The Preventive Effects of Asparagus officinalis Extract on Sodium Selenite-Induced Cataractogenesis in Experimental Animal Models

Materials and Methods Neonatal rats received a single dose of sodium selenite as an intraperitoneal injection (30 μmol/kg) on day 10 postnatal to induce cataract. Animals were then posttreated with various oral solutions of A. officinalis extract at 200 mg/kg or 400 mg/kg once daily on days 10–16 postnatal. Cataract was evaluated by slit-lamp, and lens opacification was analyzed in each group 24 hours after the last treatment at day seven postadministration of the extracts or vehicle. The total protein concentration of lenses, glutathione reductase activity as the glutathione antioxidant capacity, and malondialdehyde content as a marker of lipid peroxidation were further assessed in removed rat lenses on day 30 postnatal. Results All lenses in the healthy and control plant groups were clear. Sodium selenite significantly increased cataract grade (2.8 ± 0.2) when compared to the healthy group (p = 0.001). However, cataract grades were decreased considerably as 1.9 ± 0.72 and 1.5 ± 0.85 in groups that received 200 mg/kg and 400 mg/kg oral extract of A. officinalis, respectively. A. officinalis extract also restored all abnormalities of biochemical markers induced by sodium selenite. Conclusion Our data suggest that A. officinalis could be a promising candidate as a safe alternative treatment in cataracts upon further clinical trials. This effect is probably associated with the antioxidant activity of A. officinalis.


Introduction
Cataract is a disease that the eye lens becomes progressively opaque, resulting in blurred vision, cloudiness, or the opacification of the lens of the human eye [1][2][3]. Cataract is responsible for 51% of the world's blindness, representing approximately 20 million people [2,4]. Surgical replacement of the opacified lens with an artificial lens is currently the only way to cure vision loss. Although cataract surgery is now considered the most successful cure in terms of visual outcome, the cost, need for trained personnel, and postsurgical complications limit the worldwide availability and accessibility to this procedure [5]. us, the development of an alternative for surgical intervention to prevent or delay the onset of cataract is needed. e pathogenesis of cataracts is multifactorial and involves mechanisms that are not fully understood; oxidative stress is the foremost cause of cataract formation [6][7][8][9].
Oxidative stress causes an imbalance between the rate of oxidant production and the rate of detoxification, so the rate of oxidant production is significantly higher than that of detoxification using antioxidants [10]. With age, oxidants accumulate while antioxidant defenses gradually diminish, which is likely the most crucial mechanism in age-related cataract formation [11]. Since oxidative stress is implicated in cataract formation, the application of antioxidant agents, which may be capable of successfully penetrating the lens tissue, may counteract oxidationrelated cataractogenesis. Much herbal medicine and natural products with antioxidant effects have been found to prevent sodium selenite-induced cataracts. Echium amoenum is an Iranian indigenous medicinal plant. In (2015) study, we showed that E. amoenum extract significantly had a protective effect on selenite-induced cataract in rat. is effect is probably associated with the antioxidant activity of this medicinal plant, resulted in inhibiting oxidative stress caused by cataract [12]. In another experiment, we assessed the preventive effects of Origanum vulgare extract, a potent antioxidant herbal medicine, on selenite-induced cataractogenesis. O. vulgare acted as a powerful therapeutic plant against selenite-induced cataract formation. e anticataract effects of O. vulgare extract could be based on the high amount of flavonoids and polyphenolic compounds, resulting in the strengthen endogenous antioxidant enzymes defenses system of the body, inhibition of oxidative stress conditions in cataract, and scavenging free radicals that caused lipid peroxidation by direct or indirect antioxidant mechanisms [13].
Asparagus officinalis L. is known as an anticataract plant in the canon of medicine in Persian Medicine and traditional medical books [14]. A. officinalis is one of the Asparagaceae family plants; it is a perennial and monocotyledonous plant that is grown for its edible young stems. Young shoots are harvested in spring and consumed as a seasonal vegetable ( Figure 1). Asparagus extracts have been traditionally used as diuretics, laxatives, and antitussives. Recent studies have shown that young shoots have antioxidant and antitumor effects on humans [15]. Young shoots of A. officinalis contain specific amounts of phytosterols, such as β-phytosterol, which reduce blood cholesterol levels. Asparagus is primarily a rich source of flavonoids, including rutin and quercetin, and it is a source of vitamin B 6 , zinc, beta-carotene, vitamin C, vitamin E, vitamin K, thiamin, riboflavin, niacin, folic acid, phosphorus, copper, manganese, and selenium. e asparagus plant is rich of asparagine amino acid [15][16][17][18]. us, it is hypothesized that A. officinalis with its antioxidant compounds and excellent free radical scavenging may be having a protective action on oxidative stress conditions in selenite-induced cataractogenesis.
In the current study, we evaluated the effect of A. officinalis against selenite-induced experimental cataract in the rat model. Cataract was measured by slit-lamp, and lens opacification was analyzed in each group on day seven after selenite administration. e total protein concentration of lenses, glutathione reductase (GR) activity as the glutathione antioxidant capacity, and MDA as a marker of lipid peroxidation were further assessed in removed rat lenses.

