SUPPLEMENTARY MATERIAL
Amelioration of diabetic nephropathy by orange peel extract in rats
Parkar Nishad1,2 and Addepalli Veeranjaneyulu.2
1
Dr. Bhanuben Nanavati College of Pharmacy, Vile Parle West, Mumbai 400056
1,2
Department of Pharmacology, SPP School of Pharmacy and Technology Management,
NMIMS University, Vile Parle (W), Mumbai, 400 056, India
Abstract:
The present study was aimed to evaluate the effect alcoholic orange peel extract (OPE) on
streptozotocin induced diabetic nephropathy. Four weeks after the induction of diabetes,
treatment with OPE (100 and 200mg/kg) was further given for four weeks. Treatment with
OPE 200 improved renal functions and significantly prevented the raise in creatinine, urea
and blood urea nitrogen (BUN) levels. Histological analysis of kidneys revealed that
Tubulointerstitial Fibrosis Index was significantly decreased in OPE 200 group. The results
indicated prevention of diabetic nephropathy in rats by OPE treatment and suggests OPE as a
potential treatment option.
Keywords: – Diabetic Nephropathy; Orange peel extract, Extracellular matrix.
Experimental
Collection of Plant material and preparation of OPE
Fresh oranges were purchased from the local market of Mumbai, Maharashtra, India. Peels of
the fresh fruits were separated and shade dried. The dried orange peels were sorted and the
good quality peels were subjected to size reduction by pulverisation. The powdered peels
were extracted using methanol as a solvent in a soxhlet apparatus and completed with six
cycles. The extract obtained was dried on a water bath followed by vacuum oven at 40 0C
until completely dried. The dried extract was refrigerated until used.
Phytochemical screening of the extract for flavonoids
The dried orange peels were exhaustively extracted using methanol in a soxhlet apparatus.
The fresh extract was subjected to preliminary qualitative phytochemical screening to
confirm the presence of flavonoids. Standard tests for flavonoids (Shinoda test, lead acetate
test and sodium hydroxide test) mentioned in literature were performed on the extract
(Harborne JB., 1998).
Total Phenolic Content
The total phenolic content (TPC) of the extract was determined spectrophotometrically by
Folin Ciocalteu’s reagent using gallic acid as standard. (Claudia Anesini, et.al 2008). The
concentration of polyphenols in samples was derived from a standard curve of gallic acid
ranging from 10-50 µg/mL. 1.0 mL of sample extract was transferred in triplicate to separate
tubes containing 5.0 mL of a 1/10 dilution of Folin-Ciocalteu’s reagent in water. Then, 4.0
mL of sodium carbonate solution (7.5% w/v) was added. The tubes were vortexed and
allowed to stand at room temperature for 60 mins. The absorbance of the resultant color
developed was measured at 765 nm against reagent blank. The TPC was expressed as gallic
acid equivalents (GAE) in mg/g of the orange peel extract (n = 3).
Total Flavonoid Content
The total flavonoid content of orange peel extract was determined using AlCl 3 method.
(Brighente et.al.2007) Briefly, 0.5 ml of 2% aluminium chloride (AlCl3) in methanol was
mixed with the same volume of methanolic solution of orange peel extract. The mixture was
vortexed and kept at room temperature for 1 h. The absorbance of the samples was measured
at 415 nm using a spectrophotometer versus reagent blank sample. Total flavonoids content
was expressed as quercetin equivalents (mg QE/g dry extract). The assay was performed in
triplicate and the mean values are reported.
Drugs and chemicals
Streptozotocin (STZ) was purchased from Sigma (St. Louis, USA). Minocycline was
obtained as a gift sample from US Vitamins (Mumbai, India). All the diagnostic kits were
purchased from Erba Diagnostics, Mumbai, India. All the other reagents used were of
analytical grade purchased from Merck (Mumbai, India).
Animals
Female Wistar rats 10-12 wks (200-240 g) were used for the study. Animals were purchased
from Haffkine Institute, Parel (Mumbai, India). The animals were housed in a clean
environment at a temperature of 25±10C, relative humidity 45-55% under a 12 hr light/ dark
cycle with food and water available ad libitum. All procedures met the guidelines of
Institutional Animal Ethics Committee (IEC) and were approved by Committee for the
Purpose of Control and Supervision of Experiments on Animals (CPCSEA). (Approval no.
CPCSEA/IAEC/SPTM/P-21/2011)
Preparation of the drug solution
Orange peel extract (OPE) and minocycline were suspended in 0.5% CMC solution before
use. STZ was dissolved in freshly prepared ice cold citrate buffer (pH 4.5).
Induction and assessment of diabetes
Female Wistar rats were fasted over night (12h) and diabetes was induced by a single dose of
STZ (50mg/kg, i.p. 1ml/kg body wt.). For biochemical estimations, blood was collected by
retro orbital bleeding under mild anesthesia in accordance with National Cancer Institute
guideline for Rodent Blood Collection (2012). Further, during the experiment, blood was
withdrawn from alternate eyes to reduce the damage and allowed timely recovery of the eye
as suggested in the guidelines (NCI Guideline, 2012). The plasma glucose levels were
monitored after 48 h of STZ injection using a glucose oxidase-peroxidase kit and weekly
during the experiment. The animals with plasma glucose level of >350mg/dl were used for
the study.
