The Effect of Dietary Antioxidant Supplementation in Patients with Cataract and Glaucoma

Background: Oxidative stress may be a key risk factor for cataract and glaucoma, and many previous reports have suggested that antioxidants could be a promising treatment. Here, we investigated the effects of a novel supplement containing three food-derived antioxidants (hesperidin, crocetin, and tamarindus indica) on markers of oxidative stress in patients with these conditions. Methods: This study had a prospective, single arm design. Fifty Japanese subjects with cataract and glaucoma were recruited and asked to refrain from the use of vitamin or carotenoid supplements for 2 weeks before the study. The subjects took 4 tablets together with ample water twice a day for 8 weeks, stopped the treatment, and were then followed for an additional 8 weeks. The subjects were examined at four-week intervals, for a total of 5 examinations. We measured biological antioxidant potential (BAP) with a free radical analyzer. Clinical laboratory data, including malondialdehyde (MDA) and superoxide dismutase (SOD), were measured in venous blood samples. Clinical parameters were also recorded. The Mann-Whitney U test and Fisher’s exact test were used to determine the signicance of differences between groups. Other comparisons used a one-way analysis of variance (ANOVA) followed by the Student’s t-test or Dunnett’s test. Results: BAP was signicantly elevated at weeks 8, 12, and 16 (P = 0.007, P = 0.035, P = 0.015). The MDA level was signicantly reduced at week 8 (P = 0.019). BAP changes were recorded by subtracting the value at week 8 from week 0. Multiple regression analysis revealed that SOD, total bilirubin and diabetes were independent contributing factors to the change in BAP (P = 0.039, P = 0.019, P = 0.013). There were no supplement-related adverse events or abnormal results in blood testing in any of the patients. Conclusion: Our study found that an 8-week oral course of antioxidant supplementation was effective in patients with a low antioxidative stress level. Dietary supplementation holds

Several reports have revealed that ROS have a key role in the pathogenesis and progression of cataracts.
ROS directly cause oxidation of the lens and also lower the capacity of the lens to remove oxidants created by ultraviolet radiation and aging. Sawada et al. revealed that cataract severity was correlated with signi cantly increased superoxide dismutase (SOD) activity [7]. Kaur et al. revealed that cataract patients had a higher serum malondialdehyde (MDA) level [1]. Cataract patients have also been shown to have a lower level of plasma thiobarbituric acid reactive substances when the plasma level of vitamin C is increased [8]. Ravindran et al. found that a lower level of vitamin C was strongly associated with cataract [9]. Such ndings have inspired past efforts to develop dietary supplements containing antioxidants to treat ocular diseases, including cataract [10,11].
Treatment for glaucoma most commonly involves therapy to reduce intraocular pressure (IOP). However, disease progression persists in some patients even after IOP is successfully reduced. This has led to past investigations of possible non-IOP risk factors for glaucoma and its key underlying pathomechanism, retinal ganglion cell (RGC) degeneration. Previously reported risk factors include genetics [12], vascular dysregulation [13], mitochondrial dysfunction [14], and in particular, oxidative stress [2]. Oxidative stress has three key effects in glaucoma. First, it raises IOP by altering the trabecular meshwork and impairing aqueous humor out ow [15,16]. Second, it disrupts autoregulation of blood ow to the optic nerve by altering the vessels that feed it [17,18]. Finally, patients with a low antioxidant level in the eye are susceptible to systemic oxidative stress, which can induce RGC death, as we have previously reported [19,20]. Harris et al. revealed that one month of oral supplementation with an antioxidant increased blood ow biomarkers in the eyes of patients with open-angle glaucoma (OAG) [21]. Park et al. found that a four-week course of daily administration of 160 mg of gingko biloba extract led to a signi cant improvement in retinal blood ow volume and velocity in 15 patients with normal-tension glaucoma (NTG).
Here, we gave a daily dietary antioxidant supplement over 8 weeks to patients with cataract and glaucoma. The supplement was developed and rst reported by Maekawa et al., who identi ed the components of the supplement through screening, and found that it had a neuroprotective effect in a mouse glaucoma model [22]. We believe that the ndings reported here will shed light on the potential of new treatments for cataract and glaucoma based on antioxidant supplementation.

