Plumbagin Protects Diabetogenic Cataract Formation by Mitigating Lens Aldose Reductase and Oxidative Stress

Abstract

Objectives

The polyol pathway plays an important pathophysiological role in diabetic-related ocular complications, including cataracts, one of the major causes of ocular blindness. The current paper investigated the protective effects of plumbagin against diabetogenic cataract formation, focusing on exploring its possible mechanism of action.

Methods

The study investigates antioxidant activity, aldose reductase inhibitory activity, and anticataract activity in experimental pharmacological models. In the ex-vivo study, goat lenses were incubated in artificial aqueous humor with high concentrations of glucose (55.5 mM) and plumbagin (20, 50, and 100 µg/mL), which was assessed against cataract control lenses.

Results

The in-vitro study showed that plumbagin inhibits 2,2-Diphenyl-1-Picrylhydrazyl free radical and lens aldose reductase activity. The ex-vivo study showed that plumbagin prevents lenticular opacity against the glucose-induced model. The plumbagin exposure significantly (p < 0.05) increased the antioxidant activity (catalase, superoxide dismutase and reduced glutathione) and reduced the malonaldehyde level. Moreover, plumbagin treatment significantly (p < 0.05) restored the lens protein content. Additionally, network pharmacological approaches suggested that SLC2A1, STAT3, and TP53 are the major target proteins for plumbagin in the prevention of cataract.

Conclusion

The results concluded that plumbagin has promising anticataract activity by inhibiting lens aldose reductase and mitigating lenticular oxidative stress, making it a potential anticataract agent for diabetic conditions.

INTRODUCTION

Cataract, defined as clouding of the lens, is the leading cause of visual impairment and blindness [1]. Generally, the light passes to the retina through the lens to form an image of the object. Therefore, the lens must be clear to focus the light on the retina. As the lens opacities develop, i.e., cataract progression; therefore, the vision becomes clouded or blurred. A study indicated that approximately 40 million people will be affected by cataract in 2025 due to a rapid increase in the older population [2]. Lens opacification in normal aging may be exacerbated by various risk factors, including diabetes [3]. Diabetes can increase the incidence of cataract by 5-fold [4]. Cataract pharmacotherapy has several research gaps. Currently, surgery is the only definitive treatment for cataract [5]. The issues regarding visual outcome, need for trained personnel, cost, and complications represent limitations of cataract surgery [6]. Multiple reports revealed that alternative non-surgical treatment for cataract shows similar effectiveness compared to surgery, and they can significantly prevent or delay the progression of cataract [7]. Natural aldose reductase inhibitors are used in the management of diabetes-associated complications, including cataract. Novel research revealed that natural phytoconstituent compounds have a potent aldose reductase inhibitory activity and might be a strong alternative to surgical therapy to reduce the risk of developing diabetic cataract [8, 9].

Many natural products or secondary metabolites have anticataract activity [10]. Plumbagin, a simple hydroxynaphthoquinone, is a secondary plant metabolite. It is a yellow compound with a melting point of 78℃-79℃. Various studies investigated plumbagin, concluding that it has various activities, including antidiabetic [11], antioxidant, anti-inflammatory [12], aldose reductase inhibitory [13], and anticancer activities [14]. Moreover, Catalani et al. (2023) [15] demonstrated that plumbagin exerts an antioxidant effect on retinal neurons, providing neuroprotection. However, the beneficial effects of plumbagin on cataract complications have never been explored. We assessed the antioxidant, aldose reductase inhibitory, and anticataract activities of plumbagin using in vitro and ex vivo pharmacological models because of the potential beneficial role of plumbagin in oxidative stress, diabetes, and neuroprotection. Moreover, we explored the possible targets and mechanisms of action by using network pharmacological approaches.

MATERIALS AND METHODS

1. Drugs and chemicals

Plumbagin was procured from P.C. Chem (Mumbai, India). Other analytical chemicals and reagents were obtained from the departmental central store.

