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Evaluation of antioxidant compounds, antioxidant activities and capsaicinoid

compounds of Chili ( Capsicum sp.) germplasms available in Malaysia

Article  in  Journal of Applied Research on Medicinal and Aromatic Plants · March 2018

DOI: 10.1016/j.jarmap.2018.02.001

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 Journal of Applied Research on Medicinal and Aromatic Plants 9 (2018) 46–54

Contents lists available at ScienceDirect

Journal of Applied Research on Medicinal and Aromatic

Plants
journal homepage: www.elsevier.com/locate/jarmap

Evaluation of antioxidant compounds, antioxidant activities and T

capsaicinoid compounds of Chili (Capsicum sp.) germplasms available in
Malaysia
⁎
Md. Amirul Alama, , Nur Farah Syazwaniea, Nor Hasima Mahmoda, Noor Afiza Badaluddina,
Kamarul ‘Ain Mustafab, Nadiawati Aliasa, Farzad Aslanic, M. Asaduzzaman Prodhand
a
School of Agriculture Science and Biotechnology, Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Besut Campus, 22200, Terengganu,
Malaysia
b
School of Food Industry, Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Besut Campus, 22200, Terengganu, Malaysia
c
Faculty of Agriculture, Department of Crop Science, Universiti Putra Malaysia, UPM Serdang, 43400, Selangor, Malaysia
d
School of Biological Sciences, The University of Western Australia, 35 Stirling Highway, Perth, Western Australia 6009, Australia

A R T I C LE I N FO A B S T R A C T

Keywords: The study was carried out to quantify and to evaluate the important chilli bioactive compounds from six different
Bioactive compounds chilli varieties such as; Wild Capsicum, Cili Padi Thai, Cili Johor, Cili Padi Rangup, Cili Padi Putih and Cili Padi
Phenolics Centil collected from different locations of Malaysia. The antioxidant compounds; Total Phenolics, Total
Flavonoids Flavonoids, Total Carotenoids, ß-carotenes and antioxidant activities were determined in these chilli varieties.
Carotenoids
Ferric Reducing Antioxidant Power assay (FRAP) and 1,1-Diphenyl-2-Picrylhydrazyl assay (DPPH) including
Capsaicin and dihydrocapsaicin
capsaicin and dihydrocapsaicin were also conducted. Results revealed that, for both antioxidant compounds and
activities, Cili Padi Rangup variety produced the highest value for total phenolic contents, total flavonoid
contents, FRAP and DPPH activities expect for total carotenoid contents which was recorded high in Cili Padi
Centil variety. Besides, the highest value for capsaicin and dihydrocapsaicin were determined in Cili Padi Putih.
The phylogenetic tree produced based on morpho-physiological attributes showed that both of the high quality
chilli varieties (Cili Padi Rangup and Cili Padi Putih) are originate from the same group. Therefore, these two
chilli varieties can be recommended as having the highest content of antioxidant compounds, antioxidant ac-
tivities, capsaicin and dihydrocapsaicin.

1. Introduction
Chili is an important agricultural vegetable crop, not only because
of its economic importance but also due to the nutritional and medic-
inal value of its fruit used for coloring and flavoring foods. It is a ve-
getable that is particularly rich in organic microcomponents with an-
tioxidant properties. Commonly, their hot sensory taste is due to
capsaicinoids as the major group of organic compounds which is closely
related to the family of alkaloids, and are known to be biosynthesized
and accumulated in the placenta of Capsicum fruits (Juangsamoot et al.,
2012). Red fruits are rich in carotenoids (beta-carotene and beta-
cryptoxanthin). They are also a good source of phenolic compounds
with antioxidant properties (Perucka and Materska, 2003). Hot and
semi-hot varieties of chili contain a group of compounds that give the
fruits a spicy taste – they are capsaicinoids. Capsaicin makes up about
60% and dihydrocapsaicin about 30% of total capsaicinoids content.
Levels of all these antioxidants and capsaicinoids can vary with a hy-
brid, maturity stage at harvest and agro-climatic conditions, as well as
during storage and processing of the fruits (Iqbal et al., 2013).
In recent years, phenolic compounds have attracted the interest of
researchers because of their antioxidant activity that can protect the
human body from free radicals. Hot peppers were found to be a good
source of phenolic compounds and are ranked fourth after broccoli,
spinach, and onion with respect to total phenolic contents (Chu et al.,
2002). A variety of vegetables are good dietary sources of carotenoids,
especially those with orange, yellow, red and dark green colors (Muller,
1997). All capsicums have carotenoids; the amounts they have are
differed mainly between hybrids and level of maturity (Gnayfeed et al.,

Abbreviations: WC, Wild Capsicum; CT, Cili Padi Thai; CJ, Cili Johor; CR, Cili Padi Rangup; CP, Cili Padi Putih; CC, Cili Padi Centil; TPC, total phenolic contents; TFC, total flavonoid
contents; TCC, total carotenoid contents; FRAP, Ferric Reducing Antioxidant Power assay; DPPH, 1,1-Diphenyl-2-Picrylhydrazyl
⁎
Corresponding author.
E-mail address: amirulalam@unisza.edu.my (Md. A. Alam).

