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Dennettia tripetala Combats Oxidative Stress, Protein and Lipid Dyshomeostasis, Inflammation, Hepatic Injury, and Glomerular Blockage in Rats
1Department of Biochemistry, Faculty of Life Sciences, University of Benin, Benin City, Edo State 300213, Nigeria
2Department of Biochemistry, College of Health Sciences, School of Basic Medical Sciences, Igbinedion University Okada, Benin City, Edo State 302110, Nigeria
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Prev Nutr Food Sci 2021; 26(2): 177-185
Published June 30, 2021 https://doi.org/10.3746/pnf.2021.26.2.177
Copyright © The Korean Society of Food Science and Nutrition.
Abstract
Keywords
INTRODUCTION
In 2016, the components of the essential oil of
In 2017,
More recently, we showed that the powder and aqueous and ethanolic extracts of
Carbon tetrachloride (CCl4) is a commonly used chemi-cal for induction of hepatorenal injuries in laboratory animals; its mechanism of action is very well understood. CCl4 is metabolized by cytochrome P450 enzymes, leading to production of free radicals that trigger oxidative stress and damage cellular constituents, including lipids, proteins, and genetic material, eventually resulting in injury to organs including the liver (Weber et al., 2003) and kidneys (Sanzgiri et al., 1997). The toxicity of CCl4 is related to its dose and the duration of the exposure; higher doses and longer exposures tend to cause more serious and permanent effects (Weber et al., 2003).
In 2002,
MATERIALS AND METHODS
Plant materials
Ripe
Phytochemical content, in vitro antioxidant properties, and toxicity study of the freeze-dried extract
The phytochemical content,
Animals
Male albino Wistar rats of approximately 5 weeks of age and weighing 100∼140 g were used in experiments. Rats were bred in wooden cages covered with barbed wire and allowed free access to feed (Growers’ mash, Bendel Feed and Flour Mill Ltd., Ewu, Edo State, Nigeria) and water. The comfort of the rats was taken into considera-tion throughout the duration of the experiment. All animal experiments were carried out in accordance with the NIH guide for the care and use of laboratory animals (NIH publication No. 8023, revised 1978) and with permission from the Animal Ethics committee of the Faculty of Life Sciences, University of Benin, Nigeria (approval number LS16106). After acclimatizing for two weeks, rats were randomized into five groups of five. The experimen-tal design is shown in Table 1.
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Table 1 . Experimental design
Groups Treatment 1 Control 2 CCl4 3 CCl4+ETDT 250 4 CCl4+ETDT 500 5 CCl4+ETDT 1000 ETDT, ethanolic extract of
Dennettia tripetala .D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw (n=5).
Rats in the control group (group 1) were only adminis-tered feed and water. Rats in groups 2∼5 were adminis-tered CCl4 dissolved in olive oil (1:1 w/v) at 3 mL/kg bw twice a week for two weeks. Plant extract was dissolved in ethanol (200 mg/mL) and administered at doses of 250, 500, and 1,000 mg/kg bw to groups 3∼5, respectively, daily for seven days from day 8 of CCl4 ad-ministration. All experimental substances were adminis-tered orally using a gavage. On day 14, rats were subjected to an overnight fast, and sacrificed on day 15 using chloroform anaesthesia. Blood (serum), liver, and kidneys were collected for biochemical assays and histopa-thology.
Biochemical assays
Rat serum was assayed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), total cholesterol, triglyceride, total protein, albumin, globulin, urea, and creatinine. Livers and kidneys were assayed for superoxide dismutase (SOD), catalase, and malondialdehyde (MDA). All but four assays were conducted using kits from Randox Laboratories (Crumlin, UK), following the manufacturer’s protocol. SOD, catalase, and MDA assays were conducted using reagents purchased from Pyrex-Ig Science/laboratory (Benin City, Nigeria), following methods described by Misra and Fridovich (1972), Góth (1991), and Buege and Aust (1978) for SOD, catalase, and MDA assays, respectively. ALP was assessed using kits from Teco Diagnostics (Anaheim, CA, USA), following manufacturer’s instructions.
Histopathology
Portions of the liver and kidneys were fixed in 10% neutral buffered formalin for histopathological analysis. A Leica TP1020 automatic tissue processor (Leica Biosys-tems, Wetzlar, Germany) was used to process the tissues, which were stained with hematoxylin and eosin and viewed under a light microscope using both 10× and 40× magnification.
Statistics
Data were analyzed using one-way ANOVA and Tukey’s post-hoc test and presented as mean±standard error of the mean (SEM). GraphPad Prism version 7 software (GraphPad Software, San Diego, CA, USA) was used for data analysis.
RESULTS
Effect on liver marker enzymes in serum
CCl4 treatment induced significant increases in the activities of the liver marker enzymes AST and ALT in serum, and a non-significant elevation of ALP (Table 2). The ethanolic extract of
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Table 2 . Effect of
Dennettia tripetala on serum activity of liver marker enzymes in rats administered CCl4 (unit: U/L)Groups AST ALT ALP Control 242.10±6.55a 52.50±4.79a 39.15±5.88a CCl4 809.00±15.6c 486.70±6.67b 45.70±1.59a ETDT 250+CCl4 493.50±19.64b 460.00±48.99b 58.06±5.11b ETDT 500+CCl4 465.30±6.36b 336.70±38.44b 44.81±3.15a ETDT 1000+CCl4 520.00±1.16b 362.50±34.73b 43.63±2.72a The values presented are the mean±SEM (n=5).
