BIOASSAY SCREENING OF THE ESSENTIAL OIL AND VARIOUS EXTRACTS FROM 4 SPICES MEDICINAL PLANTS

BIOASSAY SCREENING OF THE ESSENTIAL OIL
AND VARIOUS EXTRACTS FROM 4 SPICES MEDICINAL PLANTS

FARIBA SHARIFIFAR*, MOHAMMAD HASSAN MOSHAFI**,
GHOLAMREZA DEHGHAN- NUDEHE**, ALIEH AMERI*, FAHIMEH ALISHAHI*
AND AMIN POURHEMATI***
Department of Pharmacognosy, Pharmaceutical Research Center, School of Pharmacy, Kerman University of Medical
Sciences, Kerman, Iran
*Department of Pharmaceutical Science, School of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran
**Department of Pharmaceutical Science, School of Pharmacy,
Kerman University of Medical Science, Kerman, Iran
***Department of Agriculture, Bahonar University, Kerman, Iran

ABSTRACT
Four commonly used spices plants in Iran were evaluated for cytotoxicity effect using Brine Shrimp Lethality
(BSL) assay. Essential oils and various extracts of Heracleum persicum, Nigella arvensis, Cinnamomum
zeylanicum and Zingiber officinale were assessed by two methods of disk and solution of BSL. Data were
processed in probit-analysis program to estimate LC50 values. All of the tested fractions have exhibited more
cytotoxicity in the solution method. Essential oils of H. persicum and C. zeylanicum have shown the most
cytotoxicity with LC50 values 0.007 and 0.03 µg/ml respectively. None of aqueous extracts showed significant
cytotoxicity. The analysis of the essential oil of H. persicum showed the hexyl butyrate and octyl acetate as the
main compounds. These results suggest some limitation for using of these spices in diet. Furthermore, these
plants could be considered as a source of cytotoxic compounds which might be studied in more details.

Keywords: Cytotoxicity; Artemia salina; spice; brine shrimp; essential oil.


INTRODUCTION
In Iran, the dietary inclusions of cinnamon, ginger,

siahdaneh, turmeric, onions, etc. have been practiced for
Spices and herbs are a part of the daily food in several
centuries. The present study was therefore designed to
parts of the world, comprise the most important products
assess the cytotoxicity of four most common spices of
used for flavoring foods and play a major role in concepts
parsnip (Heracleum persicum Desf. ex Fischer,)
of illness and curing. Next to their importance for general
cinnamon (Cinnamomum zeylanicum Blume., Lauraceae),
well-being, they are quite often parts of traditional ginger (Zingiber officinale Rosc., Zingiberaceae) and
formulae (Mandeel and Al-Laith, 2007). Furthermore
siahdaneh (Nigella arvensis L., Ranunculaceae) using
diets rich in bioactive phytochemicals reduce the risk of
Brine Shrimp Lethality (BSL) assay. These four plants
degenerative disorders such as cancer, diabetes, include parts of a common daily diet in Iran, and this will
cardiovascular disease and oxidative dysfunction provide a rational basis for their use in phytomedicine as a
(Bazzano et al., 2003; Lee et al., 2004; Sherry et al.,
positive health food supplement or with some precaution
2003). Foods containing these phytochemicals not only
as food supplements.
can provide our diet with certain antioxidant vitamins like

