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JOURNAL OF MEDICINAL FOOD
J Med Food 11 (1) 2008, 184–189
© Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2007.638
Short Communication
Influence of Ripening Stage on Health Benefits Properties of
Capsicum annuum Var. acuminatum L.: In Vitro Studies
Monica R. Loizzo, Rosa Tundis, Federica Menichini, Giancarlo A. Statti, and Francesco Menichini
Department of Pharmaceutical Sciences, Nutrition and Health Science, Faculty of Pharmacy,
University of Calabria, Rende (CS), Italy
ABSTRACT In the present study the in vitro hypoglycemic and anti-acetylcholinesterase activities of hot pepper fruits (Cap-
sicum annuum var. acuminatum L.) at different ripening stages were investigated. The mature, green-stage fruits had the high-
est activity against -amylase and -glucosidase with 50% inhibitory concentration (IC
50
) values of 55.88 and 76.11 g/mL,
respectively, while C. annuum var. acuminatum in the prematurity green stage exhibited the highest acetylcholinesterase in-
hibition property (IC
50
84.30 g/mL), using the Ellman method. This study highlights the biochemical rationale for chemo-
preventive significance in health benefits when consuming this variety of pepper.
KEY WORDS:
•
acetylcholinesterase inhibition
•
Alzheimer’s disease
•
-amylase
•
Capsicum annuum var. acumi-
natum
•
-glucosidase
•
hyperglycemia
184
INTRODUCTION
H
ERBS AND SPICES
have been used for generations by hu-
mans as food and to treat ailments. Scientific evidence
is accumulating that many of these herbs and spices do have
medicinal properties that alleviate symptoms or prevent dis-
ease. Demonstrating the health benefits of foods based on
sound scientific remains a challenge, particularly when com-
pared with standards applied for assessing pharmaceutical
agents.
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 frutescens L., Capsicum chinense Jacq., and Cap-
sicum annuum L. This last one, originating from Mexico,
was the first to be introduced worldwide, and nowadays it
is the most widely spread in familiar consumption and in-
dustrial processing.
1
Hot pepper is a well-known spice. It is
used dried or fresh in various pharmacological preparations
and has many applications in cookery to enrich foods with
its unique flavor. It has a long history as a source of healthy
and biologically active compounds, such as flavonoids, phe-
nols, carotenoids, capsaicinoids, and vitamins.
2
Diabetes mellitus is a serious complex chronic condition
that is a major source of health problems worldwide. In fact,
at least 171 million people worldwide have diabetes, and the
numbers are projected to increase to 366 million by 2030.
3
The World Health Organization recognizes three main
forms of diabetes mellitus—type 1, type 2, and gestational
diabetes—which have similar signs, symptoms, and conse-
quences but different causes and population distributions.
While, ultimately, all forms are due to the -cells of the pan-
creas being unable to produce sufficient insulin to prevent
hyperglycemia, the causes are different. Type 1 diabetes is
usually due to an autoimmune destruction of the pancreatic
-cells. Type 2 diabetes is characterized by insulin resis-
tance in target tissues; this causes the need for abnormally
high amounts of insulin, and therefore diabetes develops
when the -cells cannot meet this demand. Gestational di-
abetes is similar to type 2 diabetes in that it involves insulin
resistance; the hormones of pregnancy can cause insulin re-
sistance in women genetically predisposed to developing
this condition.
4
One therapeutic approach for treating type 2 diabetes is
to decrease the postprandial hyperglycemia. This is done by
retarding the absorption of glucose through the inhibition of
the carbohydrate-hydrolyzing enzymes, -amylase and -
glucosidase, in the digestive tract. Inhibitors of these en-
Manuscript received 22 October 2007. Revision accepted 7 December 2007.
Address reprint requests to: Dr. Monica R. Loizzo, Department of Pharmaceutical Sci-
ences, Nutrition and Health Science, Faculty of Pharmacy, University of Calabria, I-
87036 Rende (CS), Italy, E-mail: loizzomr@unical.it
zymes, like acarbose and voglibose, delay carbohydrate di-
gestion and prolong overall carbohydrate digestion time,
causing a reduction in the rate of glucose absorption and
consequently reducing the postprandial plasma glucose
rise.
