Biochemistry and Cell Biology

Biological Activity of spinosus on Inhibition of Paracetamol- Induced Renal Inflammation

Journal: Biochemistry and Cell Biology

Manuscript ID bcb-2018-0212.R2

Manuscript Type: Article

Date Submitted by the 20-Sep-2018 Author:

Complete List of Authors: Hegazy, Marwa; Ain Shams University Faculty of Science Emam, Manal; Ain Shams University Faculty of Science Khattab, Hemmat; Ain Shams University Faculty of Science Helal, Nesma;Draft Ain Shams University Faculty of Science Nephrotoxicity, Phytochemicals, oxidative stress, gas chromatography Keyword: mass spectrometry, acetaminophen

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

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1 Biological Activity of Echinops spinosus on Inhibition of Paracetamol-

2 Induced Renal Inflammation

3 Short title: Echinops spinosus attenuate nephrotoxicity

4 Marwa Hegazya*, Manal Emama, Hemmat Khattabb, Nesma Helalb

5 a Biochemistry Department, Faculty of Science, Ain Shams University, Abbassia, 11566, Cairo,

6 Egypt.

7 b Botany Department, Faculty of Science, Ain Shams University, Abbassia, 11566, Cairo, Egypt.

8 *Corresponding author: Draft

9 Marwa Hegazy

10 Email: [email protected], [email protected]

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11

12 Abstract

13 This study was designed to evaluate the possible mechanisms through which Echinops Spinosus

14 (ES) extract demonstrates nephroprotective effect on paracetamol (APAP)-

15 induced nephrotoxicity in rat. Twenty-Four Swiss albino rats were divided into four groups (six

16 rats each). Placebo group was orally administered sterile saline; APAP group received APAP (200

17 mg/kg/day i.p) daily; ES group was given orally ES extract (250 mg/kg); (APAP+ES) group: received

18 APAP as for APAP group and administrated ES extract as for ES group. Pretreatment of methyl

19 alcohol extract of ES reduced the protein expression of inflammatory parameters including 20 cyclooxygenase-2 (COX-2) and nuclearDraft factor kappa B (NF-κB) in kidney. It also reduced the 21 mRNA gene expression of tumor necrosis factor-α (TNF-α) and Interleukin-1β (IL-1β). ES

22 extract compensated deficits in the total antioxidant activity, suppressed lipid peroxidation and

23 amended the APAP induced histopathological kidney alterations. Moreover, ES treatment

24 restored the elevated levels of urea nitrogen in blood and creatinine in serum by acetaminophen.

25 ES extract attenuated the acetaminophen-induced elevations in renal nitric oxide levels. We

26 clarified that ES extract has the potential to defend kidney from APAP-induced inflammation,

27 and protection mechanism might by through decreasing oxidative stress and regulating the

28 inflammatory signaling pathway through modulating key signaling inflammatory biomarkers.

29 Key Words: Nephrotoxicity; Phytochemicals; oxidative stress; gas chromatography mass

30 spectrometry; acetaminophen.

31 Introduction

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32 A wide range of medicinal have been used in different countries and cultures as a

33 prophylactic and curative agent for urolithiasis (Gülçin et al., 2006). One of the most cited

34 families are (Ahmed et al. 2016). Genus Echinops family Asteraceae (Compositae)

35 comprises about 120 distributed through Mediterranean region to central Asia

36 and Tropical Africa (Kadereit and Jeffrey 2007). It is common throughout the Sahara including

37 Sinai and the Red Sea coast. In Egypt, Echinops spinosus L is among five other species

38 representing this genus (Boulos 2009). Echinops spinosus (ES) is a perennial herb growing 1

39 meter or more and locally named Tassekra (Khedher 2014). Stems, and roots of ES are

40 used as diuretic (Boulos 1983).

41 As a medicinal , ES is also used as a curative plant, it was frequently employed in

42 traditional medicine as an abortifacient,Draft a diuretic, for blood circulation, diabetes, gastric pain,

43 indigestion and spasmolytic problems (Khedher 2014). ES displayed several therapeutic

44 properties like anti-oxidant, anti-inflammatory and anti-microbial activities (Mujawar et al. 2015;

45 Maurya et al. 2015; Bouattour et al. 2016).

46 Secondary metabolites produced by plants are a great source of drug due to their safety, easy

47 availability, valuable effects on human body and high medicinal values. These metabolites have

48 been well-studied to not only their antioxidant properties but also, they have been proven to have

49 nephroprotective effects (Katanić et al. 2017; Gülçin, 2012). The Echinops characteristic

50 medicative uses could be due to the incidence of phenols (Khedher et al. 2014), quinoline

51 alkaloids Chevrier et al. 1976), flavanoids and sesquiterpenes (Boumaraf et al. 2016), acetylated

52 terpenoids and sterols (Bouattour et al. 2016), moreover as fatty acids and alkanes (Chevrier et

53 al. 1975).

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54 Acetaminophen (N-acetyl-p-aminophenol; APAP), which is known as paracetamol, is the

55 most commonly used analgesic and antipyretic medication. However, its overdose leads to

56 kidney and liver damage. numerous scientific reports have focused majorly on APAP

57 hepatotoxicity. Alternatively, not many works approach APAP nephrotoxicity focusing on both

58 its mechanisms of action and therapeutic exploration (Karthivashan et al. 2016).

59 Acetaminophen toxicity generates acute tubular necrosis; one of the main causes of acute

60 renal failure. This toxicity initially happens by APAP metabolism to N-acetyl-p-benzoquinone

61 by the microsomal P-450 enzyme system that depletes reduced glutathione (GSH) and forms

62 APAP-protein adducts. Later on, the reactive nitrogen species peroxynitrite is created from nitric

63 oxide (NO) and superoxide resulting in 3-nitrotyrosine (Banerjee et al. 2017). Free radicals are

64 produced by exposure to drug toxicity inDraft an organism, and oxidative damage plays a significant

65 role in acetaminophen-induced hepatorenal injuries (Kandemir et al. 2017). Therefore, Medicinal

66 plants and phytomedicine are the prime choice of research as they possess better activity than

67 synthetic drugs and lesser side effects (Parameshappa et al. 2012).