https://doi.org/10.1016/j.jarmap.2018.02.001
Received 20 September 2017; Received in revised form 15 January 2018; Accepted 8 February 2018
Available online 14 March 2018
2214-7861/ © 2018 Elsevier GmbH. All rights reserved.
Md. A. Alam et al.
2001). Capsaicinoids possess physiological, pharmacological and anti-
microbial activities, used in the treatment of several painful and in-
flammatory conditions (Dorantes et al., 2000; Perucka and Materska,
2001). Capsaicin and other members of the capsaicinoids group pro-
duce a large number of physiological and pharmacological effects on
the gastrointestinal tract, the cardiovascular and respiratory system as
well as the sensory and thermoregulation systems (Al Othman et al.,
2011). Capsaicin has been used as an analgesic against arthritis pain
and inflammation (Deal et al., 1991). It has also been reported to show
anticancer effect (Moore and Moore, 2003) and to be active against
neurogenic inflammation (burning and stinging of hands, mouth, and
eyes) (Szolcsanyi, 2004).
Understanding the morpho-physiological variation and relationship
among chili germplasms is helpful in providing correct information
about the diversity of chili germplasms, especially for antioxidant
properties and capsaicinoid compounds. Many varieties of peppers have
been commercialized all over the world while only six in Malaysia; Wild
Capsicum (WC), Cili Padi Thai (CT), Cili Johor (CJ), Cili Padi Rangup
(CR), Cili Padi Putih (CP) and Cili Padi Centil (CC). Here, this study was
conducted to evaluate the antioxidant properties and capsaicinoid
compounds among these six chili varieties in Malaysia and to re-
commend the best one through the variability analysis.
2. Materials and methods
2.1. Plant material
Six hot chili variety seeds/seedlings were collected from different
locations in Malaysia and grown in UniSZA farm field until harvesting.
The harvested fruits were dried and cleaned properly and preserved in
refrigerator for analysis later. The details of the collected chili varieties
are presented in Table 1 and Fig. 1.
2.2. Morphological traits
Plant height (cm), number of branches per plant, average leaf area
(cm2), average number of fruits per plant, fruit color, fruit length (cm),
fruit diameter (mm), average fruit weight (g) and total fruit fresh and
dry weight per plant (g) were determined in this study. Leaf area was
measured by leaf area meter (LI-Cor, Model LI-3100 Area Meter, LI-Cor
Inc. Lincoln, Nebraska, USA).
2.3. Physiological traits
2.3.1. Chlorophyll contents
Leaf chlorophyll content was measured at 60 days after trans-
planting the plants using a portable chlorophyll meter (MINOLTA™
SPAD-502, Minolta Camera CO., Osaka, Japan). Five readings were
taken per plant.
2.3.2. Leaf stomatal conductance
The stomatal conductance (cm s−1) was determined using LI 6400
Portable Photosynthesis System (LI-Cor, Inc., Lincoln, NE, USA).
Table 1
Collection locations of the studied Chili varieties in Malaysia.
Acc. No. Variety/local name Location of collection
1 Wild Capsicum (WC) Johor Bahru
2 Cili Padi Thai (CT) Selangor
3 Cili Johor (CJ) Selangor
4 Cili Padi Rangup (CR) Selangor
5 Cili Padi Putih (CP) Terengganu
6 Cili Padi Centil (CC) Terengganu
47
Journal of Applied Research on Medicinal and Aromatic Plants 9 (2018) 46–54
2.4. Extraction
The samples were extracted as described in Alam et al. (2014,
2015). An aliquot of 2 g chili powder was mixed with 20 ml methanol in
a 100 ml conical flask followed by incubation in 40 ± 1 °C for 2 h in a
water bath shaker (100 rpm). The supernatant was collected using filter
paper (Whatman No. 1) and the residue was extracted again as de-
scribed above. The supernatants were pooled together and subjected to
a rotary evaporator (BuchiRotavapor R-210, Switzerland) to remove the
methanol. The concentrated extract was stored in −20 ± 1 °C for
analysis later.
2.5. Determination of antioxidant compounds
2.5.1. Determination of total phenolic content
The TPC was determined using Folin-Ciocalteu method as described
by Alam et al. (2014, 2015) with some modifications. An aliquot of
0.5 ml sample extract was mixed with 0.5 ml Folin-Ciocalteu reagent,
followed by addition of 10 ml of 7% Na2CO3 solution. The mixture was
incubated for 1 h at 25 ± 2 °C in the dark and then absorbance was
measured at 765 nm using a UV–vis Spectrophotometer (UV-1650 PC