5,6
Neurodegenerative disease is a generic term applied to a
variety of conditions arising from a chronic breakdown and
deterioration of the neurons, particularly those of the cen-
tral nervous system. The discovery of cholinesterase in-
hibitors has been a challenging area of pharmaceutical re-
search due to the involvement of cholinesterase enzymes in
Alzheimer’s disease and other related dementias. Recently
some studies reported a number of new natural inhibitors of
cholinesterase enzymes, isolated from medicinally impor-
tant plants.
7
In our continuing search of biological properties of spices,
fruits, and vegetables,
8–11
the present work was designed to
determine the influence of ripening stage of C. annuum var.
acuminatum on hypoglycemic and anti-acetylcholinesterase
(anti-AChE) inhibitory properties.
MATERIALS AND METHODS
Chemicals
The following chemicals were obtained from Sigma-
Aldrich S.p.a. (Milan, Italy): potato starch, sodium phosphate,
sodium chloride, -amylase from porcine pancreas (EC
3.2.1.1), -glucosidase from Saccharomyces cerevisiae (EC
3.2.1.20), maltose, sodium acetate, sodium potassium tartrate,
3,5-dinitrosalicylic acid, o-dianisidine color reagent (DIAN),
PGO (peroxidase/glucose oxidase) enzyme preparation, elec-
tric eel AChE (EC 3.1.1.7, type VI-S), acetylthiocholine io-
dide, 5,5-dithionitrobenzene, bovine serum albumin, and
anisaldehyde. Methanol, chloroform, acetic acid, perchloric
acid, dimethyl sulfoxide, basic bismuth nitrate, potassium io-
dide, phosphoric acid, silica gel 70–230 mesh, and thin-layer
chromatography (TLC) plates were obtained from VWR In-
ternational s.r.l. (Milan). Acarbose from Actinoplanes sp. was
obtained from Serva (Heidelberg, Germany).
Sample preparation
The fruits of C. annuum var. acuminatum L. were har-
vested in September 2004 and purchased in a local market
at three successive stages of technological maturity on the
basis of their color as one ripening stage: premature green
pepper (1), mature green pepper (2), and mature red pepper
(3). Five hundred grams of fresh fruits at different maturity
stages had been washed, cut into small pieces, and extracted
with methanol through maceration (1 week, repeated three
times). The resultant extract was dried under reduced pres-
sure to give 31.35 g of 1, 9.13 g of 2, and 41.51 g of 3.
Assay for -amylase inhibition
The bioassay method was adopted and modified
11
from
that of Sigma-Aldrich. Briefly, a starch solution (0.5%
wt/vol) was obtained by stirring potato starch in 20 mM
sodium phosphate buffer with 6.7 mM sodium chloride. The
enzyme solution was prepared by mixing 25.3 mg of -amy-
lase in 100 mL of cold distilled water. C. annuum var. acumi-
natum extracts were dissolved in buffer to give final
concentration from 500 g/mL to 31.25 g/mL. The col-
orimetric reagent was prepared mixing a sodium potassium
tartrate solution and 96 mM 3,5-dinitrosalicylic acid solu-
tion. Both control and hot pepper extracts were added with
starch solution and left to react with -amylase solution in
alkaline condition at 25°C. The generation of maltose was
quantified by the reduction of 3,5-dinitrosalicylic acid to 3-
amino-5-nitrosalicylic acid. Acarbose was used as the pos-
itive control. This reaction is detectable at 540 nm. The -
amylase inhibition was expressed as a percentage of
inhibition and calculated by the following equations:
% Reaction 100
% Inhibition (100 % Reaction) SD
Assay for -glucosidase inhibition
The -glucosidase inhibition was measured through a
modified Sigma-Aldrich bioassay method.
12
Briefly, a mal-
tose solution (4% wt/vol) was prepared by dissolving 12 g
of maltose in 300 mL of 50 mM sodium acetate buffer. The
enzyme solution was prepared by mixing 1 mg of -glu-
cosidase in 10 mL of ice-cold distilled water. Samples are
dissolved in dimethyl sulfoxide to give final concentrations
from 5 g/mL to 1 mg/mL. The DIAN solution was pre-
pared by dissolving one tablet in 25 mL of distilled water,
while the PGO enzyme color reagent solution was freshly
prepared by dissolving one capsule in 100 mL of ice-cold
distilled water. In the first step both control and extracts
were added with maltose solution and left to equilibrate at
37°C. The reaction was started adding -glucosidase solu-
tion, and tubes were left to incubate at 37°C for 30 minutes.
