DraftDraft Nephroprotective and antioxidant effect of green tea (Camellia sinensis) against...
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Nephroprotective and antioxidant effect of green tea (Camellia sinensis) against nicotine-induced nephrotoxicity in rats and characterization of its bioactive compounds by
HPLC-DAD
Journal: Applied Physiology, Nutrition, and Metabolism
Manuscript ID apnm-2017-0834.R4
Manuscript Type: Article
Date Submitted by the Author: 12-Jul-2018
Complete List of Authors: Ben Saad, Anouar; 1Faculty of Sciences of Gafsa, 2112, University of Gafsa, Tunisia.Ncib, Sana; Unit of common services, Faculty of Sciences Gafsa, 2112, University of Gafsa, Tunisia., Rjeibi, Ilhem; 1Faculty of Sciences of Gafsa, 2112, University of Gafsa, Tunisia.Saidi, Issam; 1Faculty of Sciences of Gafsa, 2112, University of Gafsa, Tunisia.Zouari, Nacim; 3Higher Institute of Applied Biology ISBAM Medenine 4119, University of Gabes, Tunisia.
Keyword: Nicotine, Green tea, kidney function, Antioxidant, oxidative damage, animal model(s) < research models
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Nephroprotective and antioxidant effect of green tea (Camellia sinensis) against nicotine-
induced nephrotoxicity in rats and characterization of its bioactive compounds by
HPLC-DAD
Anouar Ben Saad1, Sana Ncib2,3, Ilhem Rjeibi1, Issam Saidi1,Nacim Zouari4
1Faculty of Sciences of Gafsa, 2112, University of Gafsa, Tunisia.
2Unit of Common Services, Faculty of Sciences Gafsa, 2112, University of Gafsa, Tunisia.
3Environment and Energy Laboratory (UR14ES26), Faculty of Sciences of Gafsa, University
of Gafsa, Gafsa, Tunisia.
4Higher Institute of Applied Biology ISBAM Medenine 4119, University of Gabes, Tunisia.
*Corresponding author: Anouar Ben Saad: Faculty of Sciences of Gafsa, 2112, University of
Gafsa, Tunisia
Phone: 00 21653383774, Fax: 00216 76 211 026;
E-mail address: [email protected]
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Abstract
Nicotine (NT) is a potential inducer of oxidative stress, through which it can damage
numerous biological molecules. Natural antioxidants that prevent or slow the progression and
severity of nicotine toxicity may have a significant health impact. The purpose of this study,
conducted on Wistar rats, was to evaluate the beneficial effects of green tea (Camellia
sinensis) extract on nicotine treatment induced damage on kidney.
Our results showed that nicotine significantly (p
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Introduction
Nicotine (3-(1-methyl-2-pyrrolidinyl) pyridine) is a volatile alkaloid extracted from the dried
leaves of Nicotiana tabacum and Nicotiana rustica plants. It is well diffused and absorbed
throughout the skin, mucosal lining of the respiratory tract, and lungs ((Mosbah et al. 2015;
Abu-awwad et al. 2017). Upon absorption, it enters circulation and distributes rapidly to
various tissues. The liver metabolizes most of nicotine while the rest is metabolized via the
lungs and kidneys. Nicotine had recognized to induce oxidative stress throughout ROS
generation, which was capable of initiating and promoting oxidative and inflammatory
stresses (Chandrasekharan and Simmons 2004; Das et al. 2016). It has been reported that
antioxidants appear to act against disease processes by increasing the levels of endogenous
antioxidant enzymes and decreasing lipid peroxidation (Bansal et al. 2005). Lots of studies
indicate that natural substances from edible and medicinal plants exhibited strong antioxidant
activity that could act against nicotine-induced kidney damage, because they contain lots of
free radical scavenger such as phenolic acids and flavonoid compounds (Neogy et al. 2008;
Al-Malki and Moselhy 2013; Yarahmadi et al. 2017; Ben Saad et al. 2018). Green tea
(Camellia sinensis) has a long history of use world-wide. Researches have investigated the
potential use of naturallyoccurring dietary substances for the control and management of
various chronic diseases. Since ancient times, green tea has been consumed to promote the
improvement of human health. A wide range of well defined phytochemicals, which are
absorbed and metabolized by the body, exhibit antinflammatory, anticarcinogenic,
antiatherosclerotic and antibacterial properties (Augustyniak et al. 2005). In particular, green
tea catechins and their derivatives have been characterized as antioxidants that scavenge free
radicals protecting cells in normal and pathological states (Khan et al. 2006). Among all tea
polyphenols, epigallocatechin-3-gallate has been shown to be responsible for great part of the
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health promoting-ability of green tea. Green tea has been shown to improve kidney function
in diabetic rats (Rhee et al. 2002) and has been linked to a reduced risk of cardiovascular
diseases, cancer (Mukhtar and Ahmad 2002), obesity and oral health problems (Liao 2001).
