SUPPLEMENTARY MATERIAL

In vitro anti-cataract evaluation of standardised Abies pindrow leaf extract using isolated

goat lenses

Suchita Dubeya, Sudipta Sahab, Shubhini A Sarafb*

aAmity University Uttar Pradesh (Lucknow Campus)

bDepartment of Pharmaceutical Sciences, School of Bioscience and Biotechnology

Babasaheb Bhimrao Ambedkar University (A Central University)

Lucknow- 200265, Uttar Pradesh

India

*Corresponding Author

Shubhini A Saraf

Email: shubhini.saraf@gmail.com

Oxidative stress is known to spark off pathogenesis of cataract. Antioxidant potential of Abies

pindrow Royle leaf extract [APE] is well established in literature. In this context, standard aqueous

leaf extract of this plant was evaluated for its role in hydrogen peroxide induced cataract in isolated

goat lenses using varying concentrations (5, 10, 15, 20 mg/ml). Total phenol and flavonoidal content

was evaluated and found to be high in concentration. Biochemical parameters viz. Superoxide

dismutase (SOD), Glutathione (GSH), Total protein content (TPC) and malondialdehyde (MDA) were

evaluated. SOD, GSH and TPC formation was found to increase proportionally with increasing

concentration. However, MDA level decreased significantly as the concentration of extract increased.

The results suggest that extract under investigation can delay the onset and/or prevent the progression

of cataract. Its anticataract potential may be attributed to the presence of high phenolics and

flavonoids in APE. Photographic evaluation, further, confirmed the observation.

Keywords: Antioxidant; Cataract; Hydrogen Peroxide; Abies pindrow.

Experimental

Plant extracts

APE was obtained from Navchetna Kendra, New Delhi (A registered manufacturer, exporter and

supplier of herbal products in India) along with the certificate of analysis of the extract

(AP/02/02/2013) confirming that extract complies with all the morphological specification of color,

odor, taste, loss on drying (LOD), ash value and microbial load of yeast, mould and E. coli by total

plate count method. Hydrogen peroxide was purchased from Loba Chemie (Mumbai, India).

Streptomycin and penicillin were obtained from Hindustan Antibiotics Ltd. (Pune, India). Marketed

formulation (eye drops) containing potassium iodide, available for treatment and prevention of

cataract, was purchased from a medical store in Lucknow, India. All the chemicals used during the

study were of analytical grade.

Preparation of Lens Culture

A total of 35 lenses, isolated from goat eye ball through extracapsular extraction, were used. These

lenses were further divided into two categories of 7 groups containing 5 lenses each. Visibly limpid

lenses were incubated in aqueous humor containing sodium bicarbonate (0.9 g/ml), streptomycin (100

µg/ml) and penicillin (100 IU/ml) at 37°C in an incubator with 95% air and 5% CO2. The lenses were

incubated initially for two hours to discard any lens that had opacified due to damage during the

dissection procedure. Two ml of H2O2 (0.5 mM) was used as cataract inducer. The quantity of the

marketed formulation (Positive control: KI- 3.3% w/v, NaCl- 0.83% w/v, CaCl2- 1% w/v) used was 1

ml. APE was added in varying concentration of 5, 10, 15, 20 mg/ml of lens culture respectively.

Control group

a. Normal control: lens + Aqueous humor + antibiotic solution

b. Negative control: lens + Aqueous humor + antibiotic solution + H2O2 solution

c. Positive control: lens + Aqueous humor + antibiotic solution + H2O2 solution + KI

solution

A. Experimental group

a. APE treated: lens + Aqueous humor + antibiotic solution + H2O2 solution + APE

solution (Varying Concentrations of 5, 10, 15, 20 mg/ml of lens culture)

In order to measure the degree of opacity of lens, photographic evaluation was performed during the

entire period of incubation at 0, 6th, 24th, 48th and 72nd hours (Table S1). A scale of opacity was

designed and developed based on the visibility of mesh from lens.

Scale of opacity

0- Absence of opacity

1- Slightly opaque

2- Presence of diffuse opacity

3- Presence of extensive thick opacity

Total Phenolic Content of APE

To the aliquot (50 μl) taken from stock solution (1 mg/ml) of the extracts, 3.5 ml distilled water and

250 μl of Folin-Ciocalteu’s reagent (FCR) was added, the mixtures were kept at room temperature for

1-8 min and 750 μl of 20% sodium carbonate solution was added to the extract. Mixtures were kept at

room temperature for 2 h and absorbance of the color developed was recorded at 765 nm with the help

of ultraviolet (UV)-visible spectrophotometer against blank. Total phenolic content was determined

using gallic acid standard curve (R2 = 0.996) and expressed in mg/g as gallic acid equivalents (GAE).

Total Flavoinoidal Content of APE

The reaction mixture contained 0.5 ml of extract in DMSO or standard solutions of quercetin, diluted

with 2 ml distilled water and 0.15 ml of 5% NaNO2. After 5 min, 0.3 ml of 10% AlCl3 was added.

After 6 min, l ml of 1 M NaOH was added and the total volume was made up to 5 ml with water. The

solution was mixed well and the absorbance was measured against a prepared reagent blank at 510

nm. The flavonoids content was expressed as mg of quercetin equivalents (QE) per g of dried extract,

by using a standard graph (y = 0.0025x, R2 = 0.9918).