Spectrophotometer, Shimadzu, Japan). The amount of TPC was ex-
pressed as milligram of Gallic acid equivalents (GAE) per g of sample
(mg GAE*g−1 DW).
2.5.2. Determination of total flavonoid content
TFC was determined using colorimetric method as described by
Alam et al. (2014, 2015). A 0.5 ml aliquot of sample extract was mixed
with 2.25 ml of distilled water in a black covered test tube followed by
addition of 0.15 ml of 5% NaNO2 solution. After 6 min, 0.3 ml of 10%
AlCl3*6H2O solution was added followed by addition of 1.0 ml 1 M
NaOH. The mixture was mixed well by vortexing. The absorbance was
measured at 510 nm by the spectrophotometer (details). Results were
expressed as milligram of Quercetin equivalent (QE) per g of sample
(mg QE*g−1 DW).
2.5.3. Determination of total carotenoid content (TCC)
TCC was determined based on the method described by Alam et al.
(2014, 2015) with some modifications. A 0.5 g of the dried powdered
sample was mixed with 15 ml hexane, vortexed and left for few min-
utes, then centrifuged for 1 min at 3000 rpm. The supernatant was
collected and re-extracted until become colorless. Collected supernatant
was evaporated until became dry using a rotary evaporator at 40 °C.
The crude extract was re-dissolved in 5 ml hexane, and absorbances will
be read 450 nm UV spectrophotometer (UV-1650 PC Spectro-
photometer, Shimadzu, Japan). Results were be expressed as milligrams
of ß-carotene equivalent (BCE) in 1 g of sample (mg BCE*g−1 DW).
2.5.4. Determination of ß-carotene content
The ß-carotene of six (6) different Capsicum varieties was de-
termined based on AOAC (Association of Analytical) method
970.64. Two (2) grams of blended powder of chili fruit samples were
transferred into the 100 ml flask covered with stopper. Then the
samples were shaken for 1 min with a mixture of 30 ml hex-
ane:acetone:ethanol:toluene (10:7:6:7). For hot saponification, 2 ml of
40% methanolic KOH was pipetted into the flask and swirled for 1 min
before placing in 56 °C water bath for 20 min. The samples were then
allowed for cooling for 1 h keeping in the dark. Then 30 ml of hexane
was pipetted into the flask and swirled for 1 min. 10% volume of an-
hydrous Na2SO4 was diluted with the sample and shaken vigorously for
1 min and 50 ml of upper phase was preserved for analysis using
spectrophotometer absorbance at a wavelength of 436 nm in equivalent
ß-carotene standard solution. Contents of ß-carotene in samples were
expressed in mg/100 g fresh weight. All samples were analyzed in tri-
plicate.
Md. A. Alam et al.
Fig. 1. Six different chilli varieties; (a) Wild Capsicum, (b) Cili Padi Thai, (c) Cili Johor, (d) Cili Padi Rangup, (e) Cili Padi Putih and (f) Cili Padi Centil.
2.6. Determination of antioxidant activity
2.6.1. 1 Ferric Reducing Antioxidant Power (FRAP) assay
FRAP assay was conducted based on the method described by Alam
et al. (2014, 2015) with minor modifications. The oxidant in the FRAP
assay was prepared by mixing 2.5 ml of 10 mM TPTZ prepares in 40 mM
HCl, 25 ml of acetate buffer and 2.5 ml of 20 mM FeCl3*H2O. The
mixture was referred to as “FRAP reagent”. A 200 μl of sample was
pipetted into a black covered test tube and mixed with 3 ml of FRAP
reagent by vortexing. The mixture was then allowed to react for 30 min
at 37 °C temperature in a water bath. The absorbance of the mixture
was read at 593 nm UV spectrophotometer (UV-1650 PC Spectro-
photometer, Shimadzu, Japan). Triplicate tubes were prepared for each
48
Journal of Applied Research on Medicinal and Aromatic Plants 9 (2018) 46–54
extract. The FRAP values were expressed as milligrams of Ferrous
Sulphate equivalent (FSE) in 1 g of sample (mg FSE*g−1 DW).
2.6.2. 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) free radical scavenging
activity assay
Diphenyl-2-Picrylhydrazyl (DPPH) assay was used to determine the
free radical scavenging activity according to the method of Alam et al.
(2014, 2015) with some modifications. Sample solutions with different
concentrations (0.156–10 mg/ml) were prepared from chili powder
extract. A 0.25 ml aliquot of each concentration was mixed with 1.75 ml
of DPPH solution (0.025 mg/ml). The mixture was then left at room
temperature for 30 min in the dark. Quercetin at a concentration of
1000 μg/ml was used as a standard, respectively. The absorbance of the
Md. A. Alam et al. Journal of Applied Research on Medicinal and Aromatic Plants 9 (2018) 46–54

