DraftDraft Nephroprotective and antioxidant effect of green tea (Camellia sinensis) against nicotine-induced nephrotoxicity in rats and characterization of its bioactive compounds

Draft

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

Is the invited manuscript for consideration in a Special

Issue? :Not applicable (regular submission)

https://mc06.manuscriptcentral.com/apnm-pubs

Applied Physiology, Nutrition, and Metabolism

Draft

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]

Page 1 of 28



Draft

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

Draft

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

Page 3 of 28



Draft

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

Page 4 of 28



Draft

(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

Page 5 of 28



Draft

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

Page 6 of 28



Draft

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)

Page 7 of 28



Draft

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.

Page 8 of 28



Draft

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

Page 9 of 28



Draft

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

Draft

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.

Page 11 of 28



Draft

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

Page 12 of 28



Draft

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

Page 13 of 28



https://www.ncbi.nlm.nih.gov/pubmed/28505110

Draft

(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

Page 14 of 28



Draft

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.

Page 15 of 28



Draft

References

Abu-awwad, A., Arafat, T., and Schmitz O.J. 2017. Study the influence of licorice and

pomegranate drinks on nicotine metabolism in human urine by LC-orbitrap MS. J. Pharm.

Biomed Anal.132: 60–65. doi: 10.1016/j.jpba.2016.09.026. PMID: 27693954.

Aebi, H. 1984. Catalase in vitro. Methods Enzymol. 105: 121–126. doi:10.1016/S0076-

6879(84)05016-3. PMID:6727660.

Afoulous, S., Ferhout, H., Raoelison, G.E., Valentin, A., Moukarzel, B., and Couderc F. 2013.

Chemical composition and anticancer, antiinflammatory, antioxidant and antimalarial

activities of leaves essential oil of Cedrelopsis grevei. Food. Chem.Toxicol. 56: 352–62. doi:

10.1016/j.fct.2013.02.008. PMID: 234659148.

Al-Malki, A.L., and Moselhy, S.S. 2013. Protective effect of vitamin E and epicatechin

against nicotine-induced oxidative stress in rats. Toxicol. Ind. Health. 29: 202-208. doi:

10.1177/0748233711430976. PMID:22287617.

Al-Awaida W., Akash M., Aburubaiha Z., Talib WH., Shehadeh H. 2014. Chinese green tea

consumption reduces oxidative stress, inflammation and tissues damage in smoke exposed

rats, Iran. J. Med. Sci.17: 740–746. PMID: 25726541.

Anwar D., Ibrahim, R., and Albadani N. 2014. Evaluation of the

Potential Nephroprotective and Antimicrobial Effect of Camellia sinensis Leaves versus

Hibiscus sabdariffa (In Vivo and In Vitro Studies). Adv. Pharmacol. Sci. 2014:389834. doi:

10.1155/2014/389834. PMID: 24949007.

Augustyniak, A., Waszkiewicz, E., Skrzydlewska, E. 2005. Preventive action of green tea

from changes in the liver antioxidant abilities of different aged rats intoxicated with ethanol,

Nutrition 21: 925–932. doi:http://dx.doi.org/ 10.1016/j.nut.2005.01.006. PMID: 16084066.

Page 16 of 28



https://www.ncbi.nlm.nih.gov/pubmed/?term=Al-Awaida%20W%5BAuthor%5D&cauthor=true&cauthor_uid=25729541https://www.ncbi.nlm.nih.gov/pubmed/?term=Akash%20M%5BAuthor%5D&cauthor=true&cauthor_uid=25729541https://www.ncbi.nlm.nih.gov/pubmed/?term=Aburubaiha%20Z%5BAuthor%5D&cauthor=true&cauthor_uid=25729541https://www.ncbi.nlm.nih.gov/pubmed/?term=Talib%20WH%5BAuthor%5D&cauthor=true&cauthor_uid=25729541https://www.ncbi.nlm.nih.gov/pubmed/?term=Shehadeh%20H%5BAuthor%5D&cauthor=true&cauthor_uid=25729541https://www.ncbi.nlm.nih.gov/pubmed/?term=Anwar%20Ibrahim%20D%5BAuthor%5D&cauthor=true&cauthor_uid=24949007https://www.ncbi.nlm.nih.gov/pubmed/?term=Noman%20Albadani%20R%5BAuthor%5D&cauthor=true&cauthor_uid=24949007https://www.ncbi.nlm.nih.gov/pubmed/24949007

