I. INTRODUCTION:

The main cause of lens transparency is crystalline lens optical failure. It reduces the amount of light that enters the eye, causing eyesight to deteriorate. Cataract affects around a quarter of the population of 65 percent of people over the age of 80¹. Due to elevated levels of glycosylated hemoglobin, cataract development is more common in diabetic patients with advanced age².

Cataract progression is usually slow and steady, but it can happen suddenly in some cases, and it usually affects both eyes¹. The activity of the lens's Na+ — K+ -ATPase plays a vital role in maintaining the lens's transparency, and its imbalance causes Na+ deposition and K+ loss, as well as water absorption by the lens fibers, leading to cataract formation³. The enzyme aldose reductase is known to play a role in the development of eye conditions such as cataracts⁴.

Fig. 1: Glucose induced cataract

Reduced vision clarity is a defining symptom of cataract. Clinically, mild cataracts that do not severely impair vision can be detected. The patient should be informed that the existence of opacity in the lenses does not always need surgical intervention. When vision loss makes it difficult to carry out daily activities, cataract extraction should be considered¹. The aldose reductase converts sugar molecules such as glucose, galactose, and xylose into their corresponding alcohols via metabolic pathways. These alcohols, known as polyols, accumulate within the lens, causing osmotic symptoms. As a result, polyols do not easily diffuse out and do not degrade quickly, causing hyper tonicity, which leads to cataract formation⁵. The oxidative pathway is also important in biological processes such as cataract development. The production of superoxide radicals in the aqueous humor and in the lens, as well as their derivatization to other strong oxidants, may be responsible for triggering a variety of metabolic damaging processes that contribute to cataract formation⁶.

The formation of lens opacities in the elderly is attributed to a variety of factors including sunshine, UV radiation, diabetes, dehydration, oxidation of lens protein and peroxidation of lipid, and hyperglycemia. It is also caused by damage to the long-lived lens protein, which occurs as a result of oxidation due to the creation of reactive radicals, as well as other risk factors such as nutritional deficiencies, sunshine, environmental variables, a lack of antioxidant consumption, and diabetes¹. Due to antioxidant and free radical scavenging properties, ascorbic acid (ACE Inhibitor) was proven to have substantial anti cataract activity in vitro^7,8. As a result, we use Ascorbic acid as a standard and test several parameters in vitro on goat lenses, such as (Na +& K+) estimate, Na+ -K + ATPase activity, Proteins (total proteins and water soluble proteins), and malondialdehyde (MDA)⁹.

Oxidative stress:

Life's uncertainty Fortunately, most other compounds react slowly with oxygen in the air around us. The superoxide radical (O2-) is formed when an additional, unpaired electron (reduction) is added to the oxygen molecule, weakening the oxygen-oxygen link, and making it somewhat more reactive. Free radicals are species that possess one or more unpaired electrons and are capable of autonomous existence. Free radical reactivity varies greatly, and some non-radical oxygen-derived chemicals are more reactive than oxygen free radicals. The term "reactive oxygen species" (ROS) is more commonly used because it encompasses both oxygen free radicals and non-radical O2 derivatives ¹.

II. MATERIAL AND METHODS:

Extraction of Anthocyanin from grapes:

Fig. 2: Grapes

Flask with a capacity of 200 mL was filled with 10 g of freeze-dried grape skin powder. The flask was filled with 100 mL of methanol and aqueous solutions of formic acid. A KQ2200B ultrasound instrument was used to sonicate the extract for a specified period before transferring it to a water bath for extraction at a specified temperature and fixed amount of time. After centrifugation, the supernatant was discarded. The same process was followed when extracting the solid residues five times. The gathered supernatants were all combined. They were vacuum-dried at 300°C using a RE-52AA rotary evaporator, and after that, their pH was adjusted to 3.7 by dissolving them in 100 mL of HPLC-grade methanol. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the final samples started right away. For each treatment, duplicates were carried out. The following table displays the full protocol¹⁰.

Table 1: Orthogonal test with 4 factors for extraction of anthocyanin from grape skins ¹⁰

Treatment

Formic acid / Methanol

(v/v)

Ultrasonic

Time

(min)

Extraction temperature

(⁰C)

Extraction

Time (hour)

0/100

2/98

1.5

Fig. 3: Extraction of Anthocyanin from grapes

Requirements¹:

Test Drug: Anthocyanin

Chemicals:

Sodium chloride (NaCl), Potassium chloride (KCl), Magnesium chloride (MgCl₂), Sodium bicarbonate (NaHCO₃), Calcium Chloride (CaCl₂), Glucose, Penicillin, Streptomycin, Ascorbic acid.

Instruments:

Incubator, Wired mesh, Petri dish.