Study design
This study had a prospective, single arm design. Japanese patients with previously diagnosed NTG or cataracts were recruited at Tohoku University Hospital between March 2018 and October 2019; all patients were followed for 16 weeks.

Participants
Inclusion criteria for participation were as follows: overall good health, age between 30 and 75 years; NTG con rmed in one or both eyes by a glaucoma specialist; a biological antioxidant potential (BAP) level between 1600 to 2200 nmol/L; a diacron-reactive oxygen metabolite (dROM) level not less than 300 Carrelli units (U. Carr), body mass index less than 26 kg/m 2 . Exclusion criteria were as follows: hyperopia (above + 3D); high myopia (below − 8D); any type of secondary glaucoma; high IOP (> 16 mmHg) despite glaucoma medication; and severe systemic disease, including cancer, hyperthyroidism, and autoimmune disease. No patients used exogenous hormones. Pregnant women, or women planning pregnancy during the study period, were also excluded. Candidates were asked to refrain from the use of vitamin or carotenoid supplements for 2 weeks before the study. If a patient had diabetes, hyperlipidemia, hypertension, or a current smoking habit, it was recorded. Cataract patients were recruited from the Tohoku University Hospital eye clinic. Figure 1 shows an overview of the study design. Participants were examined 5 times, at four-week intervals. At the examination, we measured clinical parameters, con rmed compliance with the study protocol of twice-daily use of the supplement, and recorded any adverse events.

Study intervention
Maekawa et al. previously reported 12 candidate neuroprotective compounds [22]. Based on these results, we developed the novel supplement used in this study, which contained the following ingredients in each daily dose of 4 tablets: hesperidin (50 mg/4 tablets), crocetin (7.5 mg/4 tablets), and tamarindus indica (25 mg/4 tablets). The supplement was manufactured and supplied by Wakamoto Pharmaceutical Co. Ltd (Tokyo, Japan). The candidates took 2 tablets with ample water twice a day (i.e., 4 tablets/day) for 8 weeks, and no supplementation for 8 weeks. Any unused supplements were returned for a pill count at the 8-week visit.

Measurements
A complete ophthalmic examination of all patients was performed by a glaucoma and cataract specialist. This comprised measurement of best-corrected visual acuity, recorded as the logarithm of the minimal angle of resolution, examinations with slit-lamp biomicroscopy and funduscopy, and evaluation of the optic disc with a 90-diopter lens. The patients with glaucoma and cataract also underwent measurement of mean deviation (MD) with the Humphrey eld analyzer (HFA). If both eyes had glaucoma, the analysis included the worse-MD eye. Similarly, for cataract subjects, the analysis included the better-VA eye.

Clinical laboratory measurements
Samples of blood were stored in containers with EDTA at weeks 0 and 8. We con rmed the supplement safety with clinical laboratory measurements. Plasma samples were stored at -80 °C for later biochemical analysis. Serum samples from the screening visit were sent to LSI Medience Co. (Tokyo, Japan), analyzed for AST, ALT, γ-GTP, total bilirubin, total protein, albumin, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, total cholesterol, triglycerides, creatinine, blood urea nitrogen, uric acid, glucose, HbA1 C , white and red blood cell count, platelet count, hematocrit, and hemoglobin.

Measurement of MDA and SOD
Venous blood samples were collected in a tube with no anticoagulant and centrifuged at 3280 rpm for 20 min. The supernatant was moved to another tube, and the samples were subsequently shipped in dry ice to Wakamoto Pharmaceutical Co. Ltd (Kanagawa, Japan). All samples were frozen at -20 °C for storage and thawed only once, before they were analyzed.
Blood sampling and oxidative stress measurement Serum samples were evaluated for oxidative stress levels and anti-oxidative potential with a free radical analyzer system (Free Carpe Diem, Wismerll Company Ltd., Tokyo, Japan). Blood samples were collected more than 3 hours after a subject ate. The samples were also evaluated for dROMs, which represent the total oxidant capacity of a serum sample towards N, N-diethyl-para-phenylendiamine, used as an indicator. The results are expressed in U. Carr. Total plasma antioxidant capacity in the samples was measured with the BAP test, which is based on the ability of a sample to reduce iron from its ferric (Fe3+) to its ferrous (Fe2+) state. The analyses were performed as previously reported [19,20].