Biochemical estimation

Total protein estimation was done by Lowry’s method (Lowry et al., 1951). It was evaluated as mg/g

of fresh weight of the lens.

MDA levels

The lenses were weighed and homogenized in 1 ml of 0.15 M potassium chloride. To the supernatant,

0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 20% w/v acetic acid (pH-3.5) and 1.5 ml of 0.81%

w/v thiobarbituric acid aqueous solution were added in succession. The mixture was then heated in

boiling water for 30 min. After cooling to room temperature, 5 ml of butanol:pyridine (15:1 v/v)

solution was added. The mixture was then centrifuged at 5000 rpm for 10 min. The upper organic

layer was separated, and the intensity of resulting pink color was read spectrophotometrically at 532

nm. Standard prepared from 1,1’,3,3’- tetramethoxypropane was used as reference.

SOD Estimation

The supernatant was assayed for SOD activity by assessing the inhibition of pyrogallol auto

oxidation. 100 µl of supernatant obtained by homogenizing three lenses in 10 ml of 0.1 M potassium

phosphate buffer further subjected to centrifugation at 10000 rpm for 45 min, was added to tris HCl

buffer, pH 8.5. The final volume was adjusted to 3 ml with the same buffer. At last, 25 µl of

pyrogallol was added. Absorbance was recorded at 420 nm at one minute interval for 3 minutes. The

increase in the absorbance at 420 nm after the addition of pyrogallol was inhibited by the presence of

SOD. One unit of SOD is described as the amount of enzyme required causing 50% inhibition of

pyrogallol auto oxidation per 3 ml of assay mixture and is given by the formula: Unit of SOD / mg of

protein = [100 x {(A-B)/ (Ax50)}]/ mg of protein, where A = Change in absorbance per minute in

control and B = Change in absorbance per minute in test sample.

GSH levels

Three of the lenses from each group were weighed and homogenized in 1 ml of 5% v/v trichloroacetic

acid (TCA), and a clear supernatant was obtained by centrifugation at 5000 rpm for 10 min. To 0.5 ml

of this supernatant, 4.0 ml of 0.3 M Na2HPO4 (Disodium hydrogen phosphate) and 0.5 ml of 0.6 mM

5,5’-dithiobis-2-nitrobenzoic acid (DTNB) in 1% w/v trisodium citrate were added in succession. The

intensity of the resulting yellow color was read spectrophotometrically at 410 nm. Reduced GSH was

used as standard.

Statistical analysis

Statistical analysis was carried out using Graph Pad Prism 5.0 (Graph Pad Software, San Diego,

CA). All results were expressed as mean ± SD. The data was analyzed by one-way ANOVA

followed by Bonferroni multiple comparison test and statistically significant data was accepted

when P<0.05. Paired t-Test was calculated between treated groups and data was considered to be

statistically significant when values were found to be P<0.05.

Certificate of analysis of APE (AP/02/02/2013)

*Supplied as snapshot of Certificate of analysis.

Table S1. Opacity Scale (in hours)

Groups Zero

hr

6 hrs 24

hrs

48

hrs

72

hrs

Time in hrs

(maintenance of vision

in test groups)

Normal (Positive control) 0 0 2 2 2 72

Toxic Control (Negative

Control)

0 1 3 3 3 18

Standard 0 0 1 1 2 33

APE (5mg/ml) 0 0 2 3 3 19

APE (10mg/ml) 0 0 1 2 3 20

APE (15mg/ml) 0 0 1 1 3 39

APE (20mg/ml) 0 0 1 1 3 39.5

Table S2. Effect of APE on TPC, MDA, SOD and GSH levels in different groups

GROUPS TPC (mg/g

of fresh wt

of lens)

MDA Levels

(Micromoles

of MDA/ g of

protein)

SOD

Levels(Unit/

mg of

protein)

GSH (µmoles/g

of protein)

Normal lens 275.77±8.3

0

0.35±0.05 1.76±0.10 2.9765±0.17

Toxic Control 200.02±1.3

3

1.57±0.04 0.23±0.01 1.045±0.10

Standard 270.32±2.3

0

0.42±0.03 1.39±0.09 2.765±0.05

APE (5mg/ml) 211.46±1.3

8

1.36±0.16 0.36±0.01 1.1125±0.04

APE (10mg/ml) 223.28±2.6

4

0.98±0.07 0.41±0.01 1.245±0.05

APE (15mg/ml) 237.31±2.0

3

0.66±0.05 0.53±0.02 1.4125±0.08

APE (20mg/ml) 239.97±1.6

9

0.53±0.03 0.57±0.01 1.5425±0.05

Figure S1. Effect of APE on Total protein content with respect to control (n=5)

Figure S2. Effect of APE on MDA levels with respect to control (n=5)

Figure S3. Effect of APE on SOD levels with respect to control (n=5)

Figure S4. Effect of APE on GSH levels with respect to control (n=5)

Figure S5

Photographic evaluation of lenses- (A)Normal Control (0 hr), (B) Normal Control (72 hrs) , (C)

Toxic control (0 hrs), (D) Toxic control (18 hrs), (E) Toxic Control Group (48 hrs), (F) Standard

group (0 hrs) , (G) Standard 24 hrs,(H) Standard (48 hrs), (I) Standard (72 hrs), (J) APE (15mg/ml)

0hrs, (K)APE (5mg/ml) 19 hrs, (L) APE (10mg/ml) 26 hrs,(M) APE (15mg/ml) 39 hrs