|Year : 2017 | Volume
| Issue : 1 | Page : 26-33
The protective effect of cloves extract against selenite-induced cataract in rat pups
Tahany El-Sayed Kholeif1, Hager Imam Mohamed2, Enas Ali Kamel1, Doaa Eid Mohamed2
1 Department of Biochemistry and Nutrition, Women's College Ain Shams University, Cairo, Egypt
2 Biochemistry Unit, Research Institute of Ophthalmology, Giza, Egypt
|Date of Web Publication||17-Jul-2017|
Doaa Eid Mohamed
Biochemistry Unit, Research Institute of Ophthalmology, Giza
Source of Support: None, Conflict of Interest: None
Aims: The present study aims to investigate the role of cloves in the prophylaxes or delay cataract development in the selenite-induced cataract in rat pups. Materials and Methods: Forty neonatal rat pups were obtained from the animal house of Research Institute of Ophthalmology, Giza, Egypt. All rats fed on basal commercial diet. Four rat mothers having rat pups (25 ± 4 g) aging 12 ± 1 days were included in this study. Each rat mother and their pups were housed in one cage and served as groups from one to four. Group 1: Normal control; Group 2: Selenite-induced cataract; Group 3: Normal control treated with cloves extract; and Group 4: Selenite-induced cataract treated with cloves extract (100 mg/kg/b.wt.). The rat pups in the experimental Groups 2, 4 received a single subcutaneous injection of sodium selenite (30 μmol/kg b.wt.), while groups (1, 3) were injected with normal saline (0.3 ml). Reduced glutathione (GSH), malondialdehyde (MDA) and nitric oxide (NO) were measured in the blood. In lens, total protein (TP), reduced GSH, MDA, NO, carbonyl protein, hydrogen peroxide, advanced glycation end products, and metalloproteinase were determined. Results: The results of ophthalmic examinations in the group treated with cloves showed that lower maturation of selenite cataract by 70% of lenses while 30% of the lenses displayed Stage 1 cataract in all treated groups. Biochemical estimation showed a signifi cant increase in MDA and NO levels which accompanied by a signifi cant decrease in reduced GSH level in a cataractous group. After rats treatment with cloves, data showed a signifi cant increase in reduced GSH levels and a significant decrease in NO and MDA levels. Conclusion: The data in the lens showed a significant improvement in all parameters after treatment with cloves. Results concluded that cloves inhibited cataract formation, and protected the lens from the development of cataracts.
Keywords: Cataract, cloves extract, lens, rat pups, selenite
|How to cite this article:|
Kholeif TE, Mohamed HI, Kamel EA, Mohamed DE. The protective effect of cloves extract against selenite-induced cataract in rat pups. Saudi J Health Sci 2017;6:26-33
|How to cite this URL:|
Kholeif TE, Mohamed HI, Kamel EA, Mohamed DE. The protective effect of cloves extract against selenite-induced cataract in rat pups. Saudi J Health Sci [serial online] 2017 [cited 2020 Mar 31];6:26-33. Available from: http://www.saudijhealthsci.org/text.asp?2017/6/1/26/210821
| Introduction|| |
A cataract occurs when the lenses of the eye become clouded or opaque, resulting in poor vision or vision loss that contributes to 50% of blindness worldwide. The lens is a closed system with the limited capability to repair or regenerate itself. The data on natural polyphenols in relation to cataract, demonstrate that flavonoids may play a role in cataract prevention.
Several spices may operate in the prevention of cataract since its flavonoids are capable of preventing multiple mechanisms or etiological factors responsible for the development of cataract.
Cloves are an aromatic herb that has many useful purposes. A clove has some medicinal purposes as well, and it tastes good in dishes. Cloves have been used as local anesthetic. The buds were used to treat dyspepsia, acute/chronic gastritis, and diarrhea. Clove oil has been used as a carminative, antispasmodic, antibacterial, and antiparasitic agent,, while in addition, anticonvulsant, anti-candidal, anticarcinogenic, and antimutagenic  have been reported. The active principles in the clove are known to have antioxidant activity.
