Food Chemistry 114 (2009) 553–560

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

The influence of fruit ripening on the phytochemical content and biological

activity of Capsicum chinense Jacq. cv Habanero
Federica Menichini c, Rosa Tundis a,*, Marco Bonesi a, Monica R. Loizzo a, Filomena Conforti a,
Giancarlo Statti a, Bruno De Cindio b, Peter J. Houghton c, Francesco Menichini a
a
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Nutritional and Health Sciences, University of Calabria, I-87036 Arcavacata di Rende (CS), Italy
b
Department of Engineering Modeling, University of Calabria, I-87036 Arcavacata di Rende (CS), Italy
c
Pharmaceutical Sciences Research Division, King’s College London, 150 Stamford Street, London SE1 9NH, UK

a r t i c l e i n f o a b s t r a c t

Article history: During the past decade, it has been reported that the consumption of certain foods and spices such as
Received 13 May 2008 pepper may have a positive effect on health. The present study evaluates the influence of fruit ripening
Received in revised form 22 July 2008 on total phenols, flavonoids, carotenoids and capsaicinoids content and antioxidant, hypoglycaemic and
Accepted 25 September 2008
anticholinesterase activities of Capsicum chinense Jacq. cv Habanero. The chemical investigation showed a
different composition between the two stages of ripening (immature and mature). Generally, the concen-
tration of carotenoids and capsaicinoids increased as the peppers reached maturity, whereas the concen-
Keywords:
tration of phenols declined. The immature fruits showed the highest radical scavenging activity (IC50 of
Capsicum chinense Jacq. cv Habanero
Phenolics
97.14 lg/ml). On the contrary, the antioxidant activity evaluated by the b-carotene bleaching test showed
Carotenoids a significant activity for mature peppers (IC50 value of 4.57 lg/ml after 30 min of incubation). Mature
Capsaicinoids peppers inhibited a-amylase with an IC50 of 130.67 lg/ml. The lipophilic fractions of both mature and
Antioxidant immature peppers exhibited an interesting and selective inhibitory activity against a-amylase with
Amylase inhibition IC50 values of 29.58 and 9.88 lg/ml, respectively. Both total extracts of mature and immature peppers
Glucosidase inhibition inhibited butyrylcholinesterase selectively. The obtained results underline the potential health benefits
Cholinesterase inhibition as a result of consuming C. chinense Habanero and suggest that it could be used as new valuable flavour
with functional properties for food or nutriceutical products on the basis of the high content of phyto-
chemicals and found biological properties.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction tion in the rate of glucose absorption and consequently blunting

A wealth of information and scientific evidence is rapidly accu-
mulating that shows the beneficial effects of a wide variety of food
components on human health. In the past few years, there has been
a renewed interest in studying and quantifying the antioxidant
constituents of fruits and vegetables for their potential health func-
tionality against various diseases such as diabetes, cancer, cardio-
vascular and neurodegenerative diseases such as Alzheimer’s
disease (Kaur & Kapoor, 2001). Diabetes is a major risk factor for
premature atherosclerosis and oxidative stress plays an important
role in its pathogenesis (Venugopal, Devaraj, Yang, & Jialal, 2002).
One therapeutic approach for treating diabetes is to decrease the
post-prandial hyperglycaemia. This is done by retarding the
absorption of glucose through the inhibition of the carbohydrate-
hydrolyzing enzymes, a-amylase and a-glucosidase, in the diges-
tive tract. Inhibitors of these enzymes delay carbohydrate digestion
and prolong overall carbohydrate digestion time, causing a reduc-
* Corresponding author. Tel.: +39 984 493246; fax: +39 984 493298.
E-mail address: tundis@unical.it (R. Tundis).
the post-prandial plasma glucose rise. Oxidative stress has been de-
scribed in the pathological changes that occur in Alzheimer’s dis-
ease (AD), the most common form of neurodegenerative disorders
(Pratico & Delanty, 2000). AD is currently treated clinically by the
use of agents which restore the level of acetylcholine through inhi-
bition of both two major forms of cholinesterase: acetylcholinester-
ase (AChE) and butyrylcholinesterase (BChE) (Loizzo, Tundis,
Menichini, & Menichini, 2008).
The genus Capsicum, which originates from tropical and humid
zones of Central and Southern America, belongs to the Solanaceae
family and includes peppers of important economic value. Several
Capsicum species exist, three of which are widely spread and have a
hot or pungent berry: Capsicum annuum, Capsicum frutescens and
Capsicum chinense. The Habanero chili pepper is the fruit of
C. chinense Jacq., a very aromatic variety and is claimed to be the
hottest chili pepper in the world. It is of great interest to know
the contribution of an individual food product in the daily
nutritional needs and how ripening affect dietary, nutrition and
therefore biological properties. The content of phytochemicals in
plant material is influenced by numerous factors such as climatic