Preparation of A. officinalis Extract.
A. officinalis was collected from Sari Asparagus gardens, Sari, Iran. Young shoots of the plant were identified by systematic plant specialist Dr. Masoud Azadbakht, the Mazandaran University of Medical Sciences, Sari, Iran. To obtain the hydroalcoholic extract of A. officinalis, we needed to dry the plant. First, we cut the young asparagus shoots into smaller pieces and dried them in an oven at 42°C for 48 hours. e dried parts of the plant were then crushed until 200 mesh using a blender. Next, 500 g of dried young shoots of the plant was soaked through the maceration method, which uses 70% hydroalcoholic extract comprising 30% distilled water and 70% ethanol. e hydroalcoholic extract was drained three times and left at room temperature for three days. e extract was then filtrated and kept in light-proof head-tight bottles in a fridge. After that, the hydroalcoholic solvent was evaporated using a rotary evaporator at 40°C and then dried using a freeze-dryer.

Determination of Total Phenol Content.
e total phenol content of the spice extract was determined using the Folin-Ciocalteu method [19].
is method is based on phosphotungstate-phosphomolybdate reduction in an alkaline medium. In this method, we followed the methods of Tankeu et al. 2016 [20]. 200 μL of 1 mg/ml of samples was first introduced to different test tubes. en, 800 μL of 10fold diluted Folin reagent and 2000 μL of sodium carbonate solution (7.5%) were added. After stirring for 5 minutes, the mixture was kept away from light for 2 h, and the absorbance was measured using a spectrophotometer at 765 nm. e phenolic content was determined from a quercetin standard curve. A concentration range from 0 mg/mL to 0.3 mg/mL of quercetin was prepared to determine the total polyphenol content expressed in mg equivalents of quercetin/g of extract.

Determination of Total Flavonoid Content.
e total flavonoid content was determined using the following method [21]. First, 100 μL of the extract was added to 300 μL of distilled water and 30 μL of NaNO 2 (5%) and then 2 Evidence-Based Complementary and Alternative Medicine incubated for 5 minutes at 25°C. Next, 30 μL of AlCl 3 (10%) was added to the solution. After another 5-minute waiting time, the reaction mixture was treated with 200 μL of 1 mM NaOH, and the reaction mixture was diluted to 1000 μL with distilled water. Quercetin served to draw the standard calibration curve in the range of 0-0.3 mg/mL, and the absorbance was measured at 510 nm. e results were expressed as mg quercetin equivalents/g of dried extract.

Experimental Treatment.
For the experiment, animals were divided into five groups (groups 1-5, n � 5 for each group) as follows: newborn rats received a single intraperitoneal (i.p) injection of NaCl 0.9% at day 10 postnatal with no further treatment as the control healthy group. A single dose of 30 μmol/kg sodium selenite dissolved in NaCl 0.9% at day 10 postnatal was intraperitoneally injected to induce cataract. Animals were then posttreated with various oral solutions of A. officinalis extract at 200 mg/kg or Evidence-Based Complementary and Alternative Medicine 3 400 mg/kg once daily on days 10-16 postnatal. Besides, rats in one group received an oral solution of A. officinalis (400 mg/kg) on days 10-16 postnatal as the control plant group. No more than 0.5 ml of sodium selenite solution was administered to each rat. e observations of lens opacification were performed on day seven after sodium selenite administration by slit-lamp. All neonatal rats were anesthetized on day 30 postnatal by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (15 mg/kg). Lenses were removed immediately after euthanasia and promptly placed on dry ice. Samples were stored at a temperature of −80°C for further analysis.

Cataract Grades.
Cataracts were graded according to these scales; grade 0, clear lens; grade 1, a lens with slight opacity; grade 2, a lens with partial nuclear opacity; and grade 3, a lens with dense nuclear opacity.