Approximately, 71.56% of the essential oil extracted from cloves have eugenol and eugenol acetate. Other essential oil ingredients of clove oil are beta-caryophyllene, crategolic acid, tannins, gallotannic acid, methyl salicylate, eugenin, kaempferol, rhamnetin, eugenitin, triterpenoids, stigmasterol and campesterol, and vanillin. These substances are also known for their antioxidant properties.
The cloves contain good amount of minerals such as potassium, manganese, iron, selenium, and magnesium. It also contains very good amounts of Vitamin A, beta carotene, Vitamin K, Vitamin B6, Vitamin B1, riboflavin and Vitamin C levels. These compounds are known to have antioxidant properties.
There is an urgent need for inexpensive, nonsurgical approaches for the treatment of cataract. Recently, considerable attention has been devoted to the search for phytochemical therapeutics. Disease prevention, including lifestyle modification, attention to dietary intake and micronutrients supplementation must become more focused on primary vision care.
The current study aims to evaluate the biological effects of cloves extract, that can protect, decrease or delay selenite-induced nuclear cataract development in rat pups.
| Materials and Methods|| |
Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). An extra pure AnalaR form of sodium selenite (Na2 SeO3) molecular weight 172.9 g, from Medex Laboratory Company, Oxon, England was used for cataract induction.
A commercial diet from the animal house of Research Institute of Ophthalmology was used as basal commercial diet that consists mainly of not >64% carbohydrates, not <21% protein, not <6% fat, not <3% fiber, and not <6% of vitamins and minerals mix, methionine, and choline chloride 
Preparation of cloves extract
Cloves were purchased from the local market. Cloves were extracted by ethanol then lyophilized and dried as possible, protected from direct sunlight, and then used. The extract materials were kept in a closed container at 8°C. Five hundred gram of dried powder of cloves was extracted with 500 ml of ethanol:water (1:1), repeated 2 times, by filtration process at room temperature (37°C). Then soaked for 3 days, after which they were decanted and filtered by filter paper no. 1. The filtrate was condensed in a rotary evaporator and stored in refrigerator then orally administrated by stomach tube at a dose (100 mg/kg b.wt. daily).
Forty Wistar rat pups weighing (25 ± 4 g) 12 ± 1 days old and their mothers were purchased and housed in the animals house of Research Institute of Ophthalmology in a polyethylene cages in a controlled environment (25°C ± 2°C, 50%–60% relative humidity and 12 h light/dark cycle).
Experimental animals model of selenite-induced nuclear cataract
Induction of selenite cataract
Rat pups in group two and four were injected subcutaneously with a single dose of sodium selenite (30 μmol/kg b.wt.) according to the method described by Orhan et al. After weaning rat mothers were taken away from the cages of their pups and doses were taken by injection for all rat pups. The progression of cataract was under observation till the end of the experiment.
Wistar rat pups and their mothers were classified into four groups. Group 1 (control) received subcutaneously with isotonic saline solution (0.9%). Group 2 was injected subcutaneously with 30 μmol/kg body weight with sodium selenite to induce cataract. Group 3 were orally administrated cloves extract (100 mg/kg b.wt. daily) by stomach tube. Group 4 also injected subcutaneously with (30 μmol/kg b.wt.) with sodium selenite and received cloves extract (100 mg/kg b.wt. daily) orally by stomach tube.
Both eyes of rats were clinically examined by slit lamp to ensure the induction of cataract. At the end of the experiment (period of 8 weeks), rats were fasted overnight, and blood is withdrawn from the eyes. Then, the eye samples were enucleated, and lenses were excised, carefully encapsulated and washed in 0.15 M isotonic sodium chloride solution. Eye lens homogenate was prepared and stored at a temperature of −50°C for further analysis.
Slit lamp biomicroscopic examination and lenticular opacification
Both eyes of all rat pups were dilated using 2% tropicamide solution eye drops. The progression of cataract was observed by slit lamp after 5 days. Cataract could be seen obviously by the naked eye in cataractous group. The progression of cataract in all cataract groups was under observation till the end of the experiment. Slit lamp biomicroscope examination (Zeiss, Japan) was carried out at regular intervals, and the stages were designated according to the method described by Kawara and Obazawa. Briefly, lenses were examined on alternate days and opacities observed were graded into five stages. A mature cataract was observed as a dense opacity in both cortex and nucleus. The rat's eyes were examined for detecting symptoms of cataract, all rats lenses were examined by the ophthalmoscope.