2. In vitro antioxidant activity

In vitro antioxidant activity was assessed through the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) radical-scavenging activity [16]. The DPPH assay is the most common technique to assess the antioxidant activity in phytochemical constituents. When DPPH radicals interact with an odd electron and produce a dark purple color, they fade in a reaction with antioxidants and form DPPHH [17]. Briefly, plumbagin (1 mL, 100 µg/mL in methanol) was added to the DPPH solution (3 mL, 40 µg/mL in methanol) to scavenge the DPPH radical. The reaction mixture was incubated at room temperature in dark conditions for 30 min. Afterward, the reaction product was read at 517 nm against a blank using a spectrophotometer (UV-1780, Shimadzu, Japan). The scavenging activity was calculated against DPPH control (without drug sample) by using the following formula:

$$\text{DPPH scavenging activity (\%) =}\frac{\text{(absorbance of DPPH control-absorbance of test sample)}}{\text{absorbance of DPPH control}}\times 100$$

3. Isolation of the lens

Fresh goat eyeballs were obtained from the slaughterhouse and transferred immediately to the laboratory in an artificial aqueous humor (NaCl 140 mM, KCl 5 mM, MgCl₂ mM, NaHCO₃ 0.5 mM, NaH₂PO₄ 0.5 mM, CaCl₂ 0.4 mM, and glucose 5.5 mM, pH 7.8). The lens was removed from the eyeballs by extracapsular extraction and incubated in the artificial aqueous humor at room temperature.

4. Aldose reductase inhibitory activity

In vitro aldose reductase inhibitory activity was performed using the isolated goat lens based on the methods of Hayman and Kinoshita (1965). Briefly, lens homogenate (10% w/v in phosphate buffer saline, 0.1 M, pH 7.4) was prepared from normal transparent lenses and centrifuged (10,000 rpm, 30 min, 4℃) to collect the lens supernatant. Furthermore, 3.5 mL of phosphate buffer saline, 0.5 mL of lens supernatant, and 0.5 mL of NADPH (25 × 10⁻⁵ M) were mixed with 0.5 mL of phosphate buffer saline (served as control) or 0.5 mL of plumbagin (100 µg/mL, served as test) in a sample tube to determine the lens aldose reductase activity. The final reaction mixture was adjusted to a pH of 6.2. The reaction was started by adding 0.5 mL of DL-glyceraldehyde (5 × 10⁻⁴ M), and the reaction rate was observed at 340 nm for 3 min. The unit activity of aldose reductase was calculated for each sample (control and test). A unit of activity was defined as a change in absorbance of 0.001 units per min [18, 19]. The percentage of aldose reductase inhibitory activity was further calculated by using the following formula:

$$\text{AR inhibitory activity (\%) =}\frac{\text{(AR activity of control-AR activity of test sample)}}{\text{AR activity of control}}\times 100$$

5. Molecular docking for lens aldose reductase

The Molegro Virtual Docker (MVD 6.0, Denmark) was used to simulate the binding affinity of plumbagin (PubChem CID: 10205) in human lens aldose reductase (RCSB PDB ID: 4JIH, Homo sapiens) and compared with the standard epalresat (PubChem CID: 1549120) [20]. The BIOVIA Discovery Studio Visualizer (Dassault Systèmes, France) was used to visualize the 3D and 2D graphical structures and H-bond interactions.

6. Ex vivo anticataract activity

A total of 55 mM glucose was used to induce cataract. At high concentrations, glucose is metabolized through the sorbitol pathway. The accumulation of sorbitol increases oxidative stress levels, which causes cataract development. This ex vivo anticataract study used 30 lenses. The lenses were incubated in the artificial aqueous humor with different glucose concentrations [21]. Goat lenses were divided into different groups, each group containing six lenses. The concentration of plumbagin was selected based on the preliminary effect of the DPPH assay and aldose reductase inhibitory activity. The following five groups were investigated.

Group I: glucose 5.5 mM (normal control)

Group II: glucose 55 mM (cataract control)

Group III: glucose 55 mM + plumbagin 20 µg/mL (plumbagin-20)

Group IV: glucose 55 mM + plumbagin 50 µg/mL (plumbagin-50)

Group V: glucose 55 mM + plumbagin 100 µg/mL (plumbagin-100)

7. Observation of lens transparency

After incubating the goat lenses in their respective incubating media for 8 h, their lens transparency was measured using the photographic method. The lenses were placed carefully on a graph paper to evaluate the visibility of the graph line, grading the transparency as 0 (transparent lens), 1 (slightly opaque lens), 2 (moderately opaque lens), and 3 (opaque lens).

8. Preparation of lens homogenate

The lens homogenate was prepared and used to determine different oxidative stress markers by the spectrophotometric method. The lens homogenate was prepared in 0.1 M potassium phosphate buffer (10% w/v, pH 7.4) and was centrifuged at 10,000 rpm for 1 h. The supernatant was used for the biochemical analysis.