Draft

Bansal, A.K., Bansal, M., Soni, G.,and Bhatnagar, D. 2005. Protective role of Vitamin E pre-

treatment on N-nitrosodiethylamine induced oxidative stress in rat liver. Chem. Biol. Interact.

156: 101-111. doi: 10.1016/j.cbi.2005.08.001. PMID:16144695.

Ben Saad, A., Rjeibi, I., Alimi, H., Ncib, S., Bouhamda, T., Zouari, N et al. 2018. Protective

effects of Mentha spicata against nicotine induced toxicity in liver and erythrocytes. Appl

Physiol Nutr Metab. 43:77-83. doi: 10.1139/apnm-2017-0144. PMID: 28892646.

Chandrasekharan, N.V., and Simmons, D.L., 2004. The cyclooxygenases, Genome. Biol.

2004: 522864. doi: 10.1186/gb-2004-5-9-241.PMID: 15345041.

Das, S., Tonelli, M., Ziedonis, D. 2016. Update on Smoking Cessation: E-Cigarettes,

Emerging Tobacco Products Trends, and New Technology-Based Interventions. Curr.

Psychiatry. Rep. 18:51. doi: 10.1007/s11920-016-0681-6. PMID: 27040275.

Delwing-Dal Magro, D., Roecker, R., Junges, G.M., Rodrigues, A.F., Delwing-de Lima,

D, da Cruz, J.G.P. 2016. Protective effect of green tea extract against proline-induced

oxidative damage in the rat kidney. Biomed. Pharmacother. 83:1422-1427 doi:

10.1016/j.biopha.2016.08.057. PMID: 16084066.

Desai, I.D. 1984. Vitamin E analysis method for animal tissue. Methods. Enzymol. 105:138–

143. doi.org/10.1016/S0076-6879(84)05019-9.

Djeridane, M., Yousfi, B., Nadjemi, S., Maamri, F., Djireb, P., and Stocker, P. 2006. Phenolic

extracts from various Algerian plants as strong inhibitors of porcine liver carboxylesterase. J.

Enzyme Inhib. Med. Chem. 21: 719–726. doi:10.1080/ 14756360600810399.

PMID:17252945.

Erkekoglou, I., Nenadis, N., Samara, E., and Mantzouridou, F.T. 2017. Functional Teas

from the Leaves of Arbutus unedo: Phenolic Content, Antioxidant Activity, and Detection of