At the end of the experiment (8 weeks), all rats were fasted overnight, anesthetized, and blood was collected from eye canthus (retro-orbital blood collection). Blood samples were collected using two separated tubes. The first tube contained ethylenediamine tetraacetic acid and the second tube was used to separate serum by centrifuging at 3500 rpm for 10 min.
Lense homogenate preparation
The eyes were enucleated, the lenses were excised, carefully decapsulated and washed in 0.15 M isotonic sodium chloride solution. All lenses of each group were homogenized in saline solution (0.2 g lens/1 ml water) and centrifuged at 6000 rpm for 30 min. The lenses homogenate of each group were collected and kept frozen until analysis.
In the blood and lens, reduced glutathione (GSH) was assessed according to the method of Beutler et al., malondialdehyde (MDA) was determined according to the method described by Draper and Hadley  and nitric oxide (NO) was assessed according to the method of Moshage et al. Furthermore, in the lens TP was determined according to the method described by Lowry et al., carbonyl protein (CP) was determined according to the method described by Levine et al. and hydrogen peroxide (H2O2) was determined according to the method described by Aebi.
Advanced glycation end products (AGEs) were assayed by enzyme immunoassay developed kits according to the method described by OxiSelect ™ Advanced Glycation End Products competitive ELISA kit (Biosource International, Inc., Camarill kits).
Apoptosis was assessed in lens by ELISA technique to determine metalloproteinase according to the manufacturer's instructions (Biosource International., Camarill kit).
SPSS program (version 9, IBM corporation, USA) for Windows. was used for data analysis. Data were analyzed using one-way ANOVA, mean and standard error were descriptive measures of data. Least significant difference multiple comparison tests were then carried out. P values were significant if <0.05
| Results|| |
Slit lamp examination and degree of cataract
Ophthalmic examination of rat eyes involves identifying the type and severity of cataract. Ophthalmic examinations 5 days after sodium selenite injection indicated the development of cataract in selenite cataract group. Low percentage signs of cataract were observed in the treated groups with cloves as shown in [Table 1] and [Figure 1].
|Figure 1: (a) Normal control group, (b) cataract group, (c) normal treated with cloves group, (d) cataract treated with cloves group|
Click here to view
As shown in [Table 1], the results of slit lamp examination indicated that all the ten rats (20 eyes, 100%) in selenite cataract group developed bilateral Stage 4 cataract, while all lenses in normal control rats were clear. The anticataract effects of cloves after sodium selenite injection were identified. The group treated with cloves manifested lower maturation of selenite cataract by 70% of lenses concerning to cloves treated, while 30% of lenses displayed Stage 1 cataract.
Effect of oral cloves administration on glutathione, serum malondialdehyde, and nitric oxide in different studied groups
[Table 2] shows the mean values ± standard deviation of blood reduced GSH, serum MDA, and NO.
|Table 2: Means±standard deviation, P values and percentage of change for blood reduced glutathione, serum malondialdehyde, and serum nitric oxide in different experimental groups|
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The result showed that there was no significant change in the level of reduced GSH in selenite-induced cataract group treated with cloves as compared to normal control group mean values were 78.31 ± 13.8. While the mean values were 47.32 ± 8.2 and 78.7 ± 13.4 in selenite-induced cataract and selenite-induced cataract treated with cloves group was a highly statistically significant difference of reduced GSH level.
[Table 2] also reveals a highly significant increase in the level of MDA in selenite-induced cataract group, 7.65 ± 0.53 nmol/ml as compared to normal control group, 2.23 ± 0.48 nmol/ml (P < 0.001). There was no significant change in the level of MDA in normal control treated with cloves group. While a highly statistical significant change in the level of MDA in cataract treated with cloves group 4.1 ± 0.32 as compared to normal or cataractous groups.