vitamin C, vitamin E and provitamin A, but also a
Simple bioassays developed for screening plant extracts
complex mixture of other natural substances with to detect plant compounds with relevant biological
antioxidant capacity. Epidemiological, biological and activities may be used as a guide for fractionating plant
clinical studies have provided various lines of evidences
extracts. As a rapid preliminary indication of possible
that dietary factors have a profound impact on etiology
adverse effects of spices medicinal remedies, the BSL
and prevention of human cancers (Surh and Ferguson,
assay has been used for screening of the plant fractions.
2003). Therefore, chemoprevention of cancers by The eggs of the brine shrimp, Artemia salina, have been
nutraceuticals and phytochemicals has become a used in a simple bench top bioassay which has yielded
flourishing research field in the past decade (Surh and
good results. A. salina, with same purine metabolism as
Ferguson, 2003; Manson, 2003; Gosslao and Chen, 2004).
that of mammalian cells, has been shown to have a good
The potential toxicity of regularly consumed spices,
correlation with anti tumor activity, although drugs that
whether as a condiment or a medicinal treatment, is an
require metabolic activation in the liver may not be
important consideration when studying the traditional use
detected by A. salina. The DNA- dependent RNA
of the plants.
polymerases of A. salina are also similar to the
*Corresponding author: Tel: 0983413220001, Fax: 00983413220799, e-mail: fsharififar@Kmu.ac.ir
Pak. J. Pharm. Sci., Vol.22, No.3, July 2009, pp.317-322
317

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Bioassay screening of the essential oil and various extracts from 4 spices medicinal plants
mammalian type (Mc Laughlin et al., 1998; Solis et al.,
chloroform, diethyl ether, methanol and water
1993). The aim of this study was to investigate and
consecutively by percolation method for 72h for each
compare the cytotoxicity of the essential oils and various
solvent. Solvent removal carried out under vaccum to
extracts of these four spices plants.
drying.


MATERIALS AND METHODS
Isolation of the essential oils

100 g of the air-dried and ground parts of the tested plants
Plant materials
were submitted for 4 h to water-distillation using a
Ginger is one of the widely used spices and has been used
Cleavenger apparatus. The obtained essential oils were
in traditional oriental medicines as antimicrobial and
dried over anhydrous sodium sulphate, then stored at 4°C
antioxidant (Habsah et al., 2000), anti-inflammatory
until tested and analyzed.
(Tjendraputra et al., 2001), anti-fungal (Ficker et al.,

2003), inhibitor of nitric oxide synthesis (Ippoushi et al., Gas chromatography/mass spectrometry analysis
2003) and an agent for protecting neuronal cells from
GC–MS analysis of the essential oil of parsnip was
amyloid insult (Kim and Kim, 2004). The plant of parsnip
performed with GC (column, oven temperature, flow rate
is native to Iran and grows in moist ground, floodlands
of the carrier gas) using a Shimadzu QP 5000 gas
and thickets. Its fruits and stems locally known as
chromatograph equipped with a Shimadzu QP 5050 mass
“golpar” in Iran, has been used in traditional medicine as
selective detector in the electron impact mode (70 eV).
antiflatulence. In traditional medicine it has been used for
Injector and MS transfer line temperatures were set at 220
treatment of epilepsy (Kermani, 1988). Its anticonvulsant
and 290°C, respectively. The components were identified
activity has been reported previously (Sayyah, et al.,
based on the comparison of their relative retention time
2005). Immunostimulant properties of Heracleum
and mass spectra with those of standards, Wiley 2001
maximum has been reported by Webster and his
library data of the GC–MS system and literature data
colleagues (2006). Cinnamon is one of the most widely
(Adams, 2001). Alkanes were used as reference points in
used spices. Its extracts and essential oil have shown a
the calculation of relative retention indices (RRI).
variety of biological activities including antioxidant
(Jayaprakasha et al., 2006), antimicrobial (Matan et al.,
Toxicity testing against the brine shrimp
2006), anti-diabetic (Kim, et al., 2006) and ovicidal
Hatching shrimp
activities (Yang et al., 2005). The seeds of N. arvensis
Brine shrimp eggs (Artemia salina leach) were prepared
known as "siahdaneh" have been used in folk medicine in
from the Schillat center in Hormozgan province and were
Iran. Its biologic activity and chemical composition has
hatched in artificial seawater which prepared by
not been known yet, but according to a study carried out
dissolving 38 g of sea salt (Sigma chemicals Co., UK) in
by Kokdil and Ylmaz (2005), the percent and composition
1 L of distilled water. The two glass compartments
of fixed oils in various species of Nigella have been
chamber with several holes on the divider was used for
shown to be similar.

hatching. One compartment was illuminated. After 48

hours incubation at room temperature (25-29°C), nauplii
Plant collection and authentication
Ginger and cinnamon were purchased from a local (larvae) were collected by pipette from the lighted side
market; siahdaneh and parsnip were collected from whereas their shells were left in the dark side.