mixture was measured at 515 nm wavelength using micro plate reader
(Model EL-800, BIOTEK Instrument, USA). IC50 value, representing the
amount of extract which scavenged/reduced 50% of DPPH radical, was
calculated from percent scavenging versus concentration curve. A
higher concentration to reduce 50% of DPPH solution show lower ac-
tivity. The calculation of percentage DPPH free radical scavenging will
be as follows:
2.9. Statistical analysis
The data were subjected to analysis of variance (ANOVA) using
Minitab (Version 16). When F was significant at the P < 0.05 level,
treatment means were compared and separated using the least sig-
nificant difference test (LSD). Pearson’s correlation coefficient analysis
was carried out to assess the association between different parameters.

Absorbance of control-Absorbance of sample

DPPH scavenging effects (%) = × 100
Absorbance of control 3. Results

All chemicals used in these studies were purchased either from 3.1. Determination of antioxidant compounds
Sigma-Aldrich Co. (St. Louis, USA), HmbG Chemical Co. (Germany),
Merck Co. (Darmstadt, Germany), Systerm Co. (USA), or Fischer 3.1.1. Total phenolic content
Scientific Co. (Leicestershire, UK). The total phenolic compounds (which are composed of phenolic
acids, flavonoids, tannins, lignans and quinones; Huang et al., 2009)
2.7. Capsaicin and dihydrocapsaicin analysis were investigated in this study. The content of total phenolic com-
pounds was determined by extrapolation from the calibration curve
Sample extraction was done following the method used by Alam prepared from gallic acid concentrations and expressed in milligram of
et al. (2014, 2015) with slight modification from the dried and blended gallic acid (mg GAE*g−1 DW). The total phenolic content of Cili Padi
powdered samples of six (6) chili varieties. For capsaicinoids extraction, Rangup was remarkably high (0.43 ± 0.102 mg*g−1) compared to that
each dried pepper sample (5 g) was placed in 5 ml ethanol in a 120 ml in other varieties. Cili Padi Rangup was followed by Cili Padi Centil
glass bottle with Teflon lined lids. Bottles were capped and placed in a (0.28 ± 0.04 mg/g), Cili Padi Putih (0.28 ± 0.33 mg/g), and Cili Padi
water bath at 80 °C for 4 h, then swirled manually every hour. Samples Thai (0.186 ± 0.02 mg/g) and Cili Johor (0.185 ± 0.02 mg/g), re-
were removed from the water bath and cooled at room temperature. spectively (Table 2). Overall, the phenolic content in Wild Capsicum
The supernatant layer of each sample (5 ml) was filtered through (0.159 ± 0.02) was found to be significantly lower than others. Fur-
0.45 μm by filter paper into vial using 5 ml syringe (Milipore, Bedford, thermore, non-significant variations were seen among Wild Capsicum,
MA, USA). The vial was capped and stored at 5 °C in a refrigerator until Chili Padi Thai and Chili Padi Jihor. At the same the time Chili Padi
analysis. Putih and Chili Padi Centil also showed non-significant variations
among them for TPC (Table 2).
Different levels of TPC contents reported in this study may be at-
2.7.1. Spectrophotometric method tributed to the different varieties of Capsicum species, procedures and
2.7.1.1. Preparation of standard stock solution. Pure capsaicin or standards used to express the total phenolic contents used by the dif-
dihydrocapsaicin was weighed and dissolved in acetonitrile up to ferent investigators. The usage of Folin-Ciocalteu reagent was also
2.0 ml to get the stock solution of 2 mg/ml. The solution then was measured based on the color measurement which was non-specific to
filtered with 0.45 μm syringe filter. The stock solution of capsaicin or phenol. Possibly there were other components that can react with the
dihydrocapsaicin was diluted as required. Calibrate daily with at least reagent such as ascorbic acid. Different phenolic compounds may also
six working standards over the range of 10–200 mg/L for each standard. have different responses to this assay. However, the measurement of
These dilutions were scanned in the UV–vis spectrometer (UV-1650 PC color changes after 30 min incubation has previously been used to de-
Spectrophotometer, Shimadzu, Japan) and capsaicin was selected as the termine the existence of phenolic compounds in different plant samples
wavelength of detection. The absorbance of the mixture was then read (Huda-Faujan et al., 2009).
at 280 nm. The capsaicinoids content in the unknown extract solutions
was calculated based on the absorbance values of known standard
3.1.2. Total flavonoid content
solutions.
Flavonoids consist of a large group of polyphenolic compounds, have a
benzo-γ-pyrone structure, and provide benefits in multiple ways to the plant
2.8. Variability analysis of chili varieties based on morpho-physiological producing them (Gurnani et al., 2016). In chili total flavonoid contents is
attributes mainly the combination of flavones, flavonols, flavanones, flavanonols, fla-
vanols or catechins and anthocyanins (Gurnani et al., 2016). In this study
2.8.1. Character coding total flavonoid content in Cili Padi Rangup (0.66 ± 0.02 mg/g) was de-
Character states were polarized using outgroup comparison method termined significantly high compared to that in other varieties followed by
(Morrison, 2012). This determines the primitive (plesiomorphic) and Cili Padi Centil (0.2 ± 0.01 mg/g), Cili Padi Putih (0.11 ± 0.01 mg/g),
derived character (apomorphic) by comparing different groups. Out-
group comparison was used in this study based on the close relation- Table 2
Total phenolic content, total flavonoid content and total carotenoid content of six vari-
ships of the study group to other taxa. Plesiomorphies are the initial or
eties of Capsicum sp.
primitive character states found in the ancestors while shared derived
character states occur in one or more terminal taxa under study are Samples TPC1 TFC2 TCC3
called apomorphies (Morrison, 2012).
WC 0.159 ± 0.016c 0.067 ± 0.011c 0.099 ± 0.01b
Outgroup was selected more than one because in certain situations,
CT 0.186 ± 0.021c 0.052 ± 0.201c 0.124 ± 0.01ab
the selected outgroup may not provide all the required information to CJ 0.185 ± 0.021c 0.050 ± 0.020c 0.095 ± 0.01b
determine the relationship between the taxa. Therefore, this is why CR 0.430 ± 0.102a 0.661 ± 0.275a 0.108 ± 0.01b
another outgroup which has a closer relationship with the taxa was CP 0.281 ± 0.326b 0.114 ± 0.011bc 0.088 ± 0.06b
recommended. CC 0.283 ± 0.039b 0.187 ± 0.010b 0.149 ± 0.07a