Determination of Total Lens
Protein. e lenses of each rat homogenized with the glass-glass homogenizer. e total protein concentration of homogenized lenses, which were obtained by enucleating the eyes of rats, was determined through the use of the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL. USA). A total of 50 μL of lens homogenate, standard bovine serum albumin (BSA), and working reagent (constituted as per the manufacturer's directive) were pipetted into a 96-well microplate. Absorbances were measured at 562 nm using a URIT-660 microplate reader (URIT Medical Electronic Co., Ltd, Guangxi, China). Each determination was in triplicate.

Determination of Glutathione Reductase (GR) Activity.
We used the previously described Ellman method to determine the glutathione antioxidant capacity of plant extracts [22]. An aliquot of PBS (580 μL), 200 μL of extract, and 200 μL of each homogenate lenses and 20 μL of inducing solution was introduced in different test tubes. e tubes were then incubated at 37°C for one hour. Finally, the test solutions, 20 μL and 3000 μL, of Ellman reagent (phosphate buffer 0.1 M; pH 6.5; 2,2-dithiol-5,5′-dibenzoïc acid) were introduced into new test tubes. Glutathione concentrations were expressed in micromoles/L and determined.

Determination of Lipid Peroxidation.
We evaluated the capacity of the extract to inhibit the lipid peroxidation process in lenses according to the following method [23]. After this, the tubes were heated again at 100°C for 20 min and transferred to an ice bath to be cooled and centrifuged at 3000 rpm for 5 min. e optical density was measured at 535 nm, and the concentration of MDA was determined.

Statistical Analysis.
e data are presented as the mean ± SD. Differences between group means were estimated using a one-way analysis of variance followed by Tukey's HSD test. A p value of less than 0.05 was considered to be significant.

Total Polyphenols and Flavonoids Content.
e mean total phenolics content of A. officinalis young shoots extract as mg gallic acid (GA) equivalent/g of dried extract was 283 ± 7.5 (GA) in mg/g. e mean total flavonoids content of A. officinalis young shoots extract as mg quercetin (QE) equivalent/g of dried extract was 14.93 ± 1.41 (QE) mg/g. Table 1 shows the results of the morphological analysis of rat eyes by slit-lamp on the 17 th day postnatal. e number of lenses of each group was sorted by the degree of opacity in four grades (grade 0-3). Cataract grades were 0 in normal, and the control plant group received only normal saline and an oral solution of A. officinalis, respectively. e mean grade of cataract in animals that received selenite was dramatically increased (2.8 ± 0.2) compared to healthy and control plant groups. In contrast, cataract grades were significantly decreased to 1.9 ± 0.72 and 1.5 ± 0.85 in groups treated by 200 mg/kg and 400 mg/kg oral extract of A. officinalis, respectively. Also, pictures of cataract grading of the lenses (with slit-lamp apparatus) on 17 th day postnatal and pictures of enucleated lenses after surgery on day 30 are shown in Figures 2 and 3, respectively. All lenses in normal and control plant groups were clear. In the single dose selenite injection group, most lenses had nuclear cataracts with highdensity grade 3, and some of them showed grade 2 nuclear cataracts. In 200 mg/kg of the A. officinalis group, cataract grades were between 1 and 2, and in 400 mg/kg of the A. officinalis group, most of the lenses showed cataract with grade 1. Furthermore, the morphological reexamination of lenses on day 30 of lens removal surgery (Figure 3) confirmed the results of the initial examination of lenses (day 17), which is shown in Figure 2.

Biochemical Examination of the Eye Lens.
e mean GSH level in the healthy group and control plant group was 141 ± 15.1 and 139.6 ± 10.3, respectively. e mean GSH level significantly decreased in the selenite group (78.6 ± 12). However, the GSH level was restored considerably as 103 ± 56 and 121.3 ± 6.11 in pretreated groups by 200 mg/kg and 400 mg/kg oral extract of A. officinalis, respectively, compared with the selenite group (Figure 4) Figure 5). e mean total protein content in the healthy group and control plant group was 631.7 ± 21.7 and 610.3 ± 18.4, respectively. e mean total protein content decreased significantly in the selenite group as 385.1 ± 26.9, and the 200 mg/kg and 400 mg/kg of A. officinalis groups increased to 452.8 ± 28.1 and 469.3 ± 19.5, respectively ( Figure 6).