In addition, a recent study demonstrated a protective role of green tea against cigarette smoke
induced oxidative stress (Al-Awaida et al. 2014).
To our knowledge, there are no data about the in vivo effect of green tea extract on male
kidney damage and oxidative stress induced by nicotine. The present study was designed to
assess in rats the protective effects of green tea (Camellia sinensis) upon nicotine induced
stress in blood and kidney and the related damages. Examined parameters were urea,
creatinine and uric acid in blood serum, and lipid peroxidation as well as on antioxidant
enzymatic and non-enzymatic activities and, subsequently, the ability of green tea extract to
improve and protect kidney function.
Material and methods
Plant material
The green tea used in this study (Lipton/Kangra-brand) was purchased from a commercial
source (Jain Pan House, New Delhi, India). The green tea extract was prepared by adding 1.25
g of green tea leaves to 25 mL of boiling water (15 min). The infusion was cooled to room
temperature and then filtered. The tea leaves were extracted a second time with 25 mL of
boiling water and filtered; the two filtrates were then combined to obtain an aqueous 2.5%
green tea extract (2.5 g of tea leaves/100 mL water) (Ben Saad et al. 2017).
2.3. Photochemical study
2.3.1. Total Phenols Content
The amount of total phenols content in green tea was determined by Folin-Ciocalteu’s
reagent method (Singleton et al.1999). 1 ml of green tea was mixed with 5 ml of Folin reagent
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(10%) and the mixture was incubated at room temperature for 5 min. Then 2 mL saturated
sodium carbonate (Na2CO3) solution was added and further incubated for 90 min at room
temperature. The absorbance was then measured at 765 nm. Gallic acid was used as a positive
control. All tests were carried out in triplicate and the results were expressed as µg of gallic
acid equivalent (µg GA)/mL GT.
Determination of flavonoids contents
To determine the total flavonoids content, 1ml of green tea samples were mixed with an equal
volume of 2% aluminum chloride solution (AlCl3). After incubation for 30 min at room
temperature, the absorbance of the reaction mixture was measured at 430 nm. The total
flavonoids content was expressed as µg of rutin equivalent (µg RE)/mL GT. All tests were
carried out in triplicate (Djeridane et al. 2006).
Determination of tannins content
Quantitative estimation of tannins was carried out using the modified vanillin–HCl in
methanol method described by Price and Butler (1977). The method is based on the ability of
condensed tannins to react with vanillin in the presence of mineral acid to produce a red color.
GT samples (1 mL) were mixed with 20 ml of 1% HCl (in methanol) for 20 min at 30 °C in a
water-bath. The samples were centrifuged at 2000 rpm for 4 min. The supernatant (1 mL) was
reacted with 5 ml vanillin solution (0.5% vanillin12% HCl in methanol) for 20 min at 30 °C.
Blanks were run with 4% HCl in methanol in place of vanillin reagent. Absorbance was read
at 500 nm on a UV spectrophotometer. A standard curve was prepared with catechin. Results
were expressed as µg of catechin equivalent (µg CE)/mL GT. Samples were analyzed in
triplicate.
Ascorbic Acid Content
The ascorbic acid content was assayed as described by Omaye et al. (1979). An amount of 1
mL of GT was mixed with 5 ml of 10% TCA, the extract was centrifuged at 3500 rpm for 20
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min. The pellet was re-extracted twice with 10% TCA and supernatant was increased 10 mL
and used for estimation. To 0.5 ml of the extract, 1 mL of DTC reagent (2,4-dinitrophenyl
hydrazine-Thiourea-CuSO4 reagent) was added and mixed thoroughly. The tubes were
incubated at 37 C for 3 h and to this a solution of 0.75 ml of ice cold 65% H2SO4 was added.