The complete dataset (shown in results section) was analyzed based 1

mg GA equivalent/gDW.
on the data scoring matrix before transferred and analyzed by MEGA4 2
mg Quercetin equivalent/gDW.
(version 4.0.2) software to construct a phylogenetic tree based on 3
mg ß-carotene equivalent/gDW. Means followed by the same letter within a column
maximum parsimony. are not significantly different at p ≤ 0.05 (LSD).

49
Md. A. Alam et al.
Fig. 2. (a) Standard curve of ß-carotene and (b) ß-carotene content (mg/g) in different varieties of Capsicum sp. Here, bar graphs indicated with different error bars ( ± SE value) are
significantly different at p ≤ 0.05.
Wild capsicum (0.067 ± 0.01 mg/g) and Cili Padi Thai (0.052 ± 0.2 mg/
g), respectively (Table 2). In contrast, the flavonoid content in Cili Padi Johor
(0.050 ± 0.02 mg/g) was found to be significantly lower compared to all
others. Furthermore, statistically non-significant (p > 0.05) variation was
observed among Wild capsicum, Cili Padi Thai and Cili Padi Johor (Table 2).
The same results were also observed for total phenolic contents in all those 3
chilli varieties (Table 2).
3.1.3. Total carotenoid content
TCC was determined by extrapolation from the calibration curve
prepared from ß-carotene concentrations and expressed in milligram of
ß-carotene (Fig. 2a). From the result shown in Table 2, it was revealed
that among the six varieties of the Capsicum sp., the highest amount of
carotenoid content was in Cili Padi Centil (0.149 ± 0.07 mg/g), fol-
lowed by Cili Padi Thai (0.124 ± 0.01 mg/g), Cili Padi Rangup
(0.108 ± 0.01 mg/g), Wild Capsicum (0.099 ± 0.01 mg/g) and Cili
Johor (0.095 ± 0.01 mg/g), respectively (Table 2). In contrast, the
carotenoid content in Cili Padi Putih (0.088 ± 0.05 mg/g) was sig-
nificantly lower compared to that in all varieties. While very interest-
ingly non-significant (p > 0.05) variations were observed among Chili
Padi Johor, Chili Padi Rangup and Chili Padi Putih for total carotenoid
contents (Table 2).
3.1.4. ß-Carotene content
ß-Carotene is not only the most abundant and important carotene
but also a dietary source of vitamin A (Jaswir et al., 2011). Besides ß-
50
Journal of Applied Research on Medicinal and Aromatic Plants 9 (2018) 46–54
carotene, the other pro-vitamin A carotenoids are alpha- and gamma-
carotene and beta-cryptoxanthin.
The content of ß-carotene was determined by extrapolation from the
calibration curve prepared from ß-carotene concentrations and ex-
pressed in milligram of ß-carotene (Fig. 2a). The ß-carotene content in
Cili Padi Rangup was remarkably higher (3.00 ± 0.04 mg/g) com-
pared to that in other varieties, followed by Cili Padi Centil
(2.63 ± 0.08 mg/g), Cili Johor (2.22 ± 0.02 mg/g), Wild Capsicum
(1.38 ± 0.04 mg/g) and Cili Padi Thai (1.17 ± 0.01 mg/ml), respec-
tively (Fig. 2b). On the other hand, the ß-carotene content in Cili Padi
Putih (0.68 ± 0.09 mg/g) was significantly lower compared to that in
any other varieties.
3.2. Antioxidant activities
3.2.1. Ferric reducing antioxidant power assay
Based on Fig. 3a and b, the absorbance (OD) of the FRAP assay in
Cili Padi Rangup (1.73 ± 0.13 mg/g) was the highest compared to that
in the other varieties followed by Cili Padi Putih (1.50 ± 0.04 mg/g),
Cili Johor (1.49 ± 0.04 mg/g), Cili Padi Centil (1.47 ± 0.09 mg/g)
and Wild Capsicum (1.04 ± 0.05 mg/g), respectively (Fig. 4b). In
contrast, the absorbance (OD) of the FRAP assay in Cili Padi Thai
(0.95 ± 0.06 mg/g) was lowest compared to that in any other vari-
eties.
Md. A. Alam et al. Journal of Applied Research on Medicinal and Aromatic Plants 9 (2018) 46–54

Fig. 3. Standard curve for Ferrous sulphate (a) for FRAP assay and FRAP value in different varieties of Capsicum sp. (4b). Here, bar graphs indicated with different error bars ( ± SE value)
are significantly different at p ≤ 0.05.

