Antioxidant and Anti-Inflammatory

The American Journal of Chinese Medicine, Vol. 40, No.
1, 97–110
© 2012 World Scientific Publishing Company
Institute for Advanced Research in Asian Science and Medicine
DOI: 10.1142/S0192415X12500085
Antioxidant and Anti-Inflammatory

Effects of a Hypoglycemic Fraction
from Cucurbita ficifolia Bouche in
Streptozotocin-Induced Diabetes Mice
Am. J. Chin. Med. 2012.40:97-110. Downloaded from www.worldscientific.com
by MICHIGAN STATE UNIVERSITY on 12/26/14. For personal use only.
R. Roman-Ramos,* J. C. Almanza-Perez,* A. Fortis-Barrera,†

S. Angeles-Mejia,† T. R. Banderas-Dorantes,§ A. Zamilpa-Alvarez,¶
M. Diaz-Flores,|| I. Jasso,* G. Blancas-Flores,†
J. Gomez‡ and F. J. Alarcon-Aguilar*
*Department of Health Sciences
†Posgrade in Experimental Biology
‡Department of Biotechnology
§Doctorate in Biological Sciences
Division of Biological and Health Sciences
Universidad Autonoma Metropolitana, Mexico, D.F. Mexico
¶
Centro de Investigación Biomedica del Sur
Instituto Mexicano del Seguro Social
Xochitepec, Morelos, Mexico
||Unity of Medical Investigation in Biochemistry
National Medical Center SXXI
Instituto Mexicano del Seguro Social, Mexico, D.F. Mexico
Abstract: Type 2 diabetes is characterized by oxidative stress and a chronic low-grade

inflammatory state, which also play roles in the pathogenesis of this disease and the
accompanying vascular complications by increasing the production of free radicals and pro-
inflammatory cytokines. Cucurbita ficifolia Bouche (C. ficifolia) is an edible Mexican plant
whose hypoglycemic activity has been demonstrated in several experimental and clinical
conditions. Recently, D-chiro-inositol has been proposed as the compound responsible for
the hypoglycemic effects; however, the antioxidant and anti-inflammatory potential of this
plant has not yet been explored. The aim of this research is to study the influence of a
hypoglycemic, D-chiro-inositol-containing fraction from the C. ficifolia fruit (AP-Fraction)
on biomarkers of oxidative stress, as well as on the inflammatory cytokines in streptozotocin-
induced diabetes. The AP-Fraction obtained from the mature fruit of C. ficifolia contained
3.31 mg of D-chiro-inositol/g of AP-Fraction. The AP-Fraction was administrated daily by
Correspondence to: Dr. R. Roman-Ramos, 186. S. Rafael Atlixco, Col. Vicentina. Iztapalapa, P.O. 09340, Mexico
D.F. Tel: (þ52) 55-5804-6483, Fax: (þ52) 55-5804-4727, E-mail: rrr@xanum.uam.mx
97
98 R. ROMAN-RAMOS et al.
gavage to normal mice for 15 days as a preventive treatment. Then these animals were given
streptozotocin, and the treatments were continued for an additional 33 days. Pioglitazone was
used as a hypoglycemic drug for comparison. Administration of the AP-Fraction significantly
increased glutathione (GSH) and decreased malondialdehyde (MDA) in the liver without
significantly affecting the levels in other tissues. The AP-Fraction reduced TNF- and
increased IL-6 and IFN- in serum. Interestingly, the AP-Fraction also increased IL-10, an
anti-inflammatory cytokine. These results suggest that C. ficifolia might be used as an
alternative medication for the control of diabetes mellitus and that it has antioxidant and anti-
inflammatory properties in addition to its hypoglycemic activity.
Keywords: Cucurbita ficifolia; Cucurbitaceae; Diabetes Mellitus; Hypoglycemic Plants;

Antioxidant Plants; Anti-Inflammatory Plants; Inflammatory Cytokines.

