Antidiabetic and Liver Histological and Ultrastructural Effects of Cynara scolymus Leaf and Flower Head Hydroethanolic Extracts in Nicotinamide/Streptozotocin-Induced Diabetic Rats

This study aims to investigate the effect of hydroethanolic extracts of Cynara scolymus (C. scolymus) leaf (CLHE) and C. scolymus flower (CFHE) on the hepatic histopathological lesions and functional biochemical changes induced by type 2 diabetes mellitus (T2DM). The rat model of T2DM was induced by intraperitoneal injection of streptozotocin (STZ) in a dose of 60 mg/kg for 15 minutes following nicotinamide (NA) (60 mg/kg). The rats were allocated into four groups: group 1 (negative control), group 2 (diabetic control), group 3 (diabetic rats supplemented with 100 mg/kg/day CLHE), and group 4 (diabetic rats supplemented with 100 mg/kg/day CFHE). Treatment with CLHE and CFHE, for the study duration of 28 days, significantly improved the deteriorated hepatic glycogen content, glycogen phosphorylase, glucose-6-phosphatase activities, serum fructosamine levels, lipid profile, aspartate transaminase activities, and alanine transaminase activities as well as serum insulin and C-peptide levels. The elevated liver lipid peroxidation and the decreased activities of superoxide dismutase and glutathione peroxidase were significantly alleviated. The elevated expression of the proinflammatory cytokine tumor necrosis factor-α in the liver of diabetic rats was significantly reduced by treatments with CLHE and CFHE. NA/STZ-induced T2DM exhibited hepatic histopathological changes in the form of disordered hepatocytes, cytoplasm dissolution, and mononuclear leukocytic infiltration. The electron microscopic ultrastructure study revealed damaged mitochondria with ill-defined cristae and fragmentation of the rough endoplasmic reticulum. Treatments with CLHE and CFHE remarkably amended these histopathological and EM ultrastructural changes. In conclusion, both CLHE and CFHE may have antidiabetic and improvement effects on the liver function and structural integrity, which may be mediated, at least in part, via suppression of inflammation and oxidative stress and enhancement of the antioxidant defence system.


Introduction
Diabetes mellitus (DM) is a chronic major disorder of carbohydrate metabolism due to impaired insulin secretion and/or insulin resistance; however, lipid and protein metabolisms are also defective [1][2][3]. It is a complex metabolic disease with increasing incidence in the adult population; the number is predicted to increase to 643 million by 2030 and 783 million by 2045 [4]. Tereby, it is one of the principal public health problems of the 21 st century that threatens populations worldwide. It was classifed into four types, with type 2 DM (T2DM) being the most common, accounting for approximately 95% of all types of DM [5,6]. Possible mechanisms of metabolic stress-induced β-cell apoptosis in T2DM include endoplasmic reticulum (ER) stress [7], oxidative stress [2,8], and ceramide production [9,10], and these culminate in the stimulation of the intrinsic or mitochondrial apoptotic pathway [11,12].
Dyslipidemia consistent with insulin resistance (IR) and T2DM is featured by elevated triacylglycerols (TAGs) and decreased high-density lipoprotein-cholesterol (HDL-C) levels [2], which are, in addition to low-density lipoproteincholesterol (LDL-C) high levels, risk factors for coronary heart disease [13]. Free fatty acids (FFAs), diacylglycerol (DAGs), and TAGs that accumulate intracellularly are the lipids responsible for the pathophysiology of IR [14,15]. Individuals with T2DM have a high incidence of liver function test abnormalities, with 23-57% prevalence [16]. Serum or plasma aminotransferases, viz aspartate transaminase (AST) and alanine transaminase (ALT), measure the intracellular hepatic enzymes leaked into the blood and serve as markers for liver injury [17].
Plant extracts and constituents with antidiabetic potentials are yet to be formulated commercially as modern medicines, although they have been attracting public approval and praise for their therapeutic activities in traditional medicines [2,3,15]. Artichoke or Cynara scolymus (C. scolymus) L., belonging to the family Asteraceae, is an ancient medicinal plant whose therapeutic potential was well-identifed by the Romans, ancient Egyptians, and Greeks [18]. Te genus Cynara (C.) originates from the Mediterranean regions and includes 2 varieties, globe artichoke (C. cardunculus var. Scolymus L.) and cardoon (C. cardunculus var. altilis DC), as well as their ancestors, wild cardoon (C. cardunculus L. var. sylvestris (Lamk) Fiori) [19]. Our previous publication reported that artichoke leaf and fower extract have antihyperglycemic actions in type 2 diabetic rats [20]. Artichoke leaf extracts have traditionally been used to treat dyspeptic symptoms, increase bile fow, and exert hepatoprotective, lipid-lowering, antioxidant, and antispasmodic efects [21].
In conductance with the previous literature, the present study was designed to assess the efects of C. scolymus leaf hydroethanolic extract (CLHE) and C. scolymus fower hydroethanolic extract (CFHE) on glycemic state, lipid profle, liver function, and histological integrity in nicotinamide (NA)/streptozotocin (STZ)-induced diabetic Wistar rats.