68 Till date, no study has been dedicated to assess the protective efficacy of E. spinosus against

69 nephrotoxicity induced by APAP in rats. Keeping this visible, this study aimed primarily to

70 assess the nephroprotective effects and modulatory mechanisms of E. spinosus upon kidney of

71 APAP treated rats.

72 Materials and methods

73 Chemicals

74 Chemicals were purchased of high analytical grade from Biodiagnostic Company for

75 diagnostic and research reagents (Dokki, Giza, Egypt). Acetaminophen (APAP) and all

76 phenolic and flavonoid reagents were purchased from Sigma Co. USA.

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77 Plant Materials

78 E. spinosus shrubs with matched size and age were collected from Wadi Hagul arid habitat

79 throughout the flowering spring season (April 2017). The plant was then identified by Professor

80 Dr. Hemmat Khattab and documented in Botany Department, Ain Shams University (Egypt).

81 Plant name has been checked with http://www.theplantlist.org. Echinops spinosus L. is

82 a synonym of Echinops spinosissimus Turra. A voucher specimen was submitted at the

83 herbarium of botany department - faculty of science - Ain shams university for future reference.

84 Preparation of plant extract

85 The aerial parts (leaves/stem), roots and flower heads were cleansed, dried at room

86 temperature within the shade then ground,Draft to a powder by mechanical mills. The dried powder

87 (100 g) was extracted with distilled water, ethyl alcohol, methyl alcohol, petroleum ether, ethyl

88 acetate, and hexane at 4º C. After 72 h the extracts were filtered and concentrated on rotary

89 evaporator under reduced pressure at 30 °C. Then, the crude concentrated extracts for every

90 solvent were completed with methanol to final volume and then subjected to phytochemical

91 analysis (Harborne 1998) after discharging their colors by using active charcoal. The

92 phytochemical analysis was carried out qualitatively to determine the suitable solvent for the

93 maximum quantitative estimation of nutraceuticals secondary metabolites.

94 Preliminary phytochemical study

95 Preliminary qualitative phytochemical screening will give idea about the chemical

96 constituents present in the extract and will help for further investigation. Phytochemical

97 screening was done as explained in literature (Ikhiri et al. 1992; Silva et al. 1993; Harborne

98 1998; Houghton and Raman 1998).

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99 Phytochemical properties of the extracts were studied using the following reagents and

100 methods: alkaloids with 10% acetic acid and ammonium hydroxide reagents; flavanoids with the

101 use of aluminum chloride colorimetric method; The total phenolics and tannins were measured

102 using Folin-Ciocalteu method; saponins by using vanillin -HC1 reagent; triterpnes and total

103 proanthocyanidins by using vanillin reagent; carbohydrate with Molish’s and anthrone reagents;

104 glycosides with Baljet’s test. Total antioxidant capacity was assessed by the

105 phosphomolybdenum method according to Prieto et al. (1999). The antioxidants soluble in water

106 such as glutathione and ascorbic acid were determined by the methods of Griffith (1980) and

107 Kampfenkel et al. (1995), respectively 108 Radical-scavenging activity assayDraft 109 The capacity to free radicals scavenge was assayed using 2,2-diphenyl-1-picrylhydrazyl

110 (DPPH) according to Brand-Williams et al. (1995). The scavenging g activity was expressed as

111 the % DPPH radical scavenged according to the equation:

112 Scavenging effect (%) = [1 - (A517 of test sample) / (A517 of control)] x 100

113 GC-MS Analysis

114 The Gas Chromatography–Mass Spectrometry (GC-MS) analysis was by using Agilent

115 7890B GC system coupled to an Agilent 5977A MSD with a capillary column (0.6 m x 100 μm

116 x 0.25 μm) (Agilent Technologies, Santa Clara, CA, USA). The carrier gas Helium was used as

117 at constant flow rate of 1.5 mL/min. The injector temperature was set at 250° C and the ion

118 source temperature was set at 230° C. The initial oven temperature was set at 40 °C for 2 min,

119 then 10 °C/min to 180° C for 5 min and then 10° C/min to 250° C for 10 min. The total GC

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120 running time was 38 min. The GC/MS was run in Scan/SIM mode and the Identification of

121 sample’s components was performed using Agilent Mass Hunter software (NIST14.L).

122 Experimental animals

123 Twenty-four adult Swiss albino male rats weighing from 120 to 150 g were acquired from

124 the Egyptian Company for Vaccines and Medicines. The animals were housed in standard

125 polypropylene cage under standard conditions of temperature (20 ± 2°C), relative humidity

126 (50 ± 15%), 12 h light/dark cycle, free access to food (standard dry rodent diet) and water ad

127 libitum. All animals were acclimatized for 7 days before experiment starts. The research was

128 conducted in accordance with the internationally accepted principles for laboratory animal use 129 and care. The experimental protocolsDraft were approved by the Institutional Animal Ethical 130 Committee of Faculty of Science, Ain Shams University, Egypt.

131 Experimental design

132 Animals were divided into four groups of 6 rats in each group. In placebo group (N), normal

133 healthy rats received orally and daily 10 ml/kg/day of normal saline for 14 days. In

134 acetaminophen control group (APAP), rats received a single daily intraperitoneal dose 200

135 mg/kg/day of APAP for 14 days (Pareta et al. 2011). In E. spinosus control group (ES), normal

136 healthy rats treated with single daily oral dose of 250 mg/kg of E. spinosus extract for 14 days

137 (Abdulrazzaq et al. 2008). In acetaminophen and E. spinosus group (APAP+ES), rats were

138 pretreated with single daily oral dose of 250 mg/kg of E. spinosus extract, 1 hour before the

139 intraperitoneal administration of 200 mg/kg/day of APAP for 14 days.

140 On the 15th day, the overnight fasted rats were sacrificed under anesthesia. Blood samples

141 and pieces of kidney from each group were collected for biochemical and histopathological

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142 examination. The kidneys were rapidly excised, weighed and spliced into pieces, where one part

143 was fixed in 10% formalin for histopathological examination while the rest was stored at -80 °C

144 for subsequent analyses.

145 Biochemical assays

146 Blood samples were withdrawn straightaway by cardiac puncture for biochemical assays into

147 clean test-tubes without anticoagulant. Serum was separated by centrifugation for 10 min at

148 1000 ×g and stored at -20°C until analysis. Serum samples obtained from each rat in each group

149 were used to determine blood urea nitrogen (BUN), creatinine (Cr), uric acid (UA) and nitric

150 oxide (NO) using commercially available kits (Bio-diagnostic kits) according to the 151 manufacturer’s protocol. Lipid peroxidationDraft was determent by method of Ohkawa et al. (1979) 152 through measuring the amount of thiobarbituric acid reactive substances (TBARS) resulted from

153 the reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA). Total antioxidant

154 activity (TAA) was determined according to the method of Koracevic et al. (2000).