Page 17 of 28



https://doi.org/10.1016/j.cbi.2005.08.001https://www.ncbi.nlm.nih.gov/pubmed/?term=Ben%20Saad%20A%5BAuthor%5D&cauthor=true&cauthor_uid=28892646https://www.ncbi.nlm.nih.gov/pubmed/?term=Rjeibi%20I%5BAuthor%5D&cauthor=true&cauthor_uid=28892646https://www.ncbi.nlm.nih.gov/pubmed/?term=Alimi%20H%5BAuthor%5D&cauthor=true&cauthor_uid=28892646https://www.ncbi.nlm.nih.gov/pubmed/?term=Ncib%20S%5BAuthor%5D&cauthor=true&cauthor_uid=28892646https://www.ncbi.nlm.nih.gov/pubmed/?term=Bouhamda%20T%5BAuthor%5D&cauthor=true&cauthor_uid=28892646https://www.ncbi.nlm.nih.gov/pubmed/?term=Zouari%20N%5BAuthor%5D&cauthor=true&cauthor_uid=28892646https://www.ncbi.nlm.nih.gov/pubmed/?term=Protective+effects+of+Mentha+spicata+against+nicotine-induced+toxicity+in+liver+and+erythrocytes+of+Wistar+ratshttps://www.ncbi.nlm.nih.gov/pubmed/?term=Protective+effects+of+Mentha+spicata+against+nicotine-induced+toxicity+in+liver+and+erythrocytes+of+Wistar+ratshttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC522864/https://doi.org/10.1186/gb-2004-5-9-241https://www.ncbi.nlm.nih.gov/pubmed/?term=Das%20S%5BAuthor%5D&cauthor=true&cauthor_uid=27040275https://www.ncbi.nlm.nih.gov/pubmed/?term=Tonelli%20M%5BAuthor%5D&cauthor=true&cauthor_uid=27040275https://www.ncbi.nlm.nih.gov/pubmed/?term=Ziedonis%20D%5BAuthor%5D&cauthor=true&cauthor_uid=27040275https://www.ncbi.nlm.nih.gov/pubmed/27040275https://www.ncbi.nlm.nih.gov/pubmed/27040275https://www.ncbi.nlm.nih.gov/pubmed/?term=Delwing-Dal%20Magro%20D%5BAuthor%5D&cauthor=true&cauthor_uid=27589827https://www.ncbi.nlm.nih.gov/pubmed/?term=Roecker%20R%5BAuthor%5D&cauthor=true&cauthor_uid=27589827https://www.ncbi.nlm.nih.gov/pubmed/?term=Junges%20GM%5BAuthor%5D&cauthor=true&cauthor_uid=27589827https://www.ncbi.nlm.nih.gov/pubmed/?term=Rodrigues%20AF%5BAuthor%5D&cauthor=true&cauthor_uid=27589827https://www.ncbi.nlm.nih.gov/pubmed/?term=Delwing-de%20Lima%20D%5BAuthor%5D&cauthor=true&cauthor_uid=27589827https://www.ncbi.nlm.nih.gov/pubmed/?term=Delwing-de%20Lima%20D%5BAuthor%5D&cauthor=true&cauthor_uid=27589827https://www.ncbi.nlm.nih.gov/pubmed/?term=da%20Cruz%20JGP%5BAuthor%5D&cauthor=true&cauthor_uid=27589827https://doi.org/10.1016/S0076-6879(84)05019-9https://www.ncbi.nlm.nih.gov/pubmed/?term=Erkekoglou%20I%5BAuthor%5D&cauthor=true&cauthor_uid=28421300https://www.ncbi.nlm.nih.gov/pubmed/?term=Nenadis%20N%5BAuthor%5D&cauthor=true&cauthor_uid=28421300https://www.ncbi.nlm.nih.gov/pubmed/?term=Samara%20E%5BAuthor%5D&cauthor=true&cauthor_uid=28421300https://www.ncbi.nlm.nih.gov/pubmed/?term=Mantzouridou%20FT%5BAuthor%5D&cauthor=true&cauthor_uid=28421300

Draft

Efficient Radical Scavengers. Plant. Foods. Hum. Nutr. 72: 176-183. doi: 10.1007/s11130-

017-0607-4. PMID: 28421300.

Flohe, L., and Gunzler, W.A. 1984. Assays of glutathione peroxidase. Methods Enzymol.

105: 114–121. doi:10.1016/S0076-6879(84)05015-1. PMID:6727659.

Ibrahim, Z.S., Alkafafy, M.E., Ahmed, M.M., Soliman, M.M. 2016. Renoprotective effect of

curcumin against the combined oxidative stress of diabetes and nicotinein rats. Mol. Med.

Rep. 13:3017-26. doi: 10.3892/mmr.2016.4922. PMID: 26936435.

Khan, N., Afaq, F., Saleem, M., Ahmad ,N., Mukhtar, H. 2006. Targeting multiple signaling

pathways by green tea polyphenol (-)-epigallocatechin-3-gallate, Cancer Res. 66: 2500–2505.

doi:10.1158/0008-5472.CAN-05-3636. PMID: 16510563.

Koleva, I.I., Van Beek, T.A., Linssen, J.P.H., De Groot, A., and Evstatieva, L.N. 2002.