Chemicals. NA and STZ were obtained from Sigma
Chemical Company (USA). Proteinase K was obtained from Bioline USA Inc., Taunton, MA, USA. All other chemicals used in this investigation were ultrapure and of an analytical grade.

Plant Extraction.
Artichoke or Cynara scolymus L. was collected from Beheira (Egypt), washed, and air-dried at room temperature for three weeks. Te plant was authenticated by Dr. Mohamed A. Abdallah Fadl, Associate Professor of Taxonomy, Botany Department, Faculty of Science, Beni-Suef University, Egypt. Plant samples were deposited in the Herbarium of Botany Department, Faculty of Science, Beni-Suef University, Egypt. By using, the National Center for Biotechnology Information (NCBI) taxonomy database, Taxonomy ID: 59895 (NCBI: txid59895) (https://www.ncbi. nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=59895), an electrical grinder later powdered the dried leaves and fower heads. Te powder was soaked in 70% ethyl alcohol separately for 72 hours at room temperature. Te mixtures were fltered via Whatman's flter papers and evaporated under reduced pressure using a rotatory evaporator to obtain crude extracts. Te hydroethanolic extracts (CLHE and CFHE) were kept at −20°C until used. Te yield of CLHE and CFHE was, respectively, 0.8% and 2% of the dry weight.

High-Performance Liquid Chromatography (HPLC)-Mass
Spectrometry (MS) Analysis. HPLC-MS analysis of the hydroethanolic extract of navel orange peels was performed in the Central Laboratory of the Faculty of Postgraduate Studies for Advanced Sciences, Beni-Suef University, Egypt, using the HPLC-MS system, Infnity 1260, Agilent Technologies, Germany, coupled with a diode array detector (DAD). Te preparations and analysis were carried out according to the method described by López-Salas et al. [22].

