Determination of Antioxidant Contents in Red Sorrel and its nticarcinogenic Potential in Azoxymethane-Induced Colonic Aberrant Crypt Foci
M. S.C. Fullerton,
L. T. Walker,
L. A. Shackelford,
C. B. Chawan
The aim of the study was to determine the antioxidant properties in sorrel
and to evaluate the effect of feeding red sorrel on azoxymethane-induced
Aberrant Crypt Foci (ACF) in Fisher 344 rats. Total phenolics and flavonoids
were spectrophotometrically determined using gallic acid and catechin
as standards and antioxidant capacity was determined using 2, 2`-azinobis
[(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)] radical scavenging assay,
with vitamin C (ascorbic acid) as standard. Twenty-four Fisher 344 rats
were divided into 3 groups and fed control-(C) diet (AIN-93G), 10 g/100
g red Sorrel Meal (SM) and Sorrel Juice (SJ). All rats received 2 subcutaneous
injections of azoxymethane (AOM) at 7 and 8 week and were killed using
CO2 euthanasia at 17 week of age. Total phenolics (mg/100 g)
as Gallic Acid Equivalents (GAE), flavonoids (mg/100 g) as Catechin Equivalents
(CE) and VCAEC ranged from 256.8"1.0 to 433.7"1.4 for fresh and dry sorrel.
Total number of ACF in rats fed C, SM and SJ were 154.4, 38.6 and 24.6,
respectively. Sorrel is attracting the attention of food manufacturers
and these results indicate that phytochemicals present in sorrel may have
many possibilities for health improvement.
to cite this article:
M. Verghese, M. S.C. Fullerton, L. T. Walker, L. A. Shackelford, E. Cebert, J. Boateng, S. Ogutu, J. Khatiwada, M. Guyton, J. Jones and C. B. Chawan, 2008. Determination of Antioxidant Contents in Red Sorrel and its nticarcinogenic Potential in Azoxymethane-Induced Colonic Aberrant Crypt Foci. Research Journal of Phytochemistry, 2: 69-76.
Colon cancer is a significant cause of morbidity and mortality in Western industrialized countries and is the second most frequent
cause of cancer deaths in the United States (Parker et al., 1997).
Human metabolic and laboratory animal model studies (Middleton et al.,
2000; Wang, 2007) indicate that the composition and physical
properties of phytochemicals influence their beneficial effects in relation
to cancer development. Based on current knowledge of the pathogenesis
of colon cancer, it is reasonable to conclude that consumption
of fruits and vegetables rich in phytochemicals are associated with a
reduced risk of colon cancer. Phytochemicals impart color and
are responsible for cranberries, strawberries and other fruits being good
sources of antioxidants.
Sorrel (Hibiscus sabdariffa) is a potentially good source of antioxidants.
Calyces of Hibiscus species contain polyphenolic acids, flavonoids
and anthocyanins. Flavonoids are widespread in the plant kingdom and are
especially common in leaves, flowering tissues and pollen (Beecher, 2003;
Liu, 2003). Known properties of flavonoids include free radical scavenging,
strong antioxidant activity, inhibition of hydrolytic and oxidative enzymes
(phospholipase A2, cycloxygenases, lipoxygenase) and anti-inflammatory
action (Frankel, 1995). Free radicals have been implicated in the inactivation
of enzymes, degradation of DNA and cell membranes (Nijveldt et al.,
2001; Heijnen et al., 2002; Chun et al., 2003) all of which
are linked to degenerative human diseases such as cancer, heart disease
and cerebrovascular diseases.
Antioxidants such as vitamins and phytochemicals which are derived from
foods have received growing attention, because they are known to function
as chemopreventive agents against oxidative damage. Vitamin C is one of
the most popular and least toxic antioxidant components in foods and is
the most used dietary supplement to prevent oxidative stress-mediated
diseases. However, the contribution of vitamin C to the total antioxidant
activity of fruits is generally <15% (29). In addition to antioxidative
properties, scientific studies have shown that phenolic phytochemicals
are also associated with anticarcinogenic, antimicrobial, antiallergic,
antimutagenic and anti-inflammatory activities (Cao and Prior, 1999).