3.2.2. 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) free radical scavenging Table 3

activity assay Inhibition concentrations (IC50) of six varieties of Capsicum sp.
The extract that can lower the initial absorbance of DPPH solution by
Variety Inhibition Concentration (IC50)
50% has been chosen as the percentage of inhibition for measuring the
antioxidant activity. In this study, the highest DPPH scavenging activity was Wild Capsicum (WC) ND
shown by the extract of Cili Padi Rangup (IC50 = 420 μg/ml) followed by Cili Padi Thai (CT) ND
Cili Padi Centil (IC50 = 491 μg/ml) and Cili Padi Putih (IC50 = 601 μg/ml), Cili Johor (CJ) ND
Cili Padi Rangup (CR) 420
respectively (Table 3 and Fig. 4) However, the lowest IC50 of the other three
Cili Padi Putih (CP) 601
varieties of Wild Capsicum, Cili Padi Thai and Cili Johor were not de- Cili Padi Centil (CC) 491
termined. The bleaching of DPPH absorption is representative of the ca-
pacity of the tested compounds to scavenge the radicals independent of any
enzymatic activity (Sannigrahi et al., 2010).

Fig. 4. DPPH free radical scavenging activity in different varieties of Capsicum sp.

51
Md. A. Alam et al. Journal of Applied Research on Medicinal and Aromatic Plants 9 (2018) 46–54

Fig. 5. Capsaicinoid content in different varieties of Capsicum sp. Here, bar graphs indicated with different error bars ( ± SE value) are significantly different at p ≤ 0.05.

3.3. Capsaicinoids compound Table 5

Scoring data matrix of six varieties of Capsicum sp. and Sweet Bell Capsicum and
The capsaicinoids content in Cili Padi Putih (3.41 ± 0.03 mg/g) Harbanero as an outgroup.

was remarkably highest compared to other varieties followed by Cili Taxa Characters
Padi Centil (3.38 ± 0.03 mg/g), Wild Capsicum (3.37 ± 0.02 mg/g),
Cili Padi Rangup (3.35 ± 0.01 mg/g) and Cili Padi Thai 1 2 3 4 5 6 7 8 9 10 11 12
(3.32 ± 0.01 mg/ml), respectively (Fig. 5). In contrast, the capsaici-
Wild Capsicum (WC) 0 1 0 1 1 0 1 1 0 0 0 1
noid content in Cili Johor (3.14 ± 0.06 mg/g) was lowest compared to Cili Padi Thai (CT) 0 1 1 1 0 1 1 1 1 1 1 1
that in all other varieties. Cili Johor (CJ) 0 0 0 0 0 1 0 0 1 1 0 1
Cili Padi Rangup (CR) 0 1 1 0 0 0 1 1 0 0 1 0
Cili Padi Putih (CP) 1 0 1 0 2 0 1 1 0 0 0 0
3.4. Variability analysis of chili varieties based on morpho-physiological Cili Padi Centil (CC) 1 1 1 0 3 0 1 1 0 0 0 1
attributes Capsicum 0 0 0 0 0 0 0 0 3 3 3 3
Harbanero 1 1 1 1 3 0 0 0 3 3 3 3
The scoring data matrix of six varieties of Capsicum sp and Sweet
Bell Capsicum and Harbanero (as an outgroup) has been presented in
Table 5 based on the scoring pattern derived from Table 4. For the Capsicum (WC) with scoring one (1) and all others were zero (0). For
characters plant height; majority of the chilli plants scored zero (0) that the fresh and dry weight of chilli fruits only Cili Padi Thai (CT) and Cili
is plant height was less than 50 cm except Cili Padi Putih (CP), Cili Padi Johor (CJ) scored one (1) for sufficient biomass production but all other
Centil (CC) and Harbanero. Regarding production of branches most of varieties scored zero (0) due to low biomass production (Table 5).
all the chilli varieties produced more than two branches so scored 1, There were a total of 10 positions in the final dataset, out of which 9
except Cili Johor (CJ), Cili Padi Putih (CP) and Capsicum. For average were parsimony informative. From the phylogenetic tree, it is evident
fruit production only the Wild Capsicum (WC), Cili Padi Thai (CT) and that the two out group chilli samples have groped in two different
Harbanero varieties produced more than 50 fruits and scored 1 and all branches and the 6 samples are in another group, though the whole tree
others produced less than 50 so scored 0 (Table 5). Regarding chilli fruit has been originated from Harbanero chilli outgroup sample. While from
length only the Cili Padi Thai (CT) and Cili Johor (CJ) produced more the 6 different chilli varieties; variety CR, CP and CC are initially
5 cm length with scoring 1 and all other fruits were less than 5 cm with grouped in one group but secondly variety CR was separated from them
scoring zero (0). Variety wise same results were observed for fruit may be due to the high contents of antioxidant properties and activities
diameter and fruit weight for the varieties; Cili Padi Centil (CC), Cili compared to others (Fig. 6). On the other hand, the variety CP which
Padi Thai (CT), Cili Padi Rangup (CR), Cili Padi Putih (CP) and Wild possessed the highest contents of capsaicin and capsaicinoid contents
was also in the same group and positioned very close to variety CP
Table 4 (Fig. 6). Furthermore, varieties; WC, CJ and CT formed another group
List of complete details for 12 morpho-physiological characters and their scoring in the where variety CT was in different position.
analysis.