Dose selection:

Anthocyanin T1 and T2- 15, 30 and 60µg/ml, Standard Ascorbic Acid: 40µg/ml.

Collection of Eyeballs:

Goat eyes were used in this experiment. They were transported to the lab at a temperature of 0-4°C, right from the slaughterhouse.

Procedure:

Lens Culture:

“A Fresh goat eyeballs were given to the lab at a temperature of 0-4°C virtually immediately after being obtained from the butcher. Extra capsular extraction was utilized to extract the lens, which was subsequently cultured at room temperature in unreal aqueous humor (NaCl 140mM, KCl 5mM, MgCl2 2mM, NaHCO3 0.5mM, NaHPO4 0.5mM, CaCl2 0.4mM, and glucose 5.5mM) with pH 7.8 maintained by NaHCO3 infusion. Penicillin G 32% and streptomycin 250mg% were added to the culture media to prevent bacterial contamination. The lens was metabolized at high doses by the sorbitol pathway, resulting in polyol accumulation, over hydration, and oxidative stress. The upshot of this is cataractogenesis”¹.

Induction of in-vitro cataract:

“Glucose at a concentration of 55mM was used to cause cataracts. The sorbitol pathway metabolizes a large amount of glucose in the lens. Polyols (sugar alcohols) build up in the body, producing oxidative stress and over hydration. As a result, cataractogenesis occurs. These lenses were cultivated in artificial aqueous humor for 72 hours with varying glucose concentrations (5.5mM as a normal control and 55mM as a hazardous control)”¹.

Study Design and Groups:

Goat lenses were divided into nine groups of nine lenses each following table:

Table No. 1: Study design and Groups ⁹

Group No.	Group Name	Treatment	Drug Dose
I	Normal control	Aqueous Humor + 5.5mM Glucose	1:1
II	Negative control	Aqueous Humor + 55mM Glucose	1:1
III	Standard (Positive control)	Aqueous Humor +55mM Glucose + Standard (AscorbicAcid)	40 µg/ml
IV	A Test I	Aqueous Humor + 55mM Glucose +Anthocyanin T1	15 µg/ml
V	A Test II	Aqueous Humor +55mM Glucose +Anthocyanin T1	30 µg/ml
VI	A Test III	Aqueous Humor +55mM Glucose + Anthocyanin T1	60 µg/ml
VII	B Test I	Aqueous Humor + 55mM Glucose +Anthocyanin T2	15 µg/ml
VIII	B Test II	Aqueous Humor +55mM Glucose +Anthocyanin T2	30 µg/ml
IX	B Test III	Aqueous Humor +55mM Glucose + Anthocyanin T2	60 µg/ml

Photographic Evaluation:

“Lens opacity was tested by placing lenses on a wired mesh with the posterior surface touching the mesh and counting the number of squares plainly visible through the lens. The degree of opacity was assessed in the following manner” ¹.

Table No. 2: Degree of Opacity ¹

Grade	Degree of Opacity
0	absence of opacity
1	slight degree of opacity
2	presence of diffuse opacity
3	presence of extensive thick opacity

Preparation of lens homogenate

“Lenses were homogenized in 0.23M tris buffer (pH 7.8) containing 0.25 10-3M EDTA after 72hours of incubation, and the homogenate was adjusted to 10% w/v, centrifuged at 10,000 G for 1 hour at 40C, and the residue was used to estimate biochemical parameters”¹. “Homogenate was produced in sodium phosphate buffer to estimate water-soluble proteins (pH-7.4)”⁹.

Biochemical Analysis:

“The electrolytes sodium and potassium (Na+ and K+) were detected using flame photometry. With the help of Unakar and Tsui method”¹¹ protein was calculated using Lowry's method after the activity of sodium potassium ATPase was determined¹². “Wilbur's approach was used to determine the level of oxidative stress”¹³.

Estimation of total protein content:

“To 0.1mL lens homogenate, 4.0mL alkaline copper solution was added and allowed to sit for 10 minutes. Then, for color development, 0.4mL of phenol reagent was quickly added and mixed, and the mixture was incubated at room temperature for 30minutes. Readings were taken in a UV-visible spectrophotometer against a blank of distilled water at 610nm. The protein content of the lens tissue was calculated using a bovine serum albumin standard curve and expressed as g/mg”¹.

Statistical Analysis:

“The mean standard deviation was used to present all data. All data was examined using the SPSS/10 student programme. The one-way analysis of variance (ANOVA) and the least significant difference (LSD) are two methods for evaluating hypotheses. If P0.05, the results were considered significantly different, and the data were expressed as mean S.D. Normal Goat lens vs Goat lens + Glucose55mM, Goat lens + Glucose55mM vs Goat lens + Glucose55mM + alpha lipoic acid are the statistical variations compared”¹.