155 Western blot analysis

156 The western blotting technique was used to evaluate the protein expression levels of COX-2

157 and NF-κB in pools of 6 rats’ kidney homogenate for each group. Kidney’s sections were

158 homogenized in TRIzol reagent (Invitrogen) according to Chomczynski (1993). The insoluble

159 material was then removed by centrifugation at 12,000 xg for 15 min at 4°C. Protein

160 concentration was measured using Bradford assay (1976). Denatured protein 20 µg per lane was

161 separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and

162 then transferred to polyvinylidene difluoride (PVDF) membranes. After being blocked with 5%

163 skimmed milk in Tris-buffered saline Tween-20 (0.1% TBST) for 1 h at room temperature,

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164 immunoblots were obtained after incubating the membranes overnight at 4°C with antibodies

165 against NF-κB, COX-2 and beta-actin as loading control (Cell Signaling Technology, Danvers,

166 USA). Following 3 washes in 10 mM Tris–Cl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20, the

167 membranes were incubated for 2 h with a horseradish peroxidase-conjugated secondary antibody

168 (cell signaling technology, Danvers, USA). The blots were visualized using the enhanced

169 chemiluminescence method (Amersham detection kit according to the manufacturer’s protocol)

170 and then exposed to X-ray film. The protein expression results were normalized to beta-actin and

171 the bands were scanned and quantified by densitometric analysis (Biomed Instrument Inc.,

172 USA). 173 RNA isolation and real-time QPCRDraft 174 Total RNA was isolated from kidney tissue samples using the Thermo Scientific GeneJET

175 RNA Purification Kit, according to the manufacturer’s instructions. Total RNA (1µg) was then

176 used for cDNA synthesis using the Thermo Scientific RevertAidTM First Strand cDNA synthesis

177 kit. The relative expression levels of mRNA encoding tumor necrosis factor alfa (TNF-α),

178 interleukin 1β (IL-1β), or β -actin were measured using the Thermo Scientific Maxima SYBR

179 Green/ROX qPCR Master Mix, according to manufacturer’s protocol, and the results were

180 computerized using Stratagene (Mx3000PTM) machine.

181 Primer sequences were: 5‵-ACTGAACTTCGGGGTGATTG-3‵ (sense) and 5‵-

182 GCTTGGTGGTTTGCTACGAC-3‵ (anti-sense) for TNF-α, 5‵-CAATCTGGCAAGGATCAGC-

183 3‵ (sense) and 5‵-GGACGGACACAAGGGTACTAA-3‵ (anti-sense) for IL-1β, 5‵-

184 GTCAGGTCATCACTATCGGCAAT-3‵ (sense) and 5‵-

185 AGAGGTCTTTACGGATGTCAACGT-3‵ (anti-sense) for β-actin. The expression levels of

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186 TNF-α and IL-1β were normalized to β-actin and presented as fold change relative to untreated

187 control.

188 Histopathological examination

189 Sections of kidneys from every group were fixed immediately in 10% neutral formalin for at

190 least 24 h, dehydrated in xylene and embedded in paraffin at 56 °C in hot air oven for 24 h.

191 Blocks of paraffin bees wax tissue were sectioned at 4 µ by sledge microtome. The obtained

192 sections were collected on glass slides, deparaffinized and stained by hematoxylin and eosin

193 (H&E) stains (Bancroft and Stevens 1996) then examined using 400x magnification electrical

194 microscope. Scoring of histopathological changes and severity of kidney damage was assessed 195 using scores of nil (-), mild (+), moderateDraft (++), severe (+++) and extensively severe (++++).

196 Statistical analysis

197 Data were analyzed using computer software, Statistical Package for the Social Sciences

198 (SPSS) version 17 software (SPSS, Chicago, IL, USA). All results were expressed as mean value

199 ± standard error (S.E.). Statistical analysis was performed using the one-way analysis of variance

200 (ANOVA), followed by post-hoc test for intergroup comparisons. A difference in the mean

201 values of P < 0.05 was considered to be statistically significant.

202 Results

203 Phytochemical study of E. spinosus

204 The phytochemical screening of crude extracts of arid inhabiting E. spinosus parts were done

205 to detect the presence of nutraceuticals bioactive metabolites and the best suited solvent. The

206 extracts of E. spinosus shoots, flower heads (), and roots indicated the presence of

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207 phenols, flavonoids, triterpenoids, steroids, alkaloids, saponins, coumarins, glucosinolates,

208 proanthocyanidines and cardiac glycosides. The best and more relevant observed results are

209 assayed in the alcoholic extracts notably methyl alcohol (Table 1) that then used in the

210 quantification of the secondary metabolites and animal experiment.

211 Moreover, the best qualitative secondary metabolites contents were detected in E. spinosus

212 aerial parts as compared with their roots and flowers heads. Therefore, the secondary metabolites

213 levels were determined within the aerial parts of E. spinosus methanolic extract. The

214 quantitative analysis of the total bioactive components of the aerial parts of E. spinosus revealed

215 the existence of 0.52 mg soluble sugars/g dry matter, 5.21 µmole ascorbic acid (ASA)/g dry 216 matter, 20.4 mmol reduced glutathioneDraft (GSH)/g dry matter, 3.13 mg of total phenols / g dry 217 matter, 61 µ g of flavonoids / g dry matte and 0.58 µg of total tannins/ g dry matter. Moreover,

218 223.5 µg of total saponins, 1.35 mg proanthocyanidines, 4. 725 µM, 9.88 µmol glucosinolates

219 and 31.2 mg total alkaloids were determined in one-gram dry matter (Table 2).

220 Meanwhile, the GC-MS analysis of the methanolic extract of E. spinosus aerial portions is

221 shown in Figure 1 and Table 3. It revealed the presence of 48 compounds. The major identified

222 bioactive constituents are 2-furanone,3-4-dihydroxy (4.3%), alpha,-D-Mannopyranoside, methyl

223 3,6-anhydro (3.2%), 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl (2.4%), 2-

224 Cyclohexen-1-one, 2-methyl-5-(1-methylethyl)-, (S) (2.4%), 5-Hydroxymethylfurfural (4.3%),

225 2-Cyclopenten-1-one, 3-methyl-2-(2-pentenyl)-, (2.1%), 1- methyl- 3-n-Propyl-2-pyrazolin-5-

226 one (1.44%), Phenol, 4-methoxy-2,3,6-trimethyl (15.9%) and Xanthoxylin (20.1%) which are

227 characterized by the anti-inflammatory and anti-oxidant properties. Identification of the

228 compounds was carried out by matching their retention times and fragmentation patterns with

229 those of reference compounds analyzed under the same conditions (Abd El-Ghaffar et al., 2017).