Values with different letters (a-c) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala ; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase.D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
Effect on lipid homeostasis
Furthermore, CCl4 significantly elevated serum total cholesterol and low-density lipoprotein (LDL)-cholesterol and significantly reduced serum high-density lipoprotein (HDL)-cholesterol and triacylglycerol (TAG) (Table 3). The ethanolic extract of
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Table 3 . Effect of
Dennettia tripetala on serum lipid concentration in rats administered CCl4 (unit: mg/dL)Groups Total cholesterol HDL-cholesterol LDL-cholesterol TAG Control 38.28±1.93a 25.45±2.20b 13.75±0.95a 127.40±25.10c CCl4 47.85±1.08b 7.24±0.64a 41.07±0.94b 84.30±4.43b ETDT 250+CCl4 47.09±0.61b 28.57±0.24b 23.08±3.12a 46.62±4.91a ETDT 500+CCl4 47.40±2.10b 24.89±2.82b 32.33±3.65b 72.00±6.79b ETDT 1000+CCl4 42.84±1.39ab 9.57±0.97a 38.30±0.58b 49.23±2.22a The values presented are the mean±SEM (n=5).
Values with different letters (a-c) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala ; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TAG, triacylglycerols.D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
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Table 4 . Effect of
Dennettia tripetala administration on liver lipid concentration in rats administered CCl4 (unit: mg/dL)Groups Total cholesterol TAG Control 41.68±0.49a 16.88±2.11a CCl4 51.44±7.59b 28.84±6.01ab ETDT 250+CCl4 44.06±0.97a 7.74±0.70a ETDT 500+CCl4 44.46±0.23a 11.96±0.70a ETDT 1000+CCl4 46.44±0.33ab 16.35±5.05a The values presented are the mean±SEM (n=5).
Values with different letters (a,b) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala ; TAG, triacylglycerols.D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
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Figure 1. Photomicrographs of liver sections from (A) control rats showing normal liver histology: clear centrioles with well fenestrated sinusoidal space and distinct hepatocytes; (B) rats treated with 250 mg/kg bw ETDT and CCl4, showing very mild fatty changes with clear centrioles; (C) rats treated with 500 mg/kg bw ETDT and CCl4, showing fatty changes and little hydropic degeneration of the hepatocytes, with centrioles appearing congested surrounded by neutrophilic infiltrates; (D) rats treated with 1,000 mg/kg bw ETDT and CCl4, showing fatty changes with partially clear centrioles surrounded by visible multifocal neutrophilic infiltrates; (E) rats treated with CCl4 only, showing fatty changes (macrovsicular steatosis) and hydropic degeneration of the hepatocytes with multifocal distortion in liver histology, congestion of the central vein, with neutrophilic infiltrates. The liver sections were stained with hematoxylin and eosin and observed with a 10× objective. ETDT,
D. tripetala ethanolic extract.
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Figure 2. Photomicrographs of liver sections from (A) control rats showing normal liver histology: clear centrioles with well fenestrated sinusoidal space and distinct hepatocytes with well differentiated nucleus; (B) rats treated with 250 mg/kg bw ETDT and CCl4, showing very mild fatty changes although with clear centrioles; (C) rats treated with 500 mg/kg bw ETDT and CCl4, showing fatty changes and little hydropic degeneration of the hepatocytes, with centriole appearing congested surrounded by neutrophilic infiltrates; (D) rats treated with 1,000 mg/kg bw ETDT and CCl4, showing fatty changes with partially clear centrioles surrounded by visible multifocal neutrophilic infiltrates; (E) rats treated with CCl4 only showing fatty changes (macrovsicular steatosis) and hydropic degeneration of the hepatocytes with multifocal distortion in liver histology, congestion of the central vein with neutrophilic infiltrates. The liver sections were stained with hematoxylin and eosin and observed with a 40× objective. ETDT,
D. tripetala ethanolic extract.
Effect on serum protein profile
CCl4 significantly elevated the total protein and globulin concentrations in serum and significantly reduced serum albumin levels and the albumin:globulin ratio (Table 5). Therefore, we concluded that
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Table 5 . Effect of
Dennettia tripetala on serum protein concentration in rats administered CCl4Groups Total protein
(g/dL)Albumin
(g/dL)Globulin
(g/dL)Albumin:globulin ratio Control 5.94±0.18a 3.24±0.04b 2.68±0.09a 1.13±0.04c CCl4 21.04±1.45c 2.96±0.03a 14.05±1.67b 0.18±0.02a ETDT 250+CCl4 13.11±0.95b 2.71±0.05a 10.42±0.91b 0.26±0.02a ETDT 500+CCl4 8.69±1.05a 3.38±0.02b 6.12±1.34a 0.31±0.10a ETDT 1000+CCl4 9.35±0.25a 2.82±0.07a 6.06±0.45a 0.46±0.05b The values presented are the mean±SEM (n=5).
Values with different letters (a-c) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala .D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
Effect on recruitment of immune cells in the liver
Histopathology images revealed that CCl4 caused immune cells to be recruited to the sites of damage in the liver (Fig. 1 and Fig. 2).
