high fat diet-simulated Metabolic Syndrome model in male Wistar rats

Biomedicine & Pharmacotherapy 125 (2020) 109968

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Ameliorative activity of Adansonia digitata fruit on high sugar/high fat dietsimulated Metabolic Syndrome model in male Wistar rats

T

Hayat Mohamed Sulimana,*, Bashier Osmana, Iman H. Abdoona, Amir Mustafa Saadb, Hassan Khalidc a

Department of Pharmacology, Faculty of Pharmacy, University of Khartoum, 1111 Al Qasr Avenue, P.O.B 1996, Khartoum, Sudan Department of Pathology, Faculty of Veterinary Medicine, University of Khartoum, Sudan c Department of Pharmacognosy, Faculty of Pharmacy, University of Khartoum, Sudan b

ARTICLE INFO

ABSTRACT

Keywords: Metabolic Syndrome Oxidative stress High sugar/high fat diet Antioxidants Adansonia digitata fruit

Metabolic syndrome is a complex of metabolic disorders characterized by oxidative stress which compromises cell functions and entails multiple organs pathologies. We investigated the therapeutic and protective potential of Adansonia digitata fruit –a potent antioxidant– in high sugar/high fat diet-simulated metabolic syndrome in Wistar rats. 42 male rats (140–200 g) were randomly divided into 7 groups. G1 was kept on standard laboratory diet (SLD) for all 9 weeks (negative control). 5 groups were fed high Sugar/high fat diet for 6 weeks then switched to SLD for another 3 weeks + oral treatment as follows: G2+ no treatment (positive control), G3-G5 + 200, 400 and 800 mg/kg/day aqueous A. digitata fruit respectively, G6 + 10 mg/kg/day Simvastatin. G7 + HS/ HFD + 400 mg/kg/day A. digitata fruit simultaneously and was terminated at W6. Our results showed that G2G6 develops dyslipidemia, hyperglycaemia, weight gain, elevated hepatic biomarkers, elevated creatinine and urea plus pathological derangements in the heart, liver and kidney tissues compared to negative control at W6. 200 mg/kg/day A. digitata fruit significantly ameliorated the induced dyslipidemia (P ≤ 0.001), hyperglycaemia (P ≤ 0.001) with a significant reduction in the Atherogenic Index of Plasma (P ≤ 0.000) after 3 weeks treatment. The fruit normalized the elevated hepatic biomarkers as well as creatinine and urea. A dose dependent partial reduction in lesion intensity was observed in the hepatic tissue while the heart and kidney showed mostly reversed to normal histology. The inflammatory infiltration was eliminated. Relevant results were observed for the two higher doses. The simultaneous treatment showed significant lower levels in all biomarkers investigated compared to positive control which could be interpreted as protective activity. A reduction of 4–11% in whole body weight was achieved. Conclusion: MetS was successfully simulated with a HS/HFD formula in male Wistar rats. Treatment with aqueous A. digitata fruit showed anti-Metabolic Syndrome potential reflected by weight loss, anti-inflammatory, hypolipidemic, hypoglycaemic, renal, hepatic and cardio-protective activities.

1. Introduction Metabolic syndrome (MetS) - a prevalent condition with a complex of cardiovascular risk factors that share a common pathophysiology - is defined as “A cluster of risk factors for cardiovascular disease and type 2 diabetes mellitus, which occur together more often than by chance alone” [1]. MetS is characterized by dyslipidemia, hypertension diabetes mellitus, and obesity [2,3]. A line of evidence highlighted the association of MetS with increased risks to pancreatic dysfunction, NonAlcoholic Fatty Liver Disease (NAFLD), cancer, and chronic kidney disease [4–6]. In addition, skin manifestations including “psoriasis,

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androgenic alopecia, hidradenitis suppurativa acne and rosacea” are reported as co-morbidities with MetS [7]. The criteria for the clinical diagnosis of MetS includes fasting glucose ≥100 mg/dL or diabetic (type II) on treatment; elevated blood pressure ≥130/85 mm Hg or on antihypertensive medications; triglycerides ≥150 mg/dL and HDL-C <40 mg/dL in men and <50 mg/dL in women or under treatment with a lipid lowering drug and waist circumference ≥40 in. (102 cm) in men and ≥35 in. (88 cm) in women. Any three of these conditions confirm the diagnosis of Mets [8]. Oxidative stress, lipotoxcity, inflammation, micro-and macro-vascular derangements are joint hallmarks that underline the syndrome [9,10]. Under inflammatory conditions one

Corresponding author. E-mail address: [email protected] (H.M. Suliman).

https://doi.org/10.1016/j.biopha.2020.109968 Received 20 November 2019; Received in revised form 20 January 2020; Accepted 24 January 2020 0753-3322/ © 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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electron reductions of molecular oxygen induce the production of reactive oxygen species (ROS) that forms the gridlock for the generation of reactive lipid aldehydes and glucose-derived glycation end-products. Lipid peroxidation -an irreversible reaction- generates a series of isomers of the highly reactive lipid radicals which entail propagating oxidation reactions in the biological system. The resultant modified products disrupt normal metabolic processes, alter substrate utilization, aggravate the inflammatory response, jeopardise the endothelium and reduces the endogenous antioxidant protection by inhibiting glutathione peroxidase [11–13]. Consequently, clinical complications including atherosclerosis and cardiovascular events could be attributed to oxidative stress, malfunctioning cholesterol system, type II diabetes, failing clearance mechanisms and inflammatory cascades within a damaged endothelium [14,15]. Increasingly, hepatic steatosis has been consistently linked to oxidants generation and disturbed β-oxidation. The progression from simple steatosis to NAFLD propagates through oxidative stress, lipotoxcity and uncontrolled shuttle of fat from the peripheries to the liver [16]. NAFLD represent MetS manifestations in hepatic tissues with increased risk of cancer and cardiovascular complications [17,18]. High levels of hepatic biomarkers due to leakage into plasma, is considered a sign of membrane integrity loss and hepatic cells injury [19]. Similarly, increased lipid peroxidation within the renal tissue causes multiple modifications in the kidney including glomerular and tubular necrosis which precipitate chronic kidney disease and could lead to renal failure [20,21]. Furthermore, chronic kidney disease (CKD) and reduced glomerular filtration rate especially in patients with diabetes mellitus has been increasingly associated with MetS. Although natural defence mechanisms initiate an inflammatory response to protect cells from damage and facilitate repair processes, yet recurrent inflammation and failing clearance mechanisms would be accompanied with variable degrees of pathological damage. However liver and kidney cells have the capacity to remain partially functional in spite of pathological lesions [22]. Serum urea is a less reliable marker of glomerular filtration than creatinine where levels are more vulnerable to change for reasons beyond glomerular filtration. Clinics may tend to use the mean of urea and creatinine clearance to calculate a more accurate estimate of glomerular filtration rate especially in cases of advanced renal failure [23]. In human, serum levels of creatinine, urea, and uric acid/urates are excreted unchanged in urine and correlate directly to the state of filtration. On the other hand in rats, plasma levels of uric acid/urates are affected by the high level of the enzyme uricase which breakdown uric acid rendering plasma levels to be more liable to fluctuations [24]. Hence, unlike humans urea is considered a relevant biomarker for kidney function assessments in rats [25]. Conditions with high-risk factors constitute a heavy burden on patients and health service providers which could be highly reduced by early recognition, detection and assessments. The risk stratification concept uses statistical measures to estimate specific disease risks within a specific community that are associated with high possibilities of unwanted clinical outcomes [26]. Accordingly, models could be created for the identification of patients under high risks to specific disease complications. Systemic manifestations of MetS represented by the high levels of inflammatory mediators in plasma including IL-6 and TNF-α, leptin, prothrombic factor (PAI-1), oxidized LDL-C and uric acid could be used for early detection of MetS. Other biomarkers that tend to be reduced in MetS include IL-10, ghrelin, adiponectin, and HDL-C enzyme PON1 [26]. Similarly, dermatological manifestations can be detected by specific markers related to specific skin conditions [7]. As hampering excessive ROS generation is considered a therapeutic target, antioxidants could play a crucial role in the management of conditions precipitated or aggravated by lipid peroxidation/ROS generations [27]. A line of evidence has shown the capacity of exogenous antioxidants as efficient molecules that can scavenge/neutralize ROS and halt lipid peroxidation. According to Kim and Park 2018; that