Experimental Animals.
Healthy adult male Wistar rats, having a body weight (BW) of 120-140 g and aged 7-9 weeks, were purchased from Helwan Station of Experimental Animals, Egyptian Holding Company for Biological Products and Vaccines (Helwan, Cairo, Egypt). Fourteen days before the experiment onset, all animals were kept under observation for adaptation and to eliminate any inter changeable diseases. Animals were inhabited in polypropylene animal research cages (3 rats/cage) with wellaerated covers under standard temperature (22°C ± 2°C), humidity (55% ± 5%), and 12-hour alternating light and dark cycles. Te animals were provided access to tap water and a standard pelleted diet ad-libitum. Te ethical standards have been adhered to according to the guidelines for IACUC (Institutional Animal Care and Use Committee), Beni-Suef University, Egypt (Ethical Approval Number: BSU/FS/2015/2). Every efort was made to lessen the number of animals and their sufering. Evidence-Based Complementary and Alternative Medicine 2.5. Induction of T2DM. Te T2DM model was induced in rats, which were fasting for 16 hours, by intraperitoneal (IP) injection of 60 mg NA/kg BW dissolved in 0.9% NaCl solution. Tis was followed 15 minutes later by injecting STZ IP at a dose level of 60 mg/kg BW in citrate bufer at pH 4.5 [2,20]. Ten days postSTZ injections, rats' blood glucose concentrations were screened. Animals that fasted overnight (10-12 hours) were supplied with glucose 3 g/kg BW by oral gavage; then, 2 hours after glucose administration, blood samples were taken from the lateral tail vein, and blood glucose concentrations were measured by a glucometer obtained from Life Scan Corporation, Canada [23]. Rats having blood glucose concentrations >200 mg/dL were assigned as diabetic and selected for further biochemical, physiological, histological, and ultrastructural studies.

Experimental Design.
After the acclimation period and induction of T2DM, rats were divided at random into four groups (n � 6) (Scheme 1).

Biochemical Studies.
Te serum concentration of fructosamine (FA) was estimated based on the procedure of Baker et al. [26]. Hepatic glycogen content was assayed according to Seifter et al. [27]. Based on the techniques developed by Begum et al. [28] and Stalman and Hers [29], the activities of the liver enzymes glucose-6-phosphatase (G6Pase) and glycogen phosphorylase (GP) were measured. Serum AST and ALT activities were measured according to Schumann and Klauke [30] using reagent kits purchased from HUMAN Gesellschaft für Biochemica und Diagnostica mbH, Magdeburg, Germany.

Histological Investigation.
After dissection, livers were rapidly removed and fxed in 10% neutral phosphatebufered formalin for 24 hours. Following a thorough rinse in tap water, the samples were dehydrated using a series of ethyl alcohol dilutions (50%, 70%, 90%, 95%, and 100%) in a furnace set at 56°C for 24 hours and then the samples were cleaned with xylene before being submerged into parafn wax. Sections of 5-μm thickness were made from parafn wax tissue blocks with a sliding microtome. For a standard examination, the tissue sections were mounted on glass slides, dewaxed, and stained with hematoxylin and eosin (H&E) [40]. Te examination was carried out using a light electric microscope.

Ultrastructural Preparations.
Te liver specimens on day 30 were cut into small specimens, about one mm 3 , and were then instantly fxed for 18 to 24 hours at 4°C in fresh 3% glutaraldehyde-formaldehyde. Tereafter, the specimens were processed for preparation and staining of semithin sections and preparation of ultrathin sections that were investigated using a Joel CX 100 transmission electron microscope according to Bozzola and Russell [41] and Ahmed et al. [20].

Immunohistochemistry of Liver.
Immunolocalization for hepatic tumor necrosis factor-α (TNF-α) was applied on sections of 5-μm thickness and stained with the streptavidinbiotin-peroxidase staining technique [20,42,43] using an antisera containing 1 ry antibodies for rat anti-TNF-α (Santa Cruz, CA, USA), biotinylated 2 ry antibodies and streptavidin horseradish peroxidase (Dako-K0690; Dako Universal LSAB Kit), and diaminobenzidine tetrahydrochloride (DAB) substrate kit (Sigma-D5905; Sigma-Aldrich Company Ltd., Gillingham, UK) for 10 min for immunolabelling. All prepared sections were treated under the same conditions with the same antibody concentrations simultaneously, so the immunostaining among the diferent groups was comparable.
2.11. Statistical Analysis. Te mean and standard deviation (SD) were used to express the results. To compare the tested groups, a one-way analysis of variance (ANOVA) with PC-STAT (version 1A (C) copyright 1985, University of Georgia, Georgia, USA) was used, followed by the least signifcant diference (LSD) test [44]. Values of p < 0.05 were statistically signifcant while values of p ≥ 0.05 were not.