Effect on antioxidant enzyme activities in liver and kidneys
CCl4 induced a reduction in the activities of the antioxidant enzymes SOD and catalase in both the liver and the kidneys (Table 6 and Table 7). In addition, CCl4 significantly elevated the concentration of the lipid peroxidation product malondialdehyde in both the liver and kidneys. Interestingly,
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Table 6 . Effect of
Dennettia tripetala on liver antioxidant enzyme activity and lipid peroxidation status in rats administered CCl4 (unit: units/g wet tissue)Groups SOD Catalase MDA Control 1,225±3c 4,683±11c 0.08±0.01a CCl4 744.0±2a 4,326±51a 0.17±0.01c ETDT 250+CCl4 891.7±36b 4,386±31a 0.09±0.01a ETDT 500+CCl4 1,500±25d 4,518±8b 0.10±0.01ab ETDT 1000+CCl4 1,125±52c 4,656±45c 0.09±0.01a The values presented are the mean±SEM (n=5).
Values with different letters (a-d) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala ; SOD, superoxide dismutase; MDA, malondialdehyde.D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
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Table 7 . Effect of
Dennettia tripetala on kidney antioxidant enzyme activity and lipid peroxidation status in rats administered CCl4Groups SOD
[units/g wet tissue (×10−2)]Catalase
(units/g wet tissue)MDA
(units/g wet tissue)Control 873±28a 5,076±294b 0.20±0.01a CCl4 644±28a 3,984±18a 0.25±0.01b ETDT 250+CCl4 1,130±346a 4,749±11b 0.21±0.01a ETDT 500+CCl4 1,027±208a 4,815±26b 0.19±0.01a ETDT 1000+CCl4 1,030±79a 4,827±7b 0.23±0.01ab The values presented are the mean±SEM (n=5).
Values with different letters (a,b) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala ; SOD, superoxide dismutase; MDA, malondialdehyde.D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
Effect on serum urea and creatinine concentrations
CCl4 elevated the concentration of urea and creatinine in serum (Table 8). This observation was supported by histopathology results (Fig. 3 and Fig. 4) showing that CCl4 severely altered the structure of the kidneys. In addition,
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Table 8 . Effect of
Dennettia tripetala on urea and creatinine concentrations in rats administered CCl4 (unit: mg/dL)Groups Urea Creatinine Control 6.43±0.83a 1.54±0.01a CCl4 10.73±0.04b 2.15±0.02b ETDT 250+CCl4 8.31±0.63b 1.36±0.03a ETDT 500+CCl4 7.53±0.72ab 1.48±0.14a ETDT 1000+CCl4 7.37±0.87ab 1.36±0.07a The values presented are the mean±SEM (n=5).
Values with different letters (a,b) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala .D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
-
Figure 3. Photomicrographs of kidney sections from (A) control rats showing normal histological features. The section indicates a detailed cortical parenchyma and the renal corpuscles appear as dense rounded structures; (B) rats treated with 250 mg/kg bw ETDT and CCl4, showing a varying degree of distortion and disruption in microanatomy of the renal cortex (atrophy), when compared to the control although with prominent renal corpuscles; (C) rats treated with 500 mg/kg bw ETDT and CCl4, showing some degree of recovery in microanatomy of the renal cortex, including queried edema, when compared to the control with some atrophied renal corpuscles; (D) rats treated with 1,000 mg/kg bw ETDT and CCl4, showing some degree of distortion in microanatomy of the renal cortex, including queried edema, when compared to the control; (E) rats treated with CCl4 only, showing severe distortion and disruption in microanatomy of the renal cortex, including queried edema. The kidney sections were stained with hematoxylin and eosin and observed with a 10× objective. ETDT,
D. tripetala ethanolic extract.
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Figure 4. Photomicrographs of kidney sections from (A) control rats showing normal histological features. The section indicates a detailed cortical parenchyma and the renal corpuscles appear as dense rounded structures; (B) rats treated with 250 mg/kg bw ETDT and CCl4 showing some degree of distortion and disruption in microanatomy of the renal cortex (atrophy), when compared to the control, although with prominent renal corpuscles; (C) rats treated with 500 mg/kg bw ETDT and CCl4 showing some varying degree of recovery in microanatomy of the renal cortex, including queried edema, when compared to the control with some atrophied renal corpuscles; (D) rats treated with 1,000 mg/kg bw ETDT and CCl4 showing some varying degree of distortion in microanatomy of the renal cortex, including queried edema, when compared to the control; (E) rats treated with CCl4 only showing severe distortion and disruption in microanatomy of the renal cortex, including queried edema. The kidney sections were stained with hematoxylin and eosin and observed with a 40× objective. ETDT,