Introduction
Cucurbita ficifolia Bouché (C. ficifolia), from the Cucurbitaceae family, is an annual
monoecious plant cultivated in Mexico, primarily in Estado de Mexico, Hidalgo, Puebla, and
Veracruz, for its edible fruit. In Mexico, it is commonly known as “chilacayote”, and its
immature fruit is utilized for preparing different dishes, while its mature fruit is used to make
a traditional candy. C. ficifolia has been used medicinally for curing wounds, hemorrhoids,
and fever (Alarcon-Aguilar and Roman-Ramos, 2008). In addition, the mature fruit macer-
ated in water is used for the treatment of diabetes (Andrade-Cetto and Heinrich, 2005). It has
been demonstrated that the mature fruit of C. ficifolia produces acute hypoglycemic effects in
healthy and diabetic rabbits and mice, as well as in alloxan- and streptozotocin-induced
diabetic rats when administered daily (Roman-Romas et al., 1992; Alarcon-Aguilar et al.,
2002). This effect has been associated with a rise in insulin level and reduced lipid per-
oxidation in the pancreas. A role in the renewal of β-cells has also been suggested (Xia and
Wang, 2007). Xia and Wang (2006) suggested that D-chiro-inositol, one of the principal
components in C. ficifolia fruit, is responsible for its hypoglycemic activity. The fruit of C.
ficifolia also caused hypoglycemic effects in patients with Type 2 diabetes (DM2), with no
signs of toxicity (Acosta-Patiño et al., 2001). While the hypoglycemic properties of C.
ficifolia fruit have been extensively studied, the antioxidant and anti-inflammatory properties
of C. ficifolia have not yet been investigated. These effects are important since obese diabetic
patients develop vascular complications due to hyperglycemia that are aggravated by oxi-
dative stress and a chronic inflammatory response (Reusch, 2003). In fact, oxidative stress
plays an important role in the pathogenesis of DM2, producing an imbalance in the gener-
ation of free radicals with a reduction in the mechanisms of antioxidant defense, resulting in
the development of insulin resistance and vascular complications (Evans, 2007). Several
biochemical substances that have been proposed as indicators of oxidant stress, such as
glutathione (-glutamylcysteinyl glycine, GSH) and malondialdehyde (MDA), are altered
in DM2; GSH is diminished, while MDA is increased (Evans, 2007). The oxidative
stress generated in diabetes exerts major effects on signaling pathways, affecting cellular
ANTIOXIDANT AND ANTI-INFLAMMATORY EFFECTS OF C. FICIFOLIA 99
metabolism and triggering a low-grade inflammatory reaction (Dominiczak, 2003). Thus,

patients with DM2 suffer from the chronic activation of certain cells in the immune system
(monocytes, macrophages, and neutrophils), as well as adipocytes in fat tissue (Reusch,
2003; Xu et al., 2003), resulting in an abnormal production of cytokines, such as tumoral
necrosis factor type-alpha (TNF-Þ, interleukin (IL)-6, and interferon-gamma (IFN-Þ,
which have also been implicated in the development of insulin resistance, obesity, and DM2
and its complications (Esposito et al., 2002 and 2003; Wexler et al., 2005). Therefore, the
modulation of these molecules by C. ficifolia might ameliorate the development of diabetic
complications in addition to its beneficial effects on diabetes.
The aim of the present study is to determine the antioxidant and anti-inflammatory
effects of a D-chiro-inositol-containing hypoglycemic fraction (AP-Fraction) of the mature
fruit of C. ficifolia on streptozotocin-induced diabetes in mice by measuring GSH and

