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Chemical Constituents of an Iranian Grown
Capsicum annuum and their Cytotoxic
Masoumeh Foroutan Koudehi1, Aria Ashja Ardalan2 and Ramin Zibaseresht1,3*
1Biomaterials and Medicinal Chemistry Research Centre, Aja University of Medical Sciences, Iran
2Department of Marine Biology, Islamic Azad University- North Tehran Branch, Iran
3 Department of Chemistry and Physics, Maritime University of Imam Khomeini, Iran
Submission: May 27, 2020; Published: June 25, 2020
*Corresponding author: Ramin Zibaseresht, Biomaterials Laboratory, Department of Chemistry and Physics, Faculty of Sciences, Maritime
University of Imam Khomeini, Nowshahr, Mazandaran, Iran
How to cite this article: Masoumeh F K, Aria A A, Ramin Z. Chemical Constituents of an Iranian Grown Capsicum annuum and their Cytotoxic Activities
Evaluation. Organic & Medicinal Chem IJ. 2020; 9(4): 555769. DOI: 10.19080/OMCIJ.2019.09.555769
A sample of cayenne pepper ,Capsicum annuum –(Family Solanaceae) was collected from a region in Hamadan, west Iran and extracted
from EtOH. GC-MS analysis showed at least 8 compounds out of which 7 compounds were isolated and characterized using 1H NMR analysis.
Cytotoxic effects of the 7 isolated and characterized compounds along with the extract were evaluated against Caco-2 cell lines using MTT assay.
The IC50 values for ethyl palmitate (1), linolelaidic acid ethyl ester (2), ethyl stearate (3), ethyl iso-allocholate (4), 2-(((2-ethylhexyl)oxy)carbonyl)
benzoic acid (5), capsaicin (6), dihydrocapsaicin (7) and the extract mixture were determined in μM (111, 112, 118, 40, 130, 91, 115, and 110,
respectively). The data indicated higher cytotoxic effects of (4) and (6) suggested as potential compounds against Caco-2 cell lines.
Many studies have shown that many species of plants contain
a variety of different types of natural products that have different
biological activities (1-4). Relatively recently, various studies have
shown that natural products extracted from medicinal plants have
been considered by the pharmaceutical industry because of their
anti-inflammatory, anti-tumor and anti-microbial properties (5-
7). Such biological activities have made them an important source
of compounds for the discovery of new drugs. Peppers are among
the oldest cultivated plants in the Americas, and archeological
remains indicate that Capsicum annuum was used by man
even before the advent of agriculture (8). The cayenne pepper
,Capsicum annuum –(Family Solanaceae), is usually a moderately
hot chili pepper and is highly flavorful.
Toxicity, carcinogenicity, and anti-tumor activities of Capsicum
annuum extracts have been evaluated earlier (9-11). Jang et al.
(10) concluded, based on their study, that red chili (Capsicum
annuum) was relatively nontoxic at the doses tested. Other studies
revealed that the extracts might have biological activities such as
antibacterial (12), antioxidant and anti-inflammatory properties
(13). Although in most studies that we explored, mainly either the
mixture of extracts or isolated capsaicin, the pungent alkaloid of
red pepper (Capsicum annuum ) have been extensively studied for
their biological effects (12,13), but less attention has been made
towards the cytotoxicity of the extract or the natural products
isolated from the extract of Capsicum annuum . Here, we report
the extraction, isolation and characterization of some natural
products obtained from Capsicum annuum and subsequently the
investigation of cytotoxic activities of the extract and isolated
natural products against Caco-2 cell line.
The 400MHz 1H NMR and 100MHz 13C NMR spectra were
acquired on a Bruker-400 spectrometer at Shahid Beheshti
University of Medical Sciences. 1H NMR and 13C NMR chemical shifts are reported relative to tetramethylsilane. CDCl3 was used
GC-MS analysis of the ethanol extract of Capsicum annuum
was performed at Shahid Beheshti University of Medical Sciences
using an Agilent 7000-Triple-Quad mass spectrometer coupled
with 7890 A gas chromatography. Separation of PAHs was
performed using a 5% phenyl-methyl siloxane (HB-5MS) bondedphase
fused-silica capillary column (Hewlett-Packard, 30 m ×
0.25 mm i.d., film thickness 0.25 μm). The carrier gas was helium
(purity 99.9995 %) which was further purified by passage through
a helium gas purifier Agilent model RMSH-2. The injection port
was run in splitless mode and the injection volume was 2 μL.
