Anal. Methods Environ. Chem. J. 6 (2) (2023) 5-17
Research Article, Issue 2
Analytical Methods in Environmental Chemi s try Journal
Journal home page: www.amecj.com/ir
AMECJ
Gas chromatography analysis of plant extracts to examine
ingredients: Turmeric extracts on Leishmania Proma s tigotes
and anti-Leishmania eect of Ginger
Hedieh Zadeh-Abbasi Zarandia, Leila Shirani-Bidabadi b, c,*, Jafar Zolalad, Abass Aghaei-Afshar c, e, f,
Ali Faghihi Zarandig, Ehsan Salarkiah, and Mo s tafa Eghbaliani
a Department of Agricultural Biotechnology, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran.
b Department of Vector Biology and Control, Faculty of Public Health, Kerman University of Medical Sciences, Kerman, Iran.
c Research Center of Tropical and Infectious Diseases, Kerman University of Medical Sciences, Kerman, Iran.
d Department of Agricultural Biotechnology, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran.
e Department of Medical Parasitology and Mycology, Afzalipour Faculty of Medicine, K.U.M.S., Kerman, Iran. Email:
f Leishmaniosis Research Center, Kerman University of Medical Sciences (K.U.M.S), Kerman, Iran.
g Department of Occupational Health and Safety Engineering, Faculty of Public Health, K.U.M.S., Kerman, Iran.
h Leishmaniosis Research Center, Kerman University of Medical Sciences, Kerman, Iran.
i Department of Bio s tati s tics and Epidemiology, Faculty of Public Health, K.U.M.S., Kerman, Iran.
AB S TRACT
Turmeric extract and aroma oil of Curcuma longa exhibit inhibition
properties again s t various bacteria, parasites, and pathogenic
fungi. We inve s tigated the eects of Ginger (Zingiber ocinale)
and Turmeric extract on Leishmania proma s tigotes and used gas
chromatography-mass spectrometry (GC-MS) for analyzing plant
extracts. The hydroalcoholic extractions obtained from the two
plants were diluted in 70% ethanol to three dierent concentrations;
12.5, 100, and 500 mg mL-1. The Leishmania signicant s trains
were propagated in an articial medium to reach sucient parasites.
The survival percentage of Leishmania proma s tigotes was aected
signicantly by the time and concentration of the extracts (P < 0.05).
The repeated measures pattern showed an interaction eect between
various time points and treatment with the extracts. s tati s tics analysis
showed a signicant dierence between dierent concentrations and
extract samples (P < 0.05). GC-MS showed that the survival rate
of Leishmania proma s tigotes treated with hydroalcoholic extract
of Ginger at 3-time points (24, 48, and 72 hours) was lower than
Glucantime and Turmeric extract. The survival rate of proma s tigotes
treated with Turmeric extract was similar to those treated with
Glucantime but lower than those treated with a combined extract of
Ginger and Turmeric at a concentration of 500 mg mL-1. The 50%
inhibitory concentration (IC50) values of Ginger and Turmeric plant
extracts were similar to that of Glucantime, indicating that these
extracts can be used as potential drug candidates for leishmaniasis.
Keywords:
Analyzing plant extracts,
Anti-Leishmanial,
Leishmania Promas tigotes,
Turmeric,
Ginger,
Gas chromatography-mass spectrometry
ARTICLE INFO:
Received 29 Jan 2023
Revised form 6 Apr 2023
Accepted 7 May 2023
Available online 29 Jun 2023
*Corresponding Author: Leila Shirani-Bidabadi
Email: lshiranibidabadi@gmail.com and l.shirani@kmu.ac.ir
https://doi.org/10.24200/amecj.v6.i02.230
------------------------
1. Introduction
Herbal extracts and medicines are s till actively
used to treat various diseases [1]. Ginger
(Zingiber ocinale) is a famous spice in Ea s t and
Southern Asia and other parts of the world. About
100,000 tons of Ginger are produced annually, of
which 80% is made in China. Ginger has long
been used to treat musculoskeletal diseases due
to its anti-inammatory properties [2]. Many
6Anal. Methods Environ. Chem. J. 6 (2) (2023) 5-17
literature reviews have been published on the
mechanisms of action of medicinal Ginger. For
example, the s tudy of Grzanna [3] concerned
using Ginger as an anti-inammatory factor. At
the same time, the literature of Shukla and Singh
[4] examined the cancer-prevention properties
of this raw drug. In addition, Chayakonaprok
[5] inve s tigated the mechanisms of the eect
of Ginger as an anti-emetic after surgery. Many
researchers have recently surveyed in vivo and
in vitro the eects of anti-parasite essential oils
and some of their extracts from native plants.
Zingiber ocinale has antifungal, antibacterial,
and antiparasitic properties [6-9]. The identied
ingredients of Zingiber ocinale consi s ted
of gingerols, shogaols, 3-dihydroshogaols,
paradols, dihydroparadols and derivatives of
acetyl gingerols, gingerdiols and isolated mono
and diacetyl gingerdiols, 1-dehydrogenadiones,
diarylheptanoids, and methyl Combinations [10].
The Zingiberaceae family consi s ts of more than
80 species, of rhizomatous and herbaceous plants;
Curcuma is one of the Plants of this family [11].