The tubes were then allowed to stand at 30 °C for 30 min. The resulting color was read at 520
nm. The ascorbic acid content was determined using a standard curve prepared with ascorbic
acid and the results were expressed as µg of ascorbic acid equivalent (µg AA)/mL GT.
Samples were analyzed in triplicate.
High-performance liquid chromatography (HPLC) analysis conditions
A Varian Prostar HPLC system equipped with a ProStar 230 ternary pump, a manual injector
and a ProStar 330 diode array detector were used. The HPLC separation was carried out on C-
18 reverse phase HPLC column (Varian, 150 mm × 4.6 mm, particle size 5mm) on an elution
gradient at 25 C. Two phases system were used as follows: mobile phase A, pure methanol,
and mobile phase B was acetic acid aqueous solution (0.05%). Gradient conditions were: first
35% A and 65% B, followed by 30 min 50% A and 50% B, followed by 40 min 90% A and
10% B. The flow rate was maintained at 1 mL/min and 20ml of sample was injected. The
detected compounds were recognized by comparing their retention times with those of
commercially standards analyzed under the same conditions.
Antioxidant activity
The Hydrogen peroxide (H2O2) scavenging activity of green tea GT was determined
according to the method reported by Liu et al. (2010). Vitamin C was used as positive control.
ABTS•+ assays were evaluated according to the method cited by Afoulous et al. (2013). The
method described by Koleva et al. (2007) was used with a slight modification.
Animals housing conditions
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This study was carried out on 32 Wistar male rats (3-4 months old), about 140-150 g body
weight, purchased from the Central Pharmacy in Tunisia (SIPHAT, Tunisia). The animals
were handled under standard laboratory conditions of a 12-h light/dark cycle in a temperature
and humidity controlled room. The rats were fed with a commercial balanced diet (SICO,
Sfax, Tunisia) and drinking water was offered ad libitum. The handling of the animals was
approved by the Medical Ethics Committee for the Care and Use of Laboratory Animals of
the Pasteur Institute of Tunis (approval number: FST/LNFP/Pro 152012) and carried out
according to the European convention for the protection of living animals used in scientific
investigations (Council of European Communities 1986).
2.7. Experimental design
One week after acclimatization to laboratory conditions, the rats were randomly divided into
four groups of eight animals each. The four groups were treated as follows:
Group 1 (C): Control rats received distilled water (0.5 mL/100 g b.w; i.p).
Group 2 (NT): injected intraperitoneally (i.p.) with nicotine (NT in aqueous solution; 1 mg/kg
body weight/day) for 30 days.
Group 3 (GT): received green tea extract (GT) by intragastric gavage at a dose of 1 mL/100 g
b.w for 30 days and followed by distilled water (0.5 mL/100 g b.w; i.p) during the last 30
days of GT treatment.
Group 3 (NT+GT): received green tea extract (GT) at a dose of 1 mL/100 g b.w and then
injected NT (1 mg/kg body weight/day i.p.) during the last 30 days of GT treatment.
Preparation of samples
At the end of experimental period animals were sacrificed by cervical decapitation under light
ether anesthesia. The serum was collected by centrifugation of the whole blood at 1500× g for
15 min at 4 °C and stored at –80 °C until analysis. Kidney samples were quickly removed,
washed in ice-cold 1.15% KCl solution, homogenized into 2 ml ice-cold lyses buffer (pH 7.4)
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and centrifuged for 30 min at 5000× g, 4 °C. The collected supernatants were stored at –80 °C
until the analysis.
Biochemical assays
The level of creatinine, uric acid and urea in serum were determined by kit methods
(Spinreact, Girona, Spain). Serum and kidney homogenate the level of lipid peroxidation was
measured as malondialdehyde content (MDA) according to the method of Ohkawa et al.