At the end perchloric acid solution (4.2% wt/vol) was added
to stop the reaction. In the second step the generation of glu-
cose was quantified by the reduction of DIAN. The super-
natant of the tube of step I was mixed with DIAN and PGO
and was left to incubate at 37°C for 30 minutes. Acarbose
was used as the positive control. The reaction was measured
at 500 nm. The -glucosidase inhibition was expressed as a
percentage of inhibition and was calculated by the follow-
ing equations:
% Reaction 100
% Inhibition (100 % Reaction) SD
AChE inhibition assay methods
AChE inhibition was assessed by modifications of the Ell-
man method, which is based on the reaction of released thio-
[Glucose]
test
[Glucose]
control
[Maltose]
test
[Maltose]
control
PROPERTIES OF C. ANNUUM VAR. ACUMINATUM 185
choline to give a colored product with a chromogenic
reagent.
11
The spectrophotometric method was used to de-
termine the activity of C. annuum var. acuminatum extracts
showing activity by preliminary TLC screening as outlined
below. AChE (40 L; 0.36 U/mL in pH 8 buffer) and C.
annuum var. acuminatum extracts (20 L) were added to 2
mL of pH 8 buffer and preincubated in an ice bath at 4°C
for 30 minutes. Duplicate tubes were also treated this way
with 20 L of physostigmine (0.1 mM) to allow interfer-
ence of the test substances in the assay to be assessed and
to control for any hydrolysis of acetylcholine not due to
AChE activity. The reaction was started by adding 5,5-
dithionitrobenzene solution (20 L of 0.05 mM in pH 7
buffer) and acetylthiocholine iodide (20 L of 0.018 mM in
pH 7 buffer), and tubes were incubated in a water bath for
20 minutes at 37°C. The reaction was stopped by placing
the assay solution tubes in an ice bath and adding physostig-
mine (20 L of 0.018 mM in pH 7 buffer). Blanks were
used of reagents without extracts, and a positive control was
set up that was the same as the blank except that physostig-
mine (20 L of 0.018 mM in pH 7 buffer) was added. The
production of yellow anion was immediately recorded on a
spectrophotometer at 412 nm. The inhibition rate (%) was
calculated by the following equation:
[(Blank Blank positive control)
Inhibition %
(Experiment Experiment control)]
(Blank Blank positive control)
An in situ TLC autobiographic method was also employed
for preliminary screening of extracts with AChE inhibitory
effects. After the development of the TLC plate in
CHCl
3
/methanol 2.5% acetic acid (aqueous) (65:35:5 by
volume) as the solvent system, the dried plate was sprayed
with AChE in bovine serum albumin and then was left in a
moist atmosphere for 20 minutes at 37°C. The plate was
sprayed with acetylthiocholine iodide solution (45 mg/2.5
mL of water) and allowed to dry in a stream of cold air. It
was then sprayed with 5,5-dithionitrobenzene solution (100
mg/5 mL of phosphate buffer, pH 7). Physostigmine was
co-chromatographed as the standard AChE inhibitor. Any
compound inhibiting hydrolysis showed as a white spot
against yellow background. A duplicate plate was run to de-
tect false-positive effects due to interaction with the com-
ponents of the extract and the chromogenic reagent.
13
Statistical analysis
All experiments were carried out in triplicate. Data were
expressed as mean SD values. Differences were evaluated
186 LOIZZO ET AL.
T
ABLE
1. -A
MYLASE AND
-G
LUCOSIDASE
I
NHIBITORY
A
CTIVITY
(IC
50
)
OF
C.
ANNUUM
V
AR
.