0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.09.086
554 F. Menichini et al. / Food Chemistry 114 (2009) 553–560
conditions, ripening time, genotype and cultivation techniques. The
maturity stage is another important factor that influences the com-
positional quality of fruit and vegetables, since, during fruit ripening,
several biochemical, physiological and structural modifications
happen and these changes determine the attributes of fruit quality.
In relation to the importance of phytochemicals and antioxidant
power for functional aspects of pepper, the aim of this work was to
evaluate the influence of ripening stage on several quality attri-
butes, such as total carotenoids, phenols and capsaicinoids. To this
purpose, the antioxidant activity, the inhibition of the two major
carbohydrate digestive enzymes, a-amylase and a-glucosidase,
and the inhibition of two enzymes frequently targeted for the
treatment of Alzheimer’s disease were investigated and were re-
lated to the different chemical composition of C. chinense Haban-
ero, widely consumed in diets, in the two stages of ripening.
2. Materials and methods
2.1. Chemicals
Methanol, ethanol, ethyl acetate, n-hexane, sodium sulfate,
DMSO, H2SO4, chloroform, sodium carbonate, perchloric acid,
HCl, KOH, NaOH, NaCl, butanol, thin-layer chromatography plates
(TLC) were obtained from VWR International s.r.l. (Milano, Italy).
Capsaicin, dihydrocapsaicin, b-carotene, quercetin, chlorogenic
acid, thiobarbituric acid (TBA), phosphate buffered saline (PBS), bo-
vine brain extract, FeCl3, AlCl3, NaCl, ascorbic acid, o-dianisine
(DIAN), peroxidase/glucose oxidase (PGO) system, butylated
hydroxytoluene (BHT), propyl gallate, 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH), linoleic acid, Tween 20, Folin–Ciocalteu re-
agent, potato starch, sodium phosphate buffer, sodium chloride,
sodium potassium tartrate tetrahydrate, 3,5-dinitrosalicylic acid,
maltose, a-amylase (EC 3.2.1.1), a-glucosidase (EC 3.2.1.20), acet-
ylthiocholine iodide (ATCI), 5,50 -dithiobis(2-nitrobenzoic-acid)
(DTNB), butyrylthiocholine iodide (BTCI), physostigmine, acetyl-
cholinesterase from Electrophorus electricus (EC 3.1.1.7, Type VI-S)
and butyrylcholinesterase from equine serum (EC 3.1.1.8) were
purchased from Sigma–Aldrich S.p.a. (Milano, Italy). Acarbose from
Actinoplanes sp. was obtained from Serva (Heidelberg, Germany).
2.2. Plant materials
The fruits of C. chinense Jacq. cv Habanero used in this study
were purchased in September 2006 from farm Miceli s.r.l.
(Calabria, Italy). The peppers were harvested at the same time
but at two successive maturity stage on the basis of their colour
as immature or green (I) and mature or red (M). The authentication
of the pepper was carried out at Natural History Museum of Cala-
bria and Botanic Garden, University of Calabria, Italy. All peppers
received similar water and fertilizer treatments. Pepper fruits were
examined for integrity and absence of dust and insect contamina-
tion, were devoid of peduncles and seeds and they were cut into
small pieces. Samples were freeze-dried and stored at 20 °C until
they were analysed.
2.3. Extraction procedure
Components in peppers were extracted from 200 g of fruits by
ethanol. Extraction was repeated until the complete exhaustion
of colour. The ethanol solutions were combined and dried over
anhydrous Na2SO4 (total extract), obtaining a yield of 3.7% and
6.5% for immature and mature fruits, respectively. In order to oper-
ate a separation of lipophilic compounds, the total extract was
solubilised with MeOH/H2O (8:2) and extracted with n-hexane.
The n-hexane solutions were combined and dried over anhydrous
Na2SO4 to obtain the lipophilic fraction (yield 0.81% and 0.88%
for immature and mature fruits, respectively).
2.4. Determination of total phenol and flavonoid content
The amount of total phenolics of pepper samples was deter-
mined by the Folin–Ciocalteu method (Gao, Ohlander, Jeppsson,
Björk, & Trajkovski, 2000). Briefly, the extract was mixed with
0.2 ml Folin–Ciocalteu reagent (Sigma–Aldrich), 2 ml of distilled
water and 1 ml of 15% Na2CO3. The absorbance was measured at
765 nm using a Jenway 6003 UV–Vis spectrophotometer after 2 h
incubation at room temperature. The levels of total phenolics con-
tent were determined in triplicate. Chlorogenic acid was used as a
standard and the total phenolics content was expressed as chloro-
genic acid equivalents in mg per 100 g of fresh materials.
The flavonoids content was determined spectrophotometrically
using a method based on the formation of a flavonoid–aluminium
complex (Yoo, Lee, Lee, Moon, & Lee, 2008). One millilitre of the ex-
tracts was added to a 10 ml volumetric flask. Distilled water was
added to make a volume of 5 ml. At zero time, 0.3 ml of 5% (w/v)
sodium nitrite was added to the flask. After 5 min, 0.6 ml of 10%
(w/v) AlCl3 was added and then at 6 min 2 ml of 1 M NaOH were
also added to the mixture, followed by the addition of 2.1 ml dis-
tilled water. Absorbance at 510 nm was read immediately. Querce-
tin was chosen as a standard and the levels of total flavonoid
content were determined in triplicate and expressed as quercetin
equivalents in mg per 100 g fresh materials.
2.5. Determination of total carotenoid content
The total carotenoid content was determined by measuring the
absorption of lipophilic fractions at 450 nm. Triplicate aliquots of
fractions were analysed in a Jenway 6003 UV–Vis spectrophotom-
eter. A 1 ml aliquot from the lipophilic layer (0.1 g/ml) was added
to 0.5 ml of 5% NaCl, vortexed for 30 s and centrifuged for 10 min at
4500 rpm. The supernatant (100 ll) was diluted with 0.9 ml of n-
hexane and measured at 460 nm. b-Carotene was used as a stan-
dard. The total carotenoids contents were determined in triplicate
and expressed as b-carotene equivalents in mg per 100 g of fresh
materials (Gao et al., 2000).
2.6. Determination of capsaicin and dihydrocapsaicin contents
Capsaicinoid content was evaluated by gas chromatography
(GC). GC analyses were performed on a Shimadzu GC17A gas chro-
matograph equipped with a flame ionisation detector (FID) and
controlled by Borwin Software. The samples were analysed on a
fused silica 30 m SE-30 capillary column with an internal diameter
of 0.25 mm and a film thickness of 0.25 lm. Nitrogen was used as