Screening of plant extracts for antioxidant: a comparative study on three testing methods.

Phytochem . Anal. 13: 7–8. doi: 10.1002/pca.611. PMID: 11899609.

Liao, S. 2001. The medicinal action of androgens and green tea epigallocatechin gallate, Hong

Kong Med. J. 7: 369–374. PMID: 11773671.

Liu, J., Luo, J., Ye, H., Sun, Y., Lu, Z., and Zeng, X., 2010. In vitro and in vivo antioxidant

activity of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3.

Carbohydr. Polym. 82: 1278–1283. doi.org/10.1016/j.carbpol.2010.07.008. PMID: 2245789.

Li, Y.S, Kawasaki, Y., Tomita, I., and Kawai, K. 2017. Antioxidant properties of green

tea aroma in mice. J.Clin. Biochem. Nutr. 6:114-117. doi: 10.3164/jcbn.16-80. PMID:

28751804.

Lowry, O.H., Rosenbrough, N.J., and Randall R. 1951. Protein measurement with the folin

phenol reagent. J Biol Chem. 193: 265–275. doi:10.1139/cjpp-2016-0366. PMID:14907713.

Page 18 of 28



https://www.ncbi.nlm.nih.gov/pubmed/?term=Ibrahim%20ZS%5BAuthor%5D&cauthor=true&cauthor_uid=26936435https://www.ncbi.nlm.nih.gov/pubmed/?term=Alkafafy%20ME%5BAuthor%5D&cauthor=true&cauthor_uid=26936435https://www.ncbi.nlm.nih.gov/pubmed/?term=Ahmed%20MM%5BAuthor%5D&cauthor=true&cauthor_uid=26936435https://www.ncbi.nlm.nih.gov/pubmed/?term=Soliman%20MM%5BAuthor%5D&cauthor=true&cauthor_uid=26936435https://www.ncbi.nlm.nih.gov/pubmed/26936435https://www.ncbi.nlm.nih.gov/pubmed/26936435https://www.ncbi.nlm.nih.gov/pubmed/?term=Khan%20N%5BAuthor%5D&cauthor=true&cauthor_uid=16510563https://www.ncbi.nlm.nih.gov/pubmed/?term=Afaq%20F%5BAuthor%5D&cauthor=true&cauthor_uid=16510563https://www.ncbi.nlm.nih.gov/pubmed/?term=Saleem%20M%5BAuthor%5D&cauthor=true&cauthor_uid=16510563https://www.ncbi.nlm.nih.gov/pubmed/?term=Ahmad%20N%5BAuthor%5D&cauthor=true&cauthor_uid=16510563https://www.ncbi.nlm.nih.gov/pubmed/?term=Mukhtar%20H%5BAuthor%5D&cauthor=true&cauthor_uid=16510563https://doi.org/10.1158/0008-5472.CAN-05-3636https://doi.org/10.1002/pca.611https://doi.org/10.1016/j.carbpol.2010.07.008https://www.ncbi.nlm.nih.gov/pubmed/?term=Li%20YS%5BAuthor%5D&cauthor=true&cauthor_uid=28751804https://www.ncbi.nlm.nih.gov/pubmed/?term=Kawasaki%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=28751804https://www.ncbi.nlm.nih.gov/pubmed/?term=Tomita%20I%5BAuthor%5D&cauthor=true&cauthor_uid=28751804https://www.ncbi.nlm.nih.gov/pubmed/?term=Kawai%20K%5BAuthor%5D&cauthor=true&cauthor_uid=28751804https://www.ncbi.nlm.nih.gov/pubmed/28751804

Draft

Mada, E.Y., Santos, A.C., Fonseca, A.C., Biguetti, C.C., Neves, F.T., Saraiva, P.P et al.

2017.Effects of green tea and bisphosphonate association on dental socket repair of rats. Arch.

Oral. Biol. 75: 1-7. doi: 10.1016/j.archoralbio.2016. PMID: 27930925.