consumption of fish in daily diet maintains total cholesterol and low density lipoprotein levels in plasma [28]. Olive leaf extract in a dietinduced obesity in rats, exhibited cardiac and hepato-protective activities and showed anti-Metabolic Syndrome potential [29]. On the other hand, polyphenols derived from cocoa powder has significantly elevated HDL-C in plasma, reduced triglycerides and C-reactive protein [30]. On the other hand, the new insights into gut-microbiota into health and disease, considered the microbiome a significant environmental factor that maintained dynamic relationships with the host [25]. Gut-microbiota including acetates producing bacteria contribute to the host wellbeing through influencing biological processes beyond food transformations. Acetates are important energy source for intestinal epithelial cells through which the intestinal mucosa is fortified. On the other hand, the microbiome/food cross signals could induce local intestinal immunity through a number of metabolic processes including systemic induction of related antibody expression. Hence, it is evident that consistent consumption of HS/HFD disrupts the microbiome system, affects the levels of acetate producing bacteria, weaken the intestinal epithelial cells and consequently induce inflammation [30]. The recent prebiotic definition as a “substrate that is selectively utilized by host microorganisms to confer a health benefit” extends the prebiotics concept to include diverse substances other than carbohydrates derivatives [31]. Consequently, antioxidants including phenolics, triterpenoids, and other phytochemicals in general are included into this definition [31]. Novel insights into the host-microflora interactions could open new outlooks for nutraceuticals as prebiotics to confer therapeutics and preventive activities for diseases related to the immune system, metabolic disorders, skin manifestations and beyond [32]. Novel and known prebiotics could be adapted to specific physiological targets, pathological risk factors and or metabolic processes [32]. Antioxidants containing food has been shown to confer beneficial remuneration by augmenting gut-microflora which in turn could positively ameliorate specific health problems like inflammatory bowel disease, cancer and the immune system disorders. Personalized dietary plans designed according to need are shaping the concept of precision diet with new treatment possibilities [33]. Rodents models for simulation of MetS includes guinea pig, Nile, Sand, Wistar and Zuker rats in addition to Swine [2,34]. Some other models that has been addressed included the spontaneously hypertensive rats and the low-capacity runner rats [35,36]. Computational approaches including the in silico perturbations has also been introduced to study and interpret metabolic states and provide an in-depth understanding of the human system biology in health and disease [37,38]. A data-driven-physiological modelling has also been developed with combinations of in vivo and in silico models [39]. Within animal models, rodents remain the most used models to simulate MetS in human. The HS/HFD in male rats was described as the best model to simulate MetS in human as reported Wong et al., 2016 [2]; In this study, we investigated the therapeutic and protective potential of aqueous Adansonia digitata fruit pulp in a high sugar/high fat diet (HS/ HFD)-simulated Metabolic Syndrome model in male Albino Wistar rats. 2. Hypothesis behind the study 2.1. Therapeutic and protective management of Metabolic Syndrome MetS is a prevalent condition that affects 10–84 % worldwide and 20–40% of the population in the West [2]. Out of 17.5 million, 7.4 million deaths are due to coronary heart diseases and stroke [40]. Moreover, about 7.6 million deaths are attributed to hypertension and ddiabetes is considered the 6th cause of death globally [41,42]. MetS management depends solely on symptomatic treatments which entail polypharmacy, increased physical work overload and high financial cost on health service providers worldwide [43]. New therapeutic targets, new active molecules and or new strategies are on demand for MetS management [25]. 2

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Adansonia digitata (A. digitata), [Baobab] fruit has been used as food by local sub-Savannah residents in Africa through history. All parts of the tree are edible including Leaves fruit pulp and seeds. It forms one of the main food and income source for the native residents. Different parts of A. digitata have been used traditionally to treat dysentery, diarrhoea and dehydration in Sudan. In other countries it is used for ulcer, urinary disorders, fever, skin problems, anaemia, and malaria [44,45). The fruit pulp is rich in: aromatic compounds, minerals, organic acids especially ascorbic acid, amino acids including alanine, arginine, glycine, lysine, methionine, proline, serine and valine, vitamins including B1, B2, B3, beta carotene and vitamin C. Tritriterpenoids including beta-sitosterol, beta-amyrin, alpha-amyrin and ursolic acid) [46–48]. Pharmacologically, Baobab has been shown as an antioxidant [44], anti-inflammatory [49], analgesic/antipyretic [49] antilipidemic [50,51), hypoglycaemic [52,53), antimicrobial [54,55) antiobesity [52–57], cardioprotective [58], and hepatoprotective [59–61]. According to our literature reviews up to the time of this work proposal, no previous reports were found looking into the effects of this important fruit on Metabolic Syndrome. This work was designed to cover 3 of MetS conditions including dyslipidemia, diabetes mellitus and obesity using a HS/HFD formula to simulate Mets in male Albino Wistar rats. As oxidized lipid products are considered a shared diagnostic marker between other MetS conditions we looked into A. digitat fruit vs Simvastatin with focus into postprandial hyperglycaemia, hepatic function, kidney function and obesity.

Table 1 Diet and treatment protocol. Groups

Diet

Treatment

Week: 0-6

Week: 6-9

Week: 6-9

G1: Negative control G2: Positive control G3: Treatment group 1

SLD HS/HFD HS/HFD

SLD SLD SLD

G4: Treatment group 2

HS/HFD

SLD

G5:Treatment group 3

HS/HFD

SLD

G6: Standard Drug

HS/HFD

SLD

G7: Simultaneous treatment, (protective treatment)

HS/HFD+400 mg/kg/ day A. digitata simultaneously

Nil Nil 200 mg/kg/day fruit (A. digitata) 400 mg/kg/day fruit (A. digitata) 800 mg/kg/day fruit (A. digitata) 10 mg/kg/day Simvastatin Terminated from the experiment

HS/HFD= high sugar/high fat diet, SLD= standard laboratory diet.

3.2. Methods 3.2.1. Experimental protocol The HS/HFD formula (a modified version of Sikarwar MS et al.) [62] is composed of 2 % cholesterol, 1 % cholic acid, 10 % peanut oil, 40 % sucrose and 47 % standard laboratory diet (SLD). The SLD is composed of 23.5 % maize flour, 2.94 % sesame oil, 5.87 % dried meat, 2.93 % dried whole milk powder, 0.01 % sodium chloride and 11.75 % wheat bran flour (which is rich in fibres, minerals, vitamin B6, thiamine, folate, vitamin E and phenolics) [63]. Diet was prepared on demand at the same facility. A. digitata doses were determined from a pilot study. Aqueous A. digitata fruit has been freshly prepared daily and doses were delivered through the oral route via an intra-gastric tube.

2.2. Ethical clearance Ethical clearance for this work was granted by the Faculty of Pharmacy Research Ethics Board and the Institutional Review Board of the Graduate College for Medical and Health Studies, University of Khartoum Sudan.

3.2.2. Diet and treatment protocol The diet and treatments protocol for different groups is shown in Table 1.

3. Material and methods 3.1. Materials

3.2.3. Blood collection and parameters investigated Blood samples of 1 ml [64], were withdrawn in heparinised tubes at baseline, at W6, W7, W8 and W9 through the orbital plexus from alternate eyes on each sampling time. Blood was centrifuged for 15 min at 3000 rpm. The separated serum was analyzed for all biomarkers under investigations by an auto analyzer (COBAS Roche Integra 400 Plus, Switzerland) according to the manufacturer’s standard protocols. The appliance provides soft copy results. Biomarkers investigated included postprandial plasma glucose (PPPG), total cholesterol (TC), triglycerides (TGs) and high density lipoprotein cholesterol (HDL-C). Low density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald equation [65]. Liver function was assessed by measuring alanine transaminase-(ALT), aspartate transaminase-(AST), alkaline phosphatase-(ALP), albumin-(ALB), total bilirubin (TBIL), direct bilirubin (DBIL) and total protein (TP). Kidney function was assessed by measuring creatinine and urea.

3.1.1. Plant A. digitata dry fruit shells were brought from Kordofan state (West Sudan). The fruit pulp was removed from the shells and separated into fine powder from the seeds by mechanical shaking at normal room temperature. Both the fruit kernels and powder were authenticated by the Aromatic and Medicinal Plants Institute, Khartoum, Sudan. 3.1.2. Chemicals Cholesterol and cholic acid powders were Adamas Beta Reagents, China. 10 mg Simvastatin tablets were SAJA (Saudi-Japanese pharmaceuticals) product. All other dietary ingredients for the HS/HFD formula were from a local market in Khartoum. 3.1.3. Animals Forty two male Wister rats weighing 140–200 g were released from the animal house - Faculty of Pharmacy, University of Khartoum Sudan with permission from the Faculty Research Board. They were housed and cared for at the same facility, divided randomly into 7 groups (n = 6), marked clearly, housed 3 rats per cage under controlled temperature/humidity and 12 hours light/dark cycles with water and diet ad libitum. To acclimatize to the new environment, the rats were placed in a separate room for one week prior to the experiment and were used according the University of Khartoum guidelines that adopt the internationally accepted 3 Rs mandate and it comply with the ARRIVE guidelines.

3.2.4. Weight records Animal whole body weights were read with a calibrated sensitive balance in grams at base line, at week 6, and at week 9. Organs weights were recorded at week 6 and week 9. 3.2.5. Pathological procedures Dissection was performed under general anaesthesia with ethylether. Three rats were selected randomly from G1, G2 and G7 at the end of week 6. Another set of 3 rats were dissected at the end of week 9 from G1, G2, G3, G4, G5 and G6. One cubic cm specimen from the 3

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heart, kidney and liver were taken for pathological examinations. Specimens were conserved in 10 % formal saline for further use. Organ specimens were sectioned embedded in paraffin and stained with haematoxylin–eosin according to standard pathological procedures [66]. The remaining rats were killed with a high dose of ethyl ether and disposed off safely with the remaining carcasses off the dissected animals.

G3–G5 showed significant dose dependent activity in the 4 lipid parameters. 200 mg/kg/day A. digitata fruit reduced TC by 3.23 vs 2.89 fold by Simvastatin (P≤0.001) (Fig. 1a). TGs by 7.37 vs 3.44 fold by Simvastatin (P ≤ 0.001) (Fig. 1b) and LDL-C by 8.07 vs 8.03 fold by Simvastatin, (P ≤ 0.001) (Fig. 1c). HDL-C was elevated by 2.27 vs 2.92 fold by Simvastatin (P ≤ 0.000) (Fig. 1d). 400 and 800 mg/kg/day mediated reductions of 6.34 and 6.83 fold (P ≤ 0.000) in TC, 10.57 and 12.36 fold (P ≤ 0.000) in TGs, 49 fold (P ≤ 0.000) in LDL-C by both doses respectively. HDL-C was increased by 2.38 and 2.66 fold (P ≤ 0.000) respectively.