Curcuma longa is a rhizomatous owering plant
widely s tudied and has excellent potential for
eectively treating many diseases [12]. Curcuma
longa L. (Zingiberaceae) is an herbaceous plant
native to tropical regions of Asia. It is commonly
used as a spice to add avor to food and impart
color. The powdered form, called turmeric, it is
also used for medicinal purposes [13]. Curcumin
is a polyphenol, non-toxic and medicinal,
antioxidant, active anti-inammatory ingredient,
and antiparasitic activities [14]. Antiprotozoal
properties of Curcumin have also been discovered
both in vitro and in vivo for Plasmodium [15],
Leishmania [16-18], Trypanosoma [19], and
Giardia lamblia [20]. A recent s tudy has also
shown that it works again s t proma s tigote forms
of Leishmania major [21]. Curcumin exerts its
anti-inammatory activity by inhibiting several
important inammatory molecules. Turmeric
powder is eective in reducing inammation after
surgery. It also helps to prevent atherosclerosis
by reducing clot formation in blood vessels [22].
Turmeric extract and essential oil of Curcuma
longa exhibit inhibitory eects again s t various
bacteria, parasites, and pathogenic fungi. In an
animal s tudy, guinea pig models of dermatophytes,
pathogenic molds, and yea s t were treated with
topically applied turmeric oil or curcumin. The
researchers indicated that dermatophytes and
pathogenic fungi were inhibited by turmeric oil,
but the yea s t isolation was neither aected by
curcumin nor turmeric oil. Dermatophyte and
pathogenic mold lesions induced on the guinea
pigs improved with treatment and disappeared
seven days’ po s t-turmeric application. Curcumin
exhibits moderate anti-plasmodium falciparum
and anti-Leishmania major eects [22].
Curcumin was found to be responsible for mo s t
of these cases’ biological eects of turmeric
[23]. Curcumin has anti-leishmania activity in
laboratory conditions [17]. Several researchers
have found the action of curcuminoids isolated
from C.longa again s t Leishmania major with
IC50 values from 22 to 60 M [17, 18, and 24].
Therefore, this s tudy evaluated the eectiveness
of Zingiber ocinale and Curcuma longa extracts
on the inhibition of proma s tigotes of Leishmania
in vitro.
Plant chromatographic analysis can be done using
plant extracts determined by GC-MS, HPLC,
HPLC-MS, and GC-FID. The pharmaceutical
indu s try uses the HPLC method, preparative
and analytical techniques to separate and purify
herbal compounds. HPLC-MS Spectroscopy is
a high-performance liquid chromatography that
is very useful for qualitative analysis. Due to
the equipment’s high sensitivity, s tability, and
eciency, GC and GC-Ms equipment are accepted
methods for s tudying volatile components of
herbal medicines. GC-FID, several detectors are
used for gas chromatography. GC-FID has a high
initial sensitivity to hydrocarbons [25].
The current project aimed to evaluate the
eects of this plant’s extracts on Leishmania
proma s tigotes and used gas chromatography
(GC-MS) and mass spectrometry to analyze plant
extracts.
7
Determination of Plant Extracts by GC-MS Analyzer Hedieh Zadeh-Abbasi Zarandi et al
2. Material and method
2.1. Plant Specimens and Preparation of
Hydroalcoholic Extractions
Dried rhizomes of ginger and turmeric were
obtained from the herbal s tore. The dried rhizomes
were pulverized entirely with a hand mortar and
ltered or passed through a No. 10 sieve. To
prepare hydroalcoholic extractions, the powdered
form of the rhizomes was mixed with 70% ethanol
in a ratio of 1:1. In addition; hydroalcoholic
extracts were obtained by mixing for a week in
the shaker incubator. Then, the extracts were then
dried in an oven at 40°C to remove the alcohol.
Hydroalcoholic extracts obtained from the two
plants were diluted in 70% ethanol in three dierent
concentrations; 12.5, 100, and 500 mg mL-1. The
solvent was mixed with the extract and s terilized
by ltration. The nal concentrations of 12.5, 100,
and 500 mg mL-1 of all extracts were prepared in 1
mL of culture medium.
2.2. Preparation of parasite culture
Parasites were cultured in an NNN medium in
laboratory conditions. Following culture in the
NNN medium, the L. major s trains were transferred
to the RPMI medium. The L. major s trains were
initially propagated in an articial medium (RPMI
1640 with 10 - 20% FBS) to reach sucient
parasites. The anti-leishmania agents of the extracts
were inve s tigated in the s tationary phase of the
growth curve of proma s tigotes.
2.3. Cytotoxic eects of hydroalcoholic extracts
and cytotoxicity Te s t
Proma s tigote cells were collected at the s tationary
phase and numbered using a cell counter. To
evaluate the anti-Leishmania eects of Ginger
and Curcuma longa extractions in RPMI 1640
culture medium, concentrations of 500, 100, and
12.5 mg mL-1 were applied. 10 µL of each dilution
series made was added to an Eppendorf tube. The
dilution used to prepare the hydroalcoholic extract
was 70% ethanol. The solvent is blended with the
extract in small amounts. The inhibitory eect of
each dilution of the extract on Leishmania was
determined in three 1.5 ml Eppendorf tubes. 80
μL of parasite suspension was added to 20 μl of
diluted ethanol for negative control. Glucan time
in 12.5, 100, and 500 mg mL-1 concentrations of
such positive control were used. After the initial
parasites count, the plates were re-incubated at a
temperature of 27°C, and the number of parasites in
10 μL for three days was counted by Neobar slide.
