued for the isolation and
identification of the bioactive components from the plant.

ACKNOWLEDGEMENTS
The authors acknowledge the research facilities provided at the Department of Life Sciences, Dibrugarh
University and the Department of Biotechnology, Govt. of India; for providing the necessary instrument facilities at the
Centre for Studies in Biotechnology, Dibrugarh University.

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