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Experimental Biology and Medicine 229:33-39 (2004)
© 2004 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Ingestion of Proanthocyanidins Derived from Cacao Inhibits Diabetes-Induced Cataract Formation in Rats

Naomi Osakabe*,1, Megumi Yamagishi*, Midori Natsume*, Akiko Yasuda* and Toshihiko Osawa{dagger}

* Health and Bioscience Laboratory, Meiji Seika Kaisha Ltd., 5-3-1, Chiyoda Sakado 350-0289, Japan; and {dagger} Laboratories of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Chikusa, Nagoya 464-8601, Japan

1To whom requests for reprints should be addressed at Health and Bioscience Laboratory, Meiji Seika Kaisha Ltd., 5-3-1, Chiyoda Sakado 350-0289, Japan. E-mail: naomi_osakabe{at}meijico.jp


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Experimental Procedures
 Results
 Discussion
 References
 
Proanthocyanidins derived from cacao (CLP) have various antipathophysiological functions. We have tested whether dietary supplementation with CLP prevents cataract formation in rats with diabetes induced by streptozotocin (STZ), using histological, histochemical, and biochemical analyses. Starting at 7 days after the streptozotocin challenge, the animals were fed either a normal diet or a diet containing 0.5% w/w CLP over 10 weeks. There were no significant differences in plasma and urine glucose concentrations, plasma fructose amines, and plasma thiobarbituric reactive substances (TBARS) between the two dietary groups. Antioxidant status as assessed by measuring lipid peroxide production in plasma in response to azocompounds was lower in the STZ-rats fed control diet than in animals fed CLP. Opacity was first detected in the lenses of the control dietary group 5 weeks after STZ injection and cataracts had developed in the majority of these animals by 10 weeks. These changes were rarely seen in the STZ/CLP diet group. Histological examinations of the eyes of the STZ-treated normal diet group revealed focal hyperplasia of the lens epithelium and liquefaction of cortical fibers. There were similar but considerably less severe changes in the animals fed CLP. Hydroxynonenal (HNE), a marker of oxidative stress, was detected immunohistochemically in the lenses of the STZ-treated normal diet group, but not of those receiving CLP. Our findings suggest that CLP inhibits diabetes-induced cataract formation possibly by virtue of its antioxidative activity.

Key Words: proanthocyanidin • cacao • diabetes-induced cataract • HNE • oxidative stress


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Experimental Procedures
 Results
 Discussion
 References
 
Oxygen radicals play an important role in the origin of a wide range of diseases (1–5). Diabetes mellitus in both experimental animal models and humans is associated with marked reductions in the levels of plasma antioxidants such as {alpha}-tocopherol, ascorbic acid, uric acid, and glutathione causing lowered plasma antioxidative capacity. Various biochemical imbalances contribute to the development of diabetic micro- and macrovascular complications, including atherosclerosis. Among these complications, hyperglycemia-induced cataract formation is thought to be caused by the accumulation of sorbitol (6, 7). Recently, many studies have suggested that oxidative stress also has a crucial role in diabetes-induced cataract formation (8–10). This has led researchers to investigate whether the vascular complications of diabetes, including cataract formation, can be reduced by dietary supplementation with antioxidants (11–13).

Cacao liquor (CLP), an ingredient of chocolate and cocoa, is rich in polyphenols (14–16), including catechins and their oligomers linked by C4, C8 bonds as B-type proanthocyanidins (17). We have reported previously that these polyphenols have potent in vitro antioxidative activity (18, 19). The procyanidin fraction of CLP reduced oxidative stress in vitamin E-deficient rats (20) and hypercholesterolemic rabbits (21). In addition, a clinical trial in healthy human volunteers found that daily intake of cocoa powder rich in proanthocyanidins reduced the susceptibility of low-density lipoproteins to oxidation (22).