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230 Antioxidant and radical-scavenging activities

231 Assessment of the whole antioxidant activity showed that, methanol extract of the aerial parts

232 exhibited a total antioxidant capacity and free radical scavenging DPPH reached about 103.4

233 nmole ascorbic acid/g and 20.0% respectively (Table 2). Consequently, the nephroprotective

234 effect of E. spinosus against APAP evoked nephrotoxicity in rats was detected.

235 Effects of methanol extract of E. spinosus on biochemical parameters

236 To investigate the consequences of E. spinosus against APAP-induced biochemical

237 alterations, kidney function indicators were determined. As shown in Table (4) significant

238 elevation (P < 0.001) in the serum levels of creatinine, BUN and uric acid were observed among

239 rats receiving APAP alone (group APAP),Draft compared to those of the placebo group (group N).

240 Notably, co-administration of E. spinosus together with APAP (group APAP + ES) significantly

241 alleviated this APAP-induced elevation, yet, the levels were still somewhat different from the

242 normal control levels. No significant change was observed in the serum levels of these tested

243 parameters when the rats were treated with E. spinosus, alone (ES group) as compared to the

244 placebo group (N group).

245 The results of NO estimations across different treatment groups have been shown in Figure

246 (2). The amount of renal nitric oxide in the APAP treated group was significantly higher (p <

247 0.001) than the normal placebo rats. Whereas, co-treatment of rats with E. spinosus along with

248 APAP (groups APAP + ES), significantly decreased (p < 0.001) renal NO than the APAP treated

249 group. In contrast, no significant change in the renal NO was observed among rats receiving E.

250 spinosus alone (ES group).

251 Effects of methanol extract of E. spinosus on oxidative stress markers

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252 Oxidative stress resulted from the metabolism of APAP in the kidney plays a critical role in

253 its damages. The effect of oral administration of E. spinosus on MDA and TAA levels of rats’

254 kidney tissues was investigated in the study to assess the protective efficiency of E. spinosus

255 against the induced oxidative-renal tissues damages.

256 As shown in Table (2), the MDA level was significantly increased (P < 0.001) among rats of

257 the APAP group compared to those of the placebo group. Concurrently, a significant reduction

258 (P < 0.001) in the level of TAA was observed among rats of group APAP, compared to the

259 placebo group. Notably, co-treatment of rats with E. spinosus, significantly suppressed the

260 APAP -induced elevation of serum MDA and improved close to control levels of TAA, as

261 compared to the APAP group. In contrast, no significant changes in the serum MDA levels or

262 TAA levels were observed among ratsDraft receiving E. spinosus alone (ES group). These results

263 showed the antioxidant and protective effects of E. spinosus against APAP -induced oxidative

264 stress and their ability to augment cellular antioxidant defenses. The current investigation

265 showed additionally that, the increments in lipid peroxidation product (MDA) level in APAP

266 treated rat’s kidney was markedly lowered by using E. spinosus crude extract as shown in APAP

267 + ES group.

268 E. spinosus extract ameliorated inflammatory markers COX-2 and NF-κB in renal

269 tissue

270 As shown in Figure 3 (A & B), APAP treatment significantly increased protein expression

271 for the pro-inflammatory mediator COX-2 and cytokine NF-κB compared to placebo group,

272 whereas animals receiving E. spinosus alone did not show elevated levels of these

273 inflammatory factors. The up regulated protein expression of COX-2 in rats revealing APAP

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274 intoxication was reduced in rats pre-treated with E. spinosus. Additionally, E. spinosus

275 administration resulted in a decrease in the APAP-induced NF-κB protein level.

276 E. spinosus extract down regulates pro-inflammatory markers TNFα and IL-1β

277 expression in renal tissue

278 The expression levels of TNFα and IL-1β mRNA were measured in normal and treated

279 groups by real time PCR. The β-actin gene; an internal standard was used for normalization of

280 target gene expression levels. As shown in Figures (4 & 5) respectively, there was a significantly

281 up regulation of TNFα and IL-1β mRNA expression (p < 0.001) in rats treated with APAP alone

282 (APAP group), as compared to rats of the placebo group. In contrast, the co-administration of E. 283 spinosus along with APAP (groups APAPDraft + ES) significantly induced the down regulation of the 284 TNFα and IL-1β expression levels in comparison to the sole administration of APAP (APAP

285 group). The differences in TNFα or IL-1β mRNA levels were insignificant between placebo

286 group and ES group.

287 Histopathological study

288 To further confirm the protective effect of E. spinosus methanol extract against the

289 acetaminophen-induced renal injury, we histopathologically examined kidney tissue after E.

290 spinosus pretreatment (figure 6).

291 The histological studies of the placebo group rats and ES group rats showed normal

292 histological structure of renal tubules and glomeruli in kidney tissues (fig 6a &6b). However, in

293 APAP treated rats a small glomerulus, severe necrosis of tubule, degeneration of vacuolar

294 tubules, epithelial desquamation, and intraluminal casts were shown (fig 6c). Meanwhile, APAP-

295 treated rats administered E. spinosus, showed marked improvement with mild tubular necrosis

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296 and degeneration of vacuoles (fig 6d). Quantitative evaluation of ES alleviative potential on

297 APAP induced nephrotoxicity based on scoring the severity of injury is presented in Table 5.

298 Discussion

299 Many wild plants are used in the traditional medicine and pharmaceutical preparations. The

300 pharmaceutical beneficial effects of the medicinal plants are usually depending on their

301 antioxidant bioactive phytochemicals levels (Abd Eldaim et al. 2017; Abdel-Daim et al. 2015).

302 Therefore, the current searching and screening for beneficial plants with potential bioactive

303 properties and the concomitant isolation and characterization of these bioactive compounds are

304 necessary for fighting diseases (Lamichhane et al. 2017). Different plant parts of E. spinosus

305 which can be used as traditional medicine and source for some nutraceutical contain antioxidant 306 phytochemicals such as phenols, flavonoids,Draft cardiac glycosides, glucosinolates, alkaloids, 307 terpenes, steroids and coumarins which characterized by free radical scavenging abilities, anti-

308 inflammatory action, anticancer, anti-aging, and protective action for diseases (Joshi et al. 2016).