3.2.6. Data analysis All biomarkers were measured in mg/dL. All data was expressed as Mean ± SEM. Statistical significance was measured with paired samples t-test on SPSS version 22. P ≤ 0.05 was considered statistically significant. Histopathology slides were read under a light microscope Nikon E200 (H&E X40), attached to an Olympus digital camera. Whole body and organ’s weight were recorded in grams with a calibrated sensitive balance. 4. Results

4.2.2. Postprandial plasma glucose before and after HS/HFD G1 showed insignificant change in postprandial plasma glucose (PPPG) at all points of assessments. G2–G6 showed a significant increase equivalent to 39.78 % (average rise in 5 groups) (P ≤ 0.000), (Fig. 2a). G7 showed a significant (P ≤ 0.016) increase in plasma glucose to 114.83 vs 102.17 mg/dL at BL which is still significantly (P ≤ 0.024) low compared to positive control at W6.(Fig. 2a).

4.1. Lipid profile before and after HS/HFD

4.3. G 2 – G6 were considered hyperglycaemic

After 6 weeks on HS/HFD, G2–G6 depicted a significant rise of 10 fold in TC (P ≤ 0.000) (Fig. 1a), 5 fold in TGs (P ≤ 0.000) (Fig. 1b), 39 fold in LDL-C (P ≤ 0.000) (Fig. 1c) and almost one fold reduction in HDL-C (P ≤ 0.003) (Fig. 1d) compared to negative control which showed no change from baseline. G7 showed a significant increase by 1.58 fold in TC (P ≤ 0.010), 0.83 fold in TGs (P ≤ 0.010) and by 6.49 fold in LDL-C (P ≤ 0.000) compared to negative control, yet all these biomarkers were significantly low (P≤0.000) compared to positive control. HDL-C was significantly increased by 0.72 fold (P≤0.010) compared to baseline and was also significantly increased compared to positive control (P0.001).

4.3.1. Therapeutic activity of A. digitata fruit on HS/HFD-induced hyperglycaemia Although G2 showed significant reductions in PPPG levels by normal resolution mechanisms after switching to SLD (P ≤ 0.023), yet levels at W9 were still significantly high compared to BL (P ≤ 0.001) (Fig. 2a). 200 mg/kg/day A. digitata fruit depicted significant dose dependent reductions in PPPG to values below BL compared to positive control. Levels at week 9 were reduced by 60.14 %. 10 mg/kg Simvastatin showed insignificant change in PPPG levels from W6 (Fig. 2a). Reductions of 61.42 % and 63.91 % (P0.000) were depicted by 400 and 800 mg/kg/day A. digitata fruit respectively.

4.2. G2–G6 was considered dyslipidemic

4.4. Liver function test

4.2.1. Curative activity of A. digitata fruit pulp on HS/HFD-induceddyslipidemia After switching to SLD, significant reductions by normal resolution mechanisms were observed by 1.51 fold in TGs (P 0.014) and by 1.64 fold in LDL-C (P≤0.010) (Fig. 1c) as shown by G2 at W9. HDL-C was further reduced by 0.56 fold (P≤0.038) (Fig. 1d).

4.4.1. Hepatic biomarkers before and after HS/HFD A significant rise of 34.55 % in ALT-(P0.004), (Fig. 2b), 90.1 % in AST-(P ≤ 0.002), (Fig. 2d) and 100 % in ALP-(P≤0.000)- (Fig. 2c), were recorded at W6 by G2–G6. A rise of 28.53 % in total protein (P0.000) (Fig. 2f) was observed and albumin (ALB) was reduced by 7.22 % (P ≤ 0.010), (Fig. 3e). In G7 a significant rise in ALT by 6.28 %,

Fig. 1. Effects of 3 weeks treatment with Adansonia digitata fruit vs 10 mg Simvastatin on lipid parameters compared to positive control: a- total cholesterol, bTriglycerides, c- LDL-C, d- HDL-C in male albino Wistar rats. Results are mean ± SEM, (n = 6). #P ≤ 0.05 represent W6 vs baseline, *P ≤ 0.05 represent W7, W8 or W9 vs W6, +P ≤ 0.05 represent W6 (G7) vs W6 positive control. 4

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Fig. 2. a- Effect of A. digitata fruit vs 10 mg Simvastatin treatments for 3 weeks on postprandial plasma glucose levels compared to positive control in male Albino Wistar rats. b: Effect of Adansonia digitata fruit vs 10 mg Simvastatin treatments for 3 weeks on hepatic biomarkers in male Albino Wistar rats compared to positive control, 2b: ALT, 2c: ALP, 2d: AST, 2e: ALB, 2f : TP. Results of all biomarkers are mean ± SEM, (n = 6). #P ≤ 0.05 represents W6 vs BL, *P ≤ 0.05 represents W7, W8 or W9 vs W6. +P ≤ 0.05 represents W6 (G7) vs W6 positive control.

Although total bilirubin and direct bilirubin were increased by 147 % (P ≤ 0.000) and 12 % (P ≤ 0.011) respectively at W6 yet both were reduced to BL by normal resolution in all treated groups by W7. Both biomarkers continued stable through the following two weeks. Significance for bilirubin couldn’t be computed as the SD was zero. 4.5.2. Hepatic biomarkers of HS/HFD pre-treated rats after treatment with A. digitata fruit G3–G5 depicted significant reductions in all hepatic biomarkers under investigation in a dose dependent course except albumin which was significantly elevated. 200, mg/kg/day reduced ALT by 46.71 % (P≤0.002) AST by 47.47 % (P≤0.000), ALP by 46.44 % (P≤0.001)Total protein was reduced by 10.36 % (P≤0.001) and albumin was elevated by 9.91 % (P≤0.042) . 400 and 800 mg/kg/day mediated reductions of 50.94 % (P ≤ 0.000) and 52.72 % (P≤0.000) in ALT, 53.14 % (P ≤ 0.000) and 52.25 % (P ≤ 0.000) in AST, 55.17 % (P ≤ 0.000) and 56.84 % (P≤0.000) in ALP respectively. Total protein was reduced by.15.44 % (P ≤ 0.002) and 18.21 % (P≤0.001) respectively. Albumin was elevated by 17.63 % (P ≤ 0.000) and 21.11 % (P ≤ 0.000) respectively. 10 mg Simvastatin showed similar effects to normal resolution in positive control. Fig. 3. Effects of A.digitata fruit vs 10 mg Simvastatin on creatinine and urea in male Albino Wistar rats compared to positive control, Fig. 3a: creatinine, Fig. 3b: urea. Results are mean ± SEM, (n = 6). #P ≤ 0.05 represents W6 vs BL, *P ≤ 0.05 represent W7, W8 or W9 vs W6, +P ≤ 0.05 represents W6 (G7) vs W6 positive control.

4.6. Kidney function 4.6.1. Kidney function before and after HS/HFD G1 showed no significant change from BL in creatinine or urea at W6. G2–G6 showed significant increase by 28.57 % (P≤0.001) and 19.04 % (P≤0.006) respectively. G7 showed insignificant elevations in creatinine and urea at week 6 compared to BL but these levels were significantly low (P≤0.001), (P≤0.025) respectively compared to G2 at W6.

(P0.020), in AST by 11.06 % (P ≤ 0.002), in ALP by11.82 % (P0.001) and TP by 4.33 % (P≤0.033) was observed compared to G1, yet these levels were significantly low (P≤0.000) compared to G2. Albumin was insignificantly reduced in G7, (Fig. 3B).

4.7. G2–G6 results reflected kidney dysfunction 4.7.1. Effects of normal resolution on elevated renal biomarkers G1 showed no change from W6. G2 showed significant 33.33 % (P ≤ 0.017) reduction in creatinine by normal mechanisms at week 9. Insignificant change was recorded for urea by normal resolution.

4.5. G2–G6 results reflected hepatic dysfunction 4.5.1. Effects of normal resolution on the elevated hepatic biomarkers At W9 G1 showed no change in any of the biomarkers under investigations from what was recorded at BL or W6. Switching to SLD only, showed significant reductions by normal resolution in the two transaminases and alkaline phosphatase by 12.06 %, (P ≤ 0.020), 13.73 % (P ≤ 0.004) and 32.57 %, (P ≤ 0.009) respectively. Insignificant changes were recorded for total protein and albumin.

4.7.2. Renal biomarkers of HS/HFD-prereated rats after treatment with A. digitata fruit At W9 G3-G5 depicted significant dose dependent reductions in creatinine equivalent to 40.00 % (P ≤ 0.005) by 200 mg/kg/day, 5

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(Fig. 3a). Similarly 400 and 800 mg/kg/day A. digitata fruit depicted 40.54 % (P ≤ 0.007), and 47.61 % (P ≤ 0.004) respectively. Urea was significantly reduced by A. digitata fruit in a dose dependent course to base line range. A reduction of 11.97 % (P ≤ 0.010) was observed by 200 mg/kg/day. 10 mg Simvastatin group showed similar effects to normal resolution in G2 (Fig. 3b). A reduction of 19.39 % (P ≤ 0.001) and 21.34 % (P ≤ 0.000) was mediated by 400 and 800 mg/ kg/day A. digitata fruit respectively.

found to have clean-smooth white coloured fur, normal movements, steady breathing and normal pellets-shaped faeces. At W6 G1 remained with no noticeable change. G2–G6 were noticed to have less dense, patchy fur with varying degree of fur loss and alopecia patches on the dorsum and near the tail. Change in fur colour to light brownish cream was noted as well as loose irregular pellet-shaped faeces and sluggish movements. All rats had normal breathing and no obesity was noticed. G7 showed similar features but with less intensity than those noticed on G2-G6. On re-examination at W9: G1 showed no noticeable change from outset or W6. G2 remained with the same features recorded at W6. G3–G6 regained normal pellets-shaped faeces; fur patches clearly reduced with some improvement in colour, normal movement, steady breathing and no obesity was noticed at W9.

4.8. Atherogenic index of plasma (AIP) The Atherogenic Index of Plasma is considered a clinical predictor for the assessment of patient’s risk to cardiac events [67]. We calculated the AIP at BL, at W6 and at W9 to evaluate the ability of A. digitata fruit to reduce the risk from cardiovascular events. The formula used was:AIP = log (triglycerides/HDL-C) mmol/L [68], Values were interpreted as:−0.3 to 0.1=low risk, 0.1–0.24=moderate risk and ≥0.24= high risk [67]. The recorded results were shown in (Table 1).