the gas vector at a constant flow of 1.0 ml/min. The injector and
detector temperatures were 250 °C and 280 °C, respectively. Anal-
yses were performed in isothermal conditions at 210 °C.
Quantitative data were obtained from the electronic integration
of the GC peak areas for three injections of each sample with the
use of commercial capsaicin and dihydrocapsaicin as external stan-
dards injected into the GC equipment under identical conditions as
above. A stock solution of capsaicin and dihydrocapsaicin was pre-
pared with acetone at a concentration of 0.05 g/5 ml. From this
stock, six 1-ml solutions were prepared to be used to obtain a stan-
dard curve of the two capsaicinoids. The results were expressed as
lg per g fresh weight.
2.7. GC–MS analysis
The C. chinense Habanero n-hexane fractions were analysed
by gas chromatography–mass spectrometry (GC–MS). GC–MS
 F. Menichini et al. / Food Chemistry 114 (2009) 553–560
analyses were carried out using a Hewlett–Packard 6890 gas chro-
matograph equipped with an HP-5 non-polar capillary column
(30 m length, 0.25 mm i.d., 0.25 lm film thickness) and interfaced
with a Hewlett–Packard 5973 Mass Selective detector. Ionisation of
the sample components was performed in electron impact mode
(EI, 70 eV). The carrier gas was helium (1 ml/min) and the analyt-
ical conditions were as follows: oven temperature was 5 min iso-
thermal at 50 °C, then 50–250 °C at a rate of 5 °C /min; then held
isothermal for 10 min. The injector and detector temperatures
were 250 °C and 280 °C, respectively. For analysis, lipophilic frac-
tions were dissolved in acetone (ca. 1 mg/ml) and aliquots (1 ll)
were directly injected. Constituents were tentatively identified by
gas chromatography by comparison of their retention indices (I)
with those of the literature (Adams, 1995) or with those of authen-
tic compounds available in our laboratory. Retention indices for all
compounds were determined by co-injection of the sample with a
solution containing a homologous series of C9–C31 n-alkanes
(Tranchant, 1995). Further tentative identification was made by
comparison of their mass spectra with those stored in Wiley 138,
Wiley 275 and NIST98 libraries.
2.8. DPPH assay
Free radical scavenging activity was determined using a rapid
TLC screening method based on the reduction of a methanolic solu-
tion of the coloured free radical DPPH. After developing and drying
TLC plates (VWR, silica gel plates, CH2Cl2/MeOH 8:2) were sprayed
with a 0.2% DPPH radical solution in MeOH. The plates containing
the extracts and lipophilic fractions were examined 30 min after
spraying. The samples with antioxidant activity appeared as yellow
spots against a purple background. In order to determine the rad-
ical scavenging potency the samples, which exhibited antioxidant
activity, were investigated with an experimental procedure that
was adapted from Wang et al. (1998). In an ethanol solution of
DPPH radical (final concentration was 1.0  104 M), test samples
were added at different concentrations. The reaction mixtures
were shaken vigorously and then kept in the dark for 30 min.
The absorbance of the resulting solutions was measured in 1 cm
cuvettes using a Perkin–Elmer Lambda 40 UV/Vis spectrophotom-
eter at 517 nm against blank (without DPPH radical). All tests were
run in triplicate and the mean values calculated. Ascorbic acid was
used as a positive control.
2.9. b-Carotene bleaching test
Antioxidant activity was determined using the b-carotene
bleaching test with some modifications. Briefly 1 ml of b-carotene
solution (0.2 mg/ml in chloroform) was added to 0.02 ml of linoleic
acid and 0.2 ml of 100% Tween 20. After evaporation of chloroform
and dilution with water (100 ml), 5 ml of the emulsion were trans-
ferred into different test tubes containing 0.2 ml of samples in 70%
ethanol at different concentrations. Standard (propyl gallate) at the
same concentration as samples was used for comparison. The
tubes were then gently shaken and placed at 45 °C in a water bath
for 60 min. The absorbance of the samples, standard and control
was measured at 470 nm using a Perkin–Elmer Lambda 40 UV/
Vis spectrophotometer against a blank, consisting of an emulsion
without b-carotene. The measurement was carried out at initial
time (t = 0) and successively at 30 and 60 min. All samples were as-
sayed in triplicate and the mean value calculated. The antioxidant
activity (AA) was measured in terms of successful bleaching b-car-
otene by using the following equation:
AA ¼ ½1  ðA0  At Þ=ðAo0  Aot Þ  100
where A0 and Ao0
are the absorbance values measured at the initial
incubation time for samples/standard and control, respectively,
555
whilst At and Aot are the absorbance values measure in the sam-
ples/standard and control, respectively, at t = 30 min and t = 60 min.
2.10. Bioassay for a-amylase inhibition
The a-amylase inhibition assay method was performed using a
method previously described ( Tundis, Loizzo, Statti, Houghton,
et al., 2007). Briefly, a starch solution (0.5% w/v) was obtained by
stirring 0.125 g of potato starch in 25 ml of 20 mM sodium phos-
phate buffer with 6.7 mM sodium chloride, pH 6.9, at 65 °C for
15 min. The a-amylase (EC 3.2.1.1) solution was prepared by mix-
ing 0.0253 g of a-amylase in 100 ml of cold distilled water. Sam-
ples were dissolved in buffer to give final concentration from
1 mg/ml to 12.50 lg/ml. The colourimetric reagent was prepared
mixing a sodium potassium tartrate solution (12.0 g of sodium
potassium tartrate, tetrahydrate in 8.0 ml of 2 M NaOH) and
96 mM 3,5-dinitrosalicylic acid solution. Control, total extracts
and lipophilic fractions were added to starch solution and left to
react with a-amylase solution at 25 °C for 5 min. The reaction
was measured over 3 min. The generation of maltose was quanti-
fied by the reduction of 3,5-dinitrosalicylic acid to 3-amino-5-
nitrosalicylic acid, the product being detectable at 540 nm. In the
presence of an a-amylase inhibitors less maltose will be produced
and the absorbance value would decrease. Preliminary experi-
ments were carried out to establish optimal conditions and these
were found to be: starch 0.25% w/v; a-amylase 1 unit/ml; inhibitor
concentration 1 mg/ml. The a-amylase inhibition was expressed as
percentage of inhibition and calculated by the following equation:
 