309 In addition, the results of GC-MS analysis confirmed that aerial portions of E. spinosus

310 contain various bioactive compounds. Forty-eight compounds were identified in the methanolic

311 extract which have been characterized by the anti-inflammatory and antioxidant properties.

312 Moreover, E. spinosus extract also contains 1- methyl- 3-n-Propyl-2-pyrazolin-5-one which has

313 been characterized by its anti-inflammatory effects (Udupi et al. 1998). The protective effects of

314 E. spinosus against nephrotoxicity could be due to presence of squalene that displayed an

315 effective anti-inflammatory effect at low concentration via stimulating the body immune system

316 and thereby competes several ailments (Han and Bakovic 2015).

317 Several studies have demonstrated that natural antioxidant plays an important role in the

318 prevention of nephrotoxicity induced by xenobiotics (Mbarki et al. 2017). Since the methanol

319 extract of E. spinosus revealed the highest polyphenolic (phenols and flavonoids compound),

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320 total tannin contents and the most effective antioxidant potential in vivo, this fraction was further

321 chosen to study its effects against APAP-induced kidneys oxidative damages. Moreover, the

322 antioxidant potential of the methanolic extract of E. spinosus was detected by estimating the

323 radical scavenging activity using DPPH radical scavenging assay. In the present study, the

324 methanolic extracts of E. spinosa exhibited DPPH radical scavenging potential reached about

325 20.1%. Meanwhile, the total antioxidant capacity reached about 103.4 nmole ascorbic acid/g.

326 Such results indicated that the methanolic extracts of E. spinosus, could be used as a potent

327 source of natural antioxidants.

328 Meanwhile, chronic doses of APAP are commonly associated with nephrotoxicity in animals

329 (Abdul Hamid et al. 2012; Adeneye, and Benebo 2008). Acetaminophen nephrotoxicity occurs

330 due to its highly reactive metabolite NDraft-acetyl-p- benzoquinoneimine (NAPQI), which acrylates

331 proteins in the proximal tubule and thus causes tubular cell death of kidney (Emeigh Hart et al.

332 1990). NAPQI increases reactive oxygen species production including superoxide anion,

333 hydrogen peroxide and hydroxyl radical and consequently, GSH depletion, lipid peoxidation

334 (LPO) induction and cell impairment (Somani et al. 2000). Therefore, we hypothesized that

335 oxidative damage and LPO induced by ROS might be involved in the nephrotoxicity of APAP in

336 rats.

337 Lipid peroxides can be break down to give rise to a variety of harmful molecules as

338 proinflammatory isoprostanoids (Liu et al., 1998) and strong oxidants as 4- hydroxynonenal

339 (Springer et al., 1997). Accordingly, the administration of compounds with antioxidant activity

340 has been successfully used to prevent or ameliorate APAP-induced nephrotoxicity (Canayakin et

341 al., 2016).

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342 Furthermore, this study showed that administration of APAP decreased TAA and increased

343 MDA level which may be resulted from the declined antioxidant and accumulation of free

344 radicals and thereby induced cells, organs and tissues damages. Notably, the co-treatment of E.

345 spinosus, to rats along with APAP restored levels of MDA near normal and improved the levels

346 of TAA, indicating that the renal protective effect of E. spinosus might be due to their ability to

347 protect bio-membranes against lipid peroxidation and to boost cellular antioxidant defenses.

348 Moreover, the observed reduction in MDA levels in rats treated with E. spinosus + APAP was

349 attributed to the protective effects of E. spinosus extract which is rich with antioxidant

350 phytochemicals. It was recorded that flavonoids, saponins, tannins, lignans, alkaloids and

351 triterpenoids are well known anti-oxidants, free radical scavengers and anti-lipo-peroxidants

352 (Eram et al. 2013). Similar previous studiesDraft on the protective effects of different plants were

353 recorded by Hegazy and Emam (2015) and Gabr et al. (2017). Similarly, flavonoids and phenolic

354 compounds and other antioxidant constituents of other medicinal plants have been reported to

355 inhibit nephrotoxicity induced by xenobiotic in experimental animal models due to their potent

356 antioxidant effects (Pareta et al. 2011). Antioxidant mechanism could be an ameliorative factor

357 in the protective effect of E. spinosus for APAP -induced toxicity of kidneys in rats.

358 In addition, APAP -induced kidney injury is characterized by increase in levels of urea, uric

359 acid, creatinine in serum as well as severe proximal necrosis of renal tubule followed by renal

360 failure (Praveen et al. 2008). The increase in the level of serum creatinine is expressive for

361 filtration rate reduction of glomerulus that is associated with increases in serum uric acid and

362 urea (Canayakin et al. 2016; Abd El-Ghffar et al. 2017). In agreement with previous studies, our

363 study also revealed that the administration of APAP caused marked impairment in renal function

364 with significant oxidative damage in the kidney. Serum uric acid, creatinine, and urea

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365 concentrations were significantly higher in APAP-treated rats. Elevation of serum creatinine

366 level was due to altered kidney function caused by APAP. However, E. spinosus crude extract

367 administration significantly decreased the elevated levels of serum creatinine, urea, and uric acid,

368 which indicates that the E. spinosus possibly could protects renal tissue against oxidative stress

369 induced by APAP and maintain renal function.

370 Additionally, amassing proof indicates that APAP overdose prompts the transcriptional

371 activation of cytokines and pro-inflammatory mediators such as inducible nitric oxide (iNOS),

372 COX-2, TNF-α and IL-1β (Song et al., 2014). The current study also revealed that acute APAP

373 overdose resulted in inflammatory reaction with increase in production of TNF-α in the renal

374 tissue. This agrees with other studies which showed that other nephrotoxic drugs can induce

375 renal inflammation with increase generationDraft of TNF-α (Zager 2007). Similarly, the APAP toxic

376 doses treatment increased production of NO in the liver tissue which was correlated with

377 expression of the iNOS protein (Gardner et al. 2002). This can be clarified by the ability of TNF-

378 α to up-regulate the iNOS that increases NO production (Zager 2007). Elevated NO levels exert

379 toxic effects through reacting with superoxide anion to give peroxynitrite radical that directly

380 cause oxidative cell damage. Likewise, excess NO depletes intracellular GSH, which increases

381 susceptibility to oxidative stress (Morris and Billiar 1994). Therefore, from these results it can be

382 speculated that E. spinosus treatment significantly mitigated the overproduction of TNF-α and

383 NO which play important role in pathogenesis of APAP-induced nephrotoxicity.

384 Reactive oxygen species (ROS) have been implicated in the pathogenesis of most

385 inflammatory diseases. Since pro-inflammatory molecules are involved in the pathogenesis of

386 these inflammatory diseases, interactions between ROS and NF-B might be a component of the

387 intracellular signaling process that leads to activation (Christman1 et al., 2000). NF-ĸB is a

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388 pleiotropic transcription factor activated by low levels of ROS and inhibited by antioxidants and

389 some antibiotics (He et al., 2008).