Eventually, the mean number of proma s tigotes
in all three plate series and the percentage of live
parasites were calculated for each concentration.
For the cytotoxicity te s t, 100 µL of parasite
suspension (1×106 Prom s tigote per mL) was
cultured in an incubator at 27°C for 72 h in a 50
mL vial. The cell suspension was placed in two
rows of wells (A-H) in a Nunc-Immuno™ 96-
well microliters plate (Maxi Surf™ surface). After
aspiration of the medium, 150 µL of the highe s t
concentration of the Ginger and Curcuma longa
extracts was added into the rows in a serial dilution.
Wells containing only parasite cell suspension
without any extract were used as controls. 10 µL
of M.T.T reagent (3-4, 5-dimethgylthiaol-2-yl-2,
5-diphenyltetrazolium bromide) was added to each
well and we incubated. After the withdrawal of the
medium and M.T.T reagent, dimethyl sulfoxide
(DMSO) (100 µL) was added to the plates and
s tirred for 5 min. The absorbance of each well
was measured using a micro titer plate reader at a
wavelength of 490 nm [26].
2.4. Gas Chromatography Mass Spectrometry
Analysis (GC-MS)
Gas chromatography-mass spectrometry analyzer
(GC-MS) was used to analyze and identify the
compounds of essential oregano oil (Hewlett-
Packard 6890, Agilent Technology, and Santa Clara,
CA, USA) and equipped with HP-5MS column
(30m×0.25mm×0.25μm). The primary temperature
used was 40 °C for 1 min and later increased to 220
°C at a rate of 3 °C per minute and nally raised
to 270 °C for 5 min at a rate of 20 °C per minute.
Other GC-MC in s trument parameters include
helium carrier gas (999.99%), injector temperature
(260°C), detector temperature (FID, 270°C), split
8
less mode, and potential ionization 70eV, scan
rate, a scan speed of 1 scan per second, scan range
m/z 40-48 was used for all analyses. Essential oil
compounds were identied by comparing their
retention indices, and fragmentation mass spectra
with those s tored in a Wiley 7n.1 mass computer
library and the National In s titute of s tandards and
Technology (NI ST) [27, 28]. The research s tage is
illu s trated in Figure 1.
2.5. s tati s tical Te s t Analysis
Data analysis was done using SPSS version
22 software. Repeated measurement te s ts and
analysis of variance te s ts were used to compare
the data. Average survival and the percentage of
proma s tigotes in dierent concentrations of extract
and times were analyzed using the Po s t Hoc te s t
and Tukey’s multiple comparison te s t. s tati s tical
signicance was discussed when P ≤ 0.05 was
signicant.
3. Results and Discussion
3.1. Growth responses of the parasite to extracts
at dierent concentrations and time Point
In the present s tudy, the anti-Leishmania eects
of Ginger and Turmeric were inve s tigated under
laboratory conditions and were demon s trated and
compared with Glucantime. One-way ANOVA
analysis showed that ginger and turmeric extracts
signicantly aected the survival percentage of
Leishmania (P < 0.05) at dierent concentrations
and time points (Table 1). In addition, no signicant
dierences in viability percentage of Leishmania
were observed when treatment with the two plant
extracts was compared with Glucantime (Table 1).
Interaction eects between dierent time points
and extractions were observed when the repeated
measures te s t was performed. The repeated
measures te s t also, a signicant dierence was
showed between various concentrations and kinds
of extracts (P < 0.05) and between extracts at
dierent time points and concentrations (P < 0.05)
(Table 1). Te s ts for comprising the mean (Tukey’s
multiple comparison te s t) was used to e s timate
the average survival percentage of proma s tigotes
of L. major in various concentrations of two plant
extracts for both extracts, a concentration of 500 mg
mL-1 was the mo s t eective in killing proma s tigotes
(P < 0.05). Tukey’s mean comparison te s t showed
no signicant dierence between the viable
percentage of proma s tigotes in 24 hours, 48 hours
and 72 hours (Table 2a, 2b, 2c).
Anal. Methods Environ. Chem. J. 6 (2) (2023) 5-17
Fig.1. Dierent s tages of plant collection, extraction of hydroalcoholic extract of plants and detection
by the device GC-MS.
9
Table1. In-vitro eects of Ginger (Zingiber ocinale) and Turmeric (Curcuma longa) extracts
on the viability percentage of Leishmania proma s tigotes at dierent time points and concentrations.
variables Sum of
squares df Mean square F Sig.
Percent of viability -24h
Between groups
Within groups
Total
8455.233
1502.785
9958.018
3
8
11
2818.411
187.848 15.004 0.001
Percent of viability -48h
Between groups
Within groups
Total
9424.207
1536.474
10960.68
3
8
11
3141.402
192.059 16.356 0.001
Percent of viability -72h
Between groups
Within groups
Total
8714.994
1700.643
10415.64
3
8
11
2904.998
212.580 13.665 0.002
Table 2a. Comparison of the mean percent viable Leishmania proma s tigotes at 24 h
for the dierent plant extract points (Tukey HSD te s t).
Groups Time
24h 48h 72h
Glucantime 28.0033 ----- -----
Curcuma longa 58.1933 58.1933 -----
Zingiber ocinale 45.5667 ----- -----
control ----- ----- 100.0000
Sig. 0.257 0.102 1.000
Table 2b. Comparison of the mean percent viable Leishmania proma s tigotes at 48 h
for the dierent plant extract points (Tukey HSD te s t).