No Characters Character state 4. Discussion

1 Plant height
2 Branch number per plant
3 Average leaf area
4 Fruit number per plant
5 Fruit colour
6 Fruit length
7 Fruit diameter
8 Fruit weight
9 Total fresh weight of fruit
10 Total dry weight of fruit
11 Stomatal conductance
12 Chlorophyll content
0 = equal or less than 50; 1 = more than 51
0 = equal or less than 2; 1 = more than 2
0 = more than 15; 1 = equal or less than 15
0 = equal or less than 50; 1 = more than 50
0 = red; 1 = orange; 2 = white; 3 = reddish-
orange
0 = equal or less than 5; 1 = more than 5
0 = more than 20; 1 = equal or less than 20
0 = more than 5; 1 = equal or less than 5
0 = equal or less than 50; 1 = more than 50
0 = equal or less than 10; 1 = more than 10
0 = equal or less than 200; 1 = more than 200
0 = equal or less than 50; 1 = more than 50
Phenols are compounds that have the ability to destroy radicals
because they contain hydroxyl groups. These important plant compo-
nents give up hydrogen atoms from their hydroxyl groups to radicals
and form stable phenoxyl radicals; hence, they play an important role in
antioxidant activity (De Gaulejac et al., 1999). Therefore, determina-
tion of the quantity of phenolic compounds is very important in order to
determine the antioxidant capacity of plant extracts (Aksoy et al.,
2013).
The high amount of phenolic compounds in Cili Padi Rangup in
sample extract was probably because of this variety was collected at
fully matured stages compared to others varieties and these are may be