Maurya, D.K., Salvi, V.P., and Nair, C.K.K. 2005. Radiation protection of DNA by ferrulic

acid under in vitro and in vivo conditions. Mol. Cell Biochem. 280: 209–217.

doi:10.1007/s11010-005-0170-4. PMID:16311925.

Megow, I., Darvin, M.E., Meinke, M.C., and Lademann. J. 2017. A Randomized Controlled

Trial of Green Tea Beverages on the in vivo Radical Scavenging Activity in Human Skin.

Skin. Pharmacol. Physiol. 30: 225-233. doi: 10.1159/000477355. PMID: 28723689.

Mukhtar, H., Ahmad, N. 2000. Tea polyphenols: prevention of cancer and optimizing health,

Am. J. Clin. Nutr. 71: 1698–1702. doi: 10.1093/ajcn/71.6.1698S. PMID: 10837321.

Misra, H.P., and Fridovich, I. 1972. The role of superoxide anion in the autooxidation of

epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247: 3170–3175.

PMID: 4623845. doi:10.1007/s11033-015-3929.

Mosbaha, R., Yousef, M.I., and Mantovani, M. 2015. Nicotine-induced reproductive toxicity,

oxidative damage, histological changes and haematotoxicity in male rats: the protective

effects of green tea extract. Exp. Toxicol. Pathol. 67: 253–259. doi:10.1016/j.etp.2015.01.001.

PMID: 25650282.

Naganuma, T., Kuriyama, S., Kakizaki, M., Sone, T., and Nakaya N. 2009. Green tea

consump-tion and hematologic malignancies in Japan: the Ohsaki study. Am. J. Epidemiol.

29:1–9. doi: 10.1093/aje/kwp187. PMID: 19640889.

Page 19 of 28



https://www.ncbi.nlm.nih.gov/pubmed/?term=Mada%20EY%5BAuthor%5D&cauthor=true&cauthor_uid=27930925https://www.ncbi.nlm.nih.gov/pubmed/?term=Santos%20AC%5BAuthor%5D&cauthor=true&cauthor_uid=27930925https://www.ncbi.nlm.nih.gov/pubmed/?term=Fonseca%20AC%5BAuthor%5D&cauthor=true&cauthor_uid=27930925https://www.ncbi.nlm.nih.gov/pubmed/?term=Biguetti%20CC%5BAuthor%5D&cauthor=true&cauthor_uid=27930925https://www.ncbi.nlm.nih.gov/pubmed/?term=Neves%20FT%5BAuthor%5D&cauthor=true&cauthor_uid=27930925https://www.ncbi.nlm.nih.gov/pubmed/?term=Saraiva%20PP%5BAuthor%5D&cauthor=true&cauthor_uid=27930925https://www.ncbi.nlm.nih.gov/pubmed/?term=Megow%20I%5BAuthor%5D&cauthor=true&cauthor_uid=28723689https://www.ncbi.nlm.nih.gov/pubmed/?term=Darvin%20ME%5BAuthor%5D&cauthor=true&cauthor_uid=28723689https://www.ncbi.nlm.nih.gov/pubmed/?term=Meinke%20MC%5BAuthor%5D&cauthor=true&cauthor_uid=28723689https://www.ncbi.nlm.nih.gov/pubmed/?term=Lademann%20J%5BAuthor%5D&cauthor=true&cauthor_uid=28723689https://www.ncbi.nlm.nih.gov/pubmed/28723689

Draft

Ohkawa, H., Ohishi, N. and Yagi, K., 1979. Assay for lipid peroxides in animal tissues by

thiobarbituric acid reaction. Anal. Biochem. 95: 351–358. doi:10.1016/S0076

6879(84)05015-1. PMID: 36810

Omaye, S.T., Turbull, T.P., and Sauberlich, H.C. 1979. Selected methods for determination of

ascorbic acid in cells, tissues and fluids. Methods. Enzymol. 6: 3–11. doi.org/10.1016/0076-

6879(79)62181-X. PMID: 440112.

Pereira, C.G., Barreira, L., Bijttebier, S., Pieters, L., Neves, V., Rodrigues M.J et al.