½maltose test
% Inhibition ¼ 100   100  SD
½maltose control
2.11. Bioassay for a-glucosidase inhibition
The a-glucosidase inhibition was measured through a modified
Sigma–Aldrich bioassay method (Kapustka, Annala, & Swanson,
1981). A maltose solution (4% w/v) was prepared by dissolving
12 g of maltose in 300 ml of 50 mM sodium acetate buffer. The en-
zyme solution (EC 3.2.1.20) was prepared by mixing 1 mg of a-glu-
cosidase in 10 ml of ice-cold distilled water. Pepper extracts and
lipophilic fractions are dissolved in DMSO to give final concentra-
tion from 1 mg/ml to 5 lg/ml. The colourimetric reagent o-dianis-
idine (DIAN) solution was prepared by dissolving one tablet in
25 ml of distilled water, whilst the peroxidase/glucose oxidase
(PGO) system-colour reagent solution was prepared fresh by dis-
solving one capsule in 100 ml of ice-cold distilled water. In the first
step both control and samples were added to maltose solution and
left to equilibrate at 37 °C. The reaction was started by adding a-
glucosidase solution and tubes were left to incubate at 37 °C for
30 min. After that time perchloric acid solution (4.2% w/v) was
added to stop reaction. In the second step the generation of glucose
was quantified by the reduction of DIAN. The supernatant of tube
of step I was mixed with DIAN and PGO and was left to incubate
at 37 °C for 30 min. The absorbance of DIAN was measured spec-
trophotometrically at 500 nm. The a-glucosidase inhibition was
expressed as percentage of inhibition and calculated by the follow-
ing equation:
 