Groups Time
24h 48h
Glucantime 29.0900 -----
Curcuma longa 34.1933 -----
Zingiber ocinale 58.6133 -----
control ----- 100.0000
Sig. 0.115 1.000
Means for groups in homogenous subsets are displayed
Subset for alpha=0.05
Table 2c. Comparison of the mean percent viable Leishmania proma s tigotes at 72 h
for the dierent plant extract points (Tukey HSD te s t).
Groups
Time
24h 48h 72h
Glucantime 30.1000 ----- -----
Curcuma longa 42.9100 42.9100 -----
Zingiber ocinale ----- 71.0967 71.0967
control ----- 100.0000
Sig. 0.713 0.161 0.149
Means for groups in homogenous subsets are displayed
Subset for alpha=0.05
Determination of Plant Extracts by GC-MS Analyzer Hedieh Zadeh-Abbasi Zarandi et al
10
3.2. Analysis of the eects of extracts on live
Proma s tigotes of Leishmania major by M.T.T assay
The results in Figure 2 show that the survival
rate of Leishmania proma s tigotes treated with
hydroalcoholic extract of ginger at 3 time points
(24, 48, 72 hours) was lower than Glucantime
(positive control) and turmeric. The survival rate
of proma s tigotes treated with turmeric extract was
similar to Glucantime but lower than those treated
with a combined extract of ginger and Curcuma
longa at a concentration of 500 mg mL-1.
3.3. Sort by Mortality
Sort by mortality followed by Ginger › Glucantime
› Curcuma longa › Ginger 500 mg mL-1 + Curcuma
longa 500 mg mL-1.
3.4. Analysis of plant extracts by gas
chromatography and mass spectrometry
Ginger extract
The chromatogram obtained from the s tudy of the
ginger hydroalcoholic extract is shown in (Fig. 3).
Gas chromatography identied 15 compounds with
specic frequencies that are li s ted in (Table 3). Gas
chromatography identied 15 bioactive compounds
corresponding to the peaks shown in (Fig.3). Among
the compounds, 1, 2-Dithiacyclohexane (30.107%)
was the mo s t abundant with a retention time of
40.008. It is present in the bites of insects such as
ants and bees and is also the main compound in nettle
leaves [29]. Among the bioactive compounds, 1,
2-Dithiacyclohexane was the mo s t abundant 30.107%
with a retention time 40. 008. This compound is present
Anal. Methods Environ. Chem. J. 6 (2) (2023) 5-17
Fig. 2. Viability of Leishmania major proma s tigotes (MRHO/IR/75/ER)
at dierent exposure times again s t hydroalcoholic extracts of Ginger, Turmeric, and Glucantime.
Each point represents the average of three independent te s ts.
11
in insect venom, especially in the venom of ants and
bees. It is also a bioactive con s tituent of nettle leaves.
The oil con s tituent Telfairic acid was also identied
with a relative abundance of 20.312% and a retention
time of 34.591. This compound is lipid in nature.
Other important bioactive compounds identied in the
extract include the phenolic compound 2-methoxy-4-
2-propenyle, with a relative abundance of 2.957%
and a retention time of 43.112. This compound has
known anti-microbial and antioxidant properties.
Also, 2, 3, 4, and 5-tetramethyl thiophene 1.61% is
another phenolic compound identied in the extract
with anticancer properties.
3.5. Curcuma longa extract
The chromatogram obtained from the s tudy of
turmeric hydroalcoholic extract is shown in
Figure 4. Based on the result, 20 compounds were
identied and are li s ted in (Table 4). Z, Z-3, 9-Cis-
6, and 7-epoxy-nonadecadiene were the mo s t
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
Time-->
Abundance
TIC: abassi101.D\data.ms
33.534
34.591
36.206
36.824
37.073
37.595
37.773
38.129
38.844
39.603
40.008
41.573
43.114
43.450
46.063
Determination of Plant Extracts by GC-MS Analyzer Hedieh Zadeh-Abbasi Zarandi et al
Table 3. Ginger extract compounds
Compound RT %Area
9,12,15-Octadecatrienoic acid, methyl e s ter 33.536 1.055
Telfairic acid $$ Grape seed oil 34.591 20.312
2-Methyl-z,z 3,13-octadecadienoic 36.205 9.602
Cyclododecyne 36.823 1.266
Phenol,2-methoxy-(CAS) 37.074 6.655
2-Naphthol,1-(p-chlorophenyLazo) 37.592 1.652
2,3,4,5-tetramethyl thiophene 37.773 1.61
N-Methyl-3,3-dimethyl-2-hydroxy butanoic acid amide 38.129 8.16
Cyclooctene,3-ethenyl 38.845 2.595
Methoxy-4-vinyl phenol 39.603 6.44
1,2-Dithia Cyclohexane 40.005 30.107
Bicyclo[10.1.0]tridec-1-ene 41.573 6.182
Phenol,2-methoxy-4-(2-prophenyl)-CAS 43.112 2.957
Vanillin 43.45 0.889
Benzene acetic acid,4-hydroxy-3-methoxy 46.06 0.518
Fig. 3. Gas chromatographic-MSanalysis of Ginger extract.