D. tripetala ethanolic extract.
DISCUSSION
Effect on liver marker enzymes in serum
ALT and AST are normally localized in the cells of the liver, but can drain into the blood and cause noticeable elevations following damage to the membranes of hepatocytes, as is the case with the metabolites of CCl4 (Singh et al., 2011). This readily occurs in the liver, which contains a high amount of cytochrome P450 enzymes responsible for metabolizing drugs and toxins. In the present study, the ethanolic extract of
Effect on lipid homeostasis
Dyslipidemia is caused by increases in total cholesterol and LDL-cholesterol and decreases HDL-cholesterol in serum, and is a risk factor for atherosclerosis and other diseases of the heart and blood vessels (Mahdy Ali et al., 2012). Certain extents of damage to the liver have been shown to alter its ability to synthesize, package, and secrete lipids into the blood (Fromenty and Pessayre, 1995; Boll et al., 2001; Khalaf et al., 2009; Al-Yahya et al., 2013; Hamed et al., 2016). Therefore, it was unsurprising that recurrent challenges with CCl4 induced dyslipidemia. However,
Effect on serum protein profiles
The elevated total protein in the serum may comprise that which leaked out of the injured liver. The decrease in albumin may indicate that the liver had difficulty synthesizing normal amounts of this protein following injury induced by CCl4. The ethanolic extract of
Effect on recruitment of immune cells in the liver
Histopathology images revealed that CCl4 caused immune cells to be recruited to the sites of damage in the liver (Fig. 1 and Fig. 2). This is unsurprising since CCl4 is a foreign substance and is capable of evoking an immune response
Effect on antioxidant enzyme activities in livers and kidneys
Metabolism of CCl4 caused oxidative stress in the liver and kidneys by decreasing SOD and catalase activities and increasing the concentration of the lipid peroxidation product malondialdehyde. CCl4 radical formed from the breakdown of CCl4 can form DNA adducts as well as protein adducts, which may lead to impairment of the function of certain genes and proteins (Weber et al., 2003). In this study, it is possible that the metabolites of CCl4 reduced antioxidant enzymes by randomly inhibiting their formation and function at both the genetic and protein levels. Our results show that in the context of this experiment,
Effect on serum urea and creatinine concentrations
Urea and creatinine are waste products of metabolism that are normally filtered by the kidneys into the urine. However, alterations to the structure of the kidneys may affect its ability to efficiently filter these waste products, leading to their elevation in the blood (Ravnskov, 2005). In this study, CCl4 increased the concentrations of urea and creatinine in serum, while
In conclusion, our study provides evidence that the ethanolic extract of
AUTHOR DISCLOSURE STATEMENT
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
SOO and NEJO designed the study. SOO and KO carried out the experiments. SOO analyzed the data and wrote the manuscript. All authors approved the final copy of the manuscript.
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Article
Original
Prev Nutr Food Sci 2021; 26(2): 177-185
Published online June 30, 2021 https://doi.org/10.3746/pnf.2021.26.2.177
Copyright © The Korean Society of Food Science and Nutrition.
Dennettia tripetala Combats Oxidative Stress, Protein and Lipid Dyshomeostasis, Inflammation, Hepatic Injury, and Glomerular Blockage in Rats
Sylvia Oghogho Omage1 , Noghayin E. Jerry Orhue1, and Kingsley Omage2
1Department of Biochemistry, Faculty of Life Sciences, University of Benin, Benin City, Edo State 300213, Nigeria
2Department of Biochemistry, College of Health Sciences, School of Basic Medical Sciences, Igbinedion University Okada, Benin City, Edo State 302110, Nigeria
Correspondence to:Sylvia Oghogho Omage, Tel: +234-526-005-53, E-mail: sylvia.iseghohi@uniben.edu
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Dennettia tripetala, better known as ‘pepperfruit’, is a medicinal plant consumed in West Africa. D. tripetala possesses strong antioxidant properties and contains uvariopsine, an alkaloid which improves bile secretion and liver function. In the present study, the ethanolic extract of D. tripetala fruits was tested for its ability to alleviate pathophysiological conditions bordering on oxidative stress, including protein and lipid dyshomeostasis, inflammation, and hepatic and glomerular injury. Male albino Wistar rats were administered carbon tetrachloride twice a week for two weeks, and the ethanolic extract of D. tripetala fruits was administered from days 8∼14. The serum, liver, and kidneys of the rats were then subjected to biochemical assays and imaging. The extract restored the activities of liver marker enzymes in serum and the concentrations of lipids and proteins in both circulation and the liver to normal. The extract also restored the activities of antioxidant enzymes in liver and kidneys, and the concentrations of urea and creatinine in the blood. The extract also repaired the altered structures of the liver and kidney. Overall, D. tripetala elicited strong medicinal effects in rats. This study showed that the fruits of D. tripetala contain substances that could be extracted or synthesized for use in drugs for the treatment of liver and kidney disease.
Keywords: antioxidant, carbon tetrachloride, Dennettia tripetala, hepatorenal damage, oxidative stress
INTRODUCTION
In 2016, the components of the essential oil of
In 2017,
More recently, we showed that the powder and aqueous and ethanolic extracts of
Carbon tetrachloride (CCl4) is a commonly used chemi-cal for induction of hepatorenal injuries in laboratory animals; its mechanism of action is very well understood. CCl4 is metabolized by cytochrome P450 enzymes, leading to production of free radicals that trigger oxidative stress and damage cellular constituents, including lipids, proteins, and genetic material, eventually resulting in injury to organs including the liver (Weber et al., 2003) and kidneys (Sanzgiri et al., 1997). The toxicity of CCl4 is related to its dose and the duration of the exposure; higher doses and longer exposures tend to cause more serious and permanent effects (Weber et al., 2003).
In 2002,
MATERIALS AND METHODS
Plant materials
Ripe
Phytochemical content, in vitro antioxidant properties, and toxicity study of the freeze-dried extract
The phytochemical content,
Animals
Male albino Wistar rats of approximately 5 weeks of age and weighing 100∼140 g were used in experiments. Rats were bred in wooden cages covered with barbed wire and allowed free access to feed (Growers’ mash, Bendel Feed and Flour Mill Ltd., Ewu, Edo State, Nigeria) and water. The comfort of the rats was taken into considera-tion throughout the duration of the experiment. All animal experiments were carried out in accordance with the NIH guide for the care and use of laboratory animals (NIH publication No. 8023, revised 1978) and with permission from the Animal Ethics committee of the Faculty of Life Sciences, University of Benin, Nigeria (approval number LS16106). After acclimatizing for two weeks, rats were randomized into five groups of five. The experimen-tal design is shown in Table 1.
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Table 1 . Experimental design.