½glucose test
% Inhibition ¼ 100   100  SD
½glucose control
2.12. Bioassay for anticholinesterase (acetyl- and
butyrylcholinesterase) activity
Acetylcholinesterase (AChE, EC 3.1.1.7, Type VI-S) and butyr-
ylcholinesterase (BChE, EC 3.1.1.8) inhibition was assessed by
556 F. Menichini et al. / Food Chemistry 114 (2009) 553–560
modifications of the Ellman method (Conforti, Statti, Tundis,
Loizzo, & Menichini, 2007), which is based on the reaction of re-
leased thiocholine to give a coloured product with a chromogenic
reagent. AChE or BChE 20 ll (0.20 U/ml in Na2HPO4 buffer pH 8)
and pepper total extracts and lipophilic fractions (25 ll) were
added to 50 ll of buffer pH 8 and pre-incubated on an ice bath
at 4 °C for 30 min. Duplicate solutions were also treated this way
with 20 ll of physostigmine (0.1 mM) to allow interference of
the test substances in the assay to be assessed, and to control for
any hydrolysis of acetylcholine not due to AChE activity. The reac-
tion was started by adding DTNB solution (125 ll of 0.05 mM in
NaH2PO4 buffer pH 7) and acetylthiocholine or butyrylthiocholine
(25 ll 0.018 mM in buffer pH 7) and tubes were incubated in water
bath for 20 min at 37 °C. The reaction was stopped by placing the
assay solution tubes in an ice bath and adding physostigmine
(20 ll 0.018 mM in buffer pH 7). Blanks were used of reagents
without samples and a positive control was set up which was the
same as the blank except that physostigmine (20 ll 0.018 mM in
buffer pH 7) was added. The production of yellow anion was imme-
diately recorded using a spectrophotometer at 405 nm. The inhibi-
tion rate (%) was calculated by equation:
½ðblank  blank positive controlÞ  ðexperiment  experiment controlÞ
% Inhibition ¼
ðblank  blank positive controlÞ
2.13. Statistical analysis
All experiments were carried out in triplicate. Data were ex-
pressed as means ± SD. The concentration giving 50% inhibition
(IC50) was calculated by nonlinear regression with the use of Prism
Graphpad Prism version 4.0 for Windows (GraphPad Software, San
Diego, CA, USA). The dose–response curve was obtained by plotting
the percentage inhibition versus concentration.
3. Results and discussion
3.1. Phytochemical content
The phenols, flavonoids, carotenoids and capsaicinoids content
of the C. chinense Habanero extracts are reported in Table 1.
Total soluble phenols were determined by Folin–Ciocoltau as-
say. The immature peppers showed 782 mg chlorogenic acid
equivalent whilst mature fruits contained 759 mg chlorogenic acid
equivalent. Generally, the concentration of these chemical constit-
uents increased as the pepper reached maturity regardless of the
analytical method employed, as previously reported (Howard,
Talcott, Brenes, & Villalon, 2000). In disagreement with Howard
et al. (2000), who investigated the total phenols of two mature C.
chinense Habanero cultivars (Francisca and Red Savina) from Texas,
but in agreement with Conforti, Statti, and Menichini (2007) the
phenols content of C. chinese Habanero extracts decreased with
the increase of maturity of the fruits. In agreement with Marín,
Ferrerei, Tomás-Barberán, and Gill (2004) the flavonoid content
followed the same trend as for the phenol content, decreasing with
maturity. In particular, the immature peppers showed 138 mg
quercetin equivalent whilst mature fruits contained 45.0 mg quer-
cetin equivalent. The content of flavonoids in immature green pep-
per was generally 4–5 times higher than that detected for other
maturity stages.
The intense and characteristic red colour of Capsicum fruits is
due to carotenoid pigments that are synthesised mainly during
fruit ripening. Table 1 shows changes in carotenoid content during
fruit ripening. According to the results, the carotenoid profile
changes during Habanero fruit development and ripening, with
values of 62.7 and 362 mg b-carotene equivalent for immature
and mature pepper fruits, respectively.
Capsaicinoids are a group of 12 or more related ‘alkaloids’
responsible for the pungent sensation in fruits of the genus Capsi-
cum. The two major capsaicinoids capsaicin and dihydrocapsaicin
are responsible for up to 90% of the total pungency of pepper fruits.
The degree of pungency depends on the Capsicum species and cul-
tivars, and the capsaicin and dihydrocapsaicin contents can be af-
fected by different factors such as the developmental stage of the
fruit (Sukrasno & Yeoman, 1993) and the environmental growth
conditions (Estrada, Pomar, Díaz, Merino, & Bernal, 1998; Lindsey
& Bosland, 1995; Zewdie & Bosland, 2000). Kozukue et al. (2005)
reported values for capsaicin and dihydrocapsaicin, respectively,
ranging from a minimum of 2.6 and 8.2 lg/g fresh weight in
canned sliced Jalapeño to maxima of 1187 and 771 lg/g of fresh
weight in Habanero fresh pepper. Capsaicin and dihydrocapsaicin
contents were recently reported in Habanero white and Habanero
orange types from Yucatan, Mexico (Cisneros-Pineda et al., 2007).
Our results give capsaicin content as 4363 lg/g and 1071 lg/g
for mature and immature C. chinense Habanero cultivated in
Calabria, Italy. Habanero hot peppers from Italy showed a capsaicin
content 4 times higher than Habanero from Yucatan. The same
trend was observed for the dihydrocapsaicin content with values
of 2498 lg/g and 357 lg/g for mature and immature stage,
respectively.
GC–MS analysis of the n-hexane fractions of C. chinense Haban-
ero showed a different composition between the two stages of rip-
ening (Table 2). The n-hexane fraction mature red peppers
compared to immature green peppers showed a major content in
vitamin E (5.90%), b-caryophyllene (1.81%), germacrene D (1.37%)
and in certain fatty acids and methyl esters such as palmitic acid
(11.20%), methyl linoleate (1.10%), methyl linolenate (0.89%) and
ethyl stearate (1.73%). Immature pepper fruits were rich in triter-
penes such as a- and b-amyrin and in certain sterols such as
b-sitosterol (2.50%), stigmasta-5,23-dien-3-ol (1.91%) and stigmas-
terol (3.43%). Moreover, Habanero green peppers possess a high
content of phytol (4.77%), myristic acid (2.68%), linoleic acid
(3.50%), ethyl stearate (2.81%), methyl behenate (0.82%), palmitic
acid, 14-methyl, methyl ester (1.63%) and 2-monolinolenin
(1.78%) (Fig. 1).
3.2. Radical scavenging and antioxidant activity
The radical scavenging effects of the total extract of two matu-
rity stages of C. chinense Habanero on DPPH radical were examined
at different concentrations (25, 50, 100, 250, 500 and 1000 lg/ml).
As shown in Table 3, the first stage of maturation (green) showed
the highest activity (IC50 of 97.1 lg/ml) whilst the later stage,
red, showed lower activity (IC50 of 287 lg/ml). When the non-polar
constituents were selected by extraction in n-hexane, the activity
was reduced, in fact the values of IC50 was 321 and 655 lg/ml
for immature and mature peppers, respectively. The high level of
phenolic components in immature pepper fruits appears to be
responsible for the antiradical scavenging activity. Several studies
have been reported on the relationship between phenolic content
and antioxidant activity (Materska & Perucka, 2005). Antioxidant
activity, which reflected the ability of the samples to inhibit the
bleaching of b-carotene, was measured and compared with that
 F. Menichini et al. / Food Chemistry 114 (2009) 553–560 557