Statistical analysis
The signi cance of differences between groups was determined with the Mann-Whitney U test and Fisher's exact test. Comparisons of groups used one-way analysis of variance (ANOVA) and the Student's t-test or Dunnett's test. All numerical ndings are mean ± SD. Statistical signi cance was set at P < 0.05. SPSS version 23.0 (SPSS Inc., Chicago, IL, USA) was used for all analyses.

Subjects
This study enrolled 50 patients with NTG and cataract. Table 1 shows the demographic characteristics of the subjects. There was no supplement-related adverse events for patients including blood tests (data not shown). Oxidative stress BAP was signi cantly elevated at weeks 8, 12, and 16 (P = 0.007, P = 0.035, P = 0.015, respectively; Table 2, Fig. 2A). MDA level was signi cantly reduced at week 8 (P = 0.019, Table 2, Fig. 2B). There were reductions in the 8OHdG level, but these did not reach statistical signi cance at any of the ve time points (Table 2, Fig. 2C). There were no signi cant differences in dROMs and SOD after supplementation. Multiple regression analysis BAP changes were recorded by subtracting the value at weeks 8 from weeks 0. A univariable regression analysis revealed that SOD, total bilirubin and diabetes independently contributed to the change in BAP (P = 0.023, P = 0.013, P = 0.007, respectively; Table 3). Thus, we selected these three variables and analyzed them with a multiple regression analysis. This revealed that SOD, total bilirubin, and diabetes remained independent contributing factors to the change in BAP (P = 0.039, P = 0.019, P = 0.013, respectively; Table 3).