4.11.2. Gross examination Organs examined after dissection at W6 included the liver, heart, kidney and spleen. G1 showed normal colour, size and shape of all 4 organs. Noticeable morphological changes were observed on G2 organs. The liver colour was changed to lightly pinkish-white with dull and greasy appearance compared to dark bright maroon red in G1. G7 liver showed slightly pinkish red colour. At W9 no change was observed in G1 and G2 livers. G3G5: a dose dependent change in colour to pinkish red was observed compared to G2. G6 showed no improvement from the image seen at W6 compared to G2. The heart, kidney and spleen of all groups looked congested with darkened dull colour compared to G1 at week 6. Less congestion and a brighter colour was observed in G3-G5 at W9.

4.9. Anti-obesity activity 4.9.1. Whole body weight before and after HS/HFD At W6 G1 showed a significant 5.27 % (P ≤ 0.001) increase in whole body weight at the end of W6. Significant weight gain of 9.35 % (P ≤ 0.002) was observed in G2-G6 compared to BL. G3 represented the highest weight gain (9.79 %) and G4 represented the lowest, (6.56 %), (Table 2). G7 showed insignificant weight gain compared to G1 (P ≤ 0.087) but was significantly low (P ≤ 0.021) compared to G2 at W6, (Table 3). At W9, G1 showed a further increase in weight by 3.95 % (P ≤ 0.018) while G2 showed insignificant change from W6, (Table 3).

4.11.3. Change in organ weight by HS/HFD Changes in organ weight of G2 were compared to G1 at W6. G3–G6 at W9 was compared to G1 and G2. G1 livers showed no significant change in weight between W6 and W9 (Table 4). G2 at W6 showed significant 2 fold increase in liver weight compared to G1 (P ≤ 0.000) at W6. Normal resolution mechanisms significantly decreased liver weight by 0.33 fold at W9 (P ≤ 0.018) (Table 4). G3–G5 showed significant dose dependent reduction of 0.51 (P ≤ 0.023), 0.74 (P ≤ 0.006) and 0.77 (P ≤ 0.005) fold with 200, 400 and 800 mg/kg/day respectively compared to G2 (Table 4). G7 showed 0.75 fold increase (P ≤ 0.000) in liver weight compared to G1 which was also significantly low by 0.67 fold (P ≤0.005) compared to G2 at W6 (Table 4). 10 mg/kg/day Simvastatin showed insignificant decrease in livers weight at W9 compared to G2 G1 spleen weight showed an increase of 14.8 %, (P ≤ 0.041) at W9. G2 was significantly increased by almost 67 % at W6 (P ≤ 0.001) and was further significantly increased by 11.10 % at W9 (P ≤ 0.015), G3-G5 showed significant dose dependent reduction in spleen weight equivalent to 13.39 % (P ≤ 0.093), 24.41 % (P ≤ 0.007) and 26.57 % (P ≤ 0.007) with 200, 400 and 800 mg/kg/day A. dgitata fruit respectively. Insignificant change in spleen weight was observed in G6 compared to positive control. G7 showed insignificant increase in spleen weight compared to G1 but was significantly low (P ≤ 0.001) compared to G2. Simvastatin showed insignificant change in spleen weight compared to positive control at W9. No significant changes were recorded in heart and kidney weight between W6 and W9 in all groups under investigation.

4.10. G2-G6 were considered overweight 4.10.1. Anti-obesity effects of A. digitata on body weight gain of HS/HFD pre-treated rats G3-G5 showed significant dose dependent weight loss at W9 of 3.98 % (P ≤ 0.048), 8.64 % (P ≤ 0.001) and 11.19 %, (P ≤ 0.000) by 200, 400 and 800 mg/kg/day A. digitata respectively. A significant 4.77 % (P ≤ 0.040) increase in whole body weight was recorded for 10 mg/kg/ day Simvastatin at W9 compared to W6, (Table 3). 4.11. Pathological results 4.11.1. Observational macroscopic signs The 42 male rats examined at outset for general features, were Table 2 Atherogenic Index of Plasma mmol/L of dyslipidemic Wister rats before and after treatment with Adansonia digitata fruit pulp. Groups G G G G G G G

1 -Negative Control 2- Positive control 3- 200 mg/kg A. digitata 4- 400 mg/kg A. digitata 5- 800 mg/kg A. digitata 6- 10 mg/kg Simvastatin 7- S/HFD+400 mg/kg A. digitata

AIP mmol/L BL levels

W6 levels

W9 levels

−0.31 −0.39 −0.43 −0.45 −0.27 −0.35 −0.34

−0.38 0.62### 0.55### 0.65### 0.71### 0.71### −0.55+

−0.37 0.10** −0.81*** −0.92*** −0.95*** −0.52*** –

4.11.4. Microscopic pathological changes in hepatic, kidney and heart tissue Microscopic image of hepatic tissues at W6: G1 showed well arranged normal hepatic cells radiating from a normal hepatic central vein, (Fig. 4a G1). G2 showed severe hydropic degeneration with cloudy and fatty cells, (Fig. 4a G2). Inflammatory cells infiltration was observed, (Fig. 4a G2a). G7 showed mild hydropic degeneration with no inflammatory infiltrations, (Fig. 4a G7). Hepatic tissue at W9: G1 showed similar picture to W6 and G2 showed no change in the HS/HFD-precipitated pathology of W6, (Fig. 4a G2b). G3G5 showed a dose dependent reduced lesion intensity of the HS/HFD-precipitated pathological changes in the hepatic tissue, (Fig. 4a G3, G4 and G5). Simvastatin showed no effects on the HS/HFD-precipitated lesions, (Fig. 4a

Results are mean ± SEM, (n= 6). ### P ≤0.000, ##P ≤0.001 and #P≤0.05: represents W6 vs BL. ***P≤0.000, **P≤0.001 and *P≤0.05: represents W7 vs W6, W8 vs W6, W9 vs W6.+P≤0.05 represents W6-G7 vs W6 positive control. 6

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Table 3 Effects of Adansonia digitata fruit vs 10 mg Simvastatin on whole body weight of HS/HFD pre-treated Wistar rats compared to BL and W6. Groups

Whole body weight/g Baseline

G1: G2: G3: G4: G5: G6: G7:

negative control positive control 200 mg/kg/day A. digitata fruit 400 mg/kg/day A. digitata fruit 800 mg/kg/day A. digitata fruit 10 mg/kg/day Simvastatin HS/HFD + 400 mg/kg/day A. digitata fruit

Week 6

167.50 ± 7.93 174.17 ± 5.4 150.83 ± 2.39 157.50 ± 4.23 155.00 ± 3.16 155.42 ± 4.20 174.17 ± 4.73

All data is mean ± SEM, (n = 6). # P ≤ 0.05 represents W6 vs baseline, *P ≤ 0.05 represent W7, W8, W9 vs W6,

+

Week 9 ##

176.33 ± 8.49 189.67 ± 5.77### 165.60 ± 5.29### 167.83 ± 6.93## 169.83 ± 4.71## 170.60 ± 3.17## 176.50 ± 5.00+

183.3 ± 13.59# 182.67 ± 5.42 159.00 ± 3.49* 153.33 ± 5.73** 150.83 ± 5.69*** 178.75 ± 2.06# –

P ≤ 0.05 represent W6 (G7) vs W6 positive control.

G6). No inflammatory infiltration was detected at W9 in A. digitata or in Simvastatin treated groups. Kidney tissues at W6: G1 showed Normal glomeruli and tubules. G2 showed Shrunken, fragmented and some lysed glomeruli (Fig. 4b G2) in addition thickened tubules and tubular necrosis was detected (Fig. 4b G2b). At W9 no change was observed in G1. No change in the HS/HFD-precipitated pathology on the kidney tissue was noticed in G2. Specimens from the three groups treated with A. digitata (G3–G5) showed mostly normal kidney tissues (glomeruli and tubules). No inflammatory cells were detected in the kidney tissue in all three doses images (Fig. 4b G3, G4 and G5). 10 mg/kg/day Simvastatin showed no change in the HS/HFD-precipitated lesions (Fig. 4b G6) but no inflammatory cells were detected. The simultaneously treated G7 showed moderate degree of tissue damage with less tubular necrosis and fewer fragmented/shrunken glomeruli. No inflammatory cells infiltration was detected, (Fig. 4b G7). Heart tissue at W6: G1 showed normal myocardium with normal myofibrils and muscle bundles, (Fig. 4c G1). G2 showed disorganised structure of myofibrils and detached muscle bundles (Fig. 4c G2), necrosis as seen in (Fig. 4c G2b). The same degree of lesion intensity was seen at W9. No inflammatory infiltration was detected. At W9 G1 and G2 showed no change from W6. The three groups treated with 200, 400, and 800 mg/kg/day A. digitata (G3–G5) showed mostly normal myocardial muscle bundle and myofibrils compared to the pathological lesions on the heart tissue of positive control G2, (Fig. 4c G3, G4 and G5). No inflammatory cells infiltration was detected. 10 mg/kg/day Simvastatin showed no effect on the HS/HFD-induced disorganisation of heart muscle tissues as shown by (Fig. 4c G6). No inflammatory cells were detected in G6 tissues. The simultaneously treated G7 showed moderate degree of disorganised heart tissue structure. No inflammatory cells infiltrations were detected (Fig. 4C G7).