Experimental design
The animals were divided into 5 groups of 6 animals each as follows: Group 1 – Diabetic
Vehicle control (DW + 0.5 % CMC), Group 2 - treated with OPE (100 mg/kg p.o.), Group 3
– treated with OPE (200mg/kg p.o.), Group 4 – treated with standard Minocycline (50 mg/kg
p.o.). Age matched normal animals in Group 5 were untreated. Four weeks after induction of
diabetes, the animals were further treated for the next four weeks daily p.o.
Assessment of renal function
At the end of eight weeks study protocol, the animals of each group were housed individually
in metabolic cages for a period of 24 hr. Urine samples were collected under a layer of
toluene. Blood was collected by retro orbital puncture, serum separated and stored at -800C
for further analysis. The renal function was assessed by measuring serum and urine levels of
creatinine, urea and albumin. Creatinine clearance was calculated as an index of glomerular
filteration rate
(GFR)
.Creatinine clearance was calculated using the formula:
[CreatinineUrine/CreatinineSerum]* [Vol. Urine (ml)/ time (hrs)*60]
Systemic Blood Pressure
After 8 weeks of treatment, rats were anesthetized with pentobarbital sodium and body
temperature was maintained at 37 ± 1°C. Systemic blood pressure was monitored via a
catheter inserted into the femoral artery. Blood pressure was recorded digitally through a data
acquisition system (Iworx systems USA). Mean arterial pressure (MAP) was calculated using
the following formula: MAP = diastolic pressure + 1/3 (systolic pressure - diastolic pressure).
Renal Hypertrophy, histopathology
The animals were sacrificed under anaesthesia and left kidney was excised from each of the
sacrificed rats. The kidney was decapsulated and rinsed in saline, blotted dry on a tissue
paper and weighed. The kidney/bodyweight ratio was calculated. Data was expressed as
relative organ weight of one kidney to 100g of total body weight. Kidney samples were then
transferred to 10% v/v formaldehyde in separate containers and subjected to histopathological
analysis using H &E and Masson’s Trichrome staining using standard procedures. Stained
sections were observed under light microscope.
Tubulointerstitial Fibrosis Index
Excess MMP-2 and MMP-9 activity resulted in remodelling of ECM. Tubulointerstitial
fibrosis index was measured to evaluated severity of injury to renal tissue (Christine Maric,
2004). On 5 cortical and 5 outer medullary fields it was scored semi quantitatively. Severity
of tubulointerstitial fibrosis was defined as tubular atrophy or dilatation, presence of
inflammatory cells, deposition of ECM, and interstitial cell proliferation. The degree of
tubulointerstitial fibrosis was graded on a scale of 0 to 4: grade 0, affected area 0% (normal);
grade 1, affected area less than 10%; grade 2, affected area 10 to 25%; grade 3, affected area
25 to 75%; grade 4, affected area greater than 75%. Estimation of tubulointerstitial fibrosis
was performed with the observer masked to the treatment groups.
Statistical analysis
Data was expressed as mean ± SD. The data was statistically analysed using Graph Pad Prism
software (Graph Pad Inc., San Diego, CA, USA). For parametric data ANOVA (One way
analysis of variance) was used for multiple comparisons. In case ANOVA showed significant
differences, post hoc analysis was performed using Dunnet test. For non-parametric
measures, Kruskal-Wallis test followed by Dunn’s post-hoc test were used. p < 0.05 was
considered statistically significant.
References
Brighente, M. Dias, L.G. Verdi, and M.G. Pizzolatti. (2007) Antioxidant Activity and Total
Phenolic Content of Some Brazilian Species. Pharmaceutical Biology, Vol. 45, No. 2, pp.
156–161.
Christine
Maric,
Kathryn
Sandberg,
and
Carmen
Hinojosa-Laborde
(2004)
Glomerulosclerosis and Tubulointerstitial Fibrosis are attenuated with 17β-Estradiol in the
Aging Dahl Salt Sensitive Rat. J Am Soc Nephrol 15: 1546–1556.
Claudia Anesini, Graciela E. Ferraro, and Rosana Filip (2008) Total Polyphenol Content and
Antioxidant Capacity of Commercially Available Tea (Camellia sinensis) in Argentina. J.
Agric. Food Chem., 56, 9225–9229.
Harborne JB., (1998) Phytochemical Methods: A Guide to Modern Techniques of Plant
Analysis. (3rd edition) Chapman and Hall Co., New York, pp.1-302.
National Cancer Institute Guideline for Rodent Blood Collection (2012). Available from
http://ncifrederick.cancer.gov/Lasp/acuc/Frederick/Media/Documents/ACUC20.pdf
Figure S1. Effect of 4-week treatment with OPE on A. Renal Tubulointerstitial Fibrosis
Index and B. Renal histopathology with H & E Staining. The values are given as mean ± SD.
*p < 0.05 when compared with vehicle treated diabetic group.
A.
B.
A
C
B
D
E
A –NORMO, B – DB + 0.5% CMC, C – OPE – 100, D – OPE 200, E- MINO