12
numerous compounds found in the extract with
a relative abundance of 20.082 % and a retention
time of 40. 011. It is an organic aldehyde with
medicinal and therapeutic properties. Tridecanedial
5.955% is a Trichloroethylene compound used as
an indu s trial solvent. Its use in the food and drug
indu s try was banned in several countries in the
1970s due to its toxicity National research, 2007.
The hydrocarbon compound Tetradecyl belonging
to the Alkyne group, was also identied in the
extract. Eicosene 5.786 % is another hydrocarbon
compound identied in he extract.
Anal. Methods Environ. Chem. J. 6 (2) (2023) 5-17
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
Time-->
Abundance
TIC: abassi102.D\data.ms
4.511
24.946 30.168
31.257
33.533
34.603
36.200
37.080
38.131
39.494
40.014
42.377
43.122
43.458
43.921
44.736
45.289
45.477
45.756
46.073
Fig. 4. Gas chromatographic analysis of Curcuma longa.
Table 4. Curcuma longa extract compounds
Compound RT %Area
3-METHYL BUTYL ACETATE 4.509 0.053
Benzen,1-methyl-3-(1-methylethyl) 24.944 0.084
Hexadecanoic acid, methyl e s ter 30.167 -0.189
Thiosulfuric acid (H2S2o3), 31.257 0.23
6-Octadecenoic acid, methyl e s ter(CAS)$$Methyl 6-octadecenoate 33.53 3.433
5-Tetradecyne$$5C14H26 34.602 21.01
Curcumin Keto from(C15H20O) 36.199 4.458
9-Eicosene,(E) 37.08 5.786
10-Undecenoic acid (CAS) 38.129 5.819
Oleine 7503 39.493 4.15
Z, Z-3, 9-Cis-6, 7-epoxy-nonadecadiene 40.011 20.082
Tridecanedial 43.124 5.956
Triuoroacetoxy hexadecane 43.456 3.745
Trans 12-Octadecadienoate 43.922 1.741
Bicyclo[10.1.0]tridec-1-ene 44738 8.09
E-11,13-Dimethyl-12-tetradecane 1-OL acetate 45.286 1.142
2-hydroxy-1-(hydroxymethyl)ethyl e s ter 45.479 1.913
7-pentadecyne 47.758 1.912
1,3 Cyclohexadecanedione 46.073 6.422
13
3.6. Discussion
Many s tudies have been done on the effect of
herbs such as Thyme, Yarrow, Propolis, Siderite,
Medlar leaves, and many other medicinal plants
in treating leishmaniasis. These s tudies showed
that herbaceous plant extracts have an inhibitory
effect on the growth of parasites in some of
them. The inhibitory effect of ginger extract on
many agents in recent years has been reported
[30-32]. Saki and et al. inve s tigated the in vitro
effects of Zingiber officinale on proma s tigotes
and ama s tigotes of Leishmania major and
Leishmania tropica [33]. The results showed
that the hydroalcoholic extract of Zingiber
officinale inhibited the growth of Leishmania
major and Leishmania tropica proma s tigotes
24, 48, and 72 hours after in vitro incubation.
The IC50 of hydroalcoholic extract of Zingiber
officinale was 56 μg mL-1 for Leishmania
major and 275 μg mL-1 for Leishmania tropica
proma s tigotes after 72 hours. The IC50 of
hydroalcoholic extract of Zingiber officinale
was 75 μg mL-1 for Leishmania major and 325
μg mL-1 for Leishmania tropica ama s tigotes
after 72 hours [33]. This s tudy proved that the
hydroalcoholic extract of Zingiber officinale has
cytotoxicity properties, and Leishmania major
has a lower resi s tance to the hydroalcoholic
extract of Zingiber officinale than Leishmania
tropica [33]. Duarte et al. s tudied the effect of
Zingiber officinale extract and F10 fraction on
proma s tigotes of L. Amazonasis. They observed
the IC50 values of 125.5 μg mL-1 for the aqueous
extract of Zingiber officinale and 49.8 µg mL-1
for the F10 fraction of Zingiber officinale F10
[34]. By Elamin resaech, the results showed
that Curcumin had a potent antileishmanial
effect, representing cytotoxicity again s t L.
major proma s tigotes. At 80M, the survival in
Curcumin treated proma s tigotes reached 22%;
however, the median lethal concentration of
Curcumin (LC50) was 35M [35]. Kumar et al.
inve s tigated different extracts of Curcuma longa
rhizome again s t Leishmania donovani. Their
results showed that the methanolic extract had
the maximum antileishmanial activity followed
by chloroform and acetone extract again s t both
proma s tigotes and ama s tigotes [36]. The present
s tudy showed that the survival rate of Leishmania
proma s tigotes treated with hydroalcoholic
extract of Ginger at 3 time points (24, 48, and 72
hours) was lower than Glucantime and Turmeric
extract. The survival rate of proma s tigotes
treated with Turmeric extract was similar to
those treated with Glucantime but lower than
those treated with a combined extract of Ginger
and Turmeric at a concentration of 500 μg mL-1.
Oleanolic acid is a plant compound with anti-
Leishmania properties derived from the marigold
plant (Calendua officinalis). This compound
exhibits its leishmanicidal effect by inducing
apoptosis. In a s tudy by Ghosh et al., oleanolic
acid was loaded on PLGA nanoparticles, and
its efficacy in treating mice infected with
Leishmania donovani was evaluated. The
researchers indicated that the Nanocomposite
reduced the parasitic burden by 78% (P <0.05)
in the spleen. In comparison, free oleanolic acid
could reduce the parasitic load in the spleen by
67% [37]. Rajesh et al. observed the chemical
and medicinal properties of Zingiberaceae.
