Cataract is the first cause of world blindness and one of the major causes of disability due to vision impairment [1]. The only available treatment is the surgical removal of the opaque lens and its replacement with an artificial one. Nevertheless, surgical treatment is not widely available and, thus, interventions which will maintain the transparency of the crystalline lens are intensively sought after. Individuals above 50 years of age are reported to have increased risk of developing most types of cataract. Hence, compounds that prevent or retard cataract formation would be of great benefit to human health. Ostadalova et al. [2], demonstrated that an overdose of sodium selenite (Na2SeO3) to suckling rat pups induces cataractogenesis, partially mimicking senile nuclear cataract in humans. Selenite exerts its effect on lens by inducing oxidative stress and damage.

Oxidative damage to the lens has been linked with development of cataract and decrease in antioxidant enzyme activities in the cataractous lens points to the importance of antioxidant enzymes in the prevention of oxidative damage to the lens and subsequent development of cataract [3]. A wide range of drugs like aldose reductase inhibitors, non-steroidal anti-inflammatory drugs (NSAIDs) are being tried for their anticataract activity [4]. There has been a growing interest in the various activities of indigenous plants. Many indigenous plants have been explored as potential promising sources of antioxidants [5,6].

Bitter kola (Garcinia kola) belongs to the family of plants called Guttiferae and the genus Garcinia. The seed, commonly known, as ‘bitter kola’ is eaten by many and it is culturally acceptable in Nigeria [7]. Extracts of the plant are employed in African herbal medicine for the treatment of ailments such as laryngitis, liver diseases, cough and hoarseness of voice. Kolaviron (KV) is a fraction of the defatted ethanol extract of Garcinia kola, containing Garcinia biflavonoids GB1, GB2 and kolaflavanone [7]. A number of studies have confirmed the antioxidative and anti-inflammatory effects of kolaviron in chemically-induced toxicity, animal models of diseases and chemoprevention of colon carcinogenesis [8-10]. Although the chemopreventive effect of kolaviron has been reported in aflatoxin B1-induced genotoxicity and hepatic oxidative damage [11], no study has addressed the effect of Kolaviron against selenite induced cataract in wistar rats. The present study was done to specifically test the local antioxidant and anticataract effects of extracts of garcinia kola (kolaviron).

Chemicals and Solvents 

Methanol n-hexane and chloroform were purchased from HiMedia Laboratories Pvt. Ltd., (Mumbai, India). Absolute alcohol was obtained from Hayman Ltd., Witham, England; sodium selenite (99%) and Gallic acid were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents used in the experiments were of analytical grade and were obtained from HiMedia Laboratories Pvt. Ltd. (Mumbai, India).

Extraction of Kolaviron

Garcinia kola seeds purchased from a local market in Yenagoa, Nigeria, were certified at the Department of Botany, Niger Delta University, Nigeria. Peeled seeds were sliced, pulverized with an electric blender and dried at 40 0C in a drying oven. Powdered seeds were extracted with light n-hexane in a soxhlet for 24 h. The defatted dried marc was repacked and extracted with methanol. The extract was concentrated with chloroform. The concentrated chloroform yielded kolaviron as a golden yellow solid [12].

In Vitro Lens Crystallin Turbidity Assay

Porcine lenses 160 randomly distributed into control containing no selenite 40 lenses. Toxic control containing selenite, kolaviron and selenite group and finally Gallic acid and selenite group were obtained from a local slaughterhouse and homogenized in 50 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl, 5 mM EDTA, 0.01% β-mercaptoethanol and 0.02% sodium azide. After centrifugation at 15,000 g for 30 min, the supernatant were mixed with Na2SeO3 10 mM, final concentration to induce turbidity. The kolaviron or Gallic acid (50 µg/mL) were added in lens homogenate. Samples were incubated at 37 °C for 24 hours. The plates were carefully closed with their plastic tops and covered with parafilm and aluminum foil prior to the incubation. New covers were used after each measurement. Sodium azide was used in buffer in order to prevent microbial or fungal contamination of the samples. At the end of 24 hours incubation biochemical analysis were carried out.