Groups Treatment 1 Control 2 CCl4 3 CCl4+ETDT 250 4 CCl4+ETDT 500 5 CCl4+ETDT 1000 ETDT, ethanolic extract of
Dennettia tripetala ..D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw (n=5)..
Rats in the control group (group 1) were only adminis-tered feed and water. Rats in groups 2∼5 were adminis-tered CCl4 dissolved in olive oil (1:1 w/v) at 3 mL/kg bw twice a week for two weeks. Plant extract was dissolved in ethanol (200 mg/mL) and administered at doses of 250, 500, and 1,000 mg/kg bw to groups 3∼5, respectively, daily for seven days from day 8 of CCl4 ad-ministration. All experimental substances were adminis-tered orally using a gavage. On day 14, rats were subjected to an overnight fast, and sacrificed on day 15 using chloroform anaesthesia. Blood (serum), liver, and kidneys were collected for biochemical assays and histopa-thology.
Biochemical assays
Rat serum was assayed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), total cholesterol, triglyceride, total protein, albumin, globulin, urea, and creatinine. Livers and kidneys were assayed for superoxide dismutase (SOD), catalase, and malondialdehyde (MDA). All but four assays were conducted using kits from Randox Laboratories (Crumlin, UK), following the manufacturer’s protocol. SOD, catalase, and MDA assays were conducted using reagents purchased from Pyrex-Ig Science/laboratory (Benin City, Nigeria), following methods described by Misra and Fridovich (1972), Góth (1991), and Buege and Aust (1978) for SOD, catalase, and MDA assays, respectively. ALP was assessed using kits from Teco Diagnostics (Anaheim, CA, USA), following manufacturer’s instructions.
Histopathology
Portions of the liver and kidneys were fixed in 10% neutral buffered formalin for histopathological analysis. A Leica TP1020 automatic tissue processor (Leica Biosys-tems, Wetzlar, Germany) was used to process the tissues, which were stained with hematoxylin and eosin and viewed under a light microscope using both 10× and 40× magnification.
Statistics
Data were analyzed using one-way ANOVA and Tukey’s post-hoc test and presented as mean±standard error of the mean (SEM). GraphPad Prism version 7 software (GraphPad Software, San Diego, CA, USA) was used for data analysis.
RESULTS
Effect on liver marker enzymes in serum
CCl4 treatment induced significant increases in the activities of the liver marker enzymes AST and ALT in serum, and a non-significant elevation of ALP (Table 2). The ethanolic extract of
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Table 2 . Effect of
Dennettia tripetala on serum activity of liver marker enzymes in rats administered CCl4 (unit: U/L).Groups AST ALT ALP Control 242.10±6.55a 52.50±4.79a 39.15±5.88a CCl4 809.00±15.6c 486.70±6.67b 45.70±1.59a ETDT 250+CCl4 493.50±19.64b 460.00±48.99b 58.06±5.11b ETDT 500+CCl4 465.30±6.36b 336.70±38.44b 44.81±3.15a ETDT 1000+CCl4 520.00±1.16b 362.50±34.73b 43.63±2.72a The values presented are the mean±SEM (n=5)..
Values with different letters (a-c) within the same column are significantly different at
P <0.05..ETDT, ethanolic extract of
D. tripetala ; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase..D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw..
Effect on lipid homeostasis
Furthermore, CCl4 significantly elevated serum total cholesterol and low-density lipoprotein (LDL)-cholesterol and significantly reduced serum high-density lipoprotein (HDL)-cholesterol and triacylglycerol (TAG) (Table 3). The ethanolic extract of
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Table 3 . Effect of
Dennettia tripetala on serum lipid concentration in rats administered CCl4 (unit: mg/dL).Groups Total cholesterol HDL-cholesterol LDL-cholesterol TAG Control 38.28±1.93a 25.45±2.20b 13.75±0.95a 127.40±25.10c CCl4 47.85±1.08b 7.24±0.64a 41.07±0.94b 84.30±4.43b ETDT 250+CCl4 47.09±0.61b 28.57±0.24b 23.08±3.12a 46.62±4.91a ETDT 500+CCl4 47.40±2.10b 24.89±2.82b 32.33±3.65b 72.00±6.79b ETDT 1000+CCl4 42.84±1.39ab 9.57±0.97a 38.30±0.58b 49.23±2.22a The values presented are the mean±SEM (n=5)..
Values with different letters (a-c) within the same column are significantly different at
P <0.05..ETDT, ethanolic extract of
D. tripetala ; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TAG, triacylglycerols..D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw..
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Table 4 . Effect of
Dennettia tripetala administration on liver lipid concentration in rats administered CCl4 (unit: mg/dL).Groups Total cholesterol TAG Control 41.68±0.49a 16.88±2.11a CCl4 51.44±7.59b 28.84±6.01ab ETDT 250+CCl4 44.06±0.97a 7.74±0.70a ETDT 500+CCl4 44.46±0.23a 11.96±0.70a ETDT 1000+CCl4 46.44±0.33ab 16.35±5.05a The values presented are the mean±SEM (n=5)..