ACUMINATUM
E
XTRACTS AT
D
IFFERENT
M
ATURITY
S
TAGES
: P
REMATURE
G
REEN
(1),
M
ATURE
G
REEN
(2),
AND
M
ATURE
R
ED
(3)
C. annuum var. acuminatum
IC
50
(g/mL)
extracts at different maturity stages -Amylase -Glucosidase
1 171.06 1.4** 133.30 1.6**
2 55.88 1.2** 76.11 1.3**
3 500 143.71 1.7**
Acarbose 50.01 0.90 35.52 1.2**
Data are mean SD values (n 3).
Differences within and between groups were evaluated by one-way analysis of variance test fol-
lowed by a multicomparison Dunnett’s test: **P .01 compared with the control experiment.
FIG. 1. Dose-dependent inhibition of (A) -amylase and (B) -glu-
cosidase by C. annuum var. acuminatum extracts at different matu-
rity stages: premature green (1), mature green (2), and mature red (3).
Data are mean SD values (n 3).
by one-way analysis of variance test followed by a Dun-
nett’s test. Differences were considered significant at P
.01. The 50% inhibitory concentration (IC
50
) was calculated
by nonlinear procedures with the use of Prism
®
version 4.0
for Windows (GraphPad Software, San Diego, CA)
(www.graphpad.com). The dose–response curve was ob-
tained by plotting the percentage of inhibition versus the
concentrations.
RESULTS
-Amylase and -glucosidase inhibitory activities
Premature green (1), mature green (2), and mature red (3)
C. annuum var. acuminatum peppers were evaluated to es-
timate if the inhibitory activity on the two major enzymes
involved in glucose digestion—-amylase and -glucosi-
dase—depended on the maturity stage. The analysis of the
results revealed that the change in C. annuum var. acumi-
natum during the course of ripening influenced its bioactiv-
ity (Table 1). A dose–response relationship was observed
for all extracts against both enzymes (Fig. 1). The mature
green stage (2) had the highest activity against -amylase
and -glucosidase with IC
50
values of 55.88 and 76.11
g/mL, respectively. Interesting, the fully mature red fruits
(3) were unable to inhibit -amylase (IC
50
550 g/mL).
AChE inhibition
A preliminary screening by TLC revealed that C. annuum
var. acuminatum exhibited a clear inhibitory effect com-
pared with the commercially available drug physostigmine.
In order to evaluate IC
50
values of C. annuum var. acumi-
natum extracts, the Ellman method was used. A dose–re-
sponse relationship was observed for all extracts (Fig. 2).
As shown in Table 2, the premature green stage (1) ex-
hibited the most promise (IC
50
84.30 g/mL). This bioac-
tivity decreased when the stage of ripening of the fruits in-
creased; in fact, mature red fruits (3) exhibited an IC
50
value
of 130.03 g/mL.
DISCUSSION
In recent years peppers have grown in popularity, and
now a wide number of varieties are now available in the
markets. For its exceptionally favorable climatic conditions,
the Calabria region in Southern Italy is one of the most im-
portant places of growth and diffusion of this spice. Pepper
contains moderate to high levels of phytochemicals such as
flavonoids, capsaicinoids, and vitamins that are important
components of a diet rich in vegetables, fruits, and spices,
other than traditional nutrients, for their ability to reduce the
risk of degenerative diseases.
14
This work has demonstrated, for the first time, the abil-
ity of C. annum var. acuminatum at different stages of ripen-
ing to exert some in vitro health benefit properties. To this
purpose, the inhibition of the two major carbohydrate di-
gestive enzymes, -amylase and -glucosidase, was inves-
tigated. -Amylase and -glucosidase inhibitors are cur-
rently used to reduce the postprandial plasma level of
glucose in type 2 diabetes and in the case of obesity.
15–17
Moreover, the C. annum var. acuminatum AChE inhibition
property was investigated. The inhibition of this enzyme is
one of the therapeutic approaches for the symptomatic treat-
ment of Alzheimer’s disease.
PROPERTIES OF C. ANNUUM VAR. ACUMINATUM 187
T
ABLE
2. AC
H
E I
NHIBITION
E
FFECT OF
C.
ANNUUM
V
AR
.