Here we have evaluated the effect of CLP on cataract formation, one of the major complications of both Type I and II diabetes, using the streptozotocin-induced diabetic rat as a model of human Type I diabetes.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Experimental Procedures
 Results
 Discussion
 References
 
Materials.
Streptozotocin, (-)-epicatechin, (+)-catechin, theobromin, caffeine, and fructoseamine calibrator were purchased from Sigma (St. Louis, MO). Lipid peroxide analysis kits (Determiner LPO) were obtained from Kyowa Medics Ltd. (Japan) and antihydroxynonenal antibody from Calbiochem (San Diego, CA).

The crude polyphenol fraction of CLP was prepared by the method we reported previously (18). Briefly, fat was removed from the liquor with n-hexane, followed by extraction with 80% (v/v) ethanol. The extracts were passed through a chromatographic column (Diaion HP 2MG; Mitsubishi, Kasei, Japan) and contaminants, including caffeine and theobromine, were removed by washing the column with 20% (v/v) ethanol. A crude polyphenol fraction was eluted with 80% ethanol, concentrated, and freeze dried. The total polyphenol content of the preparation was measured using the Prussian blue method with epicatechin as standard (23). Catechins, procyanidins, and xanthine derivatives were analyzed by high-performance liquid chromatography (HPLC) as described previously (24). The analysis of CLP is shown in Table 1Go.


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  		Table 1. Composition of Polyphenol Fraction Derived from Cacao Liquor (CLP)
 
Animals.
Eight-week-old male Sprague-Dawley rats from Clea, Japan, were housed in a controlled environment at 23°C and 55% humidity under a 12:12-hr light:dark cycle. The Animal Committee of Meiji Seika Kaisha Ltd. Health and Bioscience Laboratory approved the study, and the animals were cared for in conformity with established guidelines.


   Experimental Procedures
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 Abstract
 Introduction
 Material and Methods
 Experimental Procedures
Results
 Discussion
 References
 
The animals were fed the AIN-76 diet for 4 days. Food was withdrawn 16 hrs before treatment. Rats received a single intraperitioneal injection of streptozotocin (STZ; 40 mg/kg) dissolved in 0.1 M citrate buffer. An equal number of age-matched nondiabetic animals was selected as untreated controls. Seven days after the STZ challenge, blood and urine glucose levels were determined and animals with elevated levels divided randomly into two groups. The first group was fed AIN-76 diet for 10 weeks (STZ/normal diet group) while the second group received AIN-76 diet containing 0.5% CLP for this period (STZ/CLP diet group). Blood was collected from the tail vein of all the animals and 24-hr urines collected using a metabolic cage each week. At the end of the study, the animals were anesthetized with diethyl ether, blood was collected from the abdominal vein with heparinized syringes, and the eyes removed for examination. Tissues and plasma samples were kept at -80°C.

Analyses.
Plasma and urine glucose concentrations were determined with a glucose assay kit (Wako Pure Chemical Industries, Tokyo, Japan). Thiobarbituric acid reactive substances (TBARS) in plasma were measured by the method of Ohkawa et al. (25) with tetraethoxypropane as standard. Fructoseamine in plasma was measured by the method of Johnson et al. (26), and the antioxidative status of the plasma was assessed by the method of Lotito and Fraga (27). Briefly, a reaction mixture containing plasma and 25 mM 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH) was incubated at 37°C for 60 mins and the lipid peroxide concentration in the oxidized plasma determined with a commercial kit (Determiner LPO; Kyowa Medics Ltd., Tokyo, Japan).

Histological Analyses.
The eyes were fixed in 3% buffered formalin and embedded in paraffin. Sections 5 lm thick were prepared and fixed to slides, deparaffinized, and stained with hematoxylin and eosin (HE) to permit histological examination of cortical fibers and lenses. The production of hydroxynonenal (HNE) was detected immunohistochemically with anti-HNE polyclonal antibody. Briefly, deparaffinized slides were placed in a blocking reagent containing H2O2 for 45 secs in order to quench endogenous peroxidase. The sections were then blocked with 10% nonimmune goat serum for 1 hr followed by incubation for 18 hrs at 4°C in a moist chamber with a 1:400 dilution of anti-HNE. This was followed by addition of a biotinylated secondary antibody and streptavidin-peroxidase conjugate. The slides were then incubated with 3,3-diaminobenzidine tetrahydrochloride to detect HNE production and counter-stained with hematoxylin.