Biochemical Efects.
Te serum FA level and hepatic GP and G6Pase activities were remarkably (p < 0.05) elevated, while hepatic glycogen content was statistically markedly (p < 0.05) depleted in the diabetic control in comparison with the negative control rats. CLHE and CFHE therapy of the diabetic rats resulted in a discernible (p < 0.05) improvement in the serum FA level, liver GP, and the G6Pase activities and glycogen content in comparison with the diabetic control. Treatments with CLHE appeared to have a stronger impact on improving the serum FA level, liver glycogen content, and the G6Pase enzyme activity (Table 1).
Serum AST and ALT activities were markedly (p < 0.05) raised in the diabetic control rats in relation to the negative control rats. At the same time, they were remarkably decreased in the diabetic group supplemented with CLHE and CFHE in relation to the diabetic control group (Table 2). While the efect of CLHE on ALT activity was statistically signifcant (p < 0.05), but the efect on AST activity was not (p ≥ 0.05).
Regarding serum lipid profle (Table 3), the rats with diabetes exhibited signifcant elevations (p < 0.05) in TC, LDL-C, VLDL-C, and FFAs levels and signifcant reduction (p < 0.05) in HDL-C levels compared to negative control ones. Te treatment with CLHE and CFHE produced a remarkable amelioration of all these lipid profle variables to various extents; and CFHE appeared to be more efective in decreasing the elevated serum lipids. Table 4 shows the efects of CLHE and CFHE on oxidative stress and antioxidant defence system biomarkers in the liver of the study groups. Liver LPO was remarkably (p < 0.05) upregulated in the diabetic control, while it was decreased (p < 0.05) in diabetic rats supplied with CLHE and CFHE in relation to the diabetic control. Te level of the nonenzymatic antioxidant (GSH) and activity of the antioxidant enzymes including SOD and GPx decreased considerably (p < 0.05) in the liver of the diabetic control. Te supplementation of the diabetic rats with CLHE and CFHE resulted in a detectable increase in the liver GSH content and SOD and GPx activities to various extents. Despite the efect of both CLHE and CFHE on GPx and SOD activities were statistically signifcant, the impact on GSH content was not (p ≥ 0.05). CLHE seemed to be more potent than CFHE in reducing the elevations in the liver LPO.
Te serum insulin and C-peptide levels were statistically signifcantly depleted in diabetic control rats compared with the negative control ones. Te treatment with CLHE and CFHE induced a statistically signifcant increase in the lowered levels, and CLHE was the most potent ( Figure 2).

Histological
Changes. H&E staining results obtained upon histological examination are shown in Figure 3. Te hepatocytes in the negative control group were distributed in an ordered fashion and displayed a typical hepatic architecture, including organized hepatocytic cords, normal hepatocyte morphology, and a portal vein with sinusoidal cords (Figures 3(a) and 3(b)). On the other hand, the most signifcant alterations in the diabetic control rats' livers were disorderly hepatocyte, cytoplasm dissolution, monocellular       Evidence-Based Complementary and Alternative Medicine leukocytic infltration, karyomegaly, hyperchromatic nuclei, nucleus karyolysis, and dilated congested portal vein. Te proliferation of bile ducts and degenerative changes in the wall of some bile ducts were also observed. Dilated hyperemic sinusoids and thickened walls were also seen (Figures 3(c)-3(f )). Treated groups with CLHE and CFHE showed amelioration of hepatocytes, sinusoid, and central vein (Figures 3(g) and 3(h)).

Efect on TNF-α Expression by Immunohistochemical
Investigation. Immunohistochemistry sections of the negative control group showed no reaction for TNF-α ( Figure 4(a)), while the diabetic control group revealed a marked increase in the intensity of TNF-α in the cytoplasm of hepatocytes (Figure 4(b)). Treatment with CLHE and CFHE showed a noticeable reduction in the TNF-α in hepatocyte intensity (Figures 4(c) and 4(d)).