Values with different letters (a,b) within the same column are significantly different at
P <0.05..ETDT, ethanolic extract of
D. tripetala ; TAG, triacylglycerols..D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw..
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Figure 1. Photomicrographs of liver sections from (A) control rats showing normal liver histology: clear centrioles with well fenestrated sinusoidal space and distinct hepatocytes; (B) rats treated with 250 mg/kg bw ETDT and CCl4, showing very mild fatty changes with clear centrioles; (C) rats treated with 500 mg/kg bw ETDT and CCl4, showing fatty changes and little hydropic degeneration of the hepatocytes, with centrioles appearing congested surrounded by neutrophilic infiltrates; (D) rats treated with 1,000 mg/kg bw ETDT and CCl4, showing fatty changes with partially clear centrioles surrounded by visible multifocal neutrophilic infiltrates; (E) rats treated with CCl4 only, showing fatty changes (macrovsicular steatosis) and hydropic degeneration of the hepatocytes with multifocal distortion in liver histology, congestion of the central vein, with neutrophilic infiltrates. The liver sections were stained with hematoxylin and eosin and observed with a 10× objective. ETDT,
D. tripetala ethanolic extract.
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Figure 2. Photomicrographs of liver sections from (A) control rats showing normal liver histology: clear centrioles with well fenestrated sinusoidal space and distinct hepatocytes with well differentiated nucleus; (B) rats treated with 250 mg/kg bw ETDT and CCl4, showing very mild fatty changes although with clear centrioles; (C) rats treated with 500 mg/kg bw ETDT and CCl4, showing fatty changes and little hydropic degeneration of the hepatocytes, with centriole appearing congested surrounded by neutrophilic infiltrates; (D) rats treated with 1,000 mg/kg bw ETDT and CCl4, showing fatty changes with partially clear centrioles surrounded by visible multifocal neutrophilic infiltrates; (E) rats treated with CCl4 only showing fatty changes (macrovsicular steatosis) and hydropic degeneration of the hepatocytes with multifocal distortion in liver histology, congestion of the central vein with neutrophilic infiltrates. The liver sections were stained with hematoxylin and eosin and observed with a 40× objective. ETDT,
D. tripetala ethanolic extract.
Effect on serum protein profile
CCl4 significantly elevated the total protein and globulin concentrations in serum and significantly reduced serum albumin levels and the albumin:globulin ratio (Table 5). Therefore, we concluded that
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Table 5 . Effect of
Dennettia tripetala on serum protein concentration in rats administered CCl4.Groups Total protein
(g/dL)Albumin
(g/dL)Globulin
(g/dL)Albumin:globulin ratio Control 5.94±0.18a 3.24±0.04b 2.68±0.09a 1.13±0.04c CCl4 21.04±1.45c 2.96±0.03a 14.05±1.67b 0.18±0.02a ETDT 250+CCl4 13.11±0.95b 2.71±0.05a 10.42±0.91b 0.26±0.02a ETDT 500+CCl4 8.69±1.05a 3.38±0.02b 6.12±1.34a 0.31±0.10a ETDT 1000+CCl4 9.35±0.25a 2.82±0.07a 6.06±0.45a 0.46±0.05b The values presented are the mean±SEM (n=5)..
Values with different letters (a-c) within the same column are significantly different at
P <0.05..ETDT, ethanolic extract of
D. tripetala ..D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw..
Effect on recruitment of immune cells in the liver
Histopathology images revealed that CCl4 caused immune cells to be recruited to the sites of damage in the liver (Fig. 1 and Fig. 2).
Effect on antioxidant enzyme activities in liver and kidneys
CCl4 induced a reduction in the activities of the antioxidant enzymes SOD and catalase in both the liver and the kidneys (Table 6 and Table 7). In addition, CCl4 significantly elevated the concentration of the lipid peroxidation product malondialdehyde in both the liver and kidneys. Interestingly,
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Table 6 . Effect of
Dennettia tripetala on liver antioxidant enzyme activity and lipid peroxidation status in rats administered CCl4 (unit: units/g wet tissue).Groups SOD Catalase MDA Control 1,225±3c 4,683±11c 0.08±0.01a CCl4 744.0±2a 4,326±51a 0.17±0.01c ETDT 250+CCl4 891.7±36b 4,386±31a 0.09±0.01a ETDT 500+CCl4 1,500±25d 4,518±8b 0.10±0.01ab ETDT 1000+CCl4 1,125±52c 4,656±45c 0.09±0.01a The values presented are the mean±SEM (n=5)..
Values with different letters (a-d) within the same column are significantly different at
P <0.05..ETDT, ethanolic extract of
D. tripetala ; SOD, superoxide dismutase; MDA, malondialdehyde..D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw..
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Table 7 . Effect of
Dennettia tripetala on kidney antioxidant enzyme activity and lipid peroxidation status in rats administered CCl4.Groups SOD
[units/g wet tissue (×10−2)]Catalase
(units/g wet tissue)MDA
(units/g wet tissue)Control 873±28a 5,076±294b 0.20±0.01a CCl4 644±28a 3,984±18a 0.25±0.01b ETDT 250+CCl4 1,130±346a 4,749±11b 0.21±0.01a ETDT 500+CCl4 1,027±208a 4,815±26b 0.19±0.01a ETDT 1000+CCl4 1,030±79a 4,827±7b 0.23±0.01ab The values presented are the mean±SEM (n=5)..
Values with different letters (a,b) within the same column are significantly different at
P <0.05..ETDT, ethanolic extract of
D. tripetala ; SOD, superoxide dismutase; MDA, malondialdehyde..D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw..