9. Biochemical analysis

Oxidative stress markers (catalase [CAT], superoxide dismutase [SOD], glutathione reduced [GSH], and malondialdehyde [MDA]) and lens protein contents were evaluated using the spectrophotometric method described in a previous study [22]. The ability of the enzyme to convert hydrogen peroxide into water was used to evaluate CAT activity, which was then represented as micromoles of hydrogen peroxide consumed per min. Based on hydrogen peroxide decomposition, the enzymatic activity was assessed by the method of Sinha 1972 [23]. SOD was measured based on nitroblue tetrazolium reduction [24]. GSH level in lenses was measured using the Ellman reagent used for this method [25]. MDA is an end-product of lipid peroxidation. The level of MDA was measured using thiobarbituric acid [26]. Alkaline copper solution and phenol reagent were used to assess the insoluble and soluble proteins in the lens homogenate. The colored product was estimated at 610 nm, with bovine serum albumin used as a standard [27].

10. Prediction of possible targets by network pharmacology tools

Various tools were used to map the network pharmacology. Smile of plumbagin was accessed from PubChem (CID 10205), which was further used to predict possible targets by the SuperPred online tool (https://prediction.charite.de/index.php). The SuperPred predicted 95 possible targets (4 strong and 91 additional targets), of which 29 targets were screened based on the highest probability (≥ 70%). Multiple protein names were identified for Homo sapiens organisms using the UniProt ID of selected targets in the STRING database (STRING 12.0, https://string-db.org). The GeneCard database (https://www.genecards.org/) was used to find the disease target genes (cataract targets). Eighty genes were selected from 9573 genes based on the gift score (> 65). Plumbagin targets (29) and cataract targets (80) were further mapped by Vinny 2.1 tools (https://bioinfogp.cnb.csic.es/tools/venny/), indicating that 6 target genes overlapped. The overlapped genes were used in the STRING database to assess the network status, as well as the protein-protein interaction (PPI), and find the KEGG pathways using the following conditions: Homo sapiens species and the reliability at a medium confidence of ≥ 0.4. Over the 24 KEGG pathways, 6 pathways that are mostly involved in cataract were selected. After mining the target genes from the KEGG pathways, a drug-target-pathway network was developed using Cytoscape v3.7.2. All online databases were accessed on 30 December 2024 and are available in Supplementary Files.

11. Statistical analysis

The Graph Pad Prism 5.0 software was used to analyze and calculate the mean and standard error of the mean (SEM). The significant (p < 0.05) difference between groups was statistically analyzed by analysis of variance (ANOVA).

RESULTS

1. In vitro antioxidant activity

The antioxidant activity of plumbagin at 100 µg/mL showed a 46.90% ± 0.69% inhibition of DPPH radical (Fig. 1). Hence, plumbagin has a potent antioxidant activity.

Figure 1.

Effect of plumbagin on DPPH assay and Aldose reductase inhibitory activity. Results were expressed as mean ± SEM (n = 3).

2. Aldose reductase inhibitory activity

Plumbagin (100 µg/mL) reduced the lens aldose reductase activity by 44.52% (Fig. 1), which was also reflected in an in silico study. The in silico study demonstrated that plumbagin has a considerable binding affinity with the selected lens aldose reductase enzyme, similar to standard epalrestat (Table 1 and Fig. 2). Conventional H-bond interaction analysis showed that Lys 78 is a favorable site for epalrestat, and Lys 22 and Asp 217 are the favorable sites for plumbagin.

Table 1.

Molecular docking data of plumbagin on human lens aldose reductase (RCSB PDB ID: 4JIH, Homo sapiens)

SN	Ligands	MolDock score	Conventional H-Bond interaction
1	Epalrestat	–148.49	Lys 78
2	Plumbagin	–93.49	Lys 22 Asp 217

Figure 2.

Graphical representation of virtual molecular docking and drug-protein interaction of (A) epalrestat and (B) plumbagin.

3. Effect on lens transparency

According to the visual observation, lenses were opaque in the cataract control group compared to transparent lenses in the normal group (Fig. 3). However, the lenses of the plumbagin-treated groups showed slight-to-moderate opacity, which was reflected in the cataract score, with Fig. 4 providing information about cataract scores. High glucose significantly induced cataract in the cataract control group compared to the normal control group (p < 0.05). However, plumbagin-50 and plumbagin-100 groups showed a significant inhibition of cataract formation compared to the cataract control group (p < 0.05).

Figure 3.