Lalehzar and dehbakri region in Kerman province, Iran
Brine shrimp assay
(June and July, 2006 respectively). Their scientific names
The collected nauplii were treated with selected
were authenticated in department of Pharmacognosy,
concentrations (three dilutions of 10,100 and 1000 µg/ml)
Faculty of Pharmacy. A voucher specimen of siahdaneh
of plant extracts and essential oils. Potassium dichromate
and parsnip (KP1143 and KP1185 respectively) was
was used as positive control. This bioassay was done in
deposited in herbarium center in Department of two modified methods of disk and solution. In the disk
Pharmacognosy, Kerman University of Medical Sciences,
method, various concentrations of the fractions were
Faculty of Pharmacy, Kerman, Iran.
loaded on paper disks (d=0.5cm), air dried and placed in

test tubes. 5 ml of artificial sea water was added to the
Phytochemical screening
tubes and were shook to give homogenous solution. 10
These 4 plants were subjected to phytochemical studies
Active larvae was placed to each tube and subjected under
for searching the flavonoids, alkaloids, tannins and light. Survivors were counted after 24h and the percent of
saponins (Trease and Evans, 1983).
deaths were determined. In solution method, various

concentrations of each fraction were dissolved in
Plant extraction
Dimethylsulfoxide (DMSO) 1%, placed in test tubes. 5 ml
Preparation of the various extracts
of artificial sea water was added and 10 active larvae was
Dried medicinal parts of the plants were ground in a
placed to the tubes and subjected under light. Survivors
grinder with a 2 mm in diameter mesh, about 500g of dry
were counted after 24h. and the percent of deaths were
powder of each plant extracted with petroleum ether,
determined.
318
Pak. J. Pharm. Sci., Vol.22, No.3, July 2009, pp.317-322

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Fariba Sharififar et al.
STATISTICAL ANALYSIS
given in table 2. Nineteen compounds were identified,

representing 97.74% of the total oil. The oil profile
The 50% lethal concentration (LC
exhibits that the hexyl butyrate and octyl acetate were the
50 value) at 95%
confidence interval was calculated for each fraction using
main compounds (38.99% and 22.34% respectively).
the probit analysis method described by finney (Meyer et

al., 1982).
The results of extraction

The results of extraction show that the most percent of H.
RESULTS
persicum seed composed of methanol (ME) and

petroleum ether (PTE) extracts (19.34% w/w and 17.47%
Results of phytochemical screening of the plant
w/w respectively). This plant has given 1.6% w/w
The results of primary phytochemical screening of tested
essential oil. Extraction of N. arvensis resulted the PTE
plants show the presence of high amounts of alkaloid and
extract with the most percent (20.63% w/w), and the
lacking the saponins in all of four tested plants. Two
essential oil about 1.3%w/w. PTE and ME extracts
plants of Z. officinale and C. zeylanicum were found to be
constitute the most percent of the bark of C. zeylanicum
rich in flavonoid content (table 1).
(17.34% w/w and 14.41% w/w respectively). In the

rhizome of Z. officinale, chloroform extract (CHE) and
Chemical composition of the essential oil
ME extracts have shown to be the most (20.88% w/w and
Air-dried fruits of H. persicum were subjected to 13.76% w/w respectively). This plant has the less percent
hydrodistillation using a Clevenger apparatus and the pale
of the essential oil and the most percent of CHE extract
yellow-colored essential oil was obtained (yield 1.6%
among the tested plants (table 3).
v/w). The results of analysis of this essential oil have