52
Md. A. Alam et al.
Fig. 6. Phylogenetic tree of six different varieties and two outgroup of Capsicum sp.
one of the factors that contributed to the highest total phenolic content
for this variety. This can be concluded that the maturation stages could
affect the content of the phenolic compounds in this plant. According to
Howard et al. (2000), total phenolics in all other types of pepper gen-
erally increased with maturation level regardless of the analytical
method employed.
High amount of flavonoid compounds in Cili Padi Rangup in sample
extract was probably because of these varieties may contain chemical
structural elements that involved for their antioxidant activities, which
is related to the ability of hydrogen donation to stabilize the phenoxyl
radicals formed (Sowndhararajan and Kang, 2013) and play their es-
sential role as antioxidant agent and scavenge the free radical reaction.
Gurnani et al. (2016) reported that the amount of the total phenolic
content and total flavonoid content ranged from 7.95-26.15 gallic acid
equivalents (GAE mg/g) and from 4.64-12.84 rutin equivalents (RU
mg/g) of dry weight of local varieties of Indian chilli fruit extracts.
Carotenoids are divided into two classes, carotenes that contain only
carbon and hydrogen atoms and oxocarotenoids (xanthophylls) that
contain at least one oxygen atom in addition to carbon and hydrogen
atoms (Stahl and Sies, 2003). Carotenoids are involved in scavenging
reactive oxygen species and peroxyl radicals (Young and Lowe, 2001).
According to Fiedor and Burda (2014), carotenoid compounds are most
efficiently reacting with peroxyl radicals that formed under oxidative
conditions and they play a crucial role in the protection of the cellular
membrane and lipoprotein against oxidative damage.
According to Del Rocío Gómez-García and Ochoa-Alejo (2013),
carotene content was highest in red and brown fruited cultivars while
the lowest in orange and yellow ones. The yellow, orange and red colors
of Capsicum sp. fruit originate from carotenoid pigments that produced
during ripening stages (Rodriguez-Uribe et al., 2012).
Chili fruits are known as nutritionally balanced with respect to the
antioxidant vitamins A, C and E. Red chili contains at least three to four
times more of vitamin A, C and E compared to those in green fruit
(Sarkar et al., 2015).
Besides ß-carotene, the other pro-vitamin A carotenoids are α- and
γ-carotene and ß-cryptoxanthin. However, beta-carotene the most ef-
ficiently converts to retinol (Delia-Irina et al., 2003). Ponnamperuma
et al. (2000) reported that ß-carotene could bind to a retinoid receptor
to form vitamin A.
According to Su et al. (2015), ß-carotene and total carotenoid
contents were lowest in yellow and orange colored fruits at the har-
vesting stages while red colored and ripen fruit contained the highest
concentration of ß-carotene and total carotenoid content (Del Rocío
Gómez-García and Ochoa-Alejo, 2013). ß-carotene is the precursor for
the predominant orange, and red pigments in chili and genotypes with
high concentrations of ß-carotene proved to be richest in total car-
otenoid content.
The highest DPPH scavenging activity of the Cili Padi Rangup
variety was due to the high percentage of the total phenolic compound
in it that affected the scavenging activities of this variety. The low IC50
value of Wild Capsicum, Cili Padi Thai and Cili Johor variety was due to
low phenolic content in it that also affected the scavenging activities of
these varieties.
53
Journal of Applied Research on Medicinal and Aromatic Plants 9 (2018) 46–54
The different values of antioxidant activities obtained with different
types of Capsicum sp. extract can be described to their different che-
mical compositions exist in each extract. Thus, the antioxidant activity
cannot be predicted based on its total phenolic content only. Otherwise,
the synergism of polyphenolic compounds, with another component
present in the extracts may contribute to the overall observed anti-
oxidant capacity (Ordonez et al., 2006). Gurnani et al. (2016) opined
that the extraction solvent play a vital role on the exhibition of anti-
oxidant activities especially on DPPH activity. In their study they ob-
served that the n-hexane extract exhibited 26.97% DPPH radical
scavenging activity at a concentration of 1 mg/mL, while for the
chloroform extract at the same concentration, 30.9% DPPH radical
scavenging activity.
Chili pepper (Capsicum sp.) contains capsaicinoids; alkaloid com-
pounds that produce the pungency while eating chilies (Usman et al.,
2014). Capsaicin compounds are prominent for its pharmaceutical and
antioxidant properties. It has been reported that the more the capsaicin,
the hotter the pepper and the higher the antioxidant level (Iida et al.,
2003). Capsaicin in peppers has been shown to slightly control appetite
and raise the body temperature (Backonja et al., 2010).
The factors that may be affected the total capsaicin amount in dif-
ferent varieties of Capsicum sp. are the temperature at which chili
peppers are grown, the position of the fruit on the plant, the stage of
fruit maturation on the plant and also the light intensity. Furthermore,
capsaicinoid levels also depend on the genotype and changes during the
development of fruit (Butcher et al., 2012).
From the result shown in Fig. 5, the total Capsaicinoid contents in
the different varieties may be related with the genotype of its variety
and the maturation stage of the fruit. According to Orellana-Escobedo
et al. (2013), the variation of the content of capsaicinoid compound in
the chili may be affected by environment and a genetic factor of the
cultivar.
As mentioned before, Cili Padi Rangup fruits were harvested at fully
ripen stage, but this variety didn’t show the highest content of capsai-
cinoid compounds. According to Deepa et al. (2007), the content of
capsaicin in the chili decreases as the maturation increases. From the
morphological appearance, Wild Capsicum, Cili Padi Thai, Cili Padi
Rangup, Cili Padi Putih and Cili Padi Centil can be classified as small
size of fruit compared to Cili Johor where the size of fruit and capsai-
cinoid content were significantly different which is the fruit size is more
bigger and had the lowest content of capsaicinoid compound than other
varieties. However, there is no research that the size of fruit interrelated
with the capsaicin content.
5. Conclusions
In general, this study demonstrated that different Capsicum varieties
had significantly different antioxidant compounds and resulted in var-
ious responses to different antioxidant activities. Overall, our results
revealed that Cili Padi Rangup (CR) variety had the highest Total
Phenolic Content (TPC), Total Flavonoid Content (TFC), Total
Carotenoid Content (TCC) and ß-carotene content compared to those in
any other varieties of Capsicum sp. This may be due to harvesting the
Md. A. Alam et al.
fruits of this variety at complete ripen stage. However, complete ri-
pening of the fruits of this variety may have caused a decrease in the
capsaicinoid and capsaicin content of its fruits in comparison to those in
other varieties. The highest capsaicinoid content was recorded in Cili
Padi Putih (CP) among six different varieties of Capsicum sp. For both
antioxidant activities, DPPH scavenging assay and FRAP assay, the best
antioxidant activity was recorded in the Cili Padi Rangup (CR) due to
the highest antioxidant compounds (TPC, TFC, TCC) in this variety.
However, the more ripen the chili fruit during harvest, the more anti-
oxidant compounds and antioxidant activities are performed and the
lower the capsaicinoid contents. So, varying fruit maturity stage may be
a factor of consideration while the determination of capsaicinoid and
antioxidant compounds in different chili verities.
Conflict of interests
The authors declare that there is no conflict of interests regarding
the publication of this article.
Acknowledgments
The authors sincerely acknowledge Faculty of Bioreseources and
Food Industry, Universiti Sultan Zainal Abidin (UniSZA) for all the
supports and facilities for conducting this research.
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