Effect of plumbagin on lens transparency. (A) Normal control, (B) Cataract control, (C) Plumbagin-20, (D) Plumbagin-50, and (E) Plumbagin-100.

Figure 4.

Effect of plumbagin on cataract grading score. Results (mean ± SEM, n = 6) were analyzed by one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs normal control group and ^#p < 0.05 vs cataract control group.

4. Effect on oxidative stress markers and protein contents

Table 2 presents the results regarding oxidative stress markers. The impact of plumbagin on lenticular oxidative stress was tested in a glucose-induced cataract model. The level of lenticular antioxidants, such as CAT, SOD, and GSH, decreased significantly (p < 0.05 vs. normal control), while the MDA levels were significantly increased (p < 0.05 vs. normal control), in the cataract control group. However, the level of lenticular antioxidants was significantly increased in the plumbagin-treated groups (p < 0.05 vs. cataract control), and the MDA level was significantly decreased (P < 0.05 vs. cataract control).

Table 2.

Effect of plumbagin on oxidative biomarkers

S.N.	Group	GSH (µmole/g)	CAT (µmole H2O2 consumed/min)	SOD (U/g)	MDA (nmole/g)
1	Normal	13.87 ± 0.42	76.97 ± 4.25	15.62 ± 1.04	2.63 ± 0.31
2	Cataract control	4.983 ± 0.41*	20.58 ± 2.52*	4.76 ± 0.48*	16.50 ± 1.19*
3	Plumbagin-20	8.707 ± 0.22#	50.48 ± 2.77#	9.18 ± 0.61#	9.98 ± 0.39#
4	Plumbagin-50	10.84 ± 0.51#	63.23 ± 3.46#	11.30 ± 0.99#	6.47 ± 0.23#
5	Plumbagin-100	13.87 ± 1.09#	65.20 ± 3.08#	14.60 ± 0.90#	3.51 ± 0.47#

Results (mean ± SEM, n = 6) were analyzed by one-way ANOVA followed by Tukey’s post hoc test.

*p < 0.05 vs normal control group and #p < 0.05 vs cataract control group.

Fig. 5 presents the result regarding lens protein content. The cataract control group showed a significantly decreased level of soluble protein (p < 0.05 vs. normal control) and a significantly increased level of insoluble protein content (p < 0.05 vs. normal control), which was restored by plumbagin (p < 0.05 vs. cataract control).

Figure 5.

Effect of plumbagin on lens soluble (A) and insoluble protein (B) contents. Results (mean ± SEM, n = 6) were analyzed by one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs normal control group and ^#p < 0.05 vs cataract control group.

5. Prediction of possible targets

Six genes were identified as potential targets for the PPI (Fig. 6): TP53 (tumor protein p53), CTSD (cathepsin D), HDAC2 (histone deacetylase 2), CDK5 (cyclin-dependent kinase 5), SLC2A1 (solute carrier family 2 member 1), and STAT3 (signal transducer and activator of transcription 3). The PPI network analysis by the STRING database showed 6 nodes, 9 edges, 3 average node degrees, 0.73 average local clustering coefficient, and 0.042 PPI enrichment p-value. Over the 24 KEGG pathways, 6 pathways were selected (Table 3). Fig. 6 shows that SLC2A1, STAT3, and TP53 are the major target proteins involved in almost all selected pathways.

Figure 6.

Identification of possible targets for protein-protein interaction. (A) Venn diagram of plumbagin and cataract-related targets, (B) protein-protein interaction of selected targets, and (C) visualization of drug-protein targets-signaling pathway.

Table 3.

Selected KEGG pathways for protein-protein-interaction prediction

SN	Pathways	Matched protein
1	Thyroid hormone signaling pathway	TP53, SLC2A1, HDAC2
2	Adipocytokine signaling pathway	STAT3, SLC2A1
3	HIF-1 signaling pathway	STAT3, SLC2A1
4	Insulin resistance	STAT3, SLC2A1
5	Sphingolipid signaling pathway	CTSD, TP53
6	Apoptosis	CTSD, TP53

DISCUSSION

Phytochemicals, which are currently the trending source of phytomedicines, biomolecules, and nutrients, have various pharmacological actions, including antioxidant, aldose reductase inhibitory [8], and anticataract activities [28]. The exacerbation of the polyol pathway/aldose reductase pathway is the most prominent pathophysiological pathway of diabetic complications, including cataract [29]. During diabetes, glucose, at high concentrations, converts into sorbitol by the aldose reductase activity within the lens. The excessively produced sorbitol (impermeable to the cell membrane of the lens) accumulates in the lens and leads to osmotic stress and swelling, resulting in the progression of lens opacification and cataract formation [30]. Moreover, the activation of the polyol pathway increases advanced glycation end products and oxidative stress, leading to diabetic ocular complications [31]. Therefore, this study assessed the aldose inhibitory, antioxidant, and anticataract activities of the selected phytoconstituent plumbagin using experimental pharmacological models.