III. RESULT:

In- vitro anti-cataract activity:

After 8 hours of incubation, lenses with glucose 55mM demonstrate opacification on the lens's periphery and posterior surface. Complete opacification is gradually increased to words the center at the end of 72hours.

Photographic evaluation:

The transparency of the Group I (normal control group) was preserved after 72hours of incubation, whereas the transparency of the Group II (negative control group) was completely lost, showing complete cataractogenesis. Squares of graph paper were visible through the lenses in Group III (Standard/Positive control), which contained lenses treated with conventional ascorbic acid. The squares of the graph paper were visible through the lenses of goat lenses in groups with increased amounts of anthocyanin (Group IV, V, VI, VII, VIII, IX), showing that cataract formation was suppressed. Group VI (60µg/ml) [Test III] was more efficient than Group IV [Test I] and Group V [Test II] in preventing cataract formation.

Table No. 3: Effect of anthocyanin on degree of opacity on lens by glucose induced cataract ¹

Sr. No.	Compound	Degree of Opacity
1	Normal	0
2	Negative control (Glucose 55 mM)	3
3	Positive control (Ascorbic acid 40µg/ml)	1
4	A Test I (Anthocyanin T1 15µg/ml)	2
5	A Test II(Anthocyanin T1 30µg/ml)	1
6	A Test III (Anthocyanin T1 60µg/ml)	0
7	B Test I (Anthocyanin T2 15µg/ml)	2
8	B Test II(Anthocyanin T2 30µg/ml)	1
9	B Test III(Anthocyanin T2 60µg/ml)	0

Table No. 4: Effect of anthocyanin on goat lenses by glucose induced cataract⁹

I	Normal Group	0-degree opacity occurred; clear lens is obtained.
II	Negative Control (Glucose 55 mM)	The presence of extensive thick opacity, because of higher concentration of glucose- induced cataractogenesis.
III	Positive Control (Ascorbic acid 40 µg/ml)	Lenses show the slight degree of opacity, clear lens was not found.
IV	A Test I (Anthocyanin T1 15µg/ml)	Lenses show the slight degree of opacity, clear lens was not found.
V	A Test II (Anthocyanin T1 30µg/ml)	Lenses show the slight degree of opacity, clear lens was not found.
VI	A Test III (Anthocyanin T1 60µg/ml)	0-degree opacity is occurred; the clear lens is obtained. Test drug inhibits cataractogenesis.
VII	B Test I (Anthocyanin T2 15µg/ml)	Lenses show the slight degree of opacity, clear lens was not found.
VIII	B Test II (Anthocyanin T2 30µg/ml)	Lenses show the slight degree of opacity, clear lens was not found.
IX	B Test III (Anthocyanin T2 60µg/ml)	0-degree opacity is occurred; the clear lens is obtained. Test drug inhibits cataractogenesis.

Table No.5: Effect of Anthocyanin on Protein levels (total proteins and water-soluble proteins) in Goat lens homogenate after 72 hours of incubation in glucose 55 mM induced cataract

Group No.	Treatment	Total proteins [mg/gm]	Water-Soluble proteins [mg/gm]
I	Normal control (Glucose 5.5mM)	203	81.33
II	Negative control (Glucose 55mM)	168.5 ##	62.16##
III	Standard (Glucose 55mM +Ascorbic Acid 40μg/ml)	208.5	72.33
IV	A Test I (Glucose 55mM + Anthocyanin T1 15μg/ml)	181.66	70.16
V	A Test II (Glucose 55mM + Anthocyanin T1 30μg/ml)	206.5	70.83
VI	A Test III (Glucose 55mM + Anthocyanin T1 60μg/ml)	213.5****	76.5****
VII	B Test I (Glucose 55mM + Anthocyanin T2 15μg/ml)	182.52	70.11
VIII	B Test II (Glucose 55Mm + Anthocyanin T2 30μg/ml)	205.8	71.81
IX	B Test III (Glucose 55mM+ Anthocyanin T2 60μg/ml)	214.7****	77.8****

IV. DISCUSSION:

Anti-cataract activity:

Proteins, malondialdehyde (MDA), and electrolytes sodium and potassium are some of the variables that are typically considered in cataractogenesis (total proteins including water soluble proteins). After being incubated in a solution with a 55mM glucose concentration, the goat lens homogenate's total protein content and water-soluble protein were depleted. Alterations in the Na+ /K+ ratio brought on by decreased Na+ /K+ ATPase activity in the lens result in hydration, inflammation, and protein composition changes in the lens. These two elements each play a role in cataract formation. In this study, groups treated with ascorbic acid and anthocyanin exhibited increased levels of total and water-soluble proteins. Treatment with ascorbic acid and anthocyanin seems to lessen the pathophysiology of cataract formation. They might be able to scavenge free radicals, which could explain this. The lens was clear due to the low quantity of glucose, which had no influence on the lens, and the number of squares could be seen through the lens after 72hours of incubation in aqueous humor with 55mM glucose. The lens had a translucent quality to it.