2017. Chemical profiling of infusions and decoctions of Helichrysum italicum subsp. picardii

by UHPLC-PDA-MS and in vitro biological activities comparatively with green tea (Camellia

sinensis) and rooibos tisane (Aspalathus linearis). J Pharm. Biomed. Anal. 145: 593-603. doi:

10.1016/j.jpba.2017.07.007. PMID: 28787672.

Perkins, K.A., Epstein, L.H., Stiller, R.L., Fernstrom, M.H., Sexton, J.E., and Jacob, R.G.

1991. Acute effects of nicotine on hunger and caloric intake in smokers and nonsmokers.

Psychophar-macology. 103: 103–109. doi:10.1016/j.biopha.2016.12.008. PMID: 2006236.

Prasad, W.L., Srilatha, C., Sailaja, N., Raju, N.K., Jayasree N. 2016. Amelioration of Gamma-

hexachlorocyclohexane (Lindane) induced renal toxicity by Camellia sinensis in Wistar rats.

Vet World. 9: 1331–1337. DOI: 10.14202/vetworld.2016.1331-1337. PMID: 27956790.

Price, M.L., and Butler, L.G. 1977. Rapid visual estimation and spectrophotometric

determination of tannin content of sorghum grain. J. Agric. Food. Chem. 25: 1268–1273.

doi: 10.1021/jf60214a034. PMID:52456110.

Rhee, S.J., Choi, J.H., Park, M.R. 2002. Green tea catechin improves microsomal

phospholipase A2 activity and the arachidonic acid cascade system in the kidney of diabetic

rats, Asia Pac. J. Clin. Nutr. 11: 226–231. PMID: 12230237.

Page 20 of 28



https://doi.org/10.1016/0076-6879(79)62181-Xhttps://doi.org/10.1016/0076-6879(79)62181-Xhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Pereira%20CG%5BAuthor%5D&cauthor=true&cauthor_uid=28787672https://www.ncbi.nlm.nih.gov/pubmed/?term=Barreira%20L%5BAuthor%5D&cauthor=true&cauthor_uid=28787672https://www.ncbi.nlm.nih.gov/pubmed/?term=Bijttebier%20S%5BAuthor%5D&cauthor=true&cauthor_uid=28787672https://www.ncbi.nlm.nih.gov/pubmed/?term=Pieters%20L%5BAuthor%5D&cauthor=true&cauthor_uid=28787672https://www.ncbi.nlm.nih.gov/pubmed/?term=Neves%20V%5BAuthor%5D&cauthor=true&cauthor_uid=28787672https://www.ncbi.nlm.nih.gov/pubmed/?term=Rodrigues%20MJ%5BAuthor%5D&cauthor=true&cauthor_uid=28787672https://www.ncbi.nlm.nih.gov/pubmed/?term=Prasad%20WL%5BAuthor%5D&cauthor=true&cauthor_uid=27956790https://www.ncbi.nlm.nih.gov/pubmed/?term=Srilatha%20C%5BAuthor%5D&cauthor=true&cauthor_uid=27956790https://www.ncbi.nlm.nih.gov/pubmed/?term=Sailaja%20N%5BAuthor%5D&cauthor=true&cauthor_uid=27956790https://www.ncbi.nlm.nih.gov/pubmed/?term=Raju%20NK%5BAuthor%5D&cauthor=true&cauthor_uid=27956790https://www.ncbi.nlm.nih.gov/pubmed/?term=Jayasree%20N%5BAuthor%5D&cauthor=true&cauthor_uid=27956790https://doi.org/10.14202/vetworld.2016.1331-1337https://www.ncbi.nlm.nih.gov/pubmed/?term=Rhee%20SJ%5BAuthor%5D&cauthor=true&cauthor_uid=12230237https://www.ncbi.nlm.nih.gov/pubmed/?term=Choi%20JH%5BAuthor%5D&cauthor=true&cauthor_uid=12230237https://www.ncbi.nlm.nih.gov/pubmed/?term=Park%20MR%5BAuthor%5D&cauthor=true&cauthor_uid=12230237

Draft

Yarahmadi A, Zal, F., and Bolouki A. 2017. Protective effects of quercetin

on nicotine induced oxidative stress in 'HepG2 cells'. Toxicol. Mech. Methods. 27: 609-614.

doi: 10.1080/15376516. PMID: 28627253.