Determination of Malondialdehyde (MDA)

The assay for lens homogenate lipid peroxidation was done by the method of Wright et al., with some modifications. The reaction mixture in a total volume of 3.0 mL contained 1.0 mL lens homogenate, 1.0 mL of TCA (10%) and 1.0 mL TBA (0.67%). All the test tubes were placed in a boiling water bath for a period of 45 min. The tubes were shifted to ice bath and then centrifuged at 3000xg for 10 min. The amount of malondialdehyde (MDA) formed in each of the tubes were assessed by measuring the optical density of the supernatant at 532 nm. The results were expressed as the nmol MDA formed/ml by using a molar extinction coefficient of 1.56 x105 M-1 cm-1.

Determination of Reduced Glutathione (GSH)

The GSH content in lens homogenate was determined by the method of Jollow et al. [13], in which 1.0 mL of homogenate was mixed with 1.0 ml of sulphosalicylic acid (4%). The samples were incubated at 4 0C for at least 1 h and then subjected to centrifugation at 1200 x g for 15 min at 4 0C. The assay mixture contained 0.4 mL filtered aliquot, 2.2 mL phosphate buffer (0.1 M, pH 7.4) and 0.4 mL DTNB (10 mM) in a total volume of 3.0 mL. The yellow color developed was read immediately at 412 nm on spectrophotometer. The GSH content was calculated as μmol of DTNB conjugate formed/mL using molar extinction coefficient of 13.6 x 103 M-1 cm-1.

Superoxide Dismutase (SOD) activity

The SOD activity was measured by the method of Marklund and Marklund [14]. The reaction mixture consisted of 2.875 ml Tris–HCl buffer (50 mM, pH 8.5), pyrogallol (24 mM in 10 mM HCl) and 100 uL of lens homogenate in a total volume of 3 ml. The enzyme activity was measured at 420 nm and was expressed as units’/mg protein. One unit of enzyme is defined as the enzyme activity that inhibits auto-oxidation of pyrogallol by 50%.

Catalase Activity

Catalase (CAT, EC 1.11.1.6) activity was assayed according to the method of Cohen et al. One milliliter of 50mM phosphate buffer (pH 7.4) and 10 uL of lens homogenate was added to the cuvette. The reaction was then initiated by the addition of 300 uL of 30mM H2O2 prepared by diluting 0.34mL of 30% H2O2 to 100mL of 50mM phosphate buffer (pH 7.4). Specific catalase activities were determined following the changes in the absorbance of H2O2 at 240nm (ε = 0.0394 mM−1 cm−1 at 240 nm).

Glutathione-S-transferase

Glutathione-S-transferase (GST, EC 2.5.1.18) activity was assayed according to the method of Habig et al. [15]. The final reaction mixture contained 1mM CDNB, 1mM GSH in 50 mM phosphate buffer pH 7.4 and the reaction was initiated by the addition of 50 uL lens homogenate. Specific GST activities were determined following the changes in the absorbance of CDNB per min at 340nm (ε = 9.6.00 mM−1 cm−1 at 340 nm).

GSH (nmol DTNB formed/ml), MDA (nmol MDA formed/ml), GST (nmol CDNB formed/ml), CAT (Units/ml), SOD (Units/ml)

• Malondialdehyde: Lipid peroxidation measured in terms of malondialdehyde (MDA) levels showed an increase in MDA levels in Group 2 (“Toxic control”) as compared to Group 1 (normal control lenses). The increase in MDA was statistically highly significant (p<0.05)

• Glutathione: Glutathione measured in terms of DTNB/mL levels showed a decrease in GSH levels in Group 2 (“Toxic control”) as compared to Group 1 (normal control lenses). The increase in GSH was statistically highly significant (p<0.05)

• Superoxide Dismutase: Group 2 (selenite induced cataract lenses) showed a decrease in the specific activity of enzyme superoxide dismutase by as compared to Group 1(Normal control lenses) (Table 1). The decrease was statistically significant (p<0.05). There was a significant increase in kolaviron and gallic acid groups