They indicated many active compounds in
medicinal ginger, including flavonoids such
as kaimferrols and gingerols [38]. Ginger has
many usual pharmaceutical applications. It
includes many biologically active chemical
groups and various minerals and rare elements.
The mo s t crucial medicinal characteri s tic of
Ginger extract that have been inve s tigated
in previous s tudies include antimicrobial,
antifungal, anti-inflammatory, anti-parasitic and
cytotoxicity, anti-diabetic, anti-cancer, and anti-
oxidant effects [39]. In addition, the synthesis
of different nano adsorbents such as activated
carbon (AC) and carbon nanotubes (CNTs)
exhibit inhibition properties again s t various
bacteria similar to Turmeric extract and aroma
oil of Curcuma longa. Also, it can be used to
remove heavy metals and VOC pollution in
various matrixes [40].
Determination of Plant Extracts by GC-MS Analyzer Hedieh Zadeh-Abbasi Zarandi et al
14
4. Conclusion
Since the hydroalcoholic extracts of ginger
and Curcuma longa are safe and have not been
found to have any toxic effects even at high
doses, they can be used as suitable supplements
to the treatment regimen of leishmaniasis and
management of Leishmania parasites. The
current s tudy raised the anti-Leishmania and
protective effects of the two plant extracts with
increased concentration. With these properties,
the hydroalcoholic extracts of ginger and
turmeric can be combined with other compounds,
such as nano-compounds, to produce effective
herbal medicines again s t the retention time of
Leishmania parasites. In our s tudy, the IC50
values of ginger and turmeric plant extracts
were similar to that of Glucantime, indicating
that these extracts can be exploited as potential
drug candidates for cutaneous leishmaniasis.
We used the gas chromatography-mass
spectrometry (GC-MS) method to analyze plant
extracts to inve s tigate plant extracts’ effect on
Leishmania proma s tigotes. This method showed
that Ginger extract kills more parasites in the
culture medium among the plant extracts than
Turmeric extract and Glucantim.
5. Acknowledgements
I need to thank the s taff of the central Laboratory
of Shiraz University s taff, the Department of
gas chromatography, who helped me perform
the desired te s ts.
6. Conflicts of intere s t, Ethical Approval,
and Funding/Support
The authors declare that they have no Conflict
of intere s t. This s tudy has an ethics committee
license (IR.KMU.REC.1398.282). This s tudy
was nancially supported (numbering project:
98000288) by the Kerman Medical University
Research Council.
7. References
[1] M. Heinrich, J. Barnes, S. Gibbons
and E. M. Williamson, Fundamentals
of Pharmacognosy and Phytotherapy,
Churchill Living s tone, Edinburgh,
3rd Edition, 2017. https://www.
elsevier.com/books/fundamentals-of-
pharmacognosy-and-phytotherapy/
heinrich/978-0-7020-7008-2
[2] R.D. Altman, K.C. Marcussen, Effects of a
ginger extract on knee pain in patients with
o s teoarthritis, Arthritis Rheum., 44 (2001)
2531-2538. https://doi.org/10.1002/1529-
0131(200111)44:11<2531::aid-
art433>3.0.co;2-j.
[3] R. Grzanna, L. Lindmark, C.G. Frondoza,
Ginger--an herbal medicinal product
with broad anti-inflammatory actions, J.
Med. Food., 8 (2005)125-132. https://doi.
org/10.1089/jmf.2005.8.125.
[4] Y. Shukla M., Singh, Cancer preventive
properties of ginger: a brief review, Food
Chem. Toxicol., 45 (2007) 683-690.
https://doi.org/10.1016/j.fct.2006.11.002.
[5] N. Chaiyakunapruk, N. Kitikannakorn,
S. Nathisuwan, K. Leeprakobboon, C.
Leelasettagool, The efficacy of ginger for
the prevention of po s toperative nausea and
vomiting: a meta-analysis, Am. J. Ob s tet.
Gynecol., 194 (2006) 95-99. https://doi.
org/10.1016/j.ajog.2005.06.046.
[6] P.S. Luize, T.S. Tiuman, L.G. Morello,
P.K. Maza, T. Ueda- Nakamura, B.P. Dias
Filho, Effects of medicinal plant extracts on
growth of Leishmania (L.) amazonensis and
Trypanosoma cruzi, Brazil. J. Pharm. Sci.,
41 (2005) 85-94. https://doi.org/10.1590/
S1516-93322005000100010.
[7] M. Arbabi, M. Delavari, Z.F. Kashan,
M. Taghizadeh, H. Hooshyar, Ginger
(Zingiber officinale) induces apoptosis
in Trichomonas vaginalis in vitro, Int.
J. Reprod. Biomed., 14 (2016) 691-
698. https://www.ncbi.nlm.nih.gov/pmc/
articles/PMC5153574/pdf/ijrb-14-691.pdf
[8] N. Shoaie, P. Mohammadi, S. Roudbar
Mohammadi, Antifungal effect of
Teucrium polium and Zingiber officinale
Anal. Methods Environ. Chem. J. 6 (2) (2023) 5-17
15
extracts on clinical isolates of Candida
species, Armaghane Danesh J., 17 (2012)
416-422. http://armaghanj.yums.ac.ir/
article-1-414-en.html.