5. Discussion Metabolic syndrome is a prevalent disorder with no specific treatment [69,70. Management depends solely on symptomatic treatment which entails polypharmacy and increased risk of side effects. Regulating elevated plasma glucose and lipids are considered therapeutic targets as dyslipidemia and diabetes are hallmarks in MetS and the consequent cardiovascular complications [10]. In this study, dyslipidemia, hyperglycaemia, weight gain, elevated hepatic and renal (creatinine and urea) biomarkers were detected. Exceptionally, albumin was significantly reduced. A line of evidence showed that low albumin in plasma correlates directly to the degree of injury in hepatocytes and it indicates disturbed liver function [60,71–73). Bilirubin was also significantly increased at W6 but was recovered to baseline one week after termination of the HS/HFD in all groups including the positive control. The efficient normalization of bilirubin could be attributed to bilirubin antioxidant activity which could be augmented by antioxidants in A. digitata fruit. It could also be attributed to bilirubin clearance/transport natural systems [74]. Similarly, increased levels of creatinine and urea were observed at the end of the HS/HFD period. High serum creatinine and urea in plasma indicates kidney malfunction [75–77]. None of the biomarkers under investigations normalize to baseline via normal resolution mechanisms when switched to SLD only. Heart, liver and kidney tissue specimens’ showed pathological lesions as well as inflammatory cells infiltration at W6. Although none of the rats were overtly obese to the bare eye, a significant increase in rat’s whole body weight was recorded at the end of the HS/HFD period. This result is in line with other findings that HFD effects on rodent’s whole body weight were not always linked to obesity but consistently linked to liver steatosis, disturbed β-oxidation, and oxidants generation [16]. Weight gain has been increasingly linked to an imbalance between calorie consumption and energy expenditure rather than just high calorie intake [78]. On the other hand, improved fur density and colour as well as reduced alopecia patches were

Table 4 Effects of Adansonia digitata vs 10 mg Simvastatin on Liver and spleen weight of HS/HFD pre-treated Wistar rats compared to BL and W6. Groups

Liver weights

Spleen weight/g

Week 6

Week 9

Week 6

Week 9 0.29 ± 0.03# 0.50 ± 002#

–

0.40 ± 0.02*

G4: 400 mg/kg/day A. digitata

–

–

G5: 800 mg/kg/day A. digitata

–

G6:10 mg/kg/day Simvastatin G7: HS/HFD + 400 mg/kg/day A. digitata fruit

– 9.47 ± 0.68++

5.80 ± 0.06 11.94 ± 0.39* 10.45 ± 0.94* 9.08 ± 0.09** 8.94 ± 0.03** 11.80 ± 0.28 –

0.25 ± 0.03 0.46 ± 0.01##

G3:200 mg/kg/day A. digitata

5.40 ± 0.21 15.83 ± 0.12### –

0.35 ± 0.01** 0.34 ± 0.01** 0.45 ± 0.02

G1: negative control G2: positive control

– – 0.26 ± 0.01++

All tables data is mean ± SEM, (n = 6). #P ≤ 0.05 represents W6 vs baseline, *P ≤ 0.05 represent W7, W8, W9 vs W6, +P ≤ 0.05 represent W6 (G7) vs W6 positive control.

7

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Fig. 4. a Pathological changes in hepatic tissue. Image/(H&E- 40X). G1 at W6: liver specimen showed normal hepatic cells radiating from a normal central vein. G2, effects of HS/ HFD on hepatic tissue: severe hydropic degeneration, cloudy and fatty cells. G2a: Inflammatory cell infiltration. G7: showed mild hydropic degeneration with no inflammatory infiltrations. At W9: G1 and G2 showed similar picture to W6. G3-G5, A. digitata fruit activity on hepatic tissue pathology: a dose dependent reduction in lesions intensity of the HS/HFD-induced pathology in the hepatic tissue. G6: Simvastatin showed no effects on the HS/HFD-induced lesions. No inflammatory infiltration was detected at W9 in A. digitata fruit or in Simvastatin treated groups. b Pathological changes in kidney tissue. Image/(H&E- 40X). At W6, G1: kidney specimen showed normal glomeruli and tubules, G2 showed necrosis of tubular epithelium, shrunken, fragmented and lysed glomeruli in some fields. G7 showed some fragmented and lysed glomeruli but no inflammatory infiltration. At W9-: No change was seen in G1. Similar lesions were seen in G2, (Fig. 4b: G2b). G3-G5 showed A. digitata fruit activity on kidney tissue pathology: mostly reversed to normal kidney tissue, (Fig. 4b: G3, G4 and G5). Simvastatin showed no change in the HS/HFD-induced lesions in kidney tissue, (Fig. 4b: G6). c Pathological changes in heart tissue. Image/(H&E- 40X). At W6: G1 showed normal myocardium, myofibrils and normal muscle cross striations. G2: showed disorganised structure of myofibrils and detached muscle bundles, G2b: necrosis of heart tissue but no inflammatory cells infiltrations were detected. G7 showed lower degree of muscle disorganisation with no inflammatory infiltration. At W9: G1 & G2 showed similar pictures to that seen at W6. G3-G5, A. digitata fruit activity on heart tissue pathology showed mostly reversed to normal myocardium with no inflammatory infiltration (Fig. 4c: G3, G4 and G5). G6: Simvastatin showed no change in the HS/HFD-induced lesions on heart muscle, (Fig. 4c: G6).

observed. According to Ellie C. Stefanadi et al. 2018; that some dermatological manifestations including alopecia are considered as one of MetS comorbidities [7]. As reported by Wong SK, et al. 2016; [2], these findings validate the model and simulate MetS in humans. Oral administration of 200, 400, and 800 mg/kg/day A. digitata fruit pulp mediated significant dose dependent reductions in TC, LDL-C and TGs with a noticeable increase in HDL-C after one week of treatment. Similarly profound reductions were detected in postprandial plasma glucose. The simultaneously treated group showed a significant increase in atherogenic lipids and glucose from baseline yet the recorded values were significantly lower compared to positive control. Moreover, this group recorded a significant increase in HDL-C in spite of the simultaneous HS/HFD which may reflect the protective potentials of A. digiata fruit. The two transamiases as well as alkaline phosphatase were significantly normalized to baseline by A. digitata fruit. Similarly, a dose dependent reduction in total protein was observed. Albumin was significantly restored to baseline with a noticeably high significance, (P ≤ 0.000). In addition, a dose dependent reduction in creatinine and urea were recorded. Examination of tissue pathology revealed a dose dependent partial reduction in lesion severity in hepatic tissues and a reversal to almost normal structures in heart and kidney. Furthermore, the inflammatory infiltration was eliminated. The simultaneously treated group showed relatively less tissue derangements with no inflammatory cells infiltration. Significant weight loss of 4 %, 8.7 % and 11 % was recorded for the 3 doses respectively. Similarly, the elevated liver weights were significantly reduced from two to one fold by normal resolution and were further reduced to less than one fold after A. digitata treatment. Nonetheless, the elevated spleen weights

were reduced to normal in a dose dependent course. No change in kidney and heart weights was recorded. According to Chess D et al., 2008; that no difference in heart mass between SLD and HFD-treated mice was observed [79]. The above set of therapeutic and protective activities of A. digitat fruit can be linked to the fruit richness of nutrients and high antioxidant capacity. The fruit has been reported to contain vitamin C, β-sitosterol, α/β-amyrin and uroslic acids [80]. These three pentacyclic triterpenoids and vitamin C have been reported to mediate antioxidant, anti-inflammatory, antilipidemic and antidiabetic activities by improving lipoprotein expression, insulin sensitivity and hepatic protection [81–83]. As a rich source of methionine –a sulphur containing amino-acid–it could inhibit lipid peroxidation as a strong antioxidant [84,85). The fruit could facilitate LDL-C reuptake into the liver, stimulate fatty acid consumption in hepatocytes and increase plasma triglycerides catabolism by increasing the production of the hepatic enzyme carnitine synthase which is made from methionine and lysine in the presence of vitamin C as a cofactor [82,84). Containing the amino acid L arginine, it could be postulated that an antioxidative, antinitrosive and antihypertensive activities could be mediated through the L arginine/NO pathway which could augment the antioxidant/anti-inflammatory efficiency of the fruit [86]. A. digitata fruit is also rich in lysine and proline which mediate endothelial repair in the presence of vitamin C. Lysine and proline hydroxylation induce collagen and elastin cross talks that maintains the vascular bed integrity [82]. According to McRae M P et al. (2008) that supplementation with 500 mg vitamin C significantly reduce TC, LDL-C and TGs in humans but its effects on HDL-C is not significant(82). Our results showed that A. digitata fruit mediated a significant increase in HDL-C which may indicate a synergistic activity to vitamin C. A highly 8

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Scheme 1. Schematic proposed pathways of activity of Adansonia digitata fruit on HS/HFD-simulated Metabolic Syndrome model in male Wistar rats. (TC: total cholesterol, TGs: triglycerides, LDL-C: low density lipoprotein cholesterol, HDL-C: high density lipoprotein Cholesterol, AIP: Atherogenic Index of plasma, ALT: alanine transaminase, AST: aspartate aminotransferase, ALP: alkaline phosphatase, ALB: albumin, TP: total protein, BIL: bilirubin).

significant reduction in the Atherogenic Index of plasma - a clinical predictor for assessing cardiovascular risk- was depicted which indicates cardioprotective activities. The profound drop of ∼ 60–64% in PPPG to values below baseline readings could be attributed to A. digitata fruit potent antioxidant activity. It has been reported that inhibiting superoxide and reactive radicals’ could block three pathways that induce hyperglycaemia [87]. Significant reductions in glycaemic response, starch digestion, and a hypoglycaemic effects on alloxan-induced diabetes has been reported [88–90]. Normalization of the hepatic biomarkers, creatinine and urea could be interpreted as a stabilizing activity on the leaking/disturbed membrane by the antioxidant activity of A. digitata fruit. According to Weitz 2010; that supplementation with omega 3-fatty acids showed significant reduction in liver steatosis and insulin resistance [91]. A line of evidence has shown the hepatoprotective activity of A. digitata fruit against acetaminophen and carbon tetrachloride poisoning [60,59). Simvastatin mediated no or insignificant effects on the elevated hepatic biomarkers nor in creatinine and urea. The dissimilar range of activities of A. digitata from Simvastatin may indicate a distinct mechanism of action from statins. With the recent prebiotic definition, it could be relevant to postulate that A. digitata fruit as a nutraceutical may confer additional activities as a prebiotic. The cross talks between the gut-microbiota and A. digitata fruit may induce changes in the gut microflra that consequently boost the immune system and confer host wellness. This may explain the traditional use of A. digitata fruit as a cure for dysentery diarrhoea and ulcer.

biological targets. The potent antioxidant/anti-inflammatory activities may be the main mechanism that drives the synergistic and interrelated therapeutic and protective benefits. Adansonia digitat fruit, a low cost nutraceutical with a wide therapeutic index, diverse and multi-target active constituents, could be proposed for the management of MetS and other complex conditions associated with oxidative stress. Further research is highly recommended and clinical trials are essential (Scheme 1).