MDA in different tissues, as well as the serum levels of TNF-, IL-6, IFN-, and IL10.
Materials and Methods
Plant Material
Fresh mature fruits of C. ficifolia with a diameter of 18–20 cm were gathered in the
Municipio of Acolman, Estado de Mexico during April and May of 2008. This material
was identified through taxonomic claves and compared to voucher specimen No. 11,119
from the Medicinal Plant Herbarium of the Mexican Institute of Social Security (Herbario
IMSS-M) at Mexico City. The endocarp, free of seeds, was dried at room temperature and
grounded using a 2-mm mesh in a Model 4 Wiley electric mill.
Preparation of the AP-Fraction from C. ficifolia Fruit
Ground material (100 g) from the C. ficifolia fruit was extracted with 300 ml of solvents
with different polarity (hexane, methylene dichloride, and methanol). The marc was then
extracted with 300 ml of distilled water over 24 h. This extract was filtered and centrifuged
at 750 g for 10 min to obtain a precipitate, which was separated and freeze-dried,
resulting in an Aqueous-Precipitate Fraction (AP-Fraction) whose yield was 35%.
Quantitative Analysis for D-Chiro-Inositol in AP-Fraction
The quantification of D-chiro-inositol in the fraction was performed using high perform-
ance liquid chromatography (HPLC Waters 2695 separation module) and a Waters 2697
Index Refractive Detector (Waters; Milford, MA, USA) using a LiChrospher 100 A NH2
column (5 m, 4 250 mm) and an isocratic mixture of acetonitrile-methanol (8:2) as the
elution system. The injection of the AP-fraction (20 l/2 mg/ml) yielded data corre-
sponding to the concentration of D-chiro-inositol. D-chiro-inositol displayed a retention
time of 8.6 min. The standard curve was linear with R 2 ¼ 0:99. We found that the water
extract of C. ficifolia contained 3.31 mg of D-chiro-inositol per g of AP-Fraction.
Experimental Animals
Male CD-1 mice (30–35 g) were obtained from the Laboratory Animal Center of the
Metropolitan Autonomous University and maintained with rodent food (Harlan Labora-
tories, Indianapolis, USA) and water ad libitum under a light/dark cycle of 12 h.
Experimental Design for Evaluating the Antioxidant

and Anti-Inflammatory Effects by AP-Fraction
All of the procedures on animals were carried out in accordance with the international rules
for the care and use of laboratory animals and in agreement with the Mexican Official
Norm (NOM-062-ZOO-1999, revised 2001). The Institutional Committee of the Metro-