Table 1: Results of the primary phytochemical screening of 4 tested plants

Plant species
alkaloid
flavonoid
saponin
tannin
H. persicum
High
Low
Negative
Medium
N. arvensis
High
Negative
Negative
Low
C. zeylanicum
High
Medium
Negative
High
Z. officinale
High
Medium
Negative
High





PTE
CHE
EE
ME
WE
EO
k-dichromate
PTE
CHE
EE
ME
WE
K-dichromate
600
6000
500
400
4000
0
5
50
300
LC
LC
200
2000
100
0
0
m
um
um
um
ale
nicu
ensis
nic
cin
ersic
rvensis
ficinale
ersic
la
ffi
p
. a
arv
eyla
of
. p
H.
N
z
Z.
H
N.
. zey
Z. o
C.
C
plant fraction
plant fraction


Fig. 1: The mean LC50 (µg/ml) confidence interval for Fig. 2: The mean LC50 (µg/ml) confidence interval for
the essential oil and various extracts (PTE: Petroleum the various extracts (PTE: Petroleum ether ex.; CHE:
ether ex.; CHE: Chloroform ex.; EE: Ether ex.; ME: Chloroform ex.; EE: Ether ex.; ME: Methanol ex.; WE:
Methanol ex.; WE: Water ex.; EO: Essential oil) from 4 Water ex.; EO: Essential oil) from 4 plants screened
plants screened using BSL assay- Solution method. The using BSL assay- Disk method. The 50% lethal
50% lethal concentration (LC50 value) at 95% concentration (LC50 value) at 95% confidence interval
confidence interval was calculated for each plant was calculated for each plant extract, using the probit
extract, using the probit analysis method described by analysis method described by finney.
finney.

Pak. J. Pharm. Sci., Vol.22, No.3, July 2009, pp.317-322
319

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Bioassay screening of the essential oil and various extracts from 4 spices medicinal plants
Table 2: Chemical composition of Heracleum persicum
most significant cytotoxicity against brine shrimp larvae
essential oil (diluted 1/100 in acetone v/v).
with LC50 values 0.007 and 0.03µg/ml respectively. PTE
extracts of C. zeylanicum and N. arvensis, ME extract of
No.
Component
% composition
KI
C. zeylanicum, and CHE extract and essential oil of Z.
officinale showed significant activity with LC
1
Hexanol
1.07
867
50 values
4.03, 7, 7.9, 9 and 10 µg/ml respectively in comparison to
2
Butanoic acid
0.84
990
k-dichromate with LC50 value of 25.5 µg/ml. None of the
butyl ester
aqueous extracts showed significant cytotoxicity (figs. 1,
3
Octanal
0.77
998
2).
4
Hexyl acetate
0.59
1005

5
Butyl isovalearate
0.51
1042
DISCUSSION
6
1- octanol octilin
1.36
1065

7
L- linalool
1.73
1092
Chemoprevention of cancers by neutraceuticals and
8
Hexyl iso butyrate 4.58
1139
phytochemicals has become a flourishing research field in
9
1- hexanol
0.68
1154
the past decade (Surh and Ferguson, 2003; Manson, 2003;
10 Hexyl butyrate
38.99
1182
Gosslao and Chen, 2004). As a part of our interest in the
11
E-4- dodecenyl
7.81
1186
safety of Iranian herbal spices and condiments, we have
acetate
evaluated cytotoxicity of various extracts and essential
12 Hexyl isovalerate
0.51
1192
oils of 4 plants which have been used widely as spices by
13 Octyl acetate
22.34
1198
using the BSL assay. None of these plants have been
14
Hexyl-2-methyl
4.27
1223
assessed for in vitro cytotoxicity. The essential oil, PT
butyrate
and CH extracts of the H. persicum showed strong
15 1- octanol
0.67
1256
cytotoxicity with LC50 value of 0.007, 38.4 and 33.8
16 Octyl isobutyrate
2.02
1326
µg/ml respectively in comparison to K-dichromate (LC50
17 Hexyl caproate
1.57
1366
= 25.5 µg/ml). The other extracts exhibited lesser activity.
18 Octyl butyrate
0.9
1369
The analysis of the chemical composition of the essential
19
N- octyl-2-methyl 2.25
1410
oil of H. persicum shows the octyl acetate and hexyl
butyrate
butyrate as the main compounds. There is a report in the