390 It was reported that APAP activated NF-κB expression in proximal and distal convoluted

391 tubules leading to renal inflammation. NF-κB is involved in expression regulation of many

392 genes, including IL-1β, TNF-α and COX-2, that play critical roles in tumorigenesis, various

393 autoimmune diseases and inflammation (Lawrence et al. 2001). The increased activation of

394 transcription factor NF-κB which is responsible for inflammatory and oxidative stress was

395 observed by Al-Belooshi et al. (2010). TNF-α, a cytokine stimulated by NF-κB, can control

396 progression of diseases and reduce the production of other pro-inflammatory cytokines.

397 Khaneghahi1 et al. (2017) suggested that COX-2 play a role in APAP-induced nephrotoxicity.

398 Thus, blockade of NF-κB, Cox-2, and TNF-αDraft can be an effective approach to treat nephrotoxicity

399 (Ko et al. 2017).

400 In the present study, APAP treatment resulted in significant increase in NF-κB protein

401 expression concurrent with induction of Cox-2 protein expression and gene expression of TNF-α.

402 However, E. spinosus pretreatment effectively inhibited APAP-induced expression of NF-κB,

403 Cox-2 and TNF-α suggesting that E. spinosus acted as an inhibitor of APAP-mediated

404 inflammatory response which agreed with Ko et al. (2017). The results of these previous studies

405 and our investigation indicate that E. spinosus oral administration provided protection to rats

406 from renal inflammation by inhibiting NF-κB expression in corticomedullary region of kidney

407 and thus down regulate IL-1β and TNF-α that was highly expressed due to APAP.

408 The most abundant phenols of E spinosus, including 4-methoxy-2,3,6-trimethyl Xanthoxylin,

409 Syringol, benzoic acid and catechol which characterized by numerous biological activities as

410 anti-inflammatory, antioxidant, and antitumor activity due to the close relationship with the NF-

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411 κB- and mitogen activated protein kinase signaling pathways (Chen et al., 2016; Mattera et al.,

412 2017). In addition, the methanol extract of E. spinosus possessed also, Pyrazolos such as 1-

413 methyl- 3-n-Propyl-2-pyrazolin-5-one which are described as nonsteroidal anti-inflammatory

414 drugs (Patar and Yahaya 2012), alkaloids, organic acid, fatty acids and terpenoids (alcohols,

415 aldehydes, esters, ketones, ethers, phenols, glycosides and epoxides) which can be used as

416 antioxidants and anti-inflammatory bioactive compounds (Bouaziz et al. 2016; Benyelles et al.

417 2014; Thosar et al. 2013) and thereby, contribute in the alleviation of kidney disorders induced

418 by APAP overdoses. In addition, the occurrence of different bioactive constituents in E spinosus

419 extract may have synergistic effects on inhibition of renal inflammation. Previous reports

420 showed that the crude extracts of plant have greater in vitro or/and in vivo biological activity

421 than isolated constituents at equivalentDraft dose (Rasoanaivo et al. 2011). In addition, 5-

422 hydroxymethylfurfural and volatile oils are abundant in E spinosus and may have synergistic

423 antiproliferative and antioxidant activities (Zhao et al. 2013) which could protect the rat kidney

424 against APAP overdose toxicity.

425 The effect of E. spinosus on the measured biochemical parameters and prevention of the

426 detrimental increases in TNF-α, NO levels and IL-1β activity is well correlated with the

427 improvement observed in renal histological picture.

428 Moreover, the nephroprotective effects of E. spinosus were also, observed by the

429 histopathological changes in the kidney tissues. The changes in histopathology of kidney tissues

430 of APAP-treated rats showed reduced oxygen perfusion, where the development of kidney

431 diseases may progress as a function of the rat’s cell death. In line with these observations,

432 Parameshappa et al. (2012), reported that renal function was markedly damaged by

433 acetaminophen-induced acute toxicity in rats’ kidneys. However, pretreatment with E. spinosus

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434 showed marked improvement with mild tubular necrosis and vacuolar degeneration. The

435 protective achievement of E. spinosus in APAP-induced histopathological alterations could be

436 attributed to the incidence of phenolics, flavonoids, tannins, ascorbic acids, GSH, amino acids,

437 and proteins which may buffer the APAP-generated free radicals and thereby alleviated the

438 oxidative damages and the consequent degeneration and necrosis of kidney tissues.

439 To the best of our knowledge this study is the first to clarify the favorable nephroprotective

440 action of methanolic extract of E. spinosus against APAP-induced oxidative damage. E. spinosus

441 crude extract improved all the molecular targets implicated in the APAP overdose toxicity of E.

442 spinosus administration promoted total antioxidant activity, repressed inflammation in kidney

443 that are the hallmark of APAP toxicity. Consequently, we can suggest that E. spinosus

444 administration in anticipation of toxicDraft insult due to APAP overdose may have gigantic

445 therapeutic potential for patients. Further studies with elucidation of actual mechanism of action

446 of E. spinosus at molecular level should be conducted before beginning of clinical trial.

447 Conclusion

448 The results of this study cleared the favorable nephroprotective action of methanolic extract

449 of E. spinosus against APAP-induced oxidative damage through its anti-oxidative and anti-

450 inflammatory effects, which was confirmed biochemically and histopathologically. E. spinosus

451 improved the structural integrity of the cell membrane and ameliorated histopathological changes

452 as well as biochemical perturbations. Therefore, E. spinosus can serve as effective therapeutic

453 agents with a low incidence of side effects and can be used as inexpensive alternative which can

454 be consumed in the daily diet to confer protection against nephrotoxicity induced by toxin.