Phytochemical content in plants may be affected by level of maturity,
cultivars, horticultural practices, geographic origin, growing season,
post harvest storage conditions and processing procedures (Srivastava
et al., 2007; De Freitas and Glories, 1999; Kalt et al.,
1999; Donavon et al., 1998). Therefore, evaluation of red sorrel
as a potential source of phenolic phytochemicals is warranted.
Aberrant Crypt Foci (ACF) induced by azoxymethane (AOM) have
been used extensively to investigate nutritional modulation
of colon carcinogenesis in rats. AOM is a metabolite of the
procarcinogen 1, 2-dimethylhydrazine and is one metabolic step
closer to the proximate carcinogen capable of inducing colonic
ACF (Bird, 1995). The glutathione S-transferases (π, μ, α,),
a family of Phase II detoxification enzymes, play a critical role in protecting
the colonic mucosa by catalyzing the conjugation of dietary carcinogens
with glutathione (Boateng et al., 2007; Kauser et al., 2003).
Overall, there is now new evidence that antioxidants in the human diet
are beneficial for health and nutrition (Wang et al., 1997). The
objectives of the study were to determine total flavonoids, phenolics
and Vitamin C Antioxidant Capacity (VCAC) in fresh and dry sorrel and
to evaluate the effect of sorrel on azoxymethane-induced Aberrant Crypt
Foci (ACF) in Fisher 344 male rats.
MATERIALS AND METHODS
Dried red sorrel calyces were purchased from a local supermarket.
Fresh red sorrel calyxes were harvested in Summer 2006 from a greenhouse
at Alabama A and M University (Normal, Alabama). Samples were ground and
stored in amber jars at -201C for further analysis.
Gallic acid, 2,2`-azinobis (3-ethylbenzothiazoline-6-sulfonic acid),
radical scavenging assay (ABTS) as diammonium salt, (+)-catechin and Folin
and Ciocalteau`s phenol reagent were obtained from Sigma Chemical Co.
(St. Louis, MO,). 2`-azobis (2-amidinopropane) hydrochloride
(AAPH), gallic acid and ascorbic acid were obtained from Fisher Scientific
Co. (Suwannee, GA). All other chemicals were of analytical grade and purchased
from Fisher Scientific Co. (Suwannee, GA).
Extraction of Phenolics
Phenolics in dried and fresh red sorrel calyces were extracted by
an ultrasound-assisted method as described by Kim et al. (2003).
Determination of Total Phenolics
Total phenolics were measured by the Folin-Ciocalteau method previously
described by Singleton and Rossi (1995) with some modification (Kim et
al., 2003). Total phenolic content of sorrel was expressed as mg Gallic
Acid Equivalents (GAE)/100 g fresh sample. All samples were analyzed five
Determination of Total Flavonoids
A method described by Kim et al. (2003) was used to determine
total flavonoids in red sorrel samples. Absorbance was determined at 510
nm versus prepared blank (distilled deionized water). Total flavonoid
of sorrel was expressed on a fresh weight basis as mg/100 g Catechin Equivalents
(CE). All samples were analyzed four times.
Vitamin C Equivalent Antioxidant Capacity (VCEAC) Assay Using 2,2`-Azinobis
(3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS) Radical
Vitamin C Equivalent Antioxidant Capacity (VCEAC) assay was
determined as described by Kim et al. (2003) with minor modifications.
Briefly, 1 mM AAPH was mixed with 2.5 mM ABTS in phosphate-buffered saline
(pH 7.4; 100 mM potassium phosphate buffer containing 150 mM NaCl). The
solution was heated in a water bath at 68°C for 13 min and cooled
to room temperature. The resulting blue-green ABTS solution was adjusted
to an absorbance of 650"0.020 at 734 nm with phosphate-buffered saline.
One hundred microliter of sample was added to 2.9 mL of the ABTS radical
solution and incubated in a 37°C water bath under restricted light
for 10 min, radical solution was prepared daily. A control consisting
of 100 μL of 50 mL/100 mL methanol and 2.9 mL of ABTS radical solution
was ran with each series of samples. The decrease in absorbance at 734
nm was measured after endpoint of 10 min. Total antioxidant capacity of
red sorrel, was expressed on a fresh weight basis as mg/100 g vitamin
C equivalents. Experiment was done in four replications.