[9] F. Feyzi, S. Moradkhani, M. Matini, F.
Parandin, A. Roshan, M. Fallah, In vitro
Scolicidal effects of methanolic extract of
artemisia (Artemisia aucheri) and ginger
(Zingiber officinale) on live protoscoleces
of hydatid cy s t, J. Arak Uni. Med. Sci.,
18 (2015) 45-52. http://jams.arakmu.ac.ir/
article-1-3756-en.html.
[10] S.D. Jolad, R.C. Lantz, G.J. Chen, R.B.
Bates, B.N. Timmermann, Commercially
processed dry ginger (Zingiber officinale):
composition and effects on LPS- s timulated
PGE2 production, J. Photochem.,
66 (2005)1614-1635. https://doi.
org/10.1016/j.phytochem.2005.05.007.
[11] B. Sasikumar, Genetic resources of
Curcuma: diversity, characterization and
utilization, Plant Genet. Res., 3(2005)230-
251. https://doi.org/10.1079/PGR200574.
[12] J. Sanjay, S. Satyaendra, N. Satish, S.
Sumbhate, Recent trends in Curcuma
longa Linn, Phcog. Rev., 1 (2007) 119-
128. https://www.phcogrev.com/sites/
default/files/PhcogRev-1-1-119_0.pdf
[13] H.P. Ammon, M.A. Wahl, Pharmacology of
Curcuma longa. Planta Med. 57 (1991)1-7.
https://doi.org/10.1055/s-2006-960004.
[14] A. Goel, K.B. Kunnumakkara, B.B.
Aggarwal, Curcumin as ‘‘Curecumin’’:
from kitchen to clinic, Biochem.
Pharmacol., 75 (2008)787-809. https://doi.
org/10.1016/j.bcp.2007.08.016.
[15] L. Cui, J. Miao, L. Cui, Cytotoxic effect of
curcumin on malaria parasite Plasmodium
falciparum: inhibition of hi s tone
acetylation and generation of reactive
oxygen species, Antimicrob. Agents
Chemother., 51 (2007) 488-494. https://
doi.org/10.1128/AAC.01238-06.
[16] R. Das, A. Roy, N. Dutta, H.K. Majumder,
Reactive oxygen species and imbalance
of calcium homeo s tasis contributes to
curcumin induced programmed cell death
in Leishmania donovani, Apoptosis, 13
(2008) 867-882. https://doi.org/10.1007/
s10495-008-0224-7.
[17] T. Koide, M. Nose, Y. Ogihara, Y. Yabu, N.
Ohta, Leishmanicidal effect of curcumin
in vitro, Biol. Pharm. Bull., 25 (2002)131-
133. https://doi.org/10.1248/bpb.25.131.
[18] D. Saleheen, S.A. Ali, K. Ashfaq, A.A.
Siddiqui, A. Agha, M.M. Yasinzai, Latent
activity of curcumin again s t Leishmaniasis
in vitro, Biol. Pharm. Bull., 25 (2002)386-
389. https://doi.org/10.1248/bpb.25.386.
[19] M. Nose, T. Koide, Y. Ogihara, Y. Yabu,
N. Ohta, Trypanocida effects of curcumin
in vitro, Biol. Pharm. Bull., 21(1998),643-
645. https://doi.org/10.1248/bpb.21.643.
[20] L. Pérez-Arriaga, M.L. Mendoza-Magana˜,
R. Cortés-Zárate, A. CoronaRivera, L.
Bobadilla-Morales, R. Troyo-Sanromán,
M.A. RamírezHerrera, Cytotoxic effect of
curcumin on Giardia lamblia trophozoites,
Acta Trop., 98 (2006)152-161. https://doi.
org/10.1016/j.actatropica.2006.03.005.
[21] H. B. Rasmussen, S. B. Chri s tensen,
L. P. Kvi s t, A. Karazmi, A simple and
efficient separation of the curcumins, the
antiprotozoal con s tituents of Curcuma
longa, Planta Medica, 66 (2000) 396–398.
https://doi.org/10.1055/s-2000-8533.
[22] M. Akram, A.A. Shahab-Uddin, K.H. A.
N. Usmanghani, A.B.D.U.L. Hannan,
E. Mohiuddin, M. Asif, Curcuma longa
and curcumin: a review article, Rom.
J. Biol. Plant. Biol., 55(2) (2010) 65-
70. https://tarjomefa.com/wp-content/
uploads/2016/11/5685-English.pdf
[23] C.K. Kokate, A.P. Purohit, S.B. Gokhale,
Pharmacognosy. 40th edition, Nirali
Prakashan, 635 pages, 2007. https://
niralibooks.com
[24] M. Haddad, M. Sauvain, E. Deharo,
Curcuma as a parasiticidal agent: a review,
Planta Medica, 77 (2011) 672–678. https://
Determination of Plant Extracts by GC-MS Analyzer Hedieh Zadeh-Abbasi Zarandi et al
16
doi.org/10.1055/s-0030-1250549.
[25] R. K. Bijauliya, S. Alok, D. K. Chanchal,
M. Kumar, A comprehensive review on
s tandardization of herbal drugs, Int. J.
