“Evaluation of in-vitro anti-cataract activity of Abies pindrow & Luffa cylindrica”: A Comparative study

Suchita Dubey, Sudipta Saha, Shubhini A Saraf*

Abstract-

Context- Cataract is the opacification of lenses resulting in blurring of vision.Oxidative stress is the major cause of many diseases including cataract.

Objective- The study was designed to evaluate and compare the in-vitro anti-cataract activity of aqueous extracts of Abiespindrowleaves (APE) and Luffacylindrica fruits (LCE) against hydrogen peroxide induced cataractogenesis, using isolated goat lenses.

Materials and Methods-Standardized extracts of APE and LCE were compared for their anti-cataract activity against marketed eye drops. Hydrogen peroxide (0.05M) was used to induce cataract in goat eye lenses. Photographic evaluation confirmed the clarity of lenses incubated in APE and LCE respectively.

Result-.MeanGSH Value in normal lenses was found to be 2.9765±0.17µg/mg of fresh weight of the lens.In the presence of APE and LCE respectively, a significant restoration of the levels of GSH as compared to the toxic control was observed. An increase in MDA level was found in the control opposed to the normal lenses (1.57±0.04μmol/g of fresh weight of lens; P<0.01). SOD level was found to be significantly higher in normal lens group (1.76±0.10unit/mg of protein) than the toxic group (0.23±0.01unit/mg of protein).

Discussion -SOD, GSH, TPC and copper-induced lipoprotein diene formation was found to increase, whereas MDA levels significantly decreased, as the concentration of APE and LCE increased.

Conclusion: The study demonstrated that monotherapy of APE and LCE respectively, has the potential to prevent cataract because of the strong antioxidant potential of each.

Keywords- Antioxidant, Cataract, Hydrogen Peroxide, Abies pindrow, Luffa cylindrica

Introduction-

Oxidative stress has been the major cause of many diseases including cataract. The free radicals that are produced as the result of daily stress borne by the human body are scavenged by a range of antioxidant enzymes and small molecule antioxidants. Cataract is the opacification of lenses resulting in the blurring of vision. Since ancient times, India’s conventional medicinal knowledge has been extraordinarily useful in treating rarest of the rare diseases. The term cataract describes lenticular opacities that may be congenital or acquired. Systemic diseases (such as galactosemia, diabetes mellitus, Wilson disease & atopic dermatitis), drugs (especially corticosteroids), radiation, trauma, and many intraocular disorders are associated with cataract. Traditional medicines have proved to be effective to an extent in curing cataract. Plants such as Embellica Officinalis (Amla) and those which are rich in gallic acid, digallic acid, ellagic acid, and tannins respectively serve as good anticataract options.[1]

The development of the disease depends on many factors.The lens Na+- K+-ATPase activity plays an important role in maintaining lens transparency, and its impairment causes accumulation of Na+ and loss of K+ with hydration and swelling of the lens fibres leading to cataractogenesis[2] . In conjunction, aldose reductase is a lens enzyme probably involved in the development of cataract [3]. It acts on the sugars like glucose, galactose, and xylose and converts them into their respective alcohols. These alcohols, also known as polyols: accumulate within the lens thereby producing osmotic effects. Since polyols are not capable of either diffusing out easily nor are metabolizes rapidly, they may cause hyper tonicity responsible for the formation of cataract [4].

Oxidative mechanism plays an important role in biological phenomena including cataract formation. The formation of superoxide radicals in the aqueous humor and in lens and its derivatization to other potent oxidants may be responsible for initiating various toxic biochemical reactions leading to the formation of cataract. Catlin is the marketed drug with considerable anticataract activity hence was taken as standard and various parameters measured, including total proteins and malondialdehyde (MDA) in vitro on goat lenses.

Materials and Methods-

Plants- Standardized extracts of “Abies pindrow leaf” and “Luffa cylindrica fruit” were obtained from Navchetna Kendra, New Delhi (A registered manufacturer, exporter and supplier of herbal products in India) along with the certificate of analysis of both the extracts confirming that extract complies with all the morphological specification of colour, odour, taste along with LOD, Ash value and microbial load (Total Plate Count, yeast and mould and E.coli).

Drug- Catlin eye drop available in the market were purchased from medical store in Lucknow, UP.

Eye Balls-Goat eye balls were used in the present study. They were obtained from the slaughterhouse and immediately transferred to laboratory at 0-4 degree Celsius in physiological salt solution containing 1% solution of antibiotic to prevent microbial contamination.

Preparation of Lens Culture- The lenses were removed by extracapsular extraction and incubated in artificial aqueous humor (NaCl – 140 mM, KCl – 5 mM, MgCl2 – 2 mM, NaHCO3 – 0.5 mM, NaH (PO4)2 – 0.5 mM, CaCl2 – 0.4 mM) at room temperature and pH 7.8 for 72 h. Penicillin 32 mg% and streptomycin 250 mg% were added to the culture media to prevent bacterial contamination [6]. H2O2 (0.05M) was used to induce cataract.

Preparation of Lens Homogenate-After incubation, lenses were homogenized in 10 volumes of 0.1M potassium phosphate buffer, pH 7.0. The homogenate was centrifuged at 10,000 rpm for 1 hour and the supernatant was used for estimation of biochemical parameters.

Drug Concentration and Groups- The standard drug Catlin was taken in the concentration of 1% v/v and the Goat’s eye lenses were incubated with Abies pindrow extract (APE) (5mg/ml, 10mg/ml, 15mg/ml & 20mg/ml) andLuffa cylindrica extract (LCE) (5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml, 25mg/ml & 30mg/ml) concentrations as mono therapy. A total of 65 lenses were divided into 13 groups of n=5.