6. Conclusion

8. Nutritional recommendations

MetS was successfully simulated by the HS/HFD formula in this study. A. digitata fruit pulp showed anti-inflammatory, antilipidemic and hypoglycaemic activities which in turn was reflected into reduced AIP (Athrogenic Index of Plasma). In addition, it normalized hepatic/ kidney biomarkers, and mediated a restorative activity on the HS/HFDinduced pathological damaged in heart, liver and kidney tissues. It is relevant to link the rich A. digitata fruit to its diverse/broad spectrum of pharmacological/therapeutic and protective activities which could be mediated synergistically by multi-active compounds through multi-

Adansonia digitata (Baobab) is an organic fruit, well known through Africa for its nutritional and medicinal values. It had been used without limitations as juice, cakes and mixed with other grains for bread [92,45). Adansoina digitata fruit - a nutraceutical of low cost- has a wide therapeutic window (LD50 = 8000 mg/kg) [49] and no or insignificant side effects. A study group at 2017; reported that 15 g of Baobab fruit powder added to breakfast of healthy volunteers has significant effects on satiety and hunger reduction which may predict a potential for weight reduction [52] Reported by Chadare et al., (2009); that 40 g of baobab

7. Limitations to the study Main limitations of the study are:

• Only male rats were included in the study as per reference of the original method • Although high blood pressure is a major diagnostic criterion for • • •

9

MetS but was not included in our study due to shortage of BPmeasuring appliance availability. The following points although relevant to the subject, has not been addressed in the current study : Assessments of A. digitata fruit effects on middle molecules, metabolites and signalling pathways within MetS settings The potential effects of A. digitata fruit as a prebiotic on the microbiome especially in relation to the traditional use as a cure for dysentery, diarrhoea and ulcer

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pulp powder provides 100 % of the recommended daily intake of vitamin C for pregnant women of age 19 years old [93]. To extrapolate the dose mg/kg body weight in rats to humans, the human equivalent dose (HED) mg/kg body weight calculated as -No Observed Adverse Effect level- (NOAEL) was used [94]. According to the dose by factor method, a conversion factor for body surface area is stated to be = 0.67 [95]. Almost similar results were obtained with 400 and 800 mg/kg/day. Hence 400 mg/kg is considered a sub-maximal dose. The NOAEL was proposed to be 400 mg/kg/day. For an average rat weight of 178 g, the equation for HED mg/kg = [Animal NOAEL mg/kg X(weight of animal in kg/weight of human in kg)(1−0.67)]/10.(95) The starting dose for A. digitata in human by applying the above formula =

[13] [14]

[15] [16] [17] [18] [19]

HED mg//kg = [400 X (0.178/60)0.33]/10 = 5.86 mg/kg/day For A 60 kg Human, initial dose is (5.86 × 60) = 351.6 mg/day of pure powder. This dose should be adjusted through clinical trials according to pharmacokinetics/dynamics studies in humans.

[20]

Author contributions

[22]

[21]

[23]

H M S: contributed to conceptualization, literature review, experimental work, data analysis, interpretation, and writing the original manuscript. B O: and H K: contributed to conceptualization, proofread and critical evaluation. A M S: contributed to pathological interpretation and critical evaluation. I H A: contributed to data analysis and proofread.

[24] [25]

[26]

Funding

[27]

This research did not receive any grant from funding agencies in the public, commercial, or not-for-profit sectors.

[28]

Declaration of Competing Interest

[29]

The authors declare no conflict of interest. A signed Conflicts of Interest Statement Form is enclosed as a separate document.

[30]

References

[31]

[1] K.G.M.M. Alberti, R.H. Eckel, S.M. Grundy, P.Z. Zimmet, J.I. Cleeman, K.A. Donato, et al., Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity, Circulation 120 (16) (2009) 1640–1645 Oct 20. [2] S.K. Wong, K.-Y. Chin, F.H. Suhaimi, A. Fairus, S. Ima-Nirwana, Animal models of metabolic syndrome: a review, Nutr. Metab. (Lond) (13) (2016) 65 Oct 4. [3] I.C. Amihăesei, L. Chelaru, Metabolic syndrome a widespread threatening condition; risk factors, diagnostic criteria, therapeutic options, prevention and controversies: an overview, Rev. Med. Soc. Med. Nat. Iasi 118 (December (4)) (2014) 896–900. [4] E. Vanni, E. Bugianesi, A. Kotronen, S. De Minicis, H. Yki-Järvinen, G. SvegliatiBaroni, From the metabolic syndrome to NAFLD or vice versa? Dig. Liver Dis. 42 (May (5)) (2010) 320–330. [5] K. Esposito, P. Chiodini, A. Colao, A. Lenzi, D. Giugliano, Metabolic syndrome and risk of cancer, Diabetes Care 35 (November (11)) (2012) 2402–2411. [6] V.D. Raikou, S. Gavriil, Metabolic syndrome and chronic renal disease, Diseases [Internet] 6 (1) (2018) Jan 24 [cited 2020 Jan 11]; Available from: https://www. ncbi.nlm.nih.gov/pmc/articles/PMC5871958/. [7] Metabolic Syndrome and the Skin: A More Than Superficial Association. Reviewing the Association Between Skin Diseases and Metabolic Syndrome and a Clinical Decision Algorithm for High Risk Patients, (2020) [Internet]. [cited 2020 Jan 7]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5822485/. [8] P.L. Huang, A comprehensive definition for metabolic syndrome, Dis. Model. Mech. 2 (5–6) (2009) 231–237. [9] A. Cozma, O. Orăşan, D. Sâmpelean, A. Fodor, C. Vlad, V. Negrean, et al., Endothelial dysfunction in metabolic syndrome, Rom. J. Intern. Med. 47 (2) (2009) 133–140. [10] V. Kain, G.V. Halade, Metabolic and biochemical stressors in diabetic cardiomyopathy, Front Cardiovasc Med [Internet] 4 (2017) May 31 [cited 2017 Sep 23]; Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449449/. [11] S. PENDYALA, J.M. WALKER, P.R. HOLT, A high-fat diet is associated with endotoxemia that originates from the gut, Gastroenterology 142 (May (5)) (2012) 1100–1101.e2. [12] T. Nishikawa, D. Edelstein, X.L. Du, S. Yamagishi, T. Matsumura, Y. Kaneda, et al.,

[32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43]