Ultrastructural Efects.
Electron microscopic examination of the negative control group revealed normalappearing hepatocytes with a normal structure of the nucleus, mitochondria, and rough endoplasmic reticulum (RER) (Figures 5(a) and 5(b)). Kupfer cells are shown in Figure 5(c). Hepatocytes of diabetic control rats revealed cytoplasm dissolution, a nucleus with irregular nuclear membrane, dilated intercellular space containing collagen fbre bundle, damaged mitochondria with ill-defned cristae, and fragmentation of RER (Figures 6(a)-6(c)). Te nucleus of the Kupfer cells appeared with heterochromatin clumps adjacent to the irregular nuclear membrane (Figure 6(d)). Hepatocytes of diabetic rats treated with CLHE showed an intact nucleus and regular Kupfer cells (Figures 7(a) and  7(b)). Diabetic rats treated with CFHE showed normal nuclei with their Kupfer cells preserved (Figures 7(c) and 7(d)).

Discussion
DM includes a group of chronic metabolic derangements featured by hyperglycemia due to absolute or relative insulin defciency and/or IR that results from insulin receptor or postreceptor defects. It afects metabolisms of carbohydrates, proteins, and fats as well as has deteriorative efects on hepatic, renal, and pancreatic islet beta cells [45]. Glucotoxicity, lipotoxicity, and oxidative stress are some of the mechanisms that contribute to the excessive formation of Evidence-Based Complementary and Alternative Medicine reactive oxygen species and reactive nitrogen species in this situation [2]. In the presented study, a signifcant rise in the serum FA levels in rats with DM was recorded. Te serum level of FA is a good and accurate biomarker of the glycemic state since it measures the average blood glucose level over 2-3 weeks. Te supplementation of diabetic rats with CLHE and CFHE showed a signifcant improvement in the FA level which is consistent with the results of Alves et al. [46]. Te improvement in serum fructosamine levels, as a result of the treatment of diabetic rats with CLHE and CFHE, was associated with the increase in serum insulin and C-peptide levels. Tus, the improvement in the glycemic state by treatment may be attributed to the insulinogenic and insulinotropic efects of CLHE and CFHE. Tis attribution was supported by previous publications [47,48]. Te protective actions of artichoke extract on the pancreatic β-cells and enhancement of their regeneration, in addition to its stimulatory efects on insulin release from cells that survived damage by the diabetes-inducing agent, may cause of the rise in serum insulin and C-peptide levels [48].
Our results showed a signifcant drop in the liver glycogen content and a profound elevation of the liver GP and G6Pase activities in the diabetic control rats. Tese results agreed with Ahmed et al. [49] and Ahmed [50], who reported a marked depletion of the liver glycogen content and elevation of the GP and G6Pase activities in NA/STZ-induced type 2 diabetic rats. Te increase in GP activity associated with glycogen depletion refects the increased glycogenolysis, which contributes to the increase in hepatic glucose output in T2DM. Glucose-6-phosphate, which serves as a metabolic hub to link glycolysis, the pentose phosphate route, glycogen synthesis, de novo lipogenesis, and the hexosamine pathway, may be depleted as a result of the increase in G6Pase activity observed in type 2 diabetic rats in the present study [51]. Te treatments of diabetic rats with   Evidence-Based Complementary and Alternative Medicine CLHE and CFHE revealed a signifcant increase in the liver glycogen content and signifcant decreases in the GP and G6Pase activities; and these improvements may, in turn, lead to a decrease in the hepatic glucose production and may attribute to the antihyperglycemic and antidiabetic efects of CLHE and CFHE (Scheme 2). In the present study, serum AST and ALT activities exhibited a signifcant elevation in the diabetic group when compared with the negative control. Tese changes agreed with Ahmed [52] and Parmar et al. [53], who indicated a marked increase in the AST and ALT levels in rats with DM. Te increase in serum AST and ALT activities in diabetic rats may be attributed to their greater need for gluconeogenic substrates and their leakage from the damaged hepatocytes [2,52]. Te treatments of NA/STZ-induced diabetic rats with CLHE and CFHE resulted in a marked decrease in these two cytoplasmic enzymes in serum, which may be secondary to the improvements in the diabetic conditions and liver function and structural integrity. Tese results go parallel with El-Deberky et al. [25] and Heidarian and Rafeian-Kopaei [54], who revealed that Cynara scolymus extracts produced a signifcant decrease in the elevated AST and ALT activities in association with amelioration of the liver's histological architecture in thioacetamide-treated rats and in lead-intoxicated rats, respectively.