Group 1- PSS + Ab 250 mg (1%) + lens

Group 2- PSS + Ab 250 mg (1%) + H2O2(0.05 M) 0.5ml+ lens

Group 3- PSS + Ab 250 mg (1%) + Catlin (KI-3.3%, NaCl-0.83%, CaCl2-1%w/v) 1ml

Group 4- PSS + Ab 250 mg (1%) + APE (5%)

Group 5-PSS + Ab 250 mg (1%) + APE (10%)

Group 6-PSS + Ab 250 mg (1%) + APE (15%)

Group 7-PSS + Ab 250 mg (1%) + APE (20%)

Group 8-PSS + Ab 250 mg (1%) + LCE (5%)

Group 9-PSS + Ab 250 mg (1%) + LCE (10%)

Group 10-PSS + Ab 250 mg (1%) + LCE (15%)

Group 11-PSS + Ab 250 mg (1%) + LCE (20%)

Group 12-PSS + Ab 250 mg (1%) + LCE (25%)

Group 13-PSS + Ab 250 mg (1%) + LCE (30%)

Homogenate preparation- After incubation, lenses were homogenized in 10 volumes of 0.1M potassium phosphate buffer, pH 7.0. The homogenate was centrifuged at 10,000 rpm for 1 hour and the supernatant was used for estimation of biochemical parameters.

Biochemical estimation- SOD and GSH levels were measured using Ellman’s method[6]. Protein estimation was done by Lowry’s method [7]. The degree of oxidative stress was assessed by measuring the MDA levels by using TCA-TBA-HCl reagent. [8] (Table-2,3).

The mean GSH Value in normal lenses was found to be 2.9765±0.17 µg/mg of fresh weight of the lens (Fig-5) . A significant decrease was observed in presence of in GSH value in presence of hydrogen peroxide in control. In the presence of APE and LCE, there was a significant restoration of the levels of GSH as compared to the toxic control. A significant increase in MDA level was found in the control opposed to the normal lenses (1.57±0.04μmol/g of fresh weight of lens; P<0.01 (Fig-2).

APE and LCE significantly protected the test group lenses from lipid peroxidation; Hydrogen peroxide treated lenses showed significantly low concentrations of proteins (total and water soluble proteins) in the lens homogenate (P<0.01) compared with normal group having normal lenses (Table-2). APE and LCE group had significantly higher concentrations of total lens proteins and water soluble proteins (P<0.01), compared to toxic control group (Fig-1). Copper induced lipoprotein diene formation was also effected in the same way for both the extracts as total protein content (Fig-3)

SOD levels as compared to the toxic control were found to be significantly more in normal lens group (1.76±0.10unit/mg of protein) which was far less than that of the toxic group (0.23±0.01unit/mg of protein). APE and LCE were found to increase the level of SOD in presence of hydrogen peroxide as well (Fig-4).

Result- Photographic evaluation confirmed the clearance of vision when the lenses were incubated in APE and LCE (Figure 6).

Photographic Evaluation: Scale of opacity-

Absence of opacity
Slightly opaque
Presence of diffuse opacity
Presence of extensive thick opacity
Results of biochemical parameters are reported as Mean±SD. (Table-2,3). Comparisons were made on the basis of one-way ANOVA and Bonferroni test was performed between test samples and data was considered to be statistically significant when p< 0.05 between the lenses incubated with drug and control on the basis of graphical analysis (Figure 1-5).

Discussion-Cataract is mostly brought about by age. It is common to older people. Inflammatory reactions to the lens material may develop as a result of the exposure of intact lens cortex by rupture of the lens capsule.

In cataractogenesis, the parameters commonly considered are malondialdehyde (MDA) and proteins (total proteins and water soluble proteins).

Oxidative stress is the main marker of cataract and is responsible for its pathogenesis.The study demonstrated that AP and LC are effective against H2O2 induced cataractogenesis in goat eye lens, used as in-vitro model.

Significant prevention of cataract was observed during the study. SOD, GSH, TPC and copper induced lipoprotein diene formation was found to increase proportionally with the concentration whereas MDA levels significantly decreased as the concentration increased and reached its saturation level at the concentration 20% and 30% respectively for AP and LC (Table 2, 3).

The photographic evaluation based on the opacity scale (Table-1) showed that highest concentrations of both the plants i.e. AP (20%) & LC (30%) maintained the vision for 39.5 & 42 hours respectively.

It was also observed that the lens incubated in Hydrogen peroxide swelled & imbibed more solution which can be a possible outcome of inflammation while the lenses incubated in plant groups were less swollen which further potentiates the anti-inflammatory activities of both the plants.

In conclusion, the study demonstrated that monotherapy of AP and LC had potential to prevent cataract due to their strong antioxidant potential. However, AP in lower concentration was more effective in treating cataract than LC according to results of the photographic evaluation.

There is no literature available for anticataract activity of AP and LC. This is the first study which reports that the monotherapy of both the plants can afford significant prevention of cataract.

Further studies can be performed with different routes and doses to evaluate the anti-cataract effect of these two drugs as future scope of the work.

Conclusion- The herbal extract of both the plants were found to significantly reduce the free radical generation in isolated goat lenses. The extracts can be further developed into a polyherbal formulation or characterisation and isolation of phenolics in the extract can prove to be a good herbal remedy for treatment and prevention of cataract because of potent antioxidant action of plant.

Acknowledgement- Suchita Dubey is thankful to University Grant Commission for providing research grant during M.Pharm Project.

References-

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