(1979). SOD activity was estimated according to the method described by Misra and
Fridovich (1972). CAT activity was determined by measuring hydrogen peroxide
decomposition at 240 nm according to the method described by Aebi (1984). GPx activity
was assayed using the method described by Flohe and Gunzler (1984). In serum and kidney
homogenate the levels of ascorbic acid (vitamin C) concentration was measured according to
the method of Omaye et al. (1979) more so vitamin E level was determined based on the
method of Desai (1984).
Protein determination
Serum, urinary and kidney homogenate protein contents were determined according to
Lowry’s method using bovine serum albumin as standard (1951).
Kidney histopathological studies
Kidney tissues were cut into about 5-cm-thick slices and fixed with 10% phosphate-buffered
formalin (pH 7.4). The tissue slices were dehydrated through ascending grades of alcohol, and
embedded in paraffin. Tissue sections of 5–8 μm were made using microtome, and stained
with hematoxylin-eosin solutions (H&E). Tissue preparations were observed and micro-
photographed under a light BH2 Olympus microscope.
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Statistical analysis
All data were presented as means ± standard deviation (SD). Determination of all parameters
was performed from six animals per group. Significant differences between treatment effects
were determined using one-way ANOVA, followed by Tukey's HSD Post- Hoc tests for
multiple comparisons with statistical significance of P< 0.05.
Results
Total phenols, flavonoids, tannins, and ascorbic acid content
The phytochemical study of GT revealed the presence of various quantities of total phenolics,
flavonoids, tannins, and ascorbic acid contents. Ours results showed that 1 mL of GT was
equivalent to 730.90±4.52 µg of gallic acid, 289.30±0.23 µg of rutin, 100.03±1.02 µg of
catechin, and 123±1.2 µg of ascorbic acid (Table 1).
HPLC analysis of phenolic compounds
The HPLC-DAD analysis of the extract of green tea revealed the presence of phenolic acids
and flavonoids. The compounds that were identified in our extract were 6 phenolic acids:
catechic, caffeic, epicatechic, vanillic, protocatechuic and coummarin with retention times of
15.10 min, 17.2 min, 21.50 min, 28.62 min, 34 min and 35.81 min, respectively. The HPLC
elution profile of phenolic acids (Fig. 1 A) also showed 2 peaks of unknown compounds. The
HPLC elution profile of flavonoids (Fig. 1B) showed a total of 9 compounds among which 3
flavonoids were identified: rutin, quercetin, and kaempferol. The absorbance was measured at
360 nm.
Antioxidant activity of green tea extract
Antioxidant activity determined using the (H2O2) scavenging assay showed results GT
exhibited dose-dependent (H2O2) scavenging ability (Fig. 2A). The effective concentrations at
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which H2O2 were scavenged by 50% (IC50) was found to be 150±2.8 µg/mL but significantly
lower than that of vitamin C (40±1.42 µg/mL). ABTS assay is shown in Fig 2.B. GT (300
μg/mL) exhibited the highest inhibition of 89 %. As can be clearly seen in Fig. 2. C. GT
extract was efficient to inhibit the oxidation of linoleic acid. The antioxidant activity of GT
extract increased with increasing extract concentration. The IC50 value of GT (1±0.07 mg/mL)
appeared significantly (p< 0.05) lower than that of BHA (IC50= 0.3±0.09 mg/ mL) used as a
positive control.
Body weight
Table 2 shows the effects of nicotine and green tea on the body and ogan weights in different
experimental groups. Nicotine alone treated rats had a significantly (p
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Antioxidant activities
Table 4 showed that nicotine administration was found to cause a significant decrease
(p< 0.01) in the levels of serum and kidney vitamin E and vitamin C when compared with the
control group. However GT supplement significantly (p< 0.01) increased vitamin E and
vitamin C levels when compared with nicotine group. The extract alone did not shown
significant effect on Vitamin E and vitamin C. The activities of SOD, CAT and GPx of the
serum and kidney tissue are shown in Table 4. Injection of nicotine led to a lower SOD, CAT
and GPx activities compared to the normal control group. However, animals treated with GT
showed significant increase (p< 0.01) in the antioxidant enzyme activities as compared to
nicotine group.