politan Autonomous University at Mexico City approved the protocol. Normal mice were
divided into four groups of six animals each. Groups 1 and 2 received saline solution
(4 ml/kg/day), while Group 3 was given the AP-Fraction (200 mg/kg/day). Group 4 was
treated with pioglitazone (45 mg/kg/day), an insulin-sensitizer that activates the peroxi-
some proliferators-activated receptor type (PPAR-) and is associated with antioxidant
and anti-inflammatory activities (Chang et al., 2007; Fujisawa et al., 2009; Marchesi and
Schiffrin, 2008; Toba et al., 2006). These treatments were given by gavage for 15 days.
Then Groups 2–4 received a single intraperitoneal administration of streptozotocin
(137 mg/kg) dissolved in a buffer of citrates (0.1 M, pH 4.5). Group 1 received the buffer
only. All treatments were continued for an additional 33 days to assess the changes in
biomarkers of stress oxidative and inflammation.
Biochemical Parameters and Quantification of Cytokines
Measurements of body weight, food and water intake, and glycemia were performing
throughout the experiment, while glycated hemoglobin and biomarkers of stress and
inflammation were only quantified at the end of the treatments. Glycemia and glycated
hemoglobin (HbA1c) were determined from blood samples drawn from the tail vein.
Glycemia was quantified on day 30 in fasted animals (12 h without food) and at day 33 in
non-fasted animals using the Accutrend Sensor system (Roche Diagnostics). Glycated
hemoglobin (HbA1c) was analyzed with a DCA 2000 (Bayer). Quantification of total
cholesterol, triglycerides, creatinine, uric acid, aspartate aminotransferase (AST or GOT),
and alanine aminotransferase (ALT or GPT) was performed with a Reflotron System
(Bayer) using the blood samples from the ocular orbital sinus taken from animals anes-
thetized with pentobarbital on day 30. At the end of the test (day 33 after streptozotocin
administration), blood samples were again obtained from the ocular orbital sinus for
cytokine analysis. Serum cytokine levels were quantified using an ELISA purchased
from Pierce Protein Research Products (Thermo Fisher Scientific, Illinois, USA) to analyze
IL-6 and TNF- and from R & D Systems (Minneapolis, USA) to analyze IFN-
and IL-10.
Determination of GSH and MDA
Animals were sacrificed by exsanguination via heart perfusion using an isotonic saline
solution. Samples were obtained from the liver, heart, kidney and pancreas. Measurements
of GSH and lipid peroxidation were made in these organs. GSH was measured using the
Sedlak and Lindsay (1968) method to determine the thiol groups in 2-nitro-benzoic acid
(DTNB); whereas lipid peroxidation was measured with MDA using the method of thio-
barbituric acid-reactive substances (Draper and Hadley, 1990). Briefly, for GSH, 0.1 g
tissue was homogenized with 1 ml of meta-phosphoric acid and centrifuged (3200 g).
Supernatant (200 l) was mixed with 800 l of meta-phosphoric acid, 2 ml of TRIS buffer
and 50 l of DTNB, and the absorbance was recorded at 412 nm. For MDA determination,
0.1 g of each organ was homogenized with 1 ml of meta-phosphoric acid, and 300 l of this
preparation were added to 200 l of distilled water, acetic acid, thiobarbituric acid and
chlorhydric acid (TCA-TBA-HCL). This mixture was submitted to a water bath for 1 h and
centrifuged at 3200 g for 10 min. The absorbance of the supernatant was recorded at
535 nm.
Statistical Analysis
Data are presented as the mean S.E.M. Statistical differences among the treatments were
determined by an analysis of variance using the Tukey-Kramer Multiple Comparison post-
hoc test ðp < 0:05Þ. All statistics were computed using the NCSS 2000 software.
Results
Effect of the AP-Fraction on Glycemia and HbA1c
The administration of the AP-Fraction during the first 15 days did not affect glycemic
levels in non-fasted normal mice nor prevent the initial streptozotocin-induced hypergly-
cemia (Fig. 1A). However, the same fraction given over the 33 days following the
administration of streptozotocin gradually diminished the glycemic levels of non-fasted
diabetic mice (Fig. 1A). The AP-Fraction also significantly decreased glycemia in fasted
diabetic mice (day 30), while pioglitazone did not show any significant effect (Fig. 1B).
The HbA1c levels were also notably decreased by the AP-Fraction, while no changes were
produced by pioglitazone (Fig. 1C).
Effect of the AP-Fraction on Body Weight, Food and Water Intake
Body weight was not affected by the treatments (Fig. 2A), however, the elevation of food and
water intakes in diabetic mice were significantly diminished at the end of the treatment by the
AP-Fraction (Figs. 2B and 2C). Pioglitazone administration did not alter these parameters.
Administration of the AP-fraction caused a significant reduction in triglycerides without
altering total cholesterol, and liver transaminases were unchanged (Table 1). On the other
(A)
(B) (C)
Figure 1. Effect of daily administrations of C. ficifolia and pioglitazone on glycemia and HbA1c in normal mice
(15 d) and mice with streptozotocin-induced diabetes (33 d). (A) Time course of the glycemic levels. (B) Gly-
cemia in fasted streptozotocin-induced animals at day 30 of the treatment. (C) Percentage of HbA1c in strepto-
zotocin-induced diabetes mice at the end of the treatments. Mean S.E.M. (n ¼ 6). *Statistically significant
compared to the diabetic control; &statistically significant compared to the normal mice (p < 0:05). Normal:
normoglycemic mice, STZ: hyperglycemic mice by streptozotocin, STZ þ C. ficifolia: hyperglycemic mice treated
with AP-Fraction of C. ficifolia, STZ þ Piogli: hyperglycemic mice treated with pioglitazone.
hand, pioglitazone showed increased liver transaminases, although the increase was only
significant for GOT.
Effect of the AP-Fraction on GSH and MDA
In general, decreased GSH levels and increased MDA levels were observed in diabetic
animals compared to the normal mice (Figs. 3A–3H). The GSH levels were significantly
increased in the liver and heart ðp < 0:05Þ in response to treatment with both the AP-
Fraction and pioglitazone (Figs. 3A and 3B), while the levels in the kidneys and pancreas
were unchanged (Figs. 3C and 3D). In addition, MDA levels were clearly reduced in the
liver and heart by both the AP-Fraction and pioglitazone (Figs. 3E and 3F), without sig-
nificant alterations in the kidneys and pancreas (Figs. 3G and 3H).
(A)
(B) (C)
Figure 2. Physiological parameters measured at the end of treatment with C. ficifolia and pioglitazone in strep-
tozotocin-induced diabetic mice (33 d). Food and water intake were recorded weekly. (A) Body weight. (B) Food
intake. (C) Water intake. Mean S.E.M. (n ¼ 6). *Statistically significant compared to the diabetic control;
&
statistically significant compared to the normal mice (p < 0:05). Normal: normoglycemic mice, STZ: hyper-
glycemic mice by streptozotocin administration, STZ þ C. ficifolia: hyperglycemic mice treated with AP-Fraction
of C. ficifolia, STZ þ Piogli: hyperglycemic mice treated with pioglitazone.
Effect of the AP-Fraction on Biomarkers of Inflammation
The serum levels of IL-6, IFN- and IL-10 in diabetic mice were similar to those in normal
mice (Figs. 4A–4D). Treatment with both the AP-Fraction and pioglitazone produced
highly significant reductions in TNF- level (Fig. 4A). Nevertheless, increased IL-6 and
IFN- levels following AP-Fraction administration were also observed, while pioglitazone
did not significantly alter these cytokines (Figs. 4B and 4D). Interestingly, IL-10 was
notably increased by both the AP-Fraction and pioglitazone (Fig. 1C).
Discussion
The administration of the AP-Fraction during the first 15 days did not affect glycemic
levels in non-fasted normal mice, without preventing the initial hyperglycemia caused by
the streptozotocin administration. In fact, the hyperglycemia increased gradually and
Table 1. Effect of the AP-Fraction from Cucurbite ficifolia Fruit on Biochemical Parameter
Normal STZ C. ficifolia Pioglitazone
HbA1c (%) 3.2 0.16 6.72 &