Effect on serum urea and creatinine concentrations
CCl4 elevated the concentration of urea and creatinine in serum (Table 8). This observation was supported by histopathology results (Fig. 3 and Fig. 4) showing that CCl4 severely altered the structure of the kidneys. In addition,
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Table 8 . Effect of
Dennettia tripetala on urea and creatinine concentrations in rats administered CCl4 (unit: mg/dL).Groups Urea Creatinine Control 6.43±0.83a 1.54±0.01a CCl4 10.73±0.04b 2.15±0.02b ETDT 250+CCl4 8.31±0.63b 1.36±0.03a ETDT 500+CCl4 7.53±0.72ab 1.48±0.14a ETDT 1000+CCl4 7.37±0.87ab 1.36±0.07a The values presented are the mean±SEM (n=5)..
Values with different letters (a,b) within the same column are significantly different at
P <0.05..ETDT, ethanolic extract of
D. tripetala ..D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw..
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Figure 3. Photomicrographs of kidney sections from (A) control rats showing normal histological features. The section indicates a detailed cortical parenchyma and the renal corpuscles appear as dense rounded structures; (B) rats treated with 250 mg/kg bw ETDT and CCl4, showing a varying degree of distortion and disruption in microanatomy of the renal cortex (atrophy), when compared to the control although with prominent renal corpuscles; (C) rats treated with 500 mg/kg bw ETDT and CCl4, showing some degree of recovery in microanatomy of the renal cortex, including queried edema, when compared to the control with some atrophied renal corpuscles; (D) rats treated with 1,000 mg/kg bw ETDT and CCl4, showing some degree of distortion in microanatomy of the renal cortex, including queried edema, when compared to the control; (E) rats treated with CCl4 only, showing severe distortion and disruption in microanatomy of the renal cortex, including queried edema. The kidney sections were stained with hematoxylin and eosin and observed with a 10× objective. ETDT,
D. tripetala ethanolic extract.
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Figure 4. Photomicrographs of kidney sections from (A) control rats showing normal histological features. The section indicates a detailed cortical parenchyma and the renal corpuscles appear as dense rounded structures; (B) rats treated with 250 mg/kg bw ETDT and CCl4 showing some degree of distortion and disruption in microanatomy of the renal cortex (atrophy), when compared to the control, although with prominent renal corpuscles; (C) rats treated with 500 mg/kg bw ETDT and CCl4 showing some varying degree of recovery in microanatomy of the renal cortex, including queried edema, when compared to the control with some atrophied renal corpuscles; (D) rats treated with 1,000 mg/kg bw ETDT and CCl4 showing some varying degree of distortion in microanatomy of the renal cortex, including queried edema, when compared to the control; (E) rats treated with CCl4 only showing severe distortion and disruption in microanatomy of the renal cortex, including queried edema. The kidney sections were stained with hematoxylin and eosin and observed with a 40× objective. ETDT,
D. tripetala ethanolic extract.
DISCUSSION
Effect on liver marker enzymes in serum
ALT and AST are normally localized in the cells of the liver, but can drain into the blood and cause noticeable elevations following damage to the membranes of hepatocytes, as is the case with the metabolites of CCl4 (Singh et al., 2011). This readily occurs in the liver, which contains a high amount of cytochrome P450 enzymes responsible for metabolizing drugs and toxins. In the present study, the ethanolic extract of
Effect on lipid homeostasis
Dyslipidemia is caused by increases in total cholesterol and LDL-cholesterol and decreases HDL-cholesterol in serum, and is a risk factor for atherosclerosis and other diseases of the heart and blood vessels (Mahdy Ali et al., 2012). Certain extents of damage to the liver have been shown to alter its ability to synthesize, package, and secrete lipids into the blood (Fromenty and Pessayre, 1995; Boll et al., 2001; Khalaf et al., 2009; Al-Yahya et al., 2013; Hamed et al., 2016). Therefore, it was unsurprising that recurrent challenges with CCl4 induced dyslipidemia. However,
Effect on serum protein profiles
The elevated total protein in the serum may comprise that which leaked out of the injured liver. The decrease in albumin may indicate that the liver had difficulty synthesizing normal amounts of this protein following injury induced by CCl4. The ethanolic extract of
Effect on recruitment of immune cells in the liver
Histopathology images revealed that CCl4 caused immune cells to be recruited to the sites of damage in the liver (Fig. 1 and Fig. 2). This is unsurprising since CCl4 is a foreign substance and is capable of evoking an immune response
Effect on antioxidant enzyme activities in livers and kidneys
Metabolism of CCl4 caused oxidative stress in the liver and kidneys by decreasing SOD and catalase activities and increasing the concentration of the lipid peroxidation product malondialdehyde. CCl4 radical formed from the breakdown of CCl4 can form DNA adducts as well as protein adducts, which may lead to impairment of the function of certain genes and proteins (Weber et al., 2003). In this study, it is possible that the metabolites of CCl4 reduced antioxidant enzymes by randomly inhibiting their formation and function at both the genetic and protein levels. Our results show that in the context of this experiment,
Effect on serum urea and creatinine concentrations
Urea and creatinine are waste products of metabolism that are normally filtered by the kidneys into the urine. However, alterations to the structure of the kidneys may affect its ability to efficiently filter these waste products, leading to their elevation in the blood (Ravnskov, 2005). In this study, CCl4 increased the concentrations of urea and creatinine in serum, while
In conclusion, our study provides evidence that the ethanolic extract of
AUTHOR DISCLOSURE STATEMENT
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
SOO and NEJO designed the study. SOO and KO carried out the experiments. SOO analyzed the data and wrote the manuscript. All authors approved the final copy of the manuscript.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

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Table 1 . Experimental design
Groups Treatment 1 Control 2 CCl4 3 CCl4+ETDT 250 4 CCl4+ETDT 500 5 CCl4+ETDT 1000 ETDT, ethanolic extract of
Dennettia tripetala .D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw (n=5).