Histopathological findings
The histopathological findings on kidney for various treatment groups are presented in Figure
3. In light microscopic examinations, histopathological changes were observed in kidney of
nicotine and green tea extract-nicotine-treated groups compared to control rats. The kidney in
the control animals showed normal histological structure (Fig. 3A). With respect to the renal
histoarchitecture of the nicotine-treated animals, showed higher degree of tubular
degeneration and hypertrophy of glomeruli cells showing reduction of Bowman’s space (Fig.
3B). The sections of the animals belonging to the green tea extract alone and their
combinations with nicotine treatment groups showed a normal histological appearance (Fig.
3C and D.
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Discussion
Plants rich in secondary metabolites, including phenolic compounds, have antioxidant activity
due to their redox properties and chemical structures. Result from this study showed that
green tea extract had a high total phenolics (730.90±4.52 µg Gallic acid equivalent/mL GT),
flavonoids ( 289.30±0.23 µg Rutin equivalent/mL GT), tannins (100.03±1.02 µg Catechin
equivalent/ml GT), and ascorbic acid contents (123±1.2 µg Ascorbic acid equivalent/mL GT)
Our results are in agreement with previously reported (Mada et al. 2017). The overload free
radicals are continuously produced and accumulated in body which cause the oxidation of
biomolecules (e.g. protein, lipid, and DNA) which leads to cell injury and apoptosis. Our
results exhibited a significant radical-scavenging activity all tested concentrations. The
effective concentrations at which H2O2 were scavenged by 50% (IC50) was found to be
150±2.8 µg/ml. As can be clearly seen in Fig. 2. C. Same results were observed for β-carotene
activity that increased with the increase of GT concentrations. GT extract was efficient to
inhibit the oxidation of linoleic acid. The IC50 value of GT (1±0.07 mg/mL) appeared
significantly (p< 0.05) lower than that of BHA (IC50= 0.3±0.09 mg/ mL) used as a positive
control. Also, Green tea extract (300 µg/mL) exhibited the highest inhibition of ABTS. Ours
results are in concordance with several studies that demonstrated the antioxidant action of
green tea extract (Erkekoglou et al. 2017; Megow et al. 2017). In the present study, changes in
rats body weight along with relative organ weights provided an imperative indication of
nicotine-induced toxicity. Nicotine-exposure for 30 consecutive days at 1 mg/kg caused a
decrease in rats body weight which might either be due to the direct cytotoxic effects (Ibrahim
et al. 2016; Mosbah et al. 2015). Moreover, the decrease in the relative kidney weights
following nicotine-treatment as observed in our study could be attributed to the relationship
between the kidney weight increase and toxicological effects such as the reduced body weight
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gain in the experimental animals (Ibrahim et al. 2016; Perkins et al. 1991). However,
Supplementation of green tea to-nicotine treated animals ameliorated the body and kidney
weights, which could be attributed either to the phytochemical content in green tea extract.
Renal failure has been reported with nicotine intoxication. Significant increase in the level of
urea, creatinine and uric acid in treated animals is indicative of kidney damage and a possible
malfunction or failure of the kidneys. In rats pretreated with green tea extract at a dose 1
mL/100 g b.w, showed a significant derease in serum urea, creatinine and uric acid levels.
This suggested an amelioration in the kidney status following nicotine treatment as compared
to nicotine treated rats. These results lend credence to the use of green tea extract as a
nephroprotective agent (Anwar Ibrahim et al. 2015). Our results corroborated with Xie et al.
(2017) which denoted that green tea phenolic compounds had a nephroprotective effect
against high-fat diet. It was obvious from our results that treatment of nicotine significantly (p
< 0.01) increased the MDA concentration in the serum and kidney tissue and attenuates the
levels of vitamins E and C. Studies have shown that nicotine may result in increased oxidative
stress by the ROS superoxide overproduction which can later initiate free radicals chain
reactions of lipid peroxidation (Mosbah et al. 2015; Maurya and Salvi 2005). As ROS
scavengers, vitamins E and C has been shown to be effectives against superoxyde radical,
hydrogen peroxide, hydroxyl radical and singlet oxygen (Al-Malki and Moselhy 2013). The
observed decrease in the levels of serum and kidney vitamin E and C in nicotine rats could be
the results of increased uses of these antioxidants in scavenging the ethanol-overproduced free
radicals. Treatment with green tea extract significantly reversed these change. Hence it may
be possible that the mechanism of nephroprotection of extract is due to its antioxidant effect.