The oven temperature program was 80˚C for 1.5 min, raised to
290˚C at a rate of 50˚C/min and maintained at this temperature
for 10 min and the total run time was 15.2 min. The MS transfer
line and ion source temperatures were adjusted at 290˚C and
230˚C, respectively. GC-MS was performed in EI mode. The mass
spectra were taken by electronic impact at 70 eV; a scan interval
of 0.5 s and fragments from 45 to 450 Da. The solvent delay was
0 to 2 min, and the total GC/MS running time was 36 min. The
relative percentage amount of each component was calculated by
comparing its average peak area to the total areas.
In vitro cytotoxicity study by MTT assay: Caco-2 cells (human
epithelial colorectal carcinoma cells) were cultured in a medium
consisting of RPMI-1640, Dulbecco’s modified Eagle medium
(DMEM), heat-inactivated fetal bovine serum (FBS), and penicillinstreptomycin
in a ratio of 50:34:15:1 at 37°C in a humidified
incubator with 5% CO2. Stock solutions of the test compounds
were prepared in DMSO solvent and diluted 100 folds with the
culture medium. The cells were seeded in 96-well transparent
plates at 10,000 cells per well. After 24 hours, the old mediumwas
removed and Caco-2 cells were exposed to the test compounds at
concentrations ranging from 0.1 - 1000μg/mL. After 48 houres
incubation, 20μL of MTT solution (5mg/mL) was added to each
well of the plates and the plates were then maintained in incubator.
After 4 hours, 100μL DMSO solvent was added to each well
to dissolve the purple formazan crystals and then the plates were
read on a Synergy HT Microplate Reader (Bio-Tek Instruments,
Winooski, VT) at 570 nm with the reference wavelength at 690nm.
The IC50 values were calculated by Sigma Plot 12.0 software.
(MTT) was purchased from Sigma (St Louis, MO, USA). Dulbecco’s
modified eagle’s medium (DMEM), RPMI 1640 medium, and
penicillin/streptomycin solution were obtained from Gibco
Invitrogen (Carlsbad, CA, USA). Human epithelial colorectal
carcinoma (Caco-2) cells were provided by Pasteur Institute,
We have begun a research program aimed at the isolation of
natural products from marine or plant sources and investigate
their cytotoxic activities for the purpose of anti-tumor properties.
In the line of these researches, we have chosen a cayenne pepper
(Capsicum annuum- Solanaceae). Although many studies have
been undertaken by researchers on this species for their biological
activities (5-7,9-13), but as far as our knowledge is concerned,
little attention has been made on the investigation of anti-tumor
activities of the extracts from this plant. Thus, given the studies on
Capsicum annuum extracts, we decided to study the cytotoxicity
of Capsicum annuum, and its isolated natural products. For this
purpose, we used a cayenne pepper fruit which is grown in
Hamadan, west Iran (Figure 1).
The plants were randomly collected from a home garden
belonged to Haji-Morad Bashiri cultivated with the assistance of
Jeiran Bashiri and Homa Khodabandelou in Hamadan, Iran. The
red mature plant fruits were collected, washed with distilled water
and air dried under shade and then pulverized into fine particles.
The materials (100g) were macerated using ethanol for 48 h. The
extracts were filtered, and solvent was evaporated in vacuum
using a rotary evaporator and stored in refrigerator at 4 ºC until
needed for analysis. TLC analysis on silica on plastic (eluting with
dichloromethane/ MeOH, 10:1) showed 8 well separated spots
suggesting, preliminarily, at least 8 compounds in the extract
mixture. The sample of extract was further subjected to column
chromatography (silica gel, eluting with dichloromethane/ MeOH,
10:1). The mixture of extract along with the eluents which were
collected, were further submitted for GC-MS analysis.
As it is shown in Figure 2, GC-MS revealed the presence of 8
compounds with retention time ranging from 20.897 to 25.873.