0.36 4.8 0.36 6.96 0.47
Tg (mg/dL) 86 10.5 251 87.3& 164 15.3* 238 41.7
Cholesterol (mg/dL) 119 12.4 147.2 3.6& 136.6 5.5 156 3.5
ALT (U/L) 41.6 10.2 53 5.33 37.5 5.5 72.3 11.6
AST (U/L) 89.9 7.31 65.7 8.1 83 7.2 161.8 21.3*
Mean S.E.M. (n ¼ 6). *Significant difference compared to the STZ controls (p < 0:05). &Significant
difference compared to the normal controls (p < 0:05). Glycated hemoglobin (HbA1c), triglycerides (Tg),
alanine-amino transferase (ALT), aspartate-amino transferase (AST).
stayed during 13 days (day 28). However, the same fraction given during the 20 days
following gradually diminished the glycemic levels of non-fasted diabetic mice. There-
fore, these data suggest that the pharmacological effects of the AP-Fraction from
C. ficifolia are due to diabetic treatment more than diabetic prevention. Neverthless, this
(A) (B)
(C) (D)
Figure 3. Levels of GSH (A–D) and MDA (E–H) after daily administrations of C. ficifolia and pioglitazone in
streptozotocin-induced diabetic mice (33 d). Mean S.E.M. (n ¼ 6). *Statistically significant compared to the
diabetic control; &statistically significant compared to the normal mice (p < 0:05). Normal: normoglycemic mice,
STZ: hyperglycemic mice by streptozotocin administration, STZ þ C. ficifolia: hyperglycemic mice treated with
AP-Fraction of C. ficifolia, STZ þ Piogli: hyperglycemic mice treated with pioglitazone.
(E) (F)
(G) (H)
Figure 3. (Continued )
must be confirmed by additional experiments without administration of the fraction in