ACUMINATUM
E
XTRACTS AT
D
IFFERENT
M
ATURITY
S
TAGES
: P
REMATURE
G
REEN
(1), M
ATURE
G
REEN
(2),
AND
M
ATURE
R
ED
(3)
C. annuum var. acuminatum
AChE inhibition
extracts at different maturity stages TLC assay
a
IC
50
(g/mL)
1 84.30 1.3**
2 96.69 1.2**
3 130.03 1.8**
Physostigmine 0.07 0.002
Data are mean SD values (n 3).
Differences within and between groups were evaluated by one-way analysis of variance test fol-
lowed by a multicomparison Dunnett’s test: **P .01 compared with the control experiment.
a
Graded on the following scale: , strong inhibitory effect; , clear inhibitory effect; ,
weak inhibitory effect.
FIG. 2. Dose-dependent inhibition of AChE by C. annuum var.
acuminatum extracts at different maturity stages: premature green (1),
mature green (2), and mature red (3). Data are mean SD values
(n 3).
On the basis of our results, C. annum var. acuminatum at
the mature green stage of ripeness (2) exerted the most
promising bioactivity against -amylase and -glucosidase.
In this stage of maturity hot pepper was characterized by a
high content of phytol and different fatty acids, such as 8,11-
octadecadienoic acid methyl ester, methyl linolenate, methyl
palmitate, methyl palmitoleate, and palmitic acid.
10
In con-
trast, fully mature red fruits (3) were unable to inhibit -
amylase. From the phytochemical point of view, in fully ripe
fruits the total polyphenolic content, including luteolin, was
lower than the other maturity stages: 43.2 mg/g in mature
red pepper (3) and 76.0 mg/g and 73.8 mg/g in premature
green pepper (1) and mature green pepper (2), respec-
tively.
10
Recently luteolin demonstrated a strong -amylase
inhibitory property with an IC
50
of 0.17 mM.
15
These data
probably could explain the reduced inhibitory activity of
fully mature red fruits. At the same time continuous dietary
consumption of red fruits is thought to be useful in the treat-
ment of type 2 diabetes. In fact, capsaicin’s ability to stim-
ulate the release of insulin in dogs was recently demon-
strated.
18
Although insulin secretion stimulation is
important, the inhibition of -amylase and -glucosidase by
C. annum var. acuminatum would be more relevant consid-
ering that in type 2 diabetes there is a insulin resistance in
target tissues.
The phytochemical modifications that occur during ripen-
ing influence also the AChE inhibitory property. In fact, the
premature green stage fruits extract exhibited the highest ac-
tivity compared to the other samples tested. This activity de-
creased in the fully ripe stage. Based on the observation that
sterol content decrease during the ripening process,
10
we
could suppose that sterols may influence the AChE activity,
since members of this class of natural products have previ-
ously been described as AChE inhibitors.
7
The AChE in-
hibitory activity and the previously demonstrated stronger
radical scavenging property of C. annuum var. acuminatum
at the premature green stage of ripeness suggest how the
consumption of this spice could be useful in the chemopre-
vention of Alzheimer’s disease. The oxidative damage, in
fact, may initiate and promote the progression of a number
of chronic diseases, including Alzheimer’s disease.
10,19
CONCLUSIONS
The in vitro hypoglycemic and AChE inhibitory activi-
ties of C. annuum var. acuminatum extracts provide a strong
biochemical rationale for further in vivo studies. These in-
vestigations are essential to consider this pepper as a func-
tional food. Moreover, ripening and maturity of hot pepper
fruits could be chosen on the basis of their potential bio-
logical properties. At this point, daily consumption of hot
pepper has not been shown to provide clinical benefits to
people with diabetes or Alzheimer’s disease, but a possible
preventive effect with high consumption of pepper cannot
be ruled out, especially in terms of diet-linked chemopre-
vention and not for specific disease treatment used as drugs.
ACKNOWLEDGMENTS
The authors are grateful to Prof. Dimitar Uzunov, Nat-
ural History Museum of Calabria and Botanic Garden, Uni-
versity of Calabria, Rende (CS), Italy, for identification of
the samples and Dr. V. Filippelli, Faculty of Pharmacy, Nu-
trition and Health Science, University of Calabria, for Eng-
lish revision of the manuscript.
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PROPERTIES OF C. ANNUUM VAR. ACUMINATUM 189