The in silico study showed that plumbagin has a considerable affinity for lens aldose reductase, which was further assessed by the in vitro aldose reductase inhibitory activity in the goat lens. Plumbagin considerably inhibited the lens aldose reductase enzyme activity, which is corroborated by a study by Demir et al. (2019) [13]. They assessed the inhibitory activity of quinones against kidney aldose reductase and found that all quinones, including plumbagin (IC50 1.05 µM), exhibited non-competitive inhibition. Thus, plumbagin demonstrated a potentially beneficial role against diabetic ocular complications.

Moreover, plumbagin exhibited a potential anticataract activity in the glucose-induced ex vivo model, as indicated by the retention of lens transparency in plumbagin-treated groups. The loss of transparency of the lens depends upon multiple risk factors. In the glucose-induced model, the incubation of lenses under a high concentration of glucose (55 mM) led to cataractogenesis by the activation of the polyol pathway and induction of oxidative stress due to excessive production of reactive oxygen species such as superoxide anion and hydrogen peroxide [32], which is reflected in this study. This study showed that the lens incubated with the plumbagin considerably retained its transparency. Furthermore, lenticular oxidative stress was reduced, as indicated by increased lens antioxidants (GSH, CAT, and SOD) and a reduced lipid peroxidant (MAD). The lens antioxidants play a crucial role in protecting the lens from oxidative damage. SOD is responsible for the conversion of superoxide anion into hydrogen peroxide [33]. CAT converts hydrogen peroxide into water and regulates the level of hydrogen peroxide within the cells. GSH maintains the -SH group on the lens protein in a reduced form and attenuates the cross-linkage of lens crystalline protein [34]. Increased reactive oxygen species (oxidants) attack cellular proteins, lipids, and nucleic acids. The enhanced MDA level in the cataract group indicated the oxidative damage of lenticular lipids. Since the lenses exposed to plumbagin showed reduced MDA level, plumbagin was anticipated to prevent the structural modification and cross-links between lens proteins and membrane lipids [35]. This was further affirmed by the restoration of protein contents (soluble and insoluble protein) through plumbagin exposure against glucose. The structural feature of the protein plays a crucial role in maintaining lens transparency. Loss of soluble proteins and increased insoluble proteins are the distinctive features of cataract lenses [36], which were observed in the cataract control group. The considerable restoration in the lens proteins in the plumbagin-treated group indicated the potential protective role of plumbagin against protein modification.

Additionally, after finding the beneficial effect of plumbagin against diabetic cataract, we explored possible other targets of plumbagin in its action against cataract. The results suggested 3 major target proteins for plumbagin: SLC2A1, STAT3, and TP53. SLC2A1 codes for the glucose transporter (GLUT1), which is expressed in the lens epithelium. Disruption of this gene can lead to impaired glucose transportation within the cell and exacerbate the glucose-associated cataract formation [37]. STAT3 signaling pathways regulate both the physiology and pathology of the retina and cause vision loss and cataract formation. Inhibiting STAT3 may alleviate diabetic retinopathy and cataract severity in streptozotocin-induced animals [38]. TP53 is mainly associated with age-related cataract and is involved in apoptosis and oxidative stress [39].

CONCLUSION

Plumbagin (100 µg/mL) showed the most potent anticataract activity in the ex vivo glucose-induced model. Plumbagin exposure prevented cataract progression, which was further reflected in pathophysiological events such as the decrease in lenticular oxidative stress levels and protein modification. The anticataract activity of plumbagin might be due to its potent antioxidant activity, which was observed in the in vitro and ex vivo models, as well as aldose reductase inhibitory activity, which was observed in the in vitro and in silico models. The current study was limited to in silico, in vitro, and ex vivo models, which may not fully replicate the complex biological environment in living organisms. Therefore, an in vivo study is needed to validate the positive effects of plumbagin against diabetic cataracts. In the future, we will explore the anticataract potential of plumbagin in in vivo animal models.

SUPPLEMENTARY MATERIALS

Supplementary data is available at https://doi.org/10.3831/KPI.2025.28.3.191.