Discussion
We investigated the protective effect of A. officinalis young shoots extract against selenite-induced cataract in rat lenses for the first time. Evidence from previous studies suggests that reactive oxygen species and oxidative damage are involved in the development of cataracts [24,25], a multifactorial disease. Oxidative stress in the lens is considered to be the most critical factor in the formation of cataracts [26]. Lens transparency is dependent on the maintenance of redox balance, which is, in part, maintained by its high GSH content [23]. Besides, studies have shown that lipid peroxidation has been associated with the formation of cataracts. Polyunsaturated fatty acids are susceptible to free radical attack, initiating a chain reaction that terminates in the creation of stable byproducts, such as MDA, which are used as markers of lipid peroxidation [27]. erefore, as an alternative method to prevent or treat cataracts, antioxidant agents can be used to reduce lens oxidative stress.
In our study, lenses in the sodium selenite group had increased levels of MDA when compared to control group lenses, indicating increased lipid peroxidation in the sodium selenite group. is observation is the same as in our and other previous studies, which reported increased lipid peroxidation upon treatment with sodium selenite [12,27,28]. A. officinalis young shoots extract increased the amount of GSH. ey also can decrease the lipid peroxidation induced by sodium selenite by providing adequate amounts of GSH, which is a substrate of the glutathione peroxidase-catalyzed reduction of hydroperoxides. In our study, there was no apparent difference between 200 mg/kg and 400 mg/kg doses of A. officinalis extract, either in biochemical results of antioxidant activity reduction or physical examination of lenses.   Evidence-Based Complementary and Alternative Medicine Selenite cataract is common in the rodent models used in the rapid screening potentiality of anticataract agents. is method was first described by Ostadalva et al. in 1987. After they receive a subcutaneous injection of 20-30 nmol/kg of sodium selenite by body weight, cataracts begin to form after 3-4 days in the lens of young rat eyes and are completed in a week [29]. Sodium selenite injection induces cataracts in young rats through various mechanisms, including calpaininduced hydrolysis and the precipitation of lenticular proteins [25]. Oxidative damage caused by selenite may involve the oxidation of critical sulfhydryl groups on calcium ATPase or ion channels [30]. Previous studies have shown that antioxidant agents can protect rat lenses from experimental selenite-induced cataract [12,13].
Asparagus officinalis L., a well-known healthy vegetable, which was named as "the king of vegetables" for its abundant bioactive compounds, now is widely consumed all over the world. Bioactive components, such as flavonoid, lignan, and steroidal saponin, were found in this plant. In pharmacological studies, the extract from asparagus was shown to have several biological activities, such as antioxidant activity. Flavonoids and phenolics compounds were dominantly contributed to the antioxidant activity of the extract from asparagus residues [18]. A. officinalis polysaccharide is another bioactive component in asparagus. Like other plant polysaccharides, A. officinalis polysaccharide possesses a wide range of pharmacological properties, such as a significant function of scavenging hydroxyl radical [31]. Green asparagus polysaccharides could dramatically scavenge DPPH, hydroxyl, and superoxide anion free radicals and inhibit erythrocyte hemolysis and mitochondrial liver swelling. Green asparagus polysaccharides have good antioxidant effects in vitro [32]. In another investigation, aqueous extracts of green asparagus also exhibited radical scavenging, chelating activities, and protected liposome against oxidative damage. e high-performance liquid chromatography analysis indicated that polyphenolic components such as rutin, quercetin, kaempferol, and isorhamnetin were present in the aqueous extracts of green asparagus [33]. It is also known that caffeic and ferulic acids are relatively potent antioxidants. ese acids were found in asparagus.
e potent antioxidant activity of asparagus extract demonstrated a linear relationship with their flavonoid content [34]. Our data show that oxidative stress plays a role in cataract formation. e data support our hypothesis that A. officinalis extracts at 200 mg/kg and 400 mg/kg doses protect lenses by increasing GSH content, reducing lipid peroxidation, and restoring enzyme activity.

Conclusion
Our present biochemical and morphological results demonstrate that A. officinal extract inhibits the formation of Seinduced cataract, probably through its potent free radical scavenging and antioxidant properties.
us, A. officinal extract is suggested to be used as an anticataract agent, although such therapeutic modality requires further clinical trials before it applies to patients. is present study and any future ones may eventually help prevent cataract formation in high-risk populations and treat early-stage cataracts without the need for surgical intervention that led to a decrease in the socioeconomic burden of blindness worldwide.

Data Availability
e data used to support this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.