The histological appearance of the lenses was evaluated in 20 random areas (0.0625 mm2/area) at x400 magnification. The slides were graded blind without knowledge of the experimental design. Cortical fiber liquefaction was graded as follows: no observable changes, slight changes, moderate changes, and severe changes. Focal hyperplasia of lenses was graded as follows: no observable changes; mild, faint changes; moderate changes; and obvious changes.

Statistical Analysis.
The analyses were carried out with SPSS statistical software and the data expressed as means ± standard deviations. When analysis of variance (ANOVA) revealed significant changes of P < 0.05, the data were further analyzed by Tukey’s multiple range test. Results of the histological examination were analyzed by the chi-square test.


   Results
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 Abstract
 Introduction
 Material and Methods
 Experimental Procedures
 Results
Discussion
 References
 
Mean body weight at 5 and 10 weeks in the diabetic rats was significantly lower than in animals not injected with STZ (Table 2Go). There was no difference in body weight between diabetic rats fed diets with and without 0.5% CLP. The concentrations of plasma and urinary glucose were markedly elevated in the STZ-treated animals regardless of the presence or absence of CLP in the diet. Likewise, fructosamine concentration, an index of plasma glucose levels during the preceding 2 weeks, increased to the same extent in both the STZ treatment groups.


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  		Table 2. Cacao Proanthocyanidins Did Not Effect Body Weight, Plasma and Urine Glucose, and Plasma Fructose Amine Levels in STZ Rata
 
There were no significant differences in plasma TBARS levels at 5 and 10 weeks after administration of STZ (Table 3Go). However, AAPH-induced plasma lipid peroxide levels increased substantially in the STZ/normal diet group, and this increase was significantly inhibited at 10 weeks by CLP supplementation.


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  		Table 3. Cacao Proanthocyanidins Decrease AAPH-Induced Production of Lipid Peroxides but Not TBARS in Plasma of STZ Rata
 
A representative photo of eyes taken from the experimental animals is shown in Figure 1aGo. In contrast with the lenses of untreated control rats, there is advanced lens opacity in the diabetic rats not receiving CLP. Histological sections of an eye that developed cataract and stained with HE are shown in Figure 1bGo. No changes were apparent in the lens fiber and epithelial cells in the posterior region in the nondiabetic animals. In the STZ-treated normal diet group, liquefaction of cortical fibers and focal hyperplasia of the lens epithelium were evident, whereas the eyes of the CLP diet group showed only slight swelling and granular degeneration of fibers. Table 4Go summarizes the histopathological findings. Moderate to severe changes characterized by liquefaction were present in the lens fibers of more than half the animals in the STZ/normal diet group. Hyperplasia of the lens epithelium was also frequently observed in these animals, and CLP supplementation substantially reduced its prevalence.



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  		Figure 1. (a) Photograph of cataract formation. (b) Histological changes associated with cataract formation stained with hematoxylin and eosin. (c) The production of HNE in a rat lens detected by immunohistochemical staining.

 

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  		Table 4. Cacao Proanthocyanidins Decrease Cortical Fiber Liquefaction and Focal Hyperplasia in STZ Rat Lensa
 
Production of HNE in the lens, detected immunohistochemically, is illustrated in Figure 1cGo. Light-brown staining indicative of the presence of HNE was not detected in the lens of nondiabetic rats. The HNE adducts were widely distributed in the lens fibers of the STZ/normal diet group. In contrast, only slight staining was apparent in any of the eyes of the CLP-treated rats.