Table 1
Total phenols, flavonoids, carotenoids and capsaicinoids content of fresh pepper fruits as affected by maturity.

Sample Phenols Flavonoids Carotenoids Capsaicin Dihydrocapsaicin

(mg/100 g fresh weight) (mg/100 g fresh weight) (mg/100 g fresh weight) (lg/g fresh weight) (lg/g fresh weight)
Immature 782 ± 5.8 138 ± 3.2 62.7 ± 5.5 1071 ± 16.7 357 ± 4.1
Mature 759 ± 2.3 45.0 ± 1.4 362 ± 7.8 4363 ± 23.1 2498 ± 23.4

Values represent means (n = 3) ± SD.

of the control which contained no antioxidant components. It was indicating that the activity does not correlate with time of heating.
demonstrated that addition of total extract of red and green pepper The high content in carotenoids and capsaicinoids may correlate
inhibited oxidation of linoleic acid. As shown in Table 3, the extract with the potent inhibition of lipid peroxidation (Henderson, Slick-
of mature peppers showed a greater antioxidative potency than man, & Henderson, 1999). Carotenoids have been recently studied
that of the immature ones with IC50 values of 4.57 lg/ml after widely and have been proven to play an important role in prevent-
30 min of incubation and 5.31 lg/ml after 60 min of incubation, ing oxidative damage caused by free radicals in age-related dis-
eases such as cancer and ageing (Tapiero, Townsend, & Tew,
2004). A lot of current research has focused on a proposed role of
Table 2 carotenoids as lipid antioxidants which can protect against
Major components of C. chinense cv Habanero lipophilic fractions.
destructive processes mediated by singlet oxygen and free radicals
Compound Ia Identificationc Immatureb Mature b
(Britton, Liaaen-Jensen, & Pfander, 1995).
b-Caryophyllene 1415 A 0.67 ± 0.03 1.81 ± 0.11 When the non-polar constituents were selected with extraction
a-Humulene 1447 A 0.17 ± 0.03 0.50 ± 0.03 in n-hexane, the antiradical activity was inverted and immature
Germacrene D 1480 B 0.58 ± 0.01 1.37 ± 0.09 peppers showed higher activity than mature peppers (IC50 was
Pentadecane 1500 A tr 0.61 ± 0.04
6.72 and 21.7 lg/ml for green and red pepper, respectively). The
c-Cadinene 1513 B 0.46 ± 0.03 0.87 ± 0.03
d-Cadinene 1522 B 0.41 ± 0.02 1.21 ± 0.15 high level of phytosterols content and of a acyclic diterpene alco-
Tetradecanal 1614 B tr 0.62 ± 0.03 hol, phytol, in immature pepper fruits is at the base of higher rad-
Heptadecane 1700 B 1.43 ± 0.01 2.66 ± 0.03 ical scavenging activity showed for this stage of ripening in
Pentadecanal 1707 B 0.66 ± 0.01 3.11 ± 0.03
comparison to mature peppers.
Hexadecanal 1811 B tr 0.60 ± 0.03
Neophytadiene 1830 B 1.62 ± 0.03 1.78 ± 0.03
Methyl pentadecanoate 1863 B 0.99 ± 0.08 1.51 ± 0.03 3.3. Hypoglycaemic activity
Pentadecanoic acid 1883 B 2.81 ± 0.07 4.40 ± 0.13
Methyl palmitate 1934 A 1.30 ± 0.03 1.97 ± 0.03 The hypoglycaemic potential of C. chinense Habanero was eval-
Phytol 1950 B 4.77 ± 0.16 1.51 ± 0.03
uated by the a-amylase and a-glucosidase inhibition assays. As a
Palmitic acid, 14-methyl-, 1955 B 1.63 ± 0.03
methyl ester monosaccharide, glucose can be readily absorbed from the gas-
Palmitic acid 1969 A 8.70 ± 0.22 11.20 ± 0.27 tro-intestinal tract into the blood stream after the hydrolysis of
Ethyl palmitoleate 1975 B 1.39 ± 0.03 3.10 ± 0.06 glycosidic bonds in digestible carbohydrate foods containing
Myristic acid 1987 B 2.68 ± 0.03 1.86 ± 0.03 starch, by the enzymes a-amylase and a-glucosidase. Inhibition
Methyl linoleate 1996 B tr 1.10 ± 0.03
Methyl linolenate 1999 B tr 0.89 ± 0.05
of these enzymes could reduce the high post-prandial blood glu-
Eicosane 2000 A 0.19 ± 0.04 0.40 ± 0.01 cose peaks in diabetics (Tundis, Loizzo, Statti, & Menichini, 2007).
Ethyl palmitate 2005 A 0.60 ± 0.03 0.71 ± 0.03 The mature Habanero total extract exhibited an IC50 value of
Octadecanal 2024 B tr 0.88 ± 0.06 131 lg/ml against a-amylase (Table 4). The a-glucosidase enzyme
Methyl stearate 2133 B 0.51 ± 0.01 0.77 ± 0.01
was inhibited more by the immature total extract (IC50 150 lg/ml).
Linoleic acid 2156 A 3.50 ± 0.11 1.50 ± 0.12
2-Monolinolenin 2160 B 1.78 ± 0.03 Previously our research group tested the C. annuum var. acumina-
Ethyl linolenate 2167 B 0.52 ± 0.03 1.61 ± 0.23 tum extract against both digestive enzymes. Comparison of data
Methyl arachidate 2186 B 1.56 ± 0.10 0.68 ± 0.01 between the two Capsicum species revealed that C. chinense
Ethyl stearate 2197 B 2.81 ± 0.03 1.73 ± 0.08 Habanero at mature stage of ripening showed an interesting activ-
5-Eicosene 2292 B 0.39 ± 0.03 0.50 ± 0.01
Methyl behenate B 0.82 ± 0.07
ity against a-amylase (Loizzo, Tundis, Menichini, Statti, & Menichi-
Linoleic acid, 2-hydroxy-1- B 0.67 ± 0.03 1.62 ± 0.19 ni, 2008). Interestingly, C. annuum var. acuminatum at mature stage
(hydroxymethyl) ethyl did not exhibit a-amylase inhibitory activity but exhibited an IC50
ester value of 144 lg/ml against a-glucosidase. Comparison of data be-
Pentacosane 2500 B tr 0.67 ± 0.02
tween the two Capsicum species revealed that C. chinense Habanero
Hexacosane 2600 B 0.41 ± 0.03 0.76 ± 0.03
Heptacosane 2700 B 3.60 ± 0.15 4.40 ± 0.21 showed an interesting activity against a-amylase. A weak activity
Nonacosane 2900 B 0.92 ± 0.03 1.59 ± 0.18 was observed on a-glucosidase inhibition in comparison with
Vitamin E A 2.77 ± 0.18 5.90 ± 0.24 C. annuum var. acuminatum. A similar pattern of hypoglycaemic
Campesterol C tr 1.31 ± 0.08 activity was observed for immature pepper fruits.
Stigmasterol C 3.43 ± 0.03 1.46 ± 0.14
Stigmast-6-en-3-ol C 2.88 ± 0.12 3.78 ± 0.15
In order to evaluate the compounds responsible for the activ-
Stigmasta-5,23-dien-3-ol C 1.91 ± 0.10 1.10 ± 0.14 ity, the lipophilic fraction was also tested. Immature C. chinense
b-Sitosterol A 2.50 ± 0.01 1.60 ± 0.09 Habanero fruits were able to selectively inhibit a-amylase with
b-Amyrin C 1.11 ± 0.07 an IC50 value of 9.88 lg/ml. At this stage of maturity hot pepper
a-Amyrin C 1.87 ± 0.09
is characterised by a high content of phytol and different fatty
a
I, retention index on MS HP-5 non-polar column. acids. At the same time continuous dietary consumption of red
b
Abundance calculated as % peak area mean values, mean ± standard deviation fruits is thought to be useful in the treatment of type 2 diabetes
(n = 3).
c
(Nettleton, Steffen, Ni, Liu, & Jacobs, 2008; Tolan, Ragoobirsingh,
The reliability of the identification proposal is indicated by the following: A,
mass spectrum and retention index agreed with standards; B, mass spectrum and
& Morrison, 2001). Previously Tolan, Ragoobirsingh, and Morrison
retention index agreed with database or literature; C, mass spectrum agreed with (2004) demonstrated that capsaicin exerted a hypoglycaemic ef-
mass spectral database; tr, <0.1%; –, not detected. fect in dogs by a complex mechanism which involved pancreas
558 F. Menichini et al. / Food Chemistry 114 (2009) 553–560