455 In conclusion, this study proves the nephroprotective activity of 250 mg/kg of E. spinosus

456 methanolic extract which has not been established before.

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457 Acknowledgments

458 The authors express their gratitude to Professor Dr Bakir A. (Cairo University, Faculty of

459 Veterinary Medicine, Egypt) for his kind cooperation in the histopathological examinations of

460 this research.

461 Competing interests

462 Authors have no competing interests. This research did not receive any specific grant from

463 funding agencies in the public, commercial, or not-for-profit sectors.

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670 antioxidant and antiproliferative activities of 5-hydroxymethylfurfural. J Agric Food

671 Chem. 61, 10604-10611. doi: 10.1021/jf403098y

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679 Figures Captions

680 Figure 1: GC-MS chromatogram of methanol extract of E. spinosus.

681 Figure 2: Effect of methanolic extract of E. spinosus on APAP-induced elevation of renal nitric

682 oxide production (NO) in rats. Values are expressed as means ± S.E. * P < 0.05,

683 compared to the placebo group (N). # P < 0.05, compared to the APAP group.

684 Figure 3: Immunoblotting analysis of inflammatory relative proteins: (A) Cox-2, (B) NF-κB.

685 Actin, as a loading control. Protein expression was detected by Western blot analysis.

686 The bottom panels represented quantification of the immunoblot by densitometry.

687 Values are expressed as mean ± S.E. * P < 0.05 compared to control, # P < 0.05 688 compared to APAP group. Draft 689 Figure 4: Effect of E. spinosus on APAP -induced elevation in renal level of tumor necrosis

690 factor-α (TNF-α) mRNA expression in rats. Values are expressed as means ± S.E. * P

691 < 0.05, compared to the placebo group (N). # P < 0.05, compared to the APAP group.

692 Figure 5: Effect of E. spinosus on APAP -induced elevation in renal level of IL-1β mRNA

693 expression in rats. Values are expressed as means ± S.E. * P < 0.05, compared to the

694 placebo group (N). # P < 0.05, compared to the APAP group.

695 Figure 6: Microphotographs of histopathological examination of randomly selected kidney

696 sections of rats stained with hematoxylin and eosin (H&E) and examined with a light

697 microscope by 400x magnification. Kidneys from normal control group (a) or E.

698 spinosus group (b) shows the normal histological structure of the glomeruli (g) and

699 tubules (t) in the cortex. Kidney from a rat treated with APAP group (c) shows

700 extensively severe necrosis of renal tubule associated with sever degeneration of

701 vacuolar tubules (d) and sever focal inflammatory cells infiltration (m) between

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702 tubules. Kidney from a rat treated with E. spinosus and APAP group (d) shows

703 moderate coagulative necrosis (n) in the lining epithelium of some tubules at the

704 cortical portion with mild degeneration of vacuolar tubules and absence of focal

705 inflammatory cells infiltration

706

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3 * 2.5

2 *# 1.5

1

0.5

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Fold change of IL-1 β mRNA N APAP ES APAP+ES DraftGroups

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Table 1. Phytochemical screening of E. spinosus

Alkaloid Flavonoi Cardiac Solvent Plant part Steroids Tannins Saponins Quinones Coumarines Terpenoid s ds glycoside + Root + + ++ + ++ ++ + + + Methanol Aerial portion + + + + +++ +++ + ++ - Flowerers + - - - + + - + Root + + ++ ++ + + + + + Ethanol Aerial portion + + ++ ++ + ++ ++ + ++ Flowerers - - - - - + + - + Root + - ++ - - + - - - Petroleum Aerial portion + - ++ - - + - + - ether Flowerers - - - - - + - - - Root + - + ------Hexane Aerial portion + - + ------Flowerers ------Root + + + + - - + - + Water Aerial portion + + + + - - + - ++ Flowerers ------+ - + – Absent and + Present: +++: Strongly present, ++: Mildly present. Draft

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Table 1. Phytochemical quantitative analysis of the methanol extract of E. spinosus.

Parameter A mount ASA µmole/g 5.21± 0.54 GSH mmol/g 20.4± 3.14 Total phenols mg/g 3.13± 0.42 Flavonoids mg/g 0.061± 0.001 Tannins µg/g 0.58± 0.002 Saponins µg/g 223.5± 2.34 % of DPPH scavenging activity 20.0± 1.60 Reducing power n mole/g 103.4± 6.84 Proanthocyanidine mg/g 1.35± 0.032 Cardiac glycosides µM/g 4.725± 0.42 Glucosinolates µmol/g 9.88± 0.022 Alkaloids mg/g 31.2± 3.34 Soluble sugar mg/g 0.52± 0.011 Each value is a mean of three replicates ±SE Draft

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Table 1. The GC-MS identified Phytocomponents of methanol extract of E. spinosus

PEAK NATURE OF THE NO RT NAME OF COMPOUND FORMULA AREA % COMPOUND

Ethanamine, 2,2'-oxybis (2-(2-  NH CH CH OCH CH OH 1 8.47 0.43 2 2 2 2 2 Aminoethoxy) ethylamine  Amine

2-furanone,3-4-dihydroxy 2 8.59 4.34 HOC H C H OH Alcohol (aroma ) (erythroascorbic acid) 2 2 2 2

alpha. -D-Mannopyranoside, C7H14O6 3 8.62 methyl3,6-anhydro- 3.23 Glycosides

4 8.93 Fluoroacetic acid 0.26 CH2FCOOH Acid

4 9.06 N-Acetyl-D-glucosamine 0.46 C8H15NO6 Lectins 5 9.39 Ethanol, 2,2'-oxybis- Draft0.92 C10H18O5 Alcohol Xylitol (polyhydric alcohol or sugar 6 9.4 6 0.59 HOCH [CH(OH)] CH OH Sugar alcohol alcohol) 2 3 2

7 9.55 Oxirane, (ethoxymethyl) 0.53 C4H8O2 Alcohol

cyclic pentamer 8 9.89 15-Crown-5 0.32 C10H20O5 of ethylene oxide

9 9.96 4-Isothiazolecarboxamide 0.28 C4H3NO2S Amides

10 10.33 N-methyl -1,3-Propanediamine 0.49 CH3NH(CH2)3NH2 simple diamine Thiazolines 11 10.67 4,5-Dihydro-2-methylthiazol 0.28 C4H7NS (Heterocyclic compound)