• Catalase: Catalase specific activity was reduced in Group 2 (selenite induced cataract lenses) as compared to Group 1 (Normal control lenses). The decrease was statistically significant (p<0.05). There was a significant increase in kolaviron and gallic acid groups

• Glutathione-s-Transferase: Glutathione-s-transferase specific activity was reduced in Group 2 (selenite induced cataract lenses) as compared to Group 1 (Normal control lenses). The decrease was statistically significant (p<0.05). There was a significant increase in kolaviron and gallic acid groups

Table 1: Glutathione and Malondialdehyde Levels, Glutathione-S-Transferase, Catalase and Superoxide Dismutase Activities in Lens Homogenate Exposed to Selenite

Each reading represents mean±SD of 10 lenses. * Significantly different from the control value, p<0.05

Bitter kola extract (kolavkron) has been mentioned in antioxidant literature for its antidiabetic properties. However, there was a need to assess the in vitro effect of this plant extract on the oxidative stress related biochemical changes happening in cataract. Selenite induced porcine cataractogenesis has been used as an experimental model in vitro. 

Increased lipid peroxidation has been strongly implicated in the mechanism of cataractogenesis [16]. Malondialdehyde, a secondary product of lipid peroxidation, is used as an indicator of tissue damage [16]. In the present investigation, disruption of lenticular membrane lipids possibly accounted for the observed higher mean malondialdehyde level in selenite-challenged, untreated (Group II) lenses than that in control (normal) lenses (Table 1). In selenite-challenged, simultaneously kolaviron-treated lenses, the mean malondialdehyde level was significantly lower than that in Group II lenses (Table 1), suggesting that the extract prevented peroxidative changes in selenite-challenged lenses, thereby preventing lenticular opacification. Collectively, these results in the present study suggest that administration of kolaviron can effectively prevent selenite-induced cataract formation.

An unusually high level of GSH in the lens is believed to maintain protein-thiol groups in the reduced state and to prevent cross-linking of soluble crystallins [17]. In the selenite cataract model, lenticular GSH is altered by a non-enzymatic reaction of GSH with selenite, which results in the formation of the selenium derivative, GS–Se–SG. Oxidation of GS–Se–SG by a single electron transfer to oxygen results in the formation of a superoxide anion as an intermediate. Administration of GSH or maintenance of lenticular GSH levels may retard age-related loss of lenticular antioxidant activity, therein delaying the onset of cataract [18]. In the present study, simultaneous treatment of selenite-challenged lenses with kolaviron resulted in a mean lenticular GSH level that was significantly higher than that in selenite-challenged, untreated lenses (Table 1). Treatment with the bitter kola extract possibly prevented the formation of the selenium derivative, GS–Se–SG, therein maintaining GSH in its active form at a stable level, ultimately preserving lenticular transparency.

Antioxidant enzymes are able to catalytically remove free radicals and other reactive species. Catalase, superoxide dismutase and Glutathione-S-transferase are important components of the innate enzymatic antioxidant defenses of the lens. In the present investigation, the mean activities of catalase, superoxide dismutase and Glutathione-S-transferase were significantly (p<0.05) lower in selenite-challenged, untreated lenses than the mean activities in normal control lenses (Table 1), such lowered activities of these enzymes in selenite-induced cataractogenesis has been previously documented in in-vitro and in-vivo experimental models [19]. However, selenite-challenged, Kolaviron treated lenses did not exhibit such lowered activities of these enzymatic antioxidants (Table 1). These observations suggest that when lenses exposed to sodium selenite are treated with an antioxidant-rich compound, antioxidant enzyme activities are maintained at near-normal levels; similar findings have been previously reported [19].

The results of the present study suggest that kolaviron possesses antioxidant potential to prevent selenite-induced cataractogenesis by maintaining a normal antioxidant status. Thus, kolaviron may be considered for pharmacological therapy to prevent or retard cataractogenesis.

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