10

Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage, Nature 404 (6779) (2000) 787–790. Apr 13. O. Ilkun, S. Boudina, Cardiac dysfunction and oxidative stress in the metabolic syndrome: an update on antioxidant therapies, Curr. Pharm. Des. 19 (27) (2013) 4806–4817. A. Ayala, M.F. Muñoz, S. Argüelles, Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-Hydroxy-2-Nonenal, Oxid Med Cell Longev [Internet] (2014) [cited 2017 Aug 20];2014. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066722/. A. Lerman, J.C. Burnett, Intact and altered endothelium in regulation of vasomotion, Circulation. 86 (December (6)) (1992) III12–19. P.A. Kakimoto, A.J. Kowaltowski, Effects of high fat diets on rodent liver bioenergetics and oxidative imbalance, Redox Biol. 8 (2016) 216–225 Jan 14. A. Lonardo, F. Nascimbeni, M. Maurantonio, A. Marrazzo, L. Rinaldi, L.E. Adinolfi, Nonalcoholic fatty liver disease: evolving paradigms, World J. Gastroenterol. 23 (36) (2017) 6571–6592 Sep 28. A.J. Sanyal, C. Campbell-Sargent, F. Mirshahi, W.B. Rizzo, M.J. Contos, R.K. Sterling, et al., Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities, Gastroenterology. 120 (April (5)) (2001) 1183–1192. P. Valva, D.A. Ríos, E. De Matteo, M.V. Preciado, Chronic hepatitis C virus infection: serum biomarkers in predicting liver damage, World J. Gastroenterol. 22 (4) (2016) 1367–1381 Jan 28. J. Chen, P. Muntner, L.L. Hamm, D.W. Jones, V. Batuman, V. Fonseca, et al., The metabolic syndrome and chronic kidney disease in U.S. Adults, Ann. Intern. Med. 140 (3) (2004) 167 Feb 3. S.Y. Kim, A.Y. Lim, S.K. Jeon, I.S. Lee, R. Choue, Effects of dietary protein and fat contents on renal function and inflammatory cytokines in rats with adriamycininduced nephrotic syndrome [Internet], Mediators Inflamm. (2011) [cited 2017 Aug 29]. Available from: https://www.hindawi.com/journals/mi/2011/945123/. C. Brenner, L. Galluzzi, O. Kepp, G. Kroemer, Decoding cell death signals in liver inflammation, J. Hepatol. 59 (3) (2013) 583–594 Sep 1. J. Traynor, R. Mactier, C.C. Geddes, J.G. Fox, How to measure renal function in clinical practice, BMJ. 333 (7571) (2006) 733–737 Oct 7. C. Chen, J.-M. Lü, Q. Yao, Hyperuricemia-related diseases and Xanthine Oxidoreductase (XOR) Inhibitors: an overview, Med. Sci. Monit. 17 (July (22)) (2016) 2501–2512. V.V. Konopelniuk, I.I. Goloborodko, T.V. Ishchuk, T.B. Synelnyk, L.I. Ostapchenko, M.Y. Spivak, et al., Efficacy of Fenugreek-based bionanocomposite on renal dysfunction and endogenous intoxication in high-calorie diet-induced obesity rat model-comparative study, EPMA J. 8 (December (4)) (2017) 377–390. J.A. Talwalkar, The concept of risk stratification, in: R. de Franchis (Ed.), Portal Hypertension VI, Springer International Publishing, Cham, 2016, pp. 9–18. M. Laguerre, J. Lecomte, P. Villeneuve, Evaluation of the ability of antioxidants to counteract lipid oxidation: existing methods, new trends and challenges, Prog. Lipid Res. 46 (September (5)) (2007) 244–282. K. Kim, J.S. Park, Impact of fish consumption by subjects with prediabetes on the metabolic risk factors: using data in the 2015 (6th) Korea National Health and Nutrition Examination Surveys, Nutr. Res. Pract. 12 (June (3)) (2018) 233–242. H. Poudyal, F. Campbell, L. Brown, Olive leaf extract attenuates cardiac, hepatic, and metabolic changes in high carbohydrate-, high fat-fed rats, J. Nutr. 140 (May (5)) (2010) 946–953. R.K. Singh, H.-W. Chang, D. Yan, K.M. Lee, D. Ucmak, K. Wong, et al., Influence of diet on the gut microbiome and implications for human health, J Transl Med [Internet] 15 (April (8)) (2017) [cited 2020 Jan 8]; Available from: https://www. ncbi.nlm.nih.gov/pmc/articles/PMC5385025/. G.R. Gibson, R. Hutkins, M.E. Sanders, S.L. Prescott, R.A. Reimer, S.J. Salminen, et al., Expert consensus document: the International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics, Nat. Rev. Gastroenterol. Hepatol. 14 (August (8)) (2017) 491–502. R. Bubnov, L. Babenko, L. Lazarenko, M. Kryvtsova, O. Shcherbakov, N. Zholobak, et al., Can tailored nanoceria act as a prebiotic? Report on improved lipid profile and gut microbiota in obese mice, EPMA J. 10 (4) (2019) 317–335 Dec 1. R.L. Hughes, M.L. Marco, J.P. Hughes, N.L. Keim, M.E. Kable, The role of the gut microbiome in predicting response to diet and the development of precision nutrition models-part I: overview of current methods, Adv. Nutr. 10 (6) (2019) 953–978 Nov 1. X. Zhang, L.O. Lerman, Investigating the metabolic syndrome: contributions of swine models, Toxicol. Pathol. 44 (April (3)) (2016) 358–366. A. Aleixandre de Artiñano, M. Miguel Castro, Experimental rat models to study the metabolic syndrome, Br. J. Nutr. 102 (9) (2009) 1246–1253 Nov 14. S. Renner, B. Dobenecker, A. Blutke, S. Zöls, R. Wanke, M. Ritzmann, et al., Comparative aspects of rodent and nonrodent animal models for mechanistic and translational diabetes research, Theriogenology. 86 (1) (2016) 406–421 Jul 1. M. Wu, C. Chan, Human metabolic network: reconstruction, simulation, and applications in systems biology, Metabolites 2 (1) (2012) 242–253 Mar 2. C. Angione, Human systems biology and metabolic modelling: a review-from disease metabolism to precision medicine, Biomed Res. Int. 2019 (2019) 8304260. Y.J.W. Rozendaal, Y. Wang, Y. Paalvast, L.L. Tambyrajah, Z. Li, K. Willems van Dijk, et al., In vivo and in silico dynamics of the development of Metabolic Syndrome, PLoS Comput. Biol. 14 (6) (2018) e1006145. P. Pyakurel, P. Karki, M. Lamsal, A. Ghimire, P.K. Pokharel, Cardiovascular risk factors among industrial workers: a cross-sectional study from eastern Nepal, J. Occup. Med. Toxicol. 11 (2016) 25. J. Kishore, N. Gupta, C. Kohli, N. Kumar, Prevalence of hypertension and determination of its risk factors in Rural Delhi [Internet], Int. J. Hypertens. (2016) [cited 2018 Nov 3]. Available from: https://www.hindawi.com/journals/ijhy/2016/7962595/. WHO | Diabetes [Internet]. WHO. [cited 2018 Mar 3]. Available from: http://www. who.int/mediacentre/factsheets/fs312/en/. A. Chaudhury, C. Duvoor, V.S. Reddy Dendi, S. Kraleti, A. Chada, R. Ravilla, et al., Clinical review of antidiabetic drugs: implications for type 2 diabetes mellitus management, Front Endocrinol (Lausanne) [Internet] 8 (January (24)) (2017)

Biomedicine & Pharmacotherapy 125 (2020) 109968

H.M. Suliman, et al.

[44] [45] [46] [47] [48] [49] [50] [51]

[52] [53] [54]

[55] [56] [57] [58] [59] [60]

[61] [62] [63] [64] [65] [66]

[67] [68]

[69] [70]

[cited 2018 Mar 3]; Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC5256065/. E. Besco, E. Braccioli, S. Vertuani, P. Ziosi, F. Brazzo, R. Bruni, et al., The use of photochemiluminescence for the measurement of the integral antioxidant capacity of baobab products, ResearchGate. 102 (4) (2007) 1352–1356 Jan 1. J. Rahul, M.K. Jain, S.P. Singh, R.K. Kamal, N.A. Anuradha, et al., Adansonia digitata L. (baobab): a review of traditional information and taxonomic description, Asian Pac. J. Trop. Biomed. 5 (January (1)) (2015) 79–84. G.P.P. Kamatou, I. Vermaak, A.M. Viljoen, An updated review of Adansonia digitata: a commercially important African tree, South Afr. J. Bot. 77 (October (4)) (2011) 908–919. A.E. Aluko, J. Kinyuru, L.M. Chove, P. Kahenya, W. Owino, Nutritional quality and functional properties of baobab (Adansonia digitata) pulp from Tanzania, J. Food Res. 5 (5) (2016) 23 Sep 26. D. Kabore, H. Sawadogo-Lingani, B. Diawara, C.S. Compaore, M.H. Dicko, M. Jakobsen, A review of baobab (Adansonia digitata) products: effect of processing techniques, medicinal properties and uses, AJFS 5 (16) (2011) 833–844 Dec 23. A. Ramadan, F.M. Harraz, S.A. El-Mougy, Anti-inflammatory, analgesic and antipyretic effects of the fruit pulp of Adansonia digitata, ResearchGate. 65 (5) (1994) 418–422 Jan 1. A.J. Alhassan, M. Ibrahim, I.K. Jarumi, A. Wudil, Evaluation of anti-hyperlipidemic potentials of aqueous fruit pulp extract of Adensonia digitata in experimental rats, Eur. Sci. J. (12) (2016) 1857–7881 May 1. Ja&# 39, Mohammed afaru S. Lipid Profile of Alloxan-induced Diabetic Wistar Rats Treated with Methanolic Extract of Adansonia digitata Fruit Pulp. [cited 2018 Nov 5]; Available from: http://www.academia.edu/15890494/Lipid_Profile_of_Alloxaninduced_Diabetic_Wistar_Rats_Treated_with_Methanolic_Extract_of_Adansonia_ digitata_Fruit_Pulp. R. Garvey, M. Clegg, S. Coe, The acute effects of baobab fruit (Adansonia digitata) on satiety in healthy adults, Nutr. Health 23 (2) (2017) 83–86 Jun 1. wjpps | ABSTRACT [Internet]. [cited 2019 Jul 10]. Available from: http://www. wjpps.com/wjpps_controller/abstract_id/7094. J.A. Seukep, A.G. Fankam, D.E. Djeussi, I.K. Voukeng, S.B. Tankeo, J.A. Noumdem, et al., Antibacterial activities of the methanol extracts of seven Cameroonian dietary plants against bacteria expressing MDR phenotypes, Springerplus [Internet] 2 (July (31)) (2013) [cited 2017 Jan 3]; Available from: http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC3738912/. K. Anani, J.B. Hudson, C. de Souza, K. Akpagana, G.H. Tower, J.T. Arnason, et al., Investigation of medicinal plants of togo for antiviral and antimicrobial activities, Pharm. Biol. 38 (1) (2000) 40–45. M. Mwale, J.F. Mupangwa, C. Mapiye, S. Happyson, J. Chimvuramahwe, Growth Performance of Guinea Fowl Keets Fed Graded Levels of Baobab Seed Cake Diets vol. 7, (2008). E. Arama, P. Michaud, R. Rouffiac, F. Rodriguez, Bioavailability of prolonged-liberation tablets of theophylline and paracetamol formulated in pulp from the fruit of baobab (Adansonia digitata L.), Pharm. Acta Helv. 64 (4) (1989) 116–120. M.A.M. Ghoneim, A.I. Hassan, M.G. Mahmoud, M.S. Asker, Protective effect of Adansonia digitata against isoproterenol-induced myocardial injury in rats, ResearchGate. 27 (2) (2016) 84–95 Feb 25. A.A. Al-Qarawi, M.A. Al-Damegh, S.A. El-Mougy, Hepatoprotective influence of Adansonia digitata pulp, J. Herbs, Spices Med.l Plants [Internet] (2008), https:// doi.org/10.1300/J044v10n03_01 Sep 22 [cited 2017 Jan 3]; Available from:. A. Hanafy, H.M. Aldawsari, J.M. Badr, A.K. Ibrahim, S.E.-S. Abdel-Hady, Evaluation of hepatoprotective activity of Adansonia digitata extract on acetaminophen-induced hepatotoxicity in rats, Evid Based Complement Alternat Med [Internet] (2016) [cited 2017 Aug 28];2016. Available from: http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC4812277/. M. Awad Mohamed, I. Tajeldin, A.-E. Hassan Mohammed, H. Hassan, hepatoprotective effect of Adansonia digitata L. (BAOBAB) fruits pulp extract on CCL4INDUCED hepatotoxicity in rats, World J. Pharm. Res. (4) (2015) 368–377. Aug 1. M.S. Sikarwar, M.B. Patil, Antihyperlipidemic activity of Salacia chinensis root extracts in triton-induced and atherogenic diet-induced hyperlipidemic rats, Indian J. Pharmacol. 44 (1) (2012) 88–92. L. Stevenson, F. Phillips, K. O’sullivan, J. Walton, Wheat bran: its composition and benefits to health, a European perspective, Int. J. Food Sci. Nutr. 63 (December (8)) (2012) 1001–1013. S. Parasuraman, R. Raveendran, R. Kesavan, Blood sample collection in small laboratory animals, J. Pharmacol. Pharmacother. 1 (2) (2010) 87–93. S. Kannan, S. Mahadevan, B. Ramji, M. Jayapaul, V. Kumaravel, LDL-cholesterol: Friedewald calculated versus direct measurement-study from a large Indian laboratory database, Indian J. Endocrinol. Metab. 18 (4) (2014) 502–504. Bancroft’s Theory and Practice of Histological Techniques E-Book - 7th Edition [Internet]. [cited 2018 Jun 21]. Available from: https://www.elsevier.com/books/ bancrofts-theory-and-practice-of-histological-techniques-e-book/suvarna/978-07020-5032-9. M. Dobiásová, [AIP–atherogenic index of plasma as a significant predictor of cardiovascular risk: from research to practice], Vnitr. Lek. 52 (January (1)) (2006) 64–71. M. Dobiásová, J. Frohlich, The plasma parameter log (TG/HDL-C) as an atherogenic index: correlation with lipoprotein particle size and esterification rate in apoB-lipoprotein-depleted plasma (FER(HDL)), Clin. Biochem. 34 (October (7)) (2001) 583–588. P.C. Deedwania, R. Gupta, Management issues in the metabolic syndrome, J. Assoc. Physicians India 54 (2006) 797–810 Oct. K.C. Ferdinand, Management of cardiovascular risk in patients with type 2 diabetes mellitus as a component of the cardiometabolic syndrome, J. Cardiometab. Syndr. 1