normal mice (before streptozotocin administration). The AP-Fraction also significantly
decreased glycemia in fasted diabetic mice (day 30), while pioglitazone did not show any
significant effect. The decreased glycemia and HbA1c levels following AP-Fraction
administration were similar to those reported in diabetic rats (Xia and Wang, 2007).
The lack of effect on glycemia by pioglitazone may be explained because it is an
insulin-sensitizer agent with anti-hyperglycemic effect that requires insulin in the
organism to exert its action. However, the experimental diabetes induced by streptozo-
tocin administration is characterized by an important deficit in the insulin levels,
impeding the effect of pioglitazone.
Body weight was not affected by the treatments, however, the higher food and water
intakes in diabetic mice were significantly diminished at the end of the treatment by the
AP-Fraction. Pioglitazone administration did not alter these parameters. The weight-loss in
diabetic animals without treatment (diabetic control group) is absent. The body weight can
be maintained in this group because they tended to intake more food, which could ame-
liorate the weight-loss. Although the differences in body weight did not show statistical
significance, it is unquestionable that with the course of time the weight-loss would
be evident. In addition, all animals that received STZ showed hyperglycemia (between
(A) (B)
(C) (D)
Figure 4. Effect of daily administrations of C. ficifolia and pioglitazone on inflammatory cytokines in strepto-
zotocin-induced diabetic mice (33 d). (A) TNF-, (B) IL-6, (C) INF- (D), IL-10 Mean S.E.M. (n ¼ 6).
*Statistically significant compared to the diabetic control; &statistically significant compared to the normal mice
(p < 0:05). Normal: normoglycemic mice, STZ: hyperglycemic mice by streptozotocin administration, STZ þ C.
ficifolia: hyperglycemic mice treated with AP-Fraction of C. ficifolia, STZ þ Piogli: hyperglycemic mice treated
with pioglitazone.
350 and 550 mg/dl with free access to food, and between 250 and 350 mg/dl in 12 h fasted
mice) seven days after of STZ administration.
Administration of the AP-fraction caused a significant reduction in triglycerides without
altering total cholesterol and liver transaminases were unchanged. On the other hand,
pioglitazone showed increased liver transaminases, although the increase was only
significant for GOT.
In general, decreased GSH levels and increased MDA levels were observed in diabetic
animals compared to the normal mice. The GSH levels were significantly increased in the
liver and heart (p < 0:05) in response to treatment with both the AP-Fraction and pio-
glitazone, while the levels in the kidneys and pancreas were unchanged. In addition, MDA
levels were clearly reduced in the liver and heart by both the AP-Fraction and pioglitazone,
without significant alterations in the kidneys and pancreas. Xia and Wang (2007) also
reported a reduction in the MDA content, which is also consistent with our results. MDA is
considered a marker of damage due to increased lipo-peroxidation, which is increased in
diabetes (Wen et al., 2004). Therefore, reduced MDA levels are indicative of cell
protection against damage from free radicals abnormally increased in hyperglycemia. The
GSH is present in most mammalian cells and plays important roles in various biological
processes. It is involved in the cellular defense system against oxidative stress by reducing
the disulfide linkage of proteins and other cellular molecules or by scavenging free radicals
and reactive oxygen intermediates. Decreased concentrations of GSH along with increased
levels of glutathione disulfide (GSSG) have been observed in patients with DM2 and
diabetic experimental animals. These conditions lead to oxidative stress that may poten-
tially be the cause of cellular damage and the development of vascular complications
(Yoshida et al., 1995). The AP-Fraction caused a significant increase in the GSH content of
the heart and liver as well as a clear reduction in the concentration of MDA in these organs.
However, to confirm the antioxidant effect of AP-Fraction, we need to determine whether
the AP-Fraction decreases the redox ratio (GSH/GSSG), which is inversely related to
oxidative stress (Serru et al., 2001), as well as to evaluate the expression and activity of the
associated enzymes, such as glutathione oxidase and peroxidase. The GSH/GSSG ratio is
often used as a sensitive index of oxidative stress in vivo, and in the future studies of the
AP-Fraction, these measurements will be considered.
Although the observed antioxidant effect of the AP-Fraction may be a direct con-
sequence of the reduction in hyperglycemia and concomitant amelioration of glycotoxicity,
we cannot yet discard the hypothesis that some compounds in the AP-Fraction may be