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Table 2 . Effect of
Dennettia tripetala on serum activity of liver marker enzymes in rats administered CCl4 (unit: U/L)Groups AST ALT ALP Control 242.10±6.55a 52.50±4.79a 39.15±5.88a CCl4 809.00±15.6c 486.70±6.67b 45.70±1.59a ETDT 250+CCl4 493.50±19.64b 460.00±48.99b 58.06±5.11b ETDT 500+CCl4 465.30±6.36b 336.70±38.44b 44.81±3.15a ETDT 1000+CCl4 520.00±1.16b 362.50±34.73b 43.63±2.72a The values presented are the mean±SEM (n=5).
Values with different letters (a-c) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala ; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase.D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
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Table 3 . Effect of
Dennettia tripetala on serum lipid concentration in rats administered CCl4 (unit: mg/dL)Groups Total cholesterol HDL-cholesterol LDL-cholesterol TAG Control 38.28±1.93a 25.45±2.20b 13.75±0.95a 127.40±25.10c CCl4 47.85±1.08b 7.24±0.64a 41.07±0.94b 84.30±4.43b ETDT 250+CCl4 47.09±0.61b 28.57±0.24b 23.08±3.12a 46.62±4.91a ETDT 500+CCl4 47.40±2.10b 24.89±2.82b 32.33±3.65b 72.00±6.79b ETDT 1000+CCl4 42.84±1.39ab 9.57±0.97a 38.30±0.58b 49.23±2.22a The values presented are the mean±SEM (n=5).
Values with different letters (a-c) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala ; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TAG, triacylglycerols.D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
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Table 4 . Effect of
Dennettia tripetala administration on liver lipid concentration in rats administered CCl4 (unit: mg/dL)Groups Total cholesterol TAG Control 41.68±0.49a 16.88±2.11a CCl4 51.44±7.59b 28.84±6.01ab ETDT 250+CCl4 44.06±0.97a 7.74±0.70a ETDT 500+CCl4 44.46±0.23a 11.96±0.70a ETDT 1000+CCl4 46.44±0.33ab 16.35±5.05a The values presented are the mean±SEM (n=5).
Values with different letters (a,b) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala ; TAG, triacylglycerols.D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
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Table 5 . Effect of
Dennettia tripetala on serum protein concentration in rats administered CCl4Groups Total protein
(g/dL)Albumin
(g/dL)Globulin
(g/dL)Albumin:globulin ratio Control 5.94±0.18a 3.24±0.04b 2.68±0.09a 1.13±0.04c CCl4 21.04±1.45c 2.96±0.03a 14.05±1.67b 0.18±0.02a ETDT 250+CCl4 13.11±0.95b 2.71±0.05a 10.42±0.91b 0.26±0.02a ETDT 500+CCl4 8.69±1.05a 3.38±0.02b 6.12±1.34a 0.31±0.10a ETDT 1000+CCl4 9.35±0.25a 2.82±0.07a 6.06±0.45a 0.46±0.05b The values presented are the mean±SEM (n=5).
Values with different letters (a-c) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala .D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
-
Table 6 . Effect of
Dennettia tripetala on liver antioxidant enzyme activity and lipid peroxidation status in rats administered CCl4 (unit: units/g wet tissue)Groups SOD Catalase MDA Control 1,225±3c 4,683±11c 0.08±0.01a CCl4 744.0±2a 4,326±51a 0.17±0.01c ETDT 250+CCl4 891.7±36b 4,386±31a 0.09±0.01a ETDT 500+CCl4 1,500±25d 4,518±8b 0.10±0.01ab ETDT 1000+CCl4 1,125±52c 4,656±45c 0.09±0.01a The values presented are the mean±SEM (n=5).
Values with different letters (a-d) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala ; SOD, superoxide dismutase; MDA, malondialdehyde.D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
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Table 7 . Effect of
Dennettia tripetala on kidney antioxidant enzyme activity and lipid peroxidation status in rats administered CCl4Groups SOD
[units/g wet tissue (×10−2)]Catalase
(units/g wet tissue)MDA
(units/g wet tissue)Control 873±28a 5,076±294b 0.20±0.01a CCl4 644±28a 3,984±18a 0.25±0.01b ETDT 250+CCl4 1,130±346a 4,749±11b 0.21±0.01a ETDT 500+CCl4 1,027±208a 4,815±26b 0.19±0.01a ETDT 1000+CCl4 1,030±79a 4,827±7b 0.23±0.01ab The values presented are the mean±SEM (n=5).
Values with different letters (a,b) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala ; SOD, superoxide dismutase; MDA, malondialdehyde.D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
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Table 8 . Effect of
Dennettia tripetala on urea and creatinine concentrations in rats administered CCl4 (unit: mg/dL)Groups Urea Creatinine Control 6.43±0.83a 1.54±0.01a CCl4 10.73±0.04b 2.15±0.02b ETDT 250+CCl4 8.31±0.63b 1.36±0.03a ETDT 500+CCl4 7.53±0.72ab 1.48±0.14a ETDT 1000+CCl4 7.37±0.87ab 1.36±0.07a The values presented are the mean±SEM (n=5).
Values with different letters (a,b) within the same column are significantly different at
P <0.05.ETDT, ethanolic extract of
D. tripetala .D. tripetala was administered in doses of 250, 500, and 1,000 mg/kg bw.
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