Te present study showed an elevation in serum FFAs levels in the diabetic control rats. Tese results are in concurrence with the data of Ahmed [50]. Te persistent elevations in circulating FFAs levels have an important role in developing IR and β-cell dysfunction in T2DM [2,55]. Supplementation of diabetic rats with CLHE and CFHE leads to a partial decrease in serum FFAs levels compared to the diabetic control. Tis improvement in FFAs levels may have an important role in the decrease of insulin resistance produced by treatments with CLHE and CFHE as indicated in our previous publication [20].
In NA/STZ-induced diabetic rats, serum levels of TC, TAGs, LDL-C, and VLDL-C increased signifcantly, whereas there was a reduction in the HDL-C levels compared to the negative control rats. Also, a signifcant reduction in TC, TAGs, LDL-C, and VLDL-C levels in diabetic rats was attained by treatment with CLHE and CFHE, which is more potent. Tese changes are consistent with Soofniya and Heidarian [56], who found that the oral supplementation of C. scolymus leaf aqueous extract for 21 days signifcantly reduced TC, TAGs, LDL-C, VLDL-C, and glucose levels in the serum of STZ-induced diabetic rats. It was reported that C. scolymus has hypocholesterolemic efcacy by reducing the cholesterol synthesis [57]. Tis action may be attributed to the fnding that artichokes contain luteolin and cynarine, which play an important role in suppressing the cholesterol and TAGs synthesis, and glucoside release. It was found that luteolin by beta-glucosidase in the digestive tract may result in a suppression of up to 60% of cholesterol biosynthesis [58]. However, a remarkable diminishment of plasma LDL-C, VLDL-C, and an elevation of HDL-C agree with Cieslik et al. [59] who revealed a declining tendency in TC, LDL-C, and VLDL-C when diets were supplied with powder of Jerusalem artichoke. Meanwhile, Taylor [60] showed a 10-15% reduction in TC/LDL-C and LDL-C/HDL-C ratios probably due to the fact that leaf contains active constituents such as favonoids and cafeoylquinic acid, which possess antihyperlipidemic efects. Tese compounds belong to polyphenols, chlorogenic acid, cynarine, luteolin scolymosides, and cynarosides [61]. Tus, based on the obtained results, it can be concluded that CLHE and CFHE have antidiabetic action by possessing glucose and cholesterollowering efects (Scheme 2).
Our results showed signifcant downregulation in the SOD and GSH content in diabetic control rats' hepatic tissue compared to the negative control rats, which agreed with Dewanjee et al. [62]. Te decreased SOD levels in diabetic rats were almost reverted to normal status after the extract treatment. Te downregulation of the liver GSH level leads to augmented oxidative stress [63]. Repression in SOD activity in diabetic rats could result from inactivation by H 2 O 2 or glycosylation of the enzyme in DM [64]. In parallel with our study, Colak et al. [65] and Ahmadi et al. [66] found that the treatment with CLHE improved the lowered liver SOD activity, respectively, in CCl 4 -and diazinon-induced liver injuries in rats.
Diabetic control rats showed an important reduction in GPx in hepatic tissue. Tese deteriorations in GPx activity were reduced after treatment with CLHE and CFHE. Tese results agreed with Kaymaz et al. [67], who reported a signifcant increase in the hepatic GPx activity by treatment with artichoke leaf extract in alpha-amanitin-induced hepatotoxicated rats. LPO activity increased in the liver homogenates in the diabetic control group compared to the negative control but decreased in the diabetic groups treated with CLHE and CFHE compared to the diabetic control. In agreement, Matkovics et al. [68] and Anwar and Meki, [69] found that in the liver of diabetic rats, LPO increased. Colak et al. [65] and Ahmadi et al. [66] revealed that the treatment with CLHE signifcantly reduced the liver LPO elevations, respectively, in CCl 4 -and diazinon-induced liver injuries in rats.
Te present study on hepatocytes from diabetic control rats showed a signifcant increase in the expression of the proinfammatory cytokine, TNF-α, in the liver. Treatment with CLHE and CFHE showed a signifcant reduction in the liver TNF-α expression consistent with the absence of infammatory cells' infltration in histological sections and refects the anti-infammatory efects of these extracts. Ahmadi et al. [66] agreed with our results of a marked decrease in hepatic TNF-α expression in the liver of diazinon-induced liver injury in rats treated with CLHE.
Histological changes may be due to the hepatotoxic efect of STZ. Diabetic livers showed disorderly hepatocytes, cytoplasm dissolution, mononuclear leukocytic infltration, a proliferation of bile ducts, and degenerative changes in the wall of some bile ducts. After treatment with CLHE and CFHE, marked amelioration was observed. Tese changes are similar to the results of Arya et al. [70].
Te present EM ultrastructure results of the NA/STZdiabetic liver showed cytoplasm dissolution, irregular nuclear membrane, collagen fbre bundle, damaged mitochondria with ill-defned cristae, and fragmentation of RER.