Fig. 1. GC chromatogram of C. chinense Habanero lipophilic fractions of (a) immature stage and (b) mature stage.

and other peripheral organs, such as the liver and the adrenal
medulla.
3.4. Cholinesterase activity
In spite of the multi-factorial nature of Alzheimer’s disease
(AD), most current agents follow one therapeutic approach, based
on the so-called cholinergic hypothesis of cognitive dysfunction.
This hypothesis postulates that at least some of the cognitive de-
cline experienced by patients of AD results from a deficiency in
neurotransmitter acetylcholine and thus in cholinergic neurotrans-
mission, which seems to play a fundamental role in memory. In
late stages of AD, levels of AChE decline by up to 85% and BChE rep-
resents the predominant cholinesterase in the brain. Such studies
have targeted BChE as a new approach to intercede in the progres-
sion of AD (Ballard, Greig, Guillozet-Bongaarts, Enz, & Darvesh,
2005; Tasker, Perry, & Ballard, 2005). Currently cholinesterase inhi-
bition is the major treatment for the symptoms of AD and inhibi-
tion of AChE and BChE are therapeutic targets for improving the
cholinergic deficit.
The inhibitory activities of C. chinense Habanero were measured
spectrophotometrically by the modified Ellman method. Both
immature and mature total extracts inhibited BChE with IC50 val-
ues of 562 and 806 lg/ml, respectively, but did not inhibit AChE