Discussion
Our study found that patients with glaucoma and cataract who took a daily antioxidant supplement showed an increase in BAP, a biomarker of the antioxidant capacity of the body. This may be an important nding, because the development and progression of cataracts are known to be promoted by oxidative damage to the proteins comprising the lens. Oxidative stress may also play an important role in glaucoma, which is an age-related chronic neurodegenerative disease. Our results are particularly interesting for patients with a low antioxidant level and suggest that antioxidant supplementation may be effective in treating them.
BAP was signi cantly higher than baseline at weeks 8, 12, and 16, while MDA level was signi cantly reduced at week 8. MDA is an end product of free radical reactions in membrane fatty acids [23]. A variety of antioxidant molecules can be found in human plasma, with the following compounds being most common: α-tocopherol, β-carotene, albumin, ascorbic acid, bilirubin, catalase, ceruloplasmin, ferritin, glutathione peroxidase, lycopene, reduced glutathione, SOD, and uric acid [24]. All these compounds can catalytically remove reactive species such as free radicals. After taking our novel supplement, the antioxidative level increased in the subjects in this study. The higher the level of antioxidative activity, the lower the level of oxidative stress.
A multiple regression analysis showed that SOD, total bilirubin, and diabetes were independent contributing factors to changes in antioxidative potential after supplementation in both the cataract and glaucoma groups. This means that our supplement was effective in patients with a low antioxidative stress level. This is especially interesting for glaucoma, which can sometimes progress even with successful IOP-lowering treatment. There is extensive clinical evidence that systemic oxidative stress contributes to glaucomatous optic neuropathy. Moreover, antioxidant therapy has shown promising results in animal-and human-based research. Antioxidant treatment can decrease the activation of NF-κβ and decrease the production of cytokines in the optic nerve and retina [25]. In a rat glaucoma model, overexpression of thioredoxins can protect the RGCs [26]. Higher green leafy vegetable intake is associated with a lower primary OAG risk [27]. Lee et al. found that ginkgo biloba extract protected against the effects of glaucoma in some patients [28]. These results suggest that insu cient serum antioxidant proteins underlie the contribution of oxidative stress to glaucoma, suggesting that antioxidants should have a neuroprotective effect. Our past research also showed that visual eld damage was strongly correlated with systemic antioxidant levels in young, male patients with OAG [19]. Antioxidant treatments might therefore be a useful therapeutic option to delay or prevent disease progression.
Previously, Maekawa et al. reported that three food-derived compounds, hesperidin, tamarindus indica, and crocetin, had a protective effect in a primary culture of retinal cells under oxidative stress. Hesperidin is effective in reducing apoptosis, oxidative stress and in ammation. Maekawa con rmed that hesperidin was effective in vivo in mice, reducing oxidative stress and preventing RGC death caused by NMDAinduced excitotoxicity [22]. Tamarindus indica is indigenous to tropical Africa. It is high in tartaric acid, B vitamins, and minerals. It has antioxidant [29,30], anti-in ammatory [31], anti-diabetic [32], and antiatherosclerotic effects [33]. Crocetin is a natural apocartenoid dicarboxylic acid that is found in the crocus ower and in Gardenia jasminoides. Crocetin has various effects, acting as an antioxidant [34] and anti-in ammatory [35], and can inhibit the caspase pathway, preventing retinal damage induced by N-methyl-D-aspartate (NMDA) [36]. These results suggest that these three food-derived compounds, used as a dietary supplement, might help reduce RGC degeneration in retinal disease.
Systemic oxidative stress may be a key factor in the development of cataracts [37,38]. Past ndings support the idea that antioxidant therapy is effective for cataract patients. Nuclear opacities have been reported to be associated with nutrients (folate, α-carotene, and dietary ber) from the intake of foods, particularly vegetables [39]. Chylack et al. found that an oral antioxidant slightly slowed cataract progression [40]. Another study found that subjects who received multivitamin/mineral supplementation had a 36% lower prevalence of nuclear cataracts [41]. Hayashi et al. compared the total amount of hydroperoxides in the aqueous humor before and after supplementation with an antioxidant, Ocuvite Lutein [42], and found that hydroperoxides decreased in female, but not in male, subjects. That study suggested that it might be possible to inhibit oxidative stress in the aqueous humor and the lens epithelium. Lutein has been investigated by several in vitro studies, and has been reported to lower the intracellular accumulation of H 2 O 2 by scavenging both H 2 O 2 and superoxides [43]. Lutein supplementation of lens epithelial cells has also been reported to decrease protein oxidation, lipid peroxidation and DNA damage induced by H 2 O 2 [44]. Based on these data, we consider that a decreased general oxidative stress level after supplementation may result in a reduced oxidative stress level in the aqueous humor, potentially delaying cataract progression.
Our study has some limitations. First, it lacked a placebo arm, which is needed to prove the e cacy of any supplement. Thus, we plan to conduct further studies with a randomized, doubled-blind, placebocontrolled design. Second, while we observed signi cant differences in BAP before and after supplementation, we did not make a similar nding for dROMs. Nevertheless, the improvement in BAP after supplementation for eight weeks was signi cant. This discrepancy might have been due to an increase in oxidative stress after supplementation, causing increased endogenous antioxidant activity. Finally, we could not include a control group, because all potential candidates already had cataracts, due to their advanced age. We were also limited in our ability to detect relatively modest associations, making longer follow-up of the subjects necessary in a future study.
Our study indicates that patients with a low systemic antioxidant level may have increased susceptibility to oxidative stress, thereby accelerating the progression of cataract and glaucoma. This raises the possibility that antioxidant supplementation may be a viable option to delay or prevent age-related cataracts and glaucoma progression.

Conclusion
Oxidative stress may be a key non-IOP risk factor for age-related cataracts and glaucoma. Our study revealed that antioxidant supplementation was effective in patients with a low antioxidative stress level, suggesting that such supplementation may be a novel way of combating diseases induced by systemic oxidative stress, and could contribute to individualized treatment for these diseases. Research Review Board of Tohoku University (study 2019-6-068), which is certi ed by the Japanese Ministry of Health, Labor and Welfare. The trial was registered with the UMIN clinical trial registry, number 000032050. Written informed consent was obtained from all subjects before the start of the study.

Consent for publication
Not applicable Availability of data and materials The datasets analyzed in this study are available from the corresponding author on reasonable request.

Competing interests
Research funding was provided by Wakamoto Pharmaceutical Co., Ltd.  Overview of the study design