Evidence-Based Complementary and Alternative Medicine
Tese results are in concurrence with Lucchesi et al. [71], who revealed similar liver ultrastructural changes in alloxaninduced diabetic rats. Moreover, Balazs and Halmos [72] showed that dilated intercellular space contains a collagen fbre bundle. Can et al. [73] observed dense degenerative mitochondria and a RER in the diabetic group.
In our opinion, the improvement efects of CLHE and CFHE on liver function and histological and ultrastructural deteriorations in diabetic rats may be attributed to their antioxidant and anti-infammatory properties (Scheme 2). In support to this attribution, Ahmadi et al. [66] stated that the ameliorative efects of CLHE against diazinon-induced liver injury are due to its antioxidant and anti-infammatory properties.
Te antidiabetic efects and the preventive efects of CLHE and CFHE against liver injury in NA-STZ-induced diabetic rats may be attributed to their constituents which have several biological activities. Tese constituents include phenolic acids, cynarine isomers, favones (luteolin and apigenin), cynarasaponin isomers, and polyunsaturated fatty acids.

Conclusions
In conclusion, the present study provides evidence that both CLHE and CFHE have antidiabetic efects. However, further clinical studies are required to assess the efcacy and safety of CLHE and CFHE in type 2 diabetic human beings. Both CLHE and CFHC have ameliorating efects on the liver function and histological and ultrastructural integrity that may be mediated via the suppression of oxidative stress and infammation as well as enhancement of the antioxidant defence system. To pinpoint the molecular mechanisms of CLHE and CFHE, more research studies are required to examine the impact on mediators of insulin resistance and infammation.  10 Evidence-Based Complementary and Alternative Medicine GPx:

Data Availability
Te data supporting the current study are included in the article.

Ethical Approval
Etihical approval was obtained from the Faculty of Science Institutional Animal Care and Use Committee (IACUC), Egypt (Ethical Approval Number: BSU/FS/2015/2).

Conflicts of Interest
Te authors declare that they have no conficts of interest.