   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Experimental Procedures
 Results
 Discussion
References
 
In this study, rats with diabetes induced by treatment with STZ had continuously elevated levels of plasma glucose and glycosuria as described in many previous investigations. While dietary supplementation with CLP did not affect hyperglycemia or plasma TBAR concentration, AAPH-induced production of LPO was decreased. Moreover, the formation of cataracts was observed in the STZ/ normal diet animals 5 weeks after STZ treatment but not in CLP-treated animals until the eighth week (data not shown). These gross changes were confirmed histochemically by the finding that HNE, a marker of oxidative stress, was only detected when the rats were fed a normal diet. These observations suggest that the CLP extract may prevent cataract formation by decreasing oxidative stress induced by hyperglycemia.

Proanthocyanidins in cacao include monomeric (+)-catechin and (-)-epicatechin, dimeric procyanidin B2, trimeric procyanidin C1, and tetrameric cinnamtannin A2. This mixture has been shown to have potent antioxidative activity (28, 29). In vitro studies have demonstrated that each of these compounds scavenges superoxides and hydroxy radicals (30) and inhibits the formation of nitrotyrosine (31, 32). A recent report also indicates that catechins are more efficiently absorbed from the gastrointestinal tract than other flavonoids (33). We found that (-)-epicatechin and its metabolites reached their highest levels in plasma 1 or 2 hrs after feeding healthy volunteers cocoa or chocolate (34, 35). Recovery of (-)-epicatechin-related compounds in the urine was 26%–28%. We confirmed similar bioavailability of the (-)-epicatechin in cocoa in rats and found a correlation between antioxidative status and (-)-epicatechin concentration in plasma (36). However, in the present study, the procyanidin B2, (-)-epicatechin dimer was poorly absorbed, with only 0.5% of the procyanidin B2-related substances that were administered being recovered in the urine (37). While the bioavailability of proanthocyanidins remains debatable, many studies in both experimental animals and humans have shown that ingestion of these compounds in cocoa, wine, and apples affects physiological activities in vivo, such as preventing the development of atherosclerosis and some types of cancer (38–40). Although monomeric proanthocyanidins such as (-)-epicatechin and (+)-catechin have relatively high antioxidative activity compared with other compounds in CLP, further investigation is needed to identify the components that contribute to these physiological changes.

Hyperglycemia increases the production of reactive oxygen species (ROS) leading to severe oxidative stress (41, 42). Superoxide radicals are produced by glucose auto-oxidation in association with the formation of glycates of plasma and cell surface proteins; they also arise as a consequence of decreased cellular levels of glutathione (43–46). The imbalance in cellular redox potential may play a role in the development of diabetic complications. Cataract results not only from osmotic stress but also from oxidative stress caused by reduced cellular levels of glutathione following activation of the sorbitol pathway (47–49). Our observations with CLP concur with other reports that dietary supplementation with antioxidants such as vitamin C (50), carotenoids (51), and {alpha}-lipoic acid (52) decreases cataract formation. On the other hand, increased ROS production in mitochondrial complex II in response to elevated glucose may also reduce diabetic complications (53). This suggests that the distribution of antioxidants within the cell organelle is likely to be important.

It has been reported recently that the production of ROS and aldehyde end products such as HNE in diabetes may lead to diacylglycerol production and activation of protein kinase C and transcription factor, nuclear factor kappa B (53). These biochemical changes may contribute to the development of chronic diabetic complications. Because proanthocyanidins including catechins inhibit PKC activity (54, 55), it is possible that the preventative effects of CLP on cataract formation are the consequence of the combined action of antioxidative activity and modulation of cellular signal transduction.

The dosage of CLP used in this study is equivalent to the content of polyphenols in 0.75 kg of chocolate. Although supplementation with chocolate would seem paradoxical in diabetic patients, we have shown that a component of chocolate has value in preventing diabetic complications. The possibility of developing a low-carbohydrate chocolate food that does not cause hyperglycemia should be considered for diabetic patients. In conclusion, this study supports the possibility that proanthocyanidins in CLP reduce oxidative stress and prevent cataract formation in the STZ rat.

Received for publication January 21, 2003. Accepted for publication July 19, 2003.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Experimental Procedures
 Results
 Discussion
 References
 

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