Table 3
Radical scavenging and antioxidant activity of C. chinense Habanero total extracts and
Table 4
lipophilic fractions.
Hypoglycaemic activity of total extracts and lipophilic fractions.
C. chinense Habanero IC50 (lg/ml)a
C. chinense Habanero IC50 (lg/ml)
Maturity stage DPPH b-Carotene bleaching test
Maturity stage a-Amylase a-Glucosidase
30 min 60 min
Total extract I 229 ± 5.7** 150 ± 1.3**
Total extract I 97.1 ± 0.97 14.4 ± 0.12 28.1 ± 0.21 M 131 ± 6.2** 265 ± 2.2**
M 287 ± 2.57 4.57 ± 0.07 5.31 ± 0.08 Lipophilic fraction I 9.88 ± 0.4** >1000
Lipophilic fraction I 321 ± 3.17 6.72 ± 0.09 8.40 ± 0.11 M 29.6 ± 0.8** >1000
M 655 ± 4.94 21.7 ± 0.18 40.2 ± 0.41 Acarbose 50.0 ± 0.9 35.5 ± 1.2
Propyl gallateb – 1.0 ± 0.01 1.0 ± 0.01
Ascorbic acidb 2.0 ± 0.01 – – Data are given as the mean ± SD (n = 3). Differences within and between groups
were evaluated by one-way analysis of variance (ANOVA) test completed with a
a
±SD (n = 3). multicomparison Dunnett’s test.
b **
Propyl gallate and ascorbic acid were used as positive control; p < 0.05. p < .01 compared with the positive control.
 F. Menichini et al. / Food Chemistry 114 (2009) 553–560
Table 5
Cholinesterase (AChE and BChE) inhibitory activity of C. chinense Habanero total
extracts and lipophilic fractions.
C. chinense Habanero IC50 (lg/ml)
Maturity stage AChE BChE
Total extract I >1000 562 ± 12.4**
M >1000 806 ± 15.9**
Lipophilic fraction I >1000 >1000
M 732.83 ± 8.96** >1000
Physostigmine 0.2 ± 0.02 2.4 ± 0.04
Data are given as the mean ± SD (n = 3). Differences within and between groups
were evaluated by one-way analysis of variance (ANOVA) test completed with a
multicomparison Dunnett’s test.
**
p < 0.01 compared with the positive control.
(Table 5). Previously it was demonstrated that C. annuum var.
acuminatum exhibited AChE inhibition with IC50 values of 84.3
and 130 lg/ml for immature and mature stage, respectively
(Loizzo, Tundis, Menichini, & Menichini, 2008; Loizzo, Tundis,
Menichini, Statti, et al., 2008). This is in disagreement with the
activity of Habanero peppers which appear to be selective against
BChE.
This activity is not reflected in lipophilic fractions
(IC50 > 1000 lg/ml). The lipophilic fraction of mature fruits weakly
inhibited AChE (IC50 733 lg/ml). The ability of C. chinense
Habanero to inhibit BChE is of interest considering that this BChE
activity is relatively high in thalamic nuclei that project to frontal
cortical structures involved in attention, executive function, and
behaviour. However, the largest pool of BChE in the brain is found
in the glia, particularly those in deeper cortical and subcortical
structures. These findings suggest that BChE may also be an impor-
tant therapeutic target in the management of symptoms due to
subcortical pathology. It may be hypothesised that pathologies
such as ‘pure’ Alzheimer’s disease (AD), AD with cerebro-vascular
disease, vascular dementia, Parkinson’s disease and dementia due
to Lewy bodies, might derive particular benefits from cholinester-
ase inhibitors that inhibit BChE in addition to AChE (Bullock &
Lane, 2007). Previously, Suganuma, Hirano, and Inakuma (1999)
demonstrated the beneficial effect of C. annum in both memory
and acquisition performance in a senescence-accelerated mouse
model. Moreover, several studies demonstrated the association be-
tween carotenoids, vitamins E and C dietary deficiency and impair-
ments in memory and learning performance (Morris et al., 2002).
From these results there is increasing evidence of the potential
beneficial effect of these compounds against the damaging effects
of neurodegenerative disorders. The cholinesterase inhibitory
activity and the stronger radical scavenging property of C. chinense
Habanero suggest how the consumption of this spice could be use-
ful in the chemoprevention of Alzheimer’s disease.
4. Conclusions
Pepper contains moderate to high levels of phytochemicals such
as phenols, capsaicinoids and vitamins which are important com-
ponents of diet because of their ability to reduce the risk of degen-
erative diseases.
This work demonstrated for the first time the ability of
C. chinense Habanero at two different stages of ripening to provide
interesting benefits to people with diabetes or Alzheimer’s disease.
The different chemical composition showed by the two ripening
stages of C. chinense Habanero fruits affect the in vitro biological
activities. In particular, mature peppers have potent antioxidant
property with b-carotene bleaching test and inhibited a-amylase
activity whilst immature peppers showed the most activity as rad-
ical scavenger and a selective inhibition of a-glucosidase and BChE
enzymes.
559
C. chinense Habanero is a vegetable widely used in human nutri-
tion. Therefore, further in vivo clinical studies are warranted to
confirm the biological activity of this pepper species with a poten-
tial pharmaceutical use, especially in terms of diet-linked
chemoprevention.
Acknowledgement
The authors are grateful to Miceli s.r.l., Italy, for supplying the
pepper fruits.
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