4H-Pyran-4-one, 2,3-dihydro-3,5- 12 10.78 2.36 C H O Flavonoids dihydroxy-6-methyl- 6 8 4

13 10.90 Benzoic acid 0.81 C6H5COOH Phenol

14 10.93 Heptaethylene glycol , 0.60 C14H30O8 Glycol

(E)-2,6-Dimethylocta-3,7-diene-2,6- Aromatic oil 15 11.15 0.24 C H O diol 10 18 2 (terpenoid)

16 11.21 Catechol 0.80 C6H6O2 Phenol

2-Cyclohexen-1-one, 2-methyl-5-(1- 17 11.44 2.35 C10H16O Monoterpenoid methylethyl)-, (S)( Carvotanacetone)

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18 11.59 5-Hydroxymethylfurfural 4.30 C6H6O3 Furans

19 11.93 1-Piperidinecarboxaldehyde 0.21 C6H11NO Alkaloids

20 12.04 1,7-Octadiene-3,6-diol, 2,6-dimethyl 0.55 C10H18O2 Terpenoids

21 12.99 Phenol, 2,6-dimethoxy (Syringol) 0.53 CH3O)2C6H3OH Phenol 2-Cyclopenten-1-one, 3-methyl-2-(2- Monoterpene 22 13.49 2.08 C H O pentenyl)-, (Z)- Jasmone 11 16 (volatile oil)

terpenoid (volatile 23 13.74 2-Cyclohexen-1-one, 4-hydroxy-3- 0.52 C10H16 methyl-6-(1-methylethyl)-, trans- oil) Alkaloids 1- methyl- 3-n-Propyl-2-pyrazolin-5- 24 13.82 1.44 C H N (heterocyclic one 7 12 pyrazolines)

Silane, Heterocyclic 25 13.94 0.90 C H Si (cyclopentylidenemethyl)trimethyl- 9 18 compound

terpenoid (volatile 26 14.12 4-t-Butyl pyridine, 1-oxide 0,62 C H NO 9 13 oil)

R-(+)-3-Isopropyl-6-oxoheptanoic terpenoid (volatile 27 14.37 0.85 C10H18O3 acid Draft oil) Ethanone, 1-[3-methyl-3-(4-methyl- 28 14.48 0.34 C11H18O2 Diphenol 3-pentenyl) oxiranyl]-

29 15.13 p-hydroxyphenyl urea 0.70 C7H8N2-O2 Diphenol)

30 15.35 m-Isopropoxyaniline 0.65 C8H20Si Organic silicon

31 15.36 p-Cymene-2,5-diol 0.94 C10H14 O2 Thymohydroquinones

32 15.46 2'-Hydroxy-5'-methoxyacetophenone 0.41 C9H10O3 Phenol

33 15.22 Benzenethiol, 4-(1,1-dimethylethyl)- 0.28 C10 H14 S C14H21OS Phenol

1,2,3,5-Cyclohexanetetrol, (1. 34 16.29 0.46 C6H12O4 Cyclohexanetetrol alpha.,2.beta.,3.alpha.,5.beta.)-

Phenol (flavonoid 35 16.45 Phenol, 4-methoxy-2,3,6-trimethyl 15.94 C H O 10 14 2 precursor)

Ethanone, 1-(2,2- 36 16.59 0.42 C9H16O Ketone dimethylcyclopentyl)

16.9& 37 Xanthoxylin 11.42&20.48 C H O Methoxyphenol 16.99 10 12 4

(3E,10 Z)-Oxacyclotrideca-3,10- 38 17.87 1.2 C H O Flavonoids diene-2,7-dione 12 16 3

39 18.12 Tetradecanoic acid (Myristic acid) 0.83 C14H28O2 Fatty acids

40 18.41 1,4-benzenediamine, N4, N4-diethyl- 0.51 …C12H22O2Si2 … Amine

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2-fluoro- C12H22O2Si2..

41 18.86 Anthracene 0.76 C14H10 or (C6H4CH)2 aromatic hydrocarbon

C H NOS Heterocyclic 42 22.18 2(3H)-Benzothiazolimine, 3-methyl- 0.73 8 7 compound (Thiazoles)

43 22.64 n-Hexadecanoic acid 3.86 C16H32O2 Fatty acid (Palmitic acid) 44 25.45 Oleic Acid 0.69 CH3(CH2)7CH=CH(CH2)7COOH. Fatty acid

45 30.13 Bis(2-ethylhexyl) phthalate 0.75 C₆H₄(C₈H₁₇COO) ₂. Ester

46 31.29 Terephthalic acid, 2-decyl octyl ester 0.36 C26H42O4 Diester (7-Bromo-2,3-dihydro-1,4- 47 33.1 0.46 C H14BrClO3 Alcohol benzodioxin-6-yl) (phenyl)methanol 16

48 35.21 Squalene 0.84 C30H50 Steroids

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Table 1. Effect of methanol extract of E. spinosus on serum renal and oxidative stress

biomarkers

Parameters Placebo APAP ES APAP + ES

Creatinine (mg/100ml) 0.56 ± 0.042 1.26 ± 0.091*** 0.63 ± 0.045ns 0.75 ± 0.053***

Urea (mg/100ml) 18.36 ± 1.30 33.13 ± 2.34*** 19.77 ± 1.40ns 24.33 ± 1.53***

Uric acid (mg/100ml) 1.36 ± 0.10 3.55 ± 0.29*** 1.41 ± 0.10ns 1.64 ± 0.12***

MDA (nmol/ml) 167.12 ± 11.88 272.57 ± 19.87*** 160.31 ± 12.74ns 186.66 ± 13.52***

TAA (mmol/L) 2.4 ± 0.18 0.82 ± 0.06*** 2.24 ± 0.21ns 1.84 ± 0.13*** Values expressed as mean ± SE (n=6) APAP group and ES were compared with placebo group. APAP + ES group was compared with APAP group. *p<0.05, **p<0.01, ***p<0.001, ns= non-significant. Draft

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Table 1. Histopathological changes of kidney of rats treated with E. spinosus and/or

paracetamol (APAPA).

tubular Focal necrosis degeneration lining intraluminal inflammatory Group of renal of vacuolar epithelium casts cells tubule tubules swelling infiltration Control - - - - - ES - - - - - APAP ++++ +++ ++++ ++ ++++ ES + APAP ++ + + + - -: nil, +: mild, ++: moderate, +++: severe, and ++++: extensively severe

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