Also, as shown in [Table 2], the result of serum NO showed a highly significant increase of NO in selenite-induced cataract group 26.3 ± 4.85 μmol/L on comparing to normal control group 4.01 ± 1.63 μmol/l (P < 0.001). On the other hand, there was no significant change in the level of serum NO in selenite-induced cataract treated with cloves, as compared to normal control group.
Effect of oral cloves extracts administration on biochemical parameters assessed in lenses of different experimental groups
[Table 3] reveals the changes of biochemical parameters in the lenses of groups treated with cloves. The data represent the mean values of duplicate or triple samples. As shown in [Table 3], the mean values of TP levels in lens of cataractous group was 0.52 mg/g lens while the mean values of normal control group was 0.71 mg/g lens. Lenses of selenite-induced cataract group demonstrated a gross reduction in lens TP levels with a percentage of changes −26.76% as compared to normal control group.
|Table 3: Total protein, reduced glutathione, malondialdehyde, nitric oxide, carbonyl protein, and hydrogen peroxide levels in rats' lenses of different experimental groups|
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The mean value of reduced GSH in the lens of normal control group was 96.4 mg/g lens. Whereas, the mean values of cataractous group were 43.4 mg/g lens. The data of reduced GSH in the lens of cataractous group showed a highly significant decreased as compared to normal control group. The percentage change of reduced GSH in cataractous group was decreased by −54.98% as compared to normal control group. Concerning to cloves administration, the percentage of GSH was improved in selenite-induced cataract group treated with cloves and the percentage was increased by 58.29% on comparing to untreated selenite-induced cataract group.
On the other hand, there was a significant increase in the levels of MDA in selenite-induced cataract group as compared to normal control group. The mean value of MDA in the lens of cataractous group was 3.50 nmol/mg lens while the mean values of normal control group was 1.91 nmol/mg lens. As shown in [Table 3], the mean values of MDA in selenite-induced cataract rat pups treated with cloves were 2.24 nmol/mg lens. The percentage of MDA was decreased by −36.0% in selenite-induced cataract rat pups treated with cloves on comparing with untreated selenite-induced cataract group.
The mean value of NO in the lens of normal control group was 4.5 μmol/mg lens and in selenite-induced cataract group was 17.2 μmol/mg lens. In selenite-induced cataract group treated with cloves, the mean value of NO was decreased by −40.4% as compared to untreated selenite-induced cataract.
Moreover, the mean value of H2O2 in the normal control group was 6.36 nmol/g lens and in selenite-induced cataract group, the mean value was 21.1 nmol/g lens. On the other hand, the mean value of H2O2 in selenite-induced cataract treated with cloves was 13.5 nmol/g lens, the percentage was decreased by −36.02% as compared with untreated selenite-induced cataract group.
In addition, CP in the lens of selenite-induced cataract group was also increased when compared to normal control group. The mean value of CP in normal control group was 4.23 nmol/mg proteins and in cataractous group was 16.4 nmol/mg proteins. In selenite-induced cataract treated with cloves the percentage of CP was decreased to −49.88% when compared to untreated selenite-induced cataract group.
Finally, as shown in [Table 4], there was an increase in the levels of AGEs in the lenses of selenite-induced cataract group as compared to normal control group. The mean value of AGEs in the lens of cataractous group was 1.2 mg/ml while normal control group was 0.42 mg/ml. In addition, the mean value of AGEs in selenite-induced cataract rat pups treated with cloves was 0.7 mg/ml. AGEs was decreased by −41.7% in selenite-induced cataract rat pups treated with cloves on comparing with untreated selenite-induced cataract group.
|Table 4: Levels of advanced glycation end products and matrix metalloproteinase-2 in the lens of different experimental groups|
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The levels of metalloproteinase in the lens were also assessed, and the results showed increase levels of metalloproteinase in the lens of cataractous group on comparing with normal control group and an improvement was also noticed in the level of metalloproteinase after rat pups treated with cloves extract.
| Discussion|| |
Many plant products exert its antioxidant effect by quenching various free radicals and the singlet form of molecular oxygen. Spices and condiments exhibit a wide range of beneficial, pharmacological, antioxidant, anticarcinogenic, and anti-inflammatory effects., Species are widely used as food adjuncts, and cloves constitute one of the major spice species. Clove oil is widely used in flavoring food items and has analgesic properties much used in teeth ache.