In the negative control, the lens was incubated for 72 hours with aqueous humor and a 55mM glucose solution to raise glucose concentrations, which cause the lens to become dehydrated and undergo oxidative stress when they are metabolized by the sorbitol route. The upshot of this is cataractogenesis. The lens is very thickly opaque. Following a 72hour incubation period in the standard medication of Aqueous humor + 55mM Glucose + 40 g/ml Ascorbic acid, the number of squares visible through the lens in the Standard group was compared to Test-3, and the lens displayed little degree of opacity.

The number of squares were not clearly visible through the lens in Test-1 and Test-2 after 72hours of incubation with Aqueous humor + 55mM Glucose + 15g/ml and 30 g/ml anthocyanin test drugs, however the lens in anthocyanin 60g/ml test-3 drug revealed a small opacity. Because the test drug inhibits cataractogenesis and oxidative stress, Test-3 measured the number of squares that could be seen through the lens after 72hours of incubation in Aqueous humor + 55mM Glucose + 60 g/ml anthocyanin. In this case, the lens was clear.

An effective in-vitro model for cataract development employing a 55mM glucose solution is achieved on an isolated goat lens. Goat lenses developed cataracts after being incubated in solution with a higher concentration of glucose (55mM), and as opacity grew, Na+/K+ - ATPase activity significantly decreased. A Na+/K+ ATPase deficiency encourages sodium accumulation and potassium loss, which leads to cataractogenesis along with hydration and inflammation of the lens fibres. The Na+/K+ ratio affects the protein content of the lens, causing a drop in total proteins and lens opacification.

Na+ and K+ imbalances were prevented by anthocyanin's ability to balance the polyol pathway by lowering aldose reductase activity, sorbitol concentration, and intracellular glucose. The adoptogenic properties of anthocyanin are responsible for this effect. In a goat's eye, a cataract caused by glucose was evaluated to see how anthocyaninmight affect it. Anthocyaninsignificantly protected the lens morphology and activity, and 50% of the eyes had nearly clear lenses. In contrast, 100% of the negative control eyes showed thick nuclear opacity. The current study suggests that anthocyanin shields the lens from oxidative damage. These results in glucose-induced cataracts in culture not only show anthocyanin's protective effects but also raise the possibility that its antioxidant qualities prevent the development of cataracts. Anthocyaninmay therefore be useful for treating or preventing cataracts. Unlike regular lenses, those that are cultured in glucose 55mM for 72hours turn completely opaque.

When lenses were incubated with anthocyaninand ascorbic acid at both doses, the opacification process seemed to move more slowly than when lenses were treated with glucose 55mM. (Negative Control). The effect of anthocyaninshowed a considerable delay in the progression of lens opacification, which was close to normal, as compared to the negative control group.

V. CONCLUSION:

The results of this study showed that treatment with anthocyaninenhanced the number of water-soluble proteins and slowed the progression of cataractogenesis brought on by high glucose levels. anthocyaninhas an anti-cataract effect because to its antioxidant properties, which may be helpful in preventing cataract formation. The following elements may contribute to anthocyanin's anti-cataract efficacy and its mechanism of action:

· Anthocyaninsuppresses advanced glycation and glyco-oxidation due to the lens's decreased oxidative stress and the end products of glycation, such as protein, lipid, and nucleic acid, produced during the development of cataract.

· The anti-oxidant qualities of anthocyaninalso inhibit the production of reactive oxygen species, which inhibits cell division, apoptosis, and malfunction while also having an anti-cataract impact.

By raising the water content of lens proteins, anthocyaninmay also aid in the prevention of cataractogenesis. Anthocyanin's method of action requires further study, which could have a big impact on how cataract patients are treated in the future.

VI. ACKNOWLEDGEMENT:

We are thankful to PDEA’s Seth Govind Raghunath College of Pharmacy, Saswad, Pune, Maharashtra, India for providing laboratory facility for this research work.

VII. CONFLICT OF INTEREST:

All authors mentioned above declare that, they have no conflict of interest.

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Received on 29.04.2023 Modified on 30.10.2023

Asian J. Pharm. Res. 2024; 14(2):141-147.

DOI: 10.52711/2231-5691.2024.00024


I. Normal Control	II. Negative Control	III. Positive Control

IV. A TI: Anthocyanin 15µg/m	V. A TII: Anthocyanin l30µg/m	VI. A TIII: Anthocyanin l60µg/ml

VII. B TI: Anthocyanin 15µg/ml	VIII. B TII: Anthocyanin 30µg/ml	IX. B TIII: Anthocyanin 60µg/ml