directly responsible for the antioxidant activity. It has been reported that this plant contains
soluble carbohydrates, including D-chiro-inositol, myo-inositol, fagopyritols (Xia and
Wang, 2006), ascorbic acid and thiamine (Andrade-Cetto and Heinrich, 2005), as well as
other substances with potential antioxidative properties, such as cucurbitacin and poly-
phenol compounds. In addition, some cucurbitacins have also been found to have anti-
inflammatory effects (Chen et al., 2005).
The D-chiro-inositol has been reported to be responsible for the hypoglycemic activity
in C. ficifolia by augmenting insulin secretion (Xia and Wang, 2006, 2007). In our study,
the content of D-chiro-inositol was quantified in the AP-fraction. However, other
uncharacterized compounds in this fraction may be responsible for the antioxidant and/or
anti-inflammatory properties.
On the other hand, insulin resistance has been associated with obesity, DM2, hyper-
tension, and heart disease (Berg and Scherer, 2005; Haffner, 2006). A link has been
proposed between insulin resistance and these chronic afflictions. This inflammatory
hypothesis argues that the elevated production of pro-inflammatory cytokines (TNF-,
IL-6, resistin, etc.) and the decreased of anti-inflammatory cytokines (IL-10, adiponectin,
etc.) are partially responsible for these dysfunctions and their complications (Taubes,
2009). Thus, TNF-, a pro-inflammatory cytokine, is directly associated with the devel-
opment of insulin-resistance and diabetes mellitus. TNF- induces phosphorylation of the
insulin receptor (IR) and the insulin receptor substrate (IRS), causing changes in the
binding of insulin to its receptor and altering signaling, which promotes the development of
insulin resistance (Rui et al., 2001).
Our results show that the serum levels of IL-6, IFN- and IL-10 in diabetic mice were
similar to those in normal mice. Treatment with the AP-Fraction and pioglitazone produced
highly significant reductions in TNF- levels. Nevertheless, the increased IL-6 and IFN-
levels following AP-Fraction administration were also observed, while pioglitazone did not
significantly alter these cytokines. Interestingly, IL-10 was notably increased by both the
AP-Fraction and pioglitazone. Thus, the alterations in TNF- and IL-10 by the AP-Fraction
may be interpreted as a single anti-inflammatory effect in streptozotocin-induced diabetes.
However, the AP-Fraction also increased IL-6 serum level, a pro-inflammatory cytokine that
is normally elevated in diabetic patients (Wen et al., 2004), although this cytokine was not
different in streptozotocin-induced diabetic mice compared to the normal mice. New
investigations have shown polymorphisms in IL-6 that may be associated with a decreased
risk for DM2 that is not associated with the body mass index (Huth et al., 2009). Fur-
thermore, IL-6 also has been mentioned as a predictor of toxicity (Lacour et al., 2005).
Therefore, this result could also be indicative of toxicity induced by the AP-Fraction.
Previous studies of acute toxicity in mice treated with a C. ficifolia fruit extract showed an
intraperitoneal LD50 of 625 mg/kg, and a LD50 of 3689 mg/kg when administered orally.
These lethal effects correlated with certain characteristic toxic effects, including nociceptive
response, hypothermia, increased blood viscosity, increased coagulation time, and death
(Hernandez-Galicia et al., 2002).
Finally, although IFN- has been reported to be elevated in diabetic patients and is
normally associated with infectious processes, the streptozotocin-induced diabetic mice did
not differ from normal mice with respect to this cytokine, while the treatments caused a
significant increase. In fact, some antioxidant flavonoids have been associated with an
increased production of IFN- in vitro (Chang et al., 2007).
In conclusion, C. ficifolia represents an alternative compound for the control of diabetes
mellitus that also has antioxidant and anti-inflammatory properties. Because both processes
are associated with the development of vascular complications, we suggest that future
studies evaluate the effects of the AP-Fraction on these complications.
Acknowledgments
This research was partially supported by the International Foundation for Science,
Stockholm, Sweden, and the Organization for the Prohibition of Chemical Weapons, The
Hague, The Netherlands, through a grant to Francisco Javier Alarcon-Aguilar PhD.
Research Grant Agreement No. F/3338-2F, the Multidisciplinary Project 8110119 of the
Metropolitan Autonomous University, and PROMEP-SEP (P/CA-15-2006-35-53). The
authors would like to acknowledge Drs. Rocio Ortiz, Edith Cortes, Concepcion Gutierrez,
Elizabeth Hernandez, and Leticia Bucio from the Metropolitan Autonomous University for
their assistance in the realization of this research.
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Antioxidant and Anti-Inflammatory