This result is in agreement with the study of Zhang and Tsao (2016) who unambiguously
shows that polyphenols, flavonoids, and catechins display significant antioxidant properties
and scavenging activities on free radicals involved in vanadium toxicity. As antioxidants
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(enzymatic and non-enzymatic) provide protection against free radical attacks, we
investigated levels of SOD, CAT, and GPx enzymatic activities in the serum and kidney.
SOD, CAT, and GPx are major enzymes protecting cells from oxidative damages. The
increased formation of reactive oxygen species due to nicotine exposure would require some
fully active protection mechanisms against oxidative damage (Mosbah et al. 2015; Zahran et
al 2017). It is thus likely that the decreased activity of these enzymes in kidney further
exacerbate the oxidative damage. These effects are reversed by the treatment with green tea
extract. However, SOD, CAT and GPx activity was significantly elevated by administration
of green tea extract to nicotine-treated rats. The decrease in the activity of antioxidant
enzymes suggested that the balance between the oxidant and pro-oxidant was disturbed by
nicotine, and treatment of experimental rats with GT effectively reverts this imbalance and
restores the level of antioxidant enzymes. The protective effect of green tea can be attributed
to the presence of phenolic acids and flavonoids as revealed by the HPLC analysis. In fact,
phenolic acids and flavonoids were able to release a hydrogen proton from their hydroxyl
group, trap free radicals and prevent nicotine induced kidney damage. Based on the HPLC
analysis of green tea extract, phenolic acids (catechic, caffeic, epicatechic, vanillic,
protocatechuic and coumaric) and flavonoids (rutin, quercetin and kaempferol) were
identified by Prasad et al. 2016 confirmed that green tea extract was capable of normalizing
the oxidative stress in kidney.
The histopathological changes in kidney showed that treatment of rats to nicotine resulted in
degenerative changes in the kidney, including tubular degeneration and hypertrophy of
glomeruli cells showing reduction of Bowman’s space. The biomarkers of kidney dysfunction
corroborated with hisotpathological lesions observed in the current study (Zahran et al 2017).
These observations indicated marked changes in the overall histoarchitecture of kidney in
response to nicotine, which could be due to its toxic effects primarily by the generation of
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reactive oxygen species causing damage to the various membrane components of the cell.
Administration of green tea extract improved the structure of renal cells, which could be
attributed to the antioxidant properties of GT in vivo ( Li et al. 2017; Pereira et al. 2017).
Conclusion
The finding of this study showed that the administration of the green tea extract appeared to
protect the kidneys due to presence of high antioxidant phytochemicals and properties of male
rats from nicotine- induced oxidative stress by reducing the intensity of LPO and by
enhancing the antioxidants activities. In future, work will be done to isolate bioactive
constituents of grren tea extract to locate potential pharmacological agents.
Acknowledgements
This research was funded by the Tunisian Ministry of Higher Education. We would like to
express our special thanks to Unit of common services, Faculty of Sciences Gafsa, Tunisia.
Competing interests
The authors declare that they have no competing interests.
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Table 1: Phytochemical compounds in green tea extract
Composition Contents
Total phenols (µg Gallic acid equivalent/ml GT)
Flavonoids (µg Rutin equivalent/ml GT)
Tannins (µg Catechin equivalent/ml GT)
Ascorbic acid (µg Ascorbic acid equivalent/ml GT)
730.90±4.52
289.30±0.23
100.03±1.02
123±1.2
Values are expressed as mean ±SD of triplicate measurement.
Table 2: The effects of green tea and nicotine on Body weight and relative organ weights.
C NT GT NT+GT
Initial body weight (g) 145.02±12.3 146±17.30 148.20±8. 18 146.30±15.3
Final body weight (g) 246.2±10.3 163.3±6.35** 248.40±20.3++ 246.89±10.3++
Body weight gain(%) 41.09 10.59 40.33 40.74
Relative kidney weight
(g/ 100g)
0.67±0.9 0.34±0.36** 0.65±0.60++ 0.56±0.63++
Values were expressed as mean ± SD of 8 rats in group.