literature about the cytotoxicity of the Heracleum
Relative percentages of the compounds were obtained
sphondylium. The major compounds of the latter oil have
electronically from FID area percent data
been reported to be similar to the H. persicum (Ugur,
KI: Kovats index on non-polar DB-5 ms column in reference
1998). However more studies are needed to determine
to n-alkanes
cytotoxic compounds of the oil of H. persicum. These

investigations have been done and their results will be
The results of bioassay
reported later. The presence of furocoumarines has also
The results of the toxicity of essential oils and various
been reported in the genus of Heracleum (Doi et al.,
extracts of 4 medicinal plants against brine shrimp in two
2004). Among the various fractions of the N. arvensis, PT
methods of disk and solution are shown in Fig: 1 and 2. In
and CH extracts have exhibited the most cytotoxicity
vitro cytotoxicity of the studied plants in the BSL assay,
(LC50 of 7.2 and 21.4µg/ml respectively). The PT extract
expressed as LC50 values, alongside the confidence limits
(LC50 of 7.2 µg/ml) constitutes high percent of the N.
at 95% interval. A total of 24 extracts and essential oils
arvensis, therefore this plant has high potency of
were tested for their toxicity using the BSL assay. The
cytotoxicity. The phytochemical screening of this plant
cytotoxicity values ranged from 0.007 to 535.6µg/ml in
has exhibited high percent of alkaloids. The low
the solution method and 28.3 to 7.3µg/ml in the disk
cytotoxicity of this plant in disk method may be attributed
method. The upper and lower LC50 in two methods are
to its fatty acid content. There are many reports in
considerable. Among the tested fractions, the essential
literature for cytotoxicity of the another species, N. sativa
oils of H. persicum and C. zeylanicum have shown the
(Islam et al., 2004; Thabrew et al., 2005). Almost all
Table 3: The percentage (w/w) of essential oil and each extract from tested plants

Essential
Petroleum
Chlorofor
Methanol
Aqueous
Plant species
Used part
Ether ex.
oil
ether ex.
m ex.
ex.
ex.
H. persicum
Fruit
1.6
17.47
1.95
12.50
19.34
1.32
N. arvensis
Seed
1.3
20.63
1.92
6.87
8.16
4.12
C. zeylanicum
Bark
1.7
1.7
3.93
2.20
14.41
8.55
Z. officinale
rhizome
0.67
1.84
1.84
6.90
13.76
1.95
320
Pak. J. Pharm. Sci., Vol.22, No.3, July 2009, pp.317-322