Yazici, K., and Goksu, B. 2017. Effects of Kaolin (M-99-099) application on antioxidant and

phenolic compounds in tea leaves (Camellia sinensis L.O.Kuntze), Biochem Genet. 55: 367-

377. doi: 10.1007/s10528-017-9805. PMID: 28639055.

Zhang, H., and Tsao, R. 2016. Dietary polyphenols, oxidative stress and antioxidant and anti-

inflammatory effects. Curr Opin in Food Sci. 8: 33–42. doi.org/10.1016/j.cofs.2016.02.002.

PMID : 28267434.

Zahran W.E, Elsonbaty S.M., and Moawed F.S.M. 2017. Erratum to: Selenium nanoparticles

with low-level ionizing radiation exposure ameliorate nicotine-induced inflammatory

impairment in rat kidney. Environ. Sci. Pollut. Res. Int, 24:19990-19991. DOI:

10.1007/s11356-017-9779-6. PMID: 28819934.

Neogy, S., Das, S, Mahapatra, S.K, Mandal, N., Roy, S. 2007. Amelioratory effect of

Andrographis paniculata Nees on liver, kidney, heart, lung and spleen

during nicotine induced oxidative stress. Environ. Toxicol. Pharmacol. doi:

10.1016/j.etap.2007.10.034. 25 :321-8. PMID: 21783869.

Page 21 of 28



https://www.ncbi.nlm.nih.gov/pubmed/?term=Zal%20F%5BAuthor%5D&cauthor=true&cauthor_uid=28627253https://doi.org/10.1016/j.cofs.2016.02.002https://www.ncbi.nlm.nih.gov/pubmed/?term=Zahran%20WE%5BAuthor%5D&cauthor=true&cauthor_uid=28819934https://www.ncbi.nlm.nih.gov/pubmed/?term=Elsonbaty%20SM%5BAuthor%5D&cauthor=true&cauthor_uid=28819934https://www.ncbi.nlm.nih.gov/pubmed/?term=Moawed%20FSM%5BAuthor%5D&cauthor=true&cauthor_uid=28819934https://doi.org/10.1007/s11356-017-9779-6https://www.ncbi.nlm.nih.gov/pubmed/?term=Neogy%20S%5BAuthor%5D&cauthor=true&cauthor_uid=21783869https://www.ncbi.nlm.nih.gov/pubmed/?term=Das%20S%5BAuthor%5D&cauthor=true&cauthor_uid=21783869https://www.ncbi.nlm.nih.gov/pubmed/?term=Mahapatra%20SK%5BAuthor%5D&cauthor=true&cauthor_uid=21783869https://www.ncbi.nlm.nih.gov/pubmed/?term=Mandal%20N%5BAuthor%5D&cauthor=true&cauthor_uid=21783869https://www.ncbi.nlm.nih.gov/pubmed/?term=Roy%20S%5BAuthor%5D&cauthor=true&cauthor_uid=21783869

Draft

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).

Page 22 of 28



Draft

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++


*p < 0.05, **p < 0.01 compared with control (C).

+p < 0.05, ++p < 0.01, compared with nicotine-treated group (NT).

Page 23 of 28



Draft

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++


*p < 0.05, **p < 0.01 compared with control (C).

+p < 0.05, ++p < 0.01, compared with nicotine-treated group (NT).

Page 24 of 28



Draft

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.

Page 25 of 28



Draft

67x50mm (300 x 300 DPI)

Page 26 of 28



Draft

67x50mm (300 x 300 DPI)

Page 27 of 28



Draft

67x50mm (300 x 300 DPI)

Page 28 of 28



Documents

DraftDraft Nephroprotective and antioxidant effect of green tea (Camellia sinensis) against nicotine-induced nephrotoxicity in rats and characterization of its bioactive compounds