(2) (2006) 133–140. [71] S.A. Sheriff, T. Devaki, Lycopene stabilizes liver function during d-galactosamine/ lipopolysaccharide induced hepatitis in rats, J. Taibah Univ. Sci. 7 (1) (2013) 8–16 Jan 1. [72] D.G. Levitt, M.D. Levitt, Human serum albumin homeostasis: a new look at the roles of synthesis, catabolism, renal and gastrointestinal excretion, and the clinical value of serum albumin measurements, Int. J. Gen. Med. (9) (2016) 229–255 Jul 15. [73] Y.-E. Cho, E.-J. Im, P.-G. Moon, E. Mezey, B.-J. Song, M.-C. Baek, Increased liverspecific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury, PLoS One [Internet]. 12 (2) (2017) Feb 22 [cited 2017 Aug 28]; Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/ PMC5321292/. [74] H. Roelofsen, C.N. Van Der Veere, R. Ottenhoff, B. Schoemaker, L.M. Jansen peter, R.P.J.O. Elferink, Decreased bilirubin transport in the perfused liver of endotoxemic rats, Gastroenterology. 107 (4) (1994) 1075–1084 Oct 1. [75] J.M. Saland, C.B. Pierce, M.M. Mitsnefes, J.T. Flynn, J. Goebel, J.C. Kupferman, et al., Dyslipidemia in children with chronic kidney disease: a report of the chronic kidney disease in children (CKiD) study, Kidney Int. 78 (December (11)) (2010) 1154–1163. [76] M. Kawabe, A. Sato, T. Hoshi, S. Sakai, D. Hiraya, H. Watabe, et al., Impact of blood urea nitrogen for long-term risk stratification in patients with coronary artery disease undergoing percutaneous coronary intervention, IJC Heart Vessel. (4) (2014) 116–121 Sep 1. [77] J.-H. Kim, H.-J. Jang, W.-Y. Cho, S.-J. Yeon, C.-H. Lee, In vitro antioxidant actions of sulfur-containing amino acids, Arabian Journal of Chemistry [Internet] (2018) Jan 9 [cited 2018 May 25]; Available from: http://www.sciencedirect.com/ science/article/pii/S1878535218300030. [78] M.G. Picchi, A.M. de Mattos, M.R. Barbosa, C.P. Duarte, M.A. Gandini, G.V. Portari, et al., A high-fat diet as a model of fatty liver disease in rats, Acta Cir. Bras. 26 (2011) 25–30. [79] D. Chess, B. Lei, B. Hoit, A.M. Azimzadeh, W. Stanley, Effects of a high saturated fat diet on Cardiac hypertrophy and dysfunction in response to pressure overload, J. Card. Fail. 14 (February (1)) (2008) 82–88. [80] D. Kaboré, H. Sawadogo-Lingani, B. Diawara, C. Compaoré, M. Dicko, M. Jakobsen, A review of baobab (Adansonia digitata) products: effect of processing techniques, medicinal properties and uses, Afr. J. Food Sci. 5 (January (1)) (2011). [81] F.A. Santos, J.T. Frota, B.R. Arruda, T.S. de Melo, A.Ade C.A. da Silva, G.Ade C. Brito, et al., Antihyperglycemic and hypolipidemic effects of α, β-amyrin, a triterpenoid mixture from Protium heptaphyllum in mice, Lipids Health Dis. 6 (August (11)) (2012) 98. [82] M.P. McRae, Vitamin C supplementation lowers serum low-density lipoprotein cholesterol and triglycerides: a meta-analysis of 13 randomized controlled trials, J. Chiropr. Med. 7 (June (2)) (2008) 48–58. [83] F.A. Oliveira, M.H. Chaves, F.R.C. Almeida, R.C.P. Lima, R.M. Silva, J.L. Maia, et al., Protective effect of alpha- and beta-amyrin, a triterpene mixture from Protium heptaphyllum (Aubl.) March. Trunk wood resin, against acetaminopheninduced liver injury in mice, J. Ethnopharmacol. 98 (1–2) (2005) 103–108. Apr 8. [84] J.C. Aledo, F.R. Cantón, F.J. Veredas, A machine learning approach for predicting methionine oxidation sites, BMC Bioinformatics [Internet] 18 (September (29)) (2017) [cited 2018 Mar 1]; Available from: https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC5622526/. [85] P. Manna, J. Das, P.C. Sil, Role of sulfur containing amino acids as an adjuvant therapy in the prevention of diabetes and its associated complications, Curr. Diabetes Rev. 9 (May (3)) (2013) 237–248. [86] P. Tripathi, S. Pandey, L-arginine attenuates oxidative stress condition during cardiomyopathy, Indian J. Biochem. Biophys. 50 (April (2)) (2013) 99–104. [87] S. Ibrahim, A. Al-Ahdal, A. Khedr, G. Mohamed, Antioxidant α-amylase inhibitors flavonoids from Iris germanica rhizomes, Rev. Bras. Farmacogn. 27 (2) (2017) 170–174 Mar 1. [88] S.A. Coe, M. Clegg, M. Armengol, L. Ryan, The polyphenol-rich baobab fruit (Adansonia digitata L.) reduces starch digestion and glycemic response in humans, Nutr. Res. 33 (November (11)) (2013) 888–896. [89] M. Gwarzo, H. Yakubu Bako, Hypoglycemic activity of methanolic fruit pulp extract of Adansonia digitata on blood glucose levels of alloxan induced diabetic rats, J. Anim. Vet. Adv. (5) (2013) 108 Jun 20. [90] M. Ibrahim, I. Jarumi, A. Alhassan, A. Wudil, M. Dangambo, Acute toxicity and hypoglycemic activity of aqueous fruit pulp extract of Adansonia digitata L. (Afpead) on alloxan induced diabetic rats, J. Adv. Med. Pharm. Sci. (6) (2016) 1–6 Jan 10. [91] D. Weitz, H. Weintraub, E. Fisher, A.Z. Schwartzbard, Fish oil for the treatment of cardiovascular disease, Cardiol. Rev. 18 (5) (2010) 258–263. [92] I.U. Muhammad, I.K. Jarumi, A.J. Alhassan, A.M. Wudil, M.A. Dangambo, Acute toxicity and hypoglycemic activity of aqueous fruit pulp extract of Adansonia digitata L. (Afpead) on alloxan induced diabetic rats, J. Adv. Med. Pharm. Sci. 6 (3) (2016) 1–6 Feb 5. [93] F.J. Chadare, A.R. Linnemann, J.D. Hounhouigan, M.J.R. Nout, M.A.J.S. Van Boekel, Baobab food products: a review on their composition and nutritional value, Crit. Rev. Food Sci. Nutr. 49 (March (3)) (2009) 254–274. [94] Guidance for Industry on Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers; Availability [Internet], Federal Register, 2005 [cited 2018 May 31]. Available from: https://www. federalregister.gov/documents/2005/07/22/05-14456/guidance-for-industry-onestimating-the-maximum-safe-starting-dose-in-initial-clinical-trials-for. [95] A.B. Nair, S. Jacob, A simple practice guide for dose conversion between animals and human, J. Basic Clin. Pharm. 7 (March (2)) (2016) 27–31.

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