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Fariba Sharififar et al.
fractions of C. zeylanicum have exhibited strong
cardiovascular disease. Current Atherosclerosis
cytotoxicity. The essential oil of the plant has shown the
Reports, 5: 492-499.
most activity (LC50 = 0.03). The presence of trans Chrubasik S, Pittler M.H, Roufogalis BD (2005).
cinnamicaldehyde in the essential oil of C. zeylanicum
Zingiberis rhizome: A comprehensive review on the
might have been responsible for the observed
ginger effect and efficacy profile. Phytomedicine, 12:
cytotoxicity. The cytotoxicity of cinnamicaldehyde acid
84-701.
has been reported previously (Kwon et al., 1998). The
Doi M, Nakamori T, Shibano M, Taniguchi M, Wang
high cytotoxicity of the PT and CH extracts of this plant
NH, Baba K and Candibirin A (2004). A
may be due to their essential oil content. The ME extract
furanocoumarin dimer isolated from Heracleum
of this plant has shown the most activity among the ME
candicans. Acta Crystallographica Section C, 60: 833-
extracts of four tested plants (LC50= 7.9 µg/ml). This
835.
activity of ME extract of C. zeylanicum can be related to
Ficker C, Smith ML, Akpagana K, Gbeassor M, Zhang J,
the presence of its high amounts of flavonoids, tannins
Durst T, Assabgui A and Arnason JT (2003).
and polyphenols. Water extract of the plant is the only
Bioassay-guided isolation and identification of
fraction with little cytotoxic activity. Finally all of the
antifungal compounds from ginger. Phytotherapy
extracts and essential oil of Z. officinale have exhibited
Research, 17: 897-902.
strong cytotoxicity except the PT and aqueous extracts.
Gosslao A and Chen, KY (2004). Neutraceuticals,
Anti tumor activity of two compounds of zingiberene and
apoptosis and disease prevention. Nutrition, 20: 95-102.
gingerol has been studied (Chrubasik et al., 2005) and it
Habsah M, Amran M, Mackeen MM, Lajis NH, Kikuzaki
seems that at least some of the toxicity of the oil of Z.
H, Nakatani N, Rahman, AA and Ghafar Ali, AM
officinale may be attributed to these compounds. The
(2000). Screening of Zingiberaceae extracts for
cytotoxicity of the species of Z. cassumunar and Z.
antimicrobial and antioxidant activities. Journal of
zerumbone has been reported (Han, 2004; Kluwer, 2003).
Ethnopharmcology, 72: 403-410.

Han AR (2004). A new cytotoxic phenyl butenoid
CONCLUSION
dimmer from the rhizomes of Z. cassumnar. Planta

Medica, 70; 1095-1097.
In general, the results drawn from the present work show
Ippoushi K, Azuma K, Ito H, Horie H and Higashio, H
that, all of these 4 tested plants have exhibited
(2003). [6]-Gingerol inhibits nitric oxide synthesis in
considerable cytotoxicity by BSL assay. Most of the
activated J774.1 mouse macrophages and prevents
activity of these plants is due to their essential oils. For
peroxynitrite-induced oxidation and nitration reactions.
determining the limits of safety of these plants, extensive
Life Science, 73: 3427-3437.
in vitro and in vivo toxicological studies and animal
Islam SN, Begum P, Ahsan T, Huque S and Ahsan M
assays must be undertaken. The results of this work
(2004). Immuno suppressive and cytotoxic properties
suggested that the use of these plants, as spices in a food
of Nigella sativa. Phytotherapy Research, 18: 395-398.
or drug for humans or as neutraceutical, should be treated
Jayaprakasha GK, Ohnishi-Kameyama M, Ono H,
cautiously and caution should be exercised in the use of
Yoshida M and Jaganmohan Rao L (2006). Phenolic
the other herbal spices preparations until exhaustive
constituents in the fruits of Cinnamomum zeylanicum
phytochemical and bioassay-guided fractionation of the
and their antioxidant activity. J. Agric. Food Chem.,
components have been achieved. It is also probable to
54: 1672-1679.
access some compounds with cytotoxicity activity from
Kermani MKK (1988). Daghayegh-ol-Eladj, Saadat Press,
these herbal sources, however more investigations are
Kerman, p.365.
needed for logical conclusion.
Kim DSHL and Kim JY (2004). Side-chain length is

important for shogaols in protecting neuronal cells
ACKNOWLEDGEMENT
from ?-amyloid insult. Bioorganic and Medicinal

Chemistry Letters, 14: 1287-1289.
The authors are so thankful to Vice-Chancellor, Kerman
Kim SH, Hyun SH and Choung SY (2006). Anti-diabetic
University of Medical Science for the research grant to
effect of cinnamon extract on blood glucose in db/db
support this project.
mice. Journal of Ethnopharmacol., 104: 119-123.

Kluwer W (2003). Guide to popular natural products. 3rd
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