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Antioxidant and Anti-Inflammatory

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The American Journal of Chinese Medicine, Vol. 40, No.

Antioxidant and Anti-Inﬂammatory

R. Roman-Ramos,* J. C. Almanza-Perez,* A. Fortis-Barrera,†

Abstract: Type 2 diabetes is characterized by oxidative stress and a chronic low-grade

Keywords: Cucurbita ﬁcifolia; Cucurbitaceae; Diabetes Mellitus; Hypoglycemic Plants;

Antioxidant Plants; Anti-Inﬂammatory Plants; Inﬂammatory Cytokines.

metabolism and triggering a low-grade inﬂammatory reaction (Dominiczak, 2003). Thus,

fruit of C. ﬁcifolia on streptozotocin-induced diabetes in mice by measuring GSH and

Materials and Methods

Preparation of the AP-Fraction from C. ﬁcifolia Fruit

Quantitative Analysis for D-Chiro-Inositol in AP-Fraction

Experimental Design for Evaluating the Antioxidant

Norm (NOM-062-ZOO-1999, revised 2001). The Institutional Committee of the Metro-

Biochemical Parameters and Quantiﬁcation of Cytokines

Determination of GSH and MDA

Effect of the AP-Fraction on Glycemia and HbA1c

Effect of the AP-Fraction on Body Weight, Food and Water Intake

Effect of the AP-Fraction on GSH and MDA

Effect of the AP-Fraction on Biomarkers of Inﬂammation

Normal STZ C. ﬁcifolia Pioglitazone

HbA1c (%) 3.2 0.16 6.72 &

must be conﬁrmed by additional experiments without administration of the fraction in

Acosta-Patiño, J.L., E. Jimenez-Balderas, M.A. Juarez-Oropeza and J.C. Diaz-Zagoya. Hypogly-

Alarcon-Aguilar, F.J., E. Hernandez-Galicia, A.E. Campos-Sepulveda, S. Xolalpa-Molina,

Ethnopharmacol. 112: 232–236, 2007.

Chem. 47: 1321–1324, 2001.

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