Pharm. Sci. Res., 8 (2017) 3663-3677.
https://www. s tudocu.com/in/document/
tamil-nadu-dr-mgr-medical-university/b-
pharmacy/6-vol/44663041
[26] M. Mozafary, M.S. Dayer, A. A. Afshar,
H.R. Mollaie, Molecular characterization
of Leishmania parasites in naturally
infected sand flies from the endemic
focus of Kerman City, Southea s tern Iran,
Asian Pac. J. Trop. Med., 6 (3) (2016)188-
192. https://doi.org/10.1016/S2222-1808
(15)61011-8.
[27] R.P. Adams, Identification of essential
oils by gas chromatography/mass
spectrometry, Carol s tream, Allured
Publishing Corporation, 2007. http://www.
juniperus.org
[28] M. Sharififard, I. Alizadeh, E. Jahanifard,
C. Wang, ME. Azemi, Chemical
Composition and Repellency of Origanum
vulgare Essential Oil again s t Cimex
lectularius under Laboratory Conditions,
J. Arthropod Borne Dis., 12 (2018) 387-
397. https://www.ncbi.nlm.nih.gov/pmc/
articles/PMC6423461/pdf/JAD-12-387.
pdf
[29] A. Montazeri, A. Goshtasebi, M.
Vahdaninia, B. Gandek, The short form
health survey (SF-36): translation and
validation s tudy of the Iranian version,
Qual. Life Res., 14 (2005) 875-882. https://
doi.org/10.1007/s11136-004-1014-5.
[30] M. Asadi, S. Bahrami, R. Ansari Samani,
N. Pakniat, Effect of hydroalcoholic
extracts of s tachys lavandulifolia Vahl and
Mespilus germanica leaves on Leishmania
major, Hormozgan Med. J., 15 (2012) 258-
263. https://hmj.hums.ac.ir/Article/88498
[31] M. Barati, I. Sharifi, F. Sharififar, In vitro
evaluation of anti-Leishmanial activities
of Zataria Multiflora Boiss, Peganum
Harmala and Myrtus Communis by
colorimetric assay, J. Kerman Uni. Med.
Sci., 16 (2010) 32-42. https://jkmu.kmu.
ac.ir
[32] L. Shirani-Bidabadi, M. Mahmoudi, S.
Saberi, A. Zolfaghari-Baghbaderani,
M. Nilforoushzadeh, H. Abdoli, The
effectiveness of mix extracts of Thyme,
Yarrow and Propolis on Cutaneous
Leishmaniasis: a comparative s tudy in
animal model (Balb/c), Tehran Uni. Med.
J., 66 (2009) 785-790. https://tumj.tums.
ac.ir/en
[33] J. Saki, E. Biranvand, R. Arjmand, The in
vitro anti-Leishmania Effect of Zingiber
officinale extract on proma s tigotes and
ama s tigotes of Leishmania major and
Leishmania tropica, Turk. Parazitol. Derg.,
46 (2022) 91-96. https://doi.org/10.4274/
tpd.galenos.2021.53825.
[34] M.C. Duarte, G.S. Tavares, D.G.
Valadares, D.P. Lage, T.G. Ribeiro,
L.M. Lage, Antileishmanial activity and
mechanism of action from a purified
fraction of Zingiber officinalis Roscoe
again s t Leishmania amazonensis, Exp.
Parasitol., 166 (2016) 21-28. https://doi.
org/10.1016/j.exppara.2016.03.026.
[35] M. Elamin, E. Al-Olayan, R. Abdel-
Gaber, R.S. Yehia, Anti-proliferative and
apoptosis induction activities of curcumin
on Leishmania major, Rev. Argent
Microbiol., 3 (2021) 240-247. https://doi.
org/10.1016/j.ram.2020.08.004.
[36] M. Kumar, K. Pal, V. Pratap, J. Kumar
Gour, Antileishmanial potential of
different extracts of Curcuma longa
rhizome again s t Leishmania donovani, J.
Sci. Res., 66 (2022) 129-141. https://doi.
org/10.37398/JSR.2022.660114
[37] A.K. Ghosh, F.K. Bhattacharyya, D.K.
Ghosh, Leishmania donovani: ama s tigote
inhibition and mode of action of berberine,
Anal. Methods Environ. Chem. J. 6 (2) (2023) 5-17
17
Exp. Parasitol., 60 (1985) 404-413. https://
doi.org/10.1016/0014-4894(85)90047-5.
[38] Y. Rajesh, Y. Nita, K. Murlidhar,
Traditional herbal remedies for health
care: a review, Int. J. Ayur. Pharm. Res.,
2 (2014) 1-14. https://ijapr.in/index.php/
ijapr/article/view/220
[39] H. Zadeh-Abbasi Zarandi, L. Shirani-
Bidabadi, A. A. Aghaei-Afshar, M.
Eghbalian, J. Zolala, S.M. Mirtadjadini,
A. Saghafipour, E. Salarkia, In vitro
evaluation of hydroalcoholic extracts of
Capparis spinosa L., Ricinus communis,
and Solanum luteum on Leishmania
major (MRHO/IR/75/ER) proma s tigotes,
Jundishapur J. Nat. Pharm. Prod., 17(2)
(2022) e115306. https://doi.org/10.5812/
jjnpp.115306.
[40] S Golkhah, H Zavvar Mousavi, Removal
of Pb (II) and Cu (II) Ions from aqueous
solutions by cadmium sulfide Nanoparticles,
Int. J. Nanosci. Nanotechnol. 13 (2017)
105-117. https://www.ijnnonline.net/?_
action=article&keywords=Mousavi
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