Animals, Housing and Diets
Following a one-week period of acclimatization, 24 Fisher 344 male
weanling rats (Harlan, IN) were randomly divided into 3 groups (8 rats
each). Light and dark cycles were held at 12 h each and the temperature
and relative humidity were held at 211C and 50%, respectively. Rats were
assigned to one of the following diets for 13 weeks: AIN 93G (Reeves
et al., 1993a, b) (Control -C), 10 g/100 g SM and 10 g/100 g SJ. Diets
were formulated based on AIN 93G diet (Table 1). Ingredients
were obtained from ICN (Costa Mesa, CA). Red sorrel calyxes were grounded
to a fine powder using a food processor (Robot coupe, Blixer RSI, BS3).
The ground sorrel was then mixed into the diet at 10 g/100 g level at
the expense of cornstarch and fiber (Table 1). Feed was
provided ad libitum. All diets were prepared weekly and refrigerated at
-4°C, until fed. Biweekly weight gains and daily feed intakes were
recorded. The Institutional Animal Care and Use Committee of Alabama A
and M University approved all protocols involving rats.
Preparation of Red Sorrel Juice
One hundred grams of dried red sorrel calyxes was added to 1 L of
distilled deionized water and allowed to boil for 10 min. The sorrel infusion
was allowed to cool to room temperature and then filtered with cheesecloth
to remove spent calyxes. One hundred milliliter of the sorrel infusion,
regarded as sorrel juice, was fed each day to the rats in place of water.
Carcinogen injections were administered at 7 week and 8 week of age.
All groups except saline group (n = 6) were subcutaneously injected with
azoxymethane (Sigma Chemicals, St Louis, MO) in saline at 16 mg kg-1
||Composition of the diets1
|1: Formulation of diets based on AIN-93G
(Reeves et al., 1993 a, b), 2: Common ingredients:
casein (>85% protein), 200; dextrose, 132; sucrose, 100; soyabean
oil, 70; fiber (Solk-Floc), 50; mineral mix (AIN-93), 35; AIN-93G,
Vitamin mix, 10; L-cystein, 3; choline batartarate, 2.5
Tissue Sample Collection and Counting Aberrant Crypt Foci
Rats were euthanized by CO2 asphyxiation at 17 week of
age and colons were collected for enumeration of Aberrant Crypt Foci (ACF).
The colons were flushed with potassium phosphate buffer (0.1 M, pH 7.2),
split open and ACF were enumerated as described by Bird (1987).
Glutathione-S-Transferase (GST) Assay
Approximately 1 g of liver samples were homogenized in 10 volumes
of potassium phosphate buffer (pH 7.0, 0.1M) and centrifuged at 10,000
x g for 30 min. Clear supernatant was collected and an aliquot was mixed
1, chloro 2, 4-dinitrobenzene, potassium phosphate buffer and reduced
glutathione. GST activity was measured using a Cary1/3 UV/VIS dual beam
spectrophotometer as outlined by Habig et al. (1974).
Data were analyzed using ANOVA (SAS, 2004). Means were separated using Turkey`s studentized range test. Differences
were considered significant at p<0.05 (28).
Determination of Phenolics, Flavonoids and Vitamin C Antioxidant
Capacity in Red Fresh and Dry Sorrel
Total phenolics expressed as Gallic Acid Equivalents (GAE) and flavonoid
contents as Catechin Equivalents (CE) of fresh and dried red sorrel calyces
were 433.7"1.4, 408.2"1.3, 279.5"1.5 and 271.3"1.3 mg/100 g, respectively.
VCAEC in fresh and dry red sorrel were 293.4"2.2 and 256.8"1.0 mg/100
g, respectively. There were no significant differences (p<0.05) in
total phenolics, flavonoids and vitamin C antioxidant capacity between
fresh and dry sorrel (Table 2).