The maximum peak was shown by capsaicin (81.55%) followed by
ethyl palmitate (6.52%), 2-(((2-ethylhexyl)oxy)carbonyl)benzoic
acid (3.7%) and (2-methoxy-4-((8-methylnonanoylamino)
methyl)phenyl) acetate (3.29%). On comparison of the mass
spectra of the constituents with the NIST library, 8 peaks were
recognized out of which 7 phytoconstituents were identified (Table
1). The retention time (RT) is in minutes. Further identification of
isolated compounds was conducted using 1H NMR method. In all
compounds, the spectra were consistent with the literature (14-
With regard to constituent 2, GC-MS analysis suggested that
the compound linolelaidic acid ethyl ester, trans-trans isomer, was
present in the extract mixture. Although the presence of other
stereoisomers such as ethyl (Z,Z)-9,12-octadecadienoate was
also possible, but the NMR spectra associated with the isolated
constituent using column chromatography suggested the presence
of trans-trans stereoisomer , i.e. linolelaidic acid ethyl ester, as the
only compound as the second constituent of the extract. In order
to prove the structure of this compound, the 1H NMR spectrum
which was obtained was compared with that of reported for ethyl
(Z,Z)-9,12-octadecadienoate isomer by Sigmaaldrich database
(15) (Figure 3). The differences between the spectra associated
with the isomers indicated that, based on the GC-MS spectrum, the
compound 2 structure be linolelaidic acid ethyl ester as it is shown
in Table 1.
Surprisingly, GC-MS analysis showed the presence of
1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester as the
fifth fraction of the constituents of the extract mixture. Given in
many phytochemical studies, compound 1,2-benzenedicarboxylic
acid, bis(2-ethylhexyl) ester were reported as one of the extract
constituents, as far as our knowledge is concerned, there is less
reports available suggesting compound 1,2-benzenedicarboxylic
acid, mono(2-ethylhexyl) ester in their studies (21); therefore,
we infer that compound 1,2-benzenedicarboxylic acid, mono(2-
ethylhexyl) ester, fraction 5, might be of a result of hydrolysis
product of 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl) ester.
This kind of decomposition is plausible for a variety of
reasons such as contamination during the process of collecting
the samples; however, less concentration of this fraction (3.7%)
along with no evidence of the existence of bis- derivative might convince us that such phenomena had not occurred in our study.
Therefore, we concluded that the mono-derivative might have
been an original compound in the extracts. Although the 1H NMR
associated with the isolated fraction 5 was not assigned because
of the impurities in the sample, the origin of the fraction 5 remains
as an ambiguity in our study. The major constituent in our analysis
found to be capsaicin, fraction 6. The 1H NMR spectrum obtained
for this fraction was consistent with the literature (18-20) which
in addition to GC-MS analysis supported the structure. The 1H
NMR spectrum also indicated that the trans-capsaicin was the
isolated isomer (19).
Biological activities of the isolated compounds in this study
individually have been studied previously. The sample compounds
in other studies, in some cases, have been extracted from other
plants sources (Table 2). As the main aim of this research was to
investigate the cytotoxicity effect of some isolated compounds
from the extract of a sample of cayenne pepper against cancer cell
line, we investigated the in vitro effect of the seven isolated and
characterized materials along with the mother extract on Caco-
2 cells (human epithelial colorectal carcinoma cells) using MTT
Table 3 shows the cytotoxic activities of the isolated compounds
1-7 and the extract mixture derivatives against Caco-2 cell lines.
The IC50 values in μM were calculated and were compared with
the values obtained for some available drugs, Fucoxanthin and
Fucoxanthinol (30) (Table 4), and Emodin (31) and Cinnamtannin
B-1 (CTB-1) (32) (Table 5) against Caco-2 cell lines.
As it was shown in Table 3, ethyl iso-allocholate (fraction 4)
followed by capsaicin (fraction 6) showed the highest cytotoxicity
against the cultured cells compared to the other compounds we
studied and compare with the drugs shown in Tables 4 & 5, we
concluded that, ethyl iso-allocholate (fraction 4) and capsaicin
(fraction 6) in the extract can be considered as potential cytotoxic
compounds against Caco-2 cell lines. Therefore, we recommend
this plant as a plant of phytopharmaceutical importance which
might provide ways for the development of several cancer
treatments. In addition, based on cytotoxicity of some fractions
obtained, it might also be interesting to investigate the effects of
the extracts on some viruses including the new SARS-COV-2 virus
in the future.
A number of 7 compounds were isolated and characterized
form a sample extract of cayenne pepper. Cytotoxic effects of the
compounds and the extract were determined against Caco-2 cell
lines using MTT assay. The IC50 values indicated cytotoxic effects of
compounds including (4) and (6) against Caco-2 cell lines.
P Kumboonma, T Senawong, K Siriwong, C Yenjai, C Phaosiri (2016) Inhibition of Capsaicin and Dihydrocapsaicin Derivatives Towards Histone Deacetylase and Molecular Docking Studies. Songklanakarin J Sci Technol 38 (4): 399-406.