The present study was designed to determine the possible protective effects of cloves against oxidative damage induced by sodium selenite. With regard to cataract, the selenite model was selected because of the rapid, effective and reproducible cataract formation. Although the rate of opacification in the selenite model is much more rapid than in human cataract, it has many general similarities to human cataract.
The effect of cloves on selenite-induced cataract rats was examined and the results noticed by lenticular opacification (Stage 1; 30%) of the group that had received a single subcutaneous injection of sodium selenite and treated with 100 mg/kg body weight cloves. As shown in [Table 1], cloves extract had effect role to reduce the stages of cataract formation from Stage 4 in the cataract group to Stage 1 in the group treated with cloves (100 mg/kg b.wt. daily) also reduced the percentage from 100% to 30% in rats after treatment with cloves. This proved the protective effect of cloves against cataract formation.
Oxidative stress is an outcome of imbalance between reactive oxygen species (ROS) production and antioxidant defenses, which in turn evokes a series of events deregulating the cellular functions. The excess oxidative stress was previously reported to induce extensive oxidative modifications on lens proteins especially α-crystalline protein resulting in the enhanced lens opacity. In addition, an enormous production of ROS takes place leading to characteristic membrane permeability and changes including leakage of structural proteins which is implicated in the opacity of the lens.
The oxidative damage was studied by assessing parameters such as reduced GSH, MDA, NO, CP, and H2O2. The effects of coadministration of cloves on the above parameters were investigated in this study.
Truscott. proposed that low concentration or loss of reduced GSH in lens may be the common and critical feature that precedes nuclear cataract formation. Therefore, the maintenance of GSH is known to be vital for lens clarity. The present study found a significant decrease in the activity of GSH content in the blood and lens of selenite-induced cataract group.
The formation of selenite-induced cataract is hypothesized to be a result of GSH loss from the lens and the sensitivity of rat lenses to oxidative stress is enhanced. In the treated group, the data revealed the antioxidant effect of cloves since there was an increase in the percentage of the GSH to 66.3%.
Explanations of possible mechanisms underlying antioxidant potential of cloves include the prevention of GSH depletion and destruction of free radicals. This effect can be attributed to the antioxidant effect of cloves. When the oxidative stress persists for a longer duration, and also lens turns to be a cataract, the inbuilt mechanism of the body fails to alleviate the damage. The administration of cloves is suggested to have triggered the secretion of antioxidant enzymes in enhanced levels which in turn stopped the oxidative damage due to injection with sodium selenite and stop the development of cataract formation.
Cloves extract is rich in flavonoids and commonly used as natural supplement, and its main properties are protection against free radical damage. The antioxidant activity of eugenol and its isomer isoeugenol was investigated by d' Avila Farias et al.
The peroxidative damage to the lens is one of the major events in the pathogenesis of cataract. Increased lenticular MDA may be the result of lipid peroxidation. Gupta et al. demonstrated that selenite can cause increased membrane damage as indicated by the level of MDA. On treatment with cloves extract the data in the present study revealed a significant reduction in MDA level in serum and lenses of cataract group to the extent of −60.7% as compared to cataract group.
Our data suggested that higher serum and lens levels of MDA as oxidative stress markers in cataractous group. This elevation in MDA was responsible for different complications in selenite-induced cataract. A similar findings obtained by different investigators such as Chang et al. and Salem et al. suggested that high levels of MDA may be involved in the development of cataract.
These findings suggested the possibility that increased oxidative stress may be associated with the development of cataract and support the hypothesis that oxidative damage plays a role in the pathogenesis of cataract.
As reported previously flavonoids prevent experimental selenite-induced cataractogenesis in rat pups and treatment induced oxidative stress and cataractogenesis by maintaining antioxidant status, reducing ROS generation, and lipid peroxidation in the lens possibly by inhibiting lipid peroxidation as reported by Rooban et al. and Muthenna et al.