Feed Intake, Body Weight, Cecal pH and Cecal Weights
Body weight gains of rats were lower (p<0.05) in the control than
in the sorrel fed rats. Rats fed 10% SJ had a significantly higher (p<0.05)
weight gain. There were also significant (p<0.05) differences in feed
intake in rats fed control (C) and SJ and SM. Cecal weights were higher
in the control than in sorrel fed rats (Table 3). Cecal
pH was significantly (p<0.05) lower in the rats fed sorrel meal and
juice compared to the control. There were however, no significant (p<0.05)
differences in cecal pH between rats fed SM and SJ.
Aberrant Crypt Foci
The rats administered saline (vehicle), showed no evidence of ACF
formation in the colon (data not shown). In rats fed control, AOM induced
an average of .150 ACF/colon. Totals ACF numbers in rats fed SJ and SM
were 24.8 and 38.6, respectively (Table 4). Compared
to the control
||Total phenolics, flavonoids and Vitamin C Antioxidant
Capacity (VCAC) in fresh and dried sorrel
|Values are Means"SEM ab: Means in rows without
a common letter(s) different significantly (p<0.05) using Tukey`s
||Weight gain, feed intake and cecal weight and cecal
pH of Fisher 344 male rats fed control, sorrel juice and sorrel meal
|Values are means"SEM. abc: Means in rows
without a common letter(s) different significantly (p<0.05) using
Tukey`s studentized test
||Effect of Sorrel juice and Sorrel meal on Aberrant Crypt
Foci (ACF) and total crypts in colon of Fisher 344 male rats
|Values are Means"SEM. abc: Means in a column
without common letter(s) different p<0.05 using Tukey`s studentized
||Glutathione S-Transferase (GST) activity in rats fed
Sorrel as compared to the controls
|Values are means"SEM. abc: Means in the column
with a different letter(s) differ significantly (p<0.05) using
Tukey=s studentized test
there was 83.8 and 78% reduction in ACF in rats fed SJ and SM, respectively.
Total number of aberrant crypts was significantly (p<0.05) higher in
rats fed control compared to sorrel fed rats. The distal colon had
higher numbers of ACF (p<0.05) than the proximal segment
(Table 4). Foci with 4 and >5 crypts were lower
(p<0.05) in rats fed SM and SJ than in the control (data not shown).
Glutathione-S-Transferase Activity (GST)
There was a significant (p<0.05) increase in liver GST (μmol
mg-1) (a crucial detoxification enzyme) in rats fed SJ (81%)
and SM (percent) compared to the control. GST activity (μmol mg-1)
increased from 9.2 in the rats fed control diet to 31.5 and 49.4 in the
rats fed sorrel meal and juice, respectively (Table 5).
Recently, attention is being focused on the protective biochemical
functions of naturally occurring antioxidants in biological systems and
on their mechanisms of action. Results from this study indicate that total
phenolics and flavonoid contents in red sorrel are greater than those
in some traditional temperate fruits. The results are comparable with
a study by Kim et al. (2003) on red plum cultivars where they reported
values of 174 to 375 mg/100 g for phenolics, expressed as gallic acid
equivalents and 118 to 237 mg/100 g for flavonoids, expressed as catechin
equivalents. Total phenolic concentrations of various apple cultivars
were reported to range from 50.9 to 140 mg GAE/100 g (Lee and Smith, 2000)
while another study reported that total phenolics content of red plums
was 144 mg/100 g, expressed as catechin equivalents (Karakaya et al.,
The flavonoid contents in red sorrel which accounts for its red color
are also recognized as powerful antioxidants (Kandaswami and Middleton,
1994) with strong scavenging effects on superoxide radicals. This may
be the most important function of flavonoids (Miller et al., 1996).
Many studies have considered fruits, vegetables and teas as the major
sources of dietary antioxidative phenolics (Williams and Elliot, 1999;
Kim et al., 2003). Grapes and red wine have been recognized for
their antioxidant activity and the improved health in populations that
are large consumers of these products (Williams and Elliot, 1999). A study
conducted on the antioxidant capacity of fresh plums and gala apples reported
values ranging from 256 to 559 mg VCEAC/100 g and 205 to 210.6 mg VCEAC
/100 g, respectively (Kim et al., 2003). The antioxidant activity
reported in this study for fresh and dried sorrel samples were 293.7 and
256.8 mg VCEAC/100 g, respectively.