**p < 0.01 compared with control (C).
++p < 0.01 compared with nicotine-treated group (NT).
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Table 3: Effects of nicotine, green tea on urea (mmol/l), creatinine (μmol/l) and uric acid
(μmol/l) levels in serum.
Groups C NT GT NT+GT
Urea 8.32±0.63 18.23±0.53* 9.36±0.23++ 14.02±0.21+
Creatinine 21.4±1.25 32.59±1.6* 19.6±0.90++ 24.31±1.06+
Uric acid 132±5.23 99.3±5.3** 129.30±6.3++ 110±2.3++
Values were expressed as mean ± SD of 8 rats in group.
*p < 0.05, **p < 0.01 compared with control (C).
+p < 0.05, ++p < 0.01, compared with nicotine-treated group (NT).
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Table 4: Changes in the level of MDA, on vitamin E, vitamin C, and antioxidant enzymes
activities (SOD, CAT, and GPx) in the serum and kidney of control and rats treated with
nicotine (NT), GT, or their combination (NT+GT). 1nmoles/mg protein ,2mg/dl, 3μg/g tissue,
4U/mg protein, 5µmol/min/mg protein and 6µmol GSH oxidized/min/mg protein.
Groups C NT GT NT+GT
MDA1 Serum
Kidney
3.60±0.3
10.2±0.24
8.03±0.5**
18.03±0.26**
2.6±0.12++
11.02±0.5++
2.9±0.28++
10.03±0.03++
Vitamin E2 Serum
Kidney
2.5±0.01
26±0.06
0.77±0.02**
12.03±0.2**
2.56±0.12++
26.03±0.13++
2.03±0.16++
20.03±0.13++
Vitamin C3 Serum
Kidney
6.02±0.36
120.23±1.03
1.03±0.21**
85.03±0.06**
6.23±0.23++
123.2±0.04++
5.03±0.5++
99±0.96*
SOD4 Serum
Kidney
7.02±0.3
10.03±0.54
3.02±0.1**
4.21±0.08**
7.92±0.23++
10.9±0.01++
8.01±0.13++
9.03±0.23++
CAT5 Serum
Kidney
22.03±0.21
40.23±1.02
18.03±0.23*
20.03±0.9**
23.03±0.1+
22.03±0.12++
21.06±0.23+
25.21±0.23++
GPx6 Serum
Kidney
3.42±0.05
5.03±0.23
1.23±0.23**
2.03±0.13**
3.05±0.12++
6.03±1.9++
3.90±0.23++
5.06±1.02++
Values were expressed as mean ± SD of 8 rats in group.
*p < 0.05, **p < 0.01 compared with control (C).
+p < 0.05, ++p < 0.01, compared with nicotine-treated group (NT).
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Figure captions
Figure1: A. HPLC profile of phenolic acids (λ = 280 nm) from green tea extract. Peaks (2)
catechic acid, (3) caffeic acid, (4) epicatechic acid, (6) vanillic acid, (7) protocatechuic acid,
(8) coumaric acid and 2 unknown compounds. B. HPLC profile of flavonoids ( λ= 360 nm)
from green tea extract. Peaks (4) rutin, (5) quercetin, (6) kaempferol and 6 unknown
compounds.
Figure 2 : A. Hydrogen peroxide scavenging activity and positive control vitamin C at
different concentrations. B. ABTS•+ Scavenging activity (%) of green tea extract. C.
Antioxidant activities (%) of GT and BHA as positive control, measured by β-carotene
bleaching assay. Data are expressed as mean ± standard deviation of the mean (n=3).
Figure 3: Representative photographs from the kidney showing the protective effect of green
tea extract on nicotine induced renal injury in rats. (A) Controls, (B) rats treated with
nicotine, (C) rats treated with green tea extract, and (D) rats treated with the combination of
green tea extract and nicotine. Kidney sections were stained using the hematoxylin–eosin
method. Original magnifications:×400;* proximal and tubular necrosis;+ tubular
degeneration; hypertrophy of many glomeruli cells; congestion.
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