In the same line, Issac et al. found Clovinol the active flavonoid component reduced the extent of lipid peroxidation. These results suggest that cloves can prevent or retard experimental selenite-induced cataractogenesis in rat pups that attributed to its high content of Clovinol and their free radical scavenging properties.
Some studies have shown that NO may have an important role in the development of cataract , Nagai et al. reported that the NO was increased in the lenses of rats with opaque lenses. The data in the present study confirmed showing a decrease in the level of NO in the blood and lens of cloves treated group as compared to the nontreated one. These previous results suggest that cloves have a significant anticataract potential effect and acts primarily by preserving the antioxidant defense system. Its protective effect was supported by the finding of lowered NO levels in the blood and lens of the group treated with cloves.
In addition to the previously mentioned parameters, the level of H2O2 was determined in the lens of all experimental groups. H2O2 is not a free radical itself but is usually included under the general heading of ROS It is a weak oxidizing agent that might directly damage proteins and enzymes containing a reactive thiol group.
The result of the current study reported that the level of H2O2 in the cataractous lens was higher than normal control group by (69.86%). H2O2 would be expected to destroy the homeostasis maintained by the lens membrane barrier, initiating events leading to the development of lens opacity. After treatment selenite-induced cataract rat pups with cloves, the level of H2O2 was decreased by −36.0%; this means a protective effect of cloves to reduce cataract development is attributed to its antioxidant activity.
Reactive carbonyl species (RCS) such as glyoxal and methylglyoxal are ubiquitous by-products of cell metabolism, which potently induce the formation of AGEs by nonenzymatic protein glycation.,
In this work, the levels of CP and AGEs in lenses of different groups. The results showed a significant inhibitory effect on the formation of CP and AGEs. Since the levels of CP and AGEs were decreased in the group treated with cloves as compared to untreated selenite-induced cataract. Antiglycation activities of cloves were not only brought by their antioxidant activities but also related to their trapping abilities of RCS which also decreased in the lens of treated clove group as shown in [Table 3].
The result of Suantawee et al. supported the result of the current study found that clove extract markedly prevented oxidation-induced protein damage by decreasing protein carbonyl formation and protecting against the loss of protein thiol group.
Cloves extract is rich in flavonoids and commonly used as a natural supplement, and its main properties are protection against free radical damage. The polyphenol-enriched clove extract, owing to its antioxidant, was capable to inhibit the formation of AGEs and protein glycation. The results of this study suggest that supplementation with cloves extracts may delay the cataract formation and may enhance antioxidant defense. These results may be applied in the future for the prevention of cataracts using cloves.
Apoptosis is known to participate in various biological processes including immune defense, development, and maintenance of tissue homeostasis and elimination of unwanted cells or tissues. Any tilt of the balance between life and death within an organism can lead to disease. Thus, the loss of essential cells of postmitotic tissues due to enhanced cell death may play an important role in a number of functional deficiencies and degenerative diseases such as cataract.
Apoptosis has often been labeled programmed cell death, cell suicide, naturally occurring cell death, or physiological cell death. Consumption of natural foods rich in flavonoids helps to protect body from many diseases. It was found that many pharmacological actions of flavonoids can act in the inhibition of cataract. Clove showed potent activities to suppress the incidence of cataract by strong antioxidant potential, act as antiglycation and antiapoptotic agent.
The treatment of selenite-induced cataract with cloves resulted in the reduction of MDA, NO, H2O2, and CP and AGEs in the lens. Cloves have anti-cataract potential may be attributed to the presence of high phenolics and flavonoids.
| Conclusion|| |
From the results of this study can be concluded that oral consumption of cloves following selenite injection is effective in preventing cataractogenesis in selenite model by enhancing antioxidant enzyme defense system of blood and protect the lens from the oxidative stress.
These results suggest that cloves are a new functional dietary agent to stop cataract progress. Diet has a significant impact on cataract development, but the individual dietary components responsible for this effect need further investigations.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]