Since sorrel is a prime source of phenolic antioxidants, we evaluated
the potential inhibitory effects of sorrel on the formation
of AOM-induced colonic ACF, which are putative preneoplastic
lesions. The results showed that red sorrel when fed to fisher 344 rats
significantly (p<0.05) reduced the number of ACF. Sorrel contains a
composite of several antioxidants, which may have been involved in the
reduction of ACF numbers. In their study, Chewonarin et al. (1999)
observed a 22% reduction in the number of AOM induced ACF when rats were
administered a gavage of sorrel extract once a day. According to the authors,
one of the mechanisms associated with the decrease of ACF may be due to
the inhibition of DNA methylation and cytochrome P450 1A2, although the
exact mechanisms are yet to be determined.
Flavonoids have beneficial effects through their impact on the bioactivation
of carcinogens. Most food-borne carcinogens require transformation by
phase I metabolizing enzymes into a more reactive form to bind to DNA.
If the resulting mutation is not repaired, it may initiate or promote
the carcinogenesis process. The reactive chemical group introduced by
phase I enzymes can be detoxified through conjugation by phase II metabolizing
enzymes into a water-soluble compound which can be eliminated from the
body (Khan et al., 1992). Various flavonoids have also been shown
to have anti-inflammatory activity by inhibiting cyclooxygenase-2 (COX
2) and inducible nitric oxide synthase (Raso et al., 2001). Chronic
inflammation is thought to play an important role in the etiology of a
number of cancers and COX 2 inhibitors are being studied as chemopreventive
agents against colon cancer (Mutoh et al., 2000).
There was a significant (p<0.05) increase in hepatic Glutathione-S-transferase
activity in rats fed SJ and SM compared to the control (Table
4). Animal and in vitro studies have shown that flavonoids
including catechins and apigenin increase the activity of several detoxifying
and antioxidant enzymes, such as glutathione reductase, glutathione peroxidase,
catalase, quinone reductase and glutathione S-transferase (Valerio et
al., 2001). The glutathione S-transferases-crucial Phase II detoxification
enzymes play a critical role in protecting the colon mucosa by catalyzing
the conjugation of dietary carcinogens with glutathione, which renders
them more water soluble and easily excreted. Hepatic GST activity may
be used in colorectal cancer chemoprevention trials to monitor the responsiveness
of colon tissue to regimens that modify Phase II detoxification enzymes
(Mutoh et al., 2000).
To present knowledge, this is the first known study to demonstrate that
dietary administration of 10 ppm red sorrel significantly reduced
ACF, suggesting that consumption of red sorrel may retard growth
and/or development of neoplastic lesions in the colon. This
suggests the usefulness of red sorrel as a chemopreventive agent
for individuals at high risk for colon cancer development.
In conclusion, this study showed that phenolic phytochemicals such as
flavonoids and phenolic acids are present in both fresh and dried sorrel
calyces. These may function as effective natural antioxidants therefore
an increased consumption of red sorrel may be beneficial. Red sorrel (fresh
and/or dry) could possibly be utilized in commercial food processing to
prevent lipid oxidation, which results in development of off-flavors and
odors. Substituting or reducing the amount of synthetic antioxidants such
as Butylated Hydroxyanisole (BHA) and Butylated Hydroxytoluene (BHT) with
red sorrel in food products may be beneficial.
The results of this study showed a significant decrease (p<0.05) in
the total crypts in the sorrel juice and sorrel meal groups compared to
the control group. The results justify the use of dietary phytochemicals
as cancer preventive agents in population with high-risk for colon cancer
in the US Based on the results, red sorrel appears to hold promise as
a potential cancer preventive agent in high-risk populations and should
be further evaluated. End point tumor model studies may provide additional
data suggestive of chemopreventive potential of red sorrel products.
This research was supported by funding from the Agricultural Experimental
Research Station, Alabama Agricultural and Mechanical University, Normal,
AL 35762, USA.
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