Antidiabetic and anti-obesity acylated avonol diglucoside from Ammannia baccifera L. subsp. aegyptiaca (Willd.) Koehne Waste

Chemical investigation of the aerial parts of Ammania aegyptiaca ethanol extract (AEEE) revealed signicant high concentrations of polyphenols and avonoids content with notable antioxidant activity in DPPH, ORAC, and reducing power assay. New acylated diglucoside avonol myricetin 3-O-β-4 C1-(6"-O-galloyl glucopyranoside) 7-O-β-4C1-glucopyranoside (MGGG) was isolated from aerial parts of AEEE along with four additional known phenolics, not characterized previously from AEEE. Moreover, powerful inhibitory effects of MGGG, AEEE, and all isolates against α-amylase, pancreatic lipase and β-glucosidase, were assessed. In addition, exible molecular docking was used to reveal the inhibition towards digestive enzymes and conrmed that the MGGG interacted strongly with the active site residues of these enzymes with the highest binding free energy against β-glucosidase (DG=-8.98 kcal/mol) compared to the commercial drug Acarbose, thus justifying its dual management of diabetes and obesity. In streptozotocin (STZ) induced diabetic rats, AEEE signicantly decreased high serum glucose, α-amylase activity, liver and kidney function markers and increased insulin level. Moreover, it improved lipid prole due to diabetes with increased SOD activity and inhibited of TBARS formation. Consequently, AEEE and MGGG are found useful in controlling the secondary complications associated with type 2 diabetes mellitus. Histopathological studies proved the decrease in the pancreas damage and agreed with the biochemical ndings. These results provide evidence that AEEE and MGGG have potent antidiabetic activity, which warrants additional investigations.


Introduction
Diabetes Mellitus (DM) is a major metabolic disorder leading to high morbidity and mortality rate all over the world [1]. The incidence of diabetes in developing countries have reached to epidemic proportions and International Diabetes Federation (IDF) expects an increase from 382 million people with diabetes to 592 million between 2013 and 2035 [2].
It is characterized by elevated plasma glucose concentrations caused by either insu cient insulin or insulin resistance, or both. In addition to, abnormalities in different metabolic pathways [3]. As the disease progresses, it results in complications like retinopathy, neuropathy, nephropathy, stroke, ischemic heart disease, peripheral vascular disease and a variety of heterogeneous diseases [4]. More than 95% of which are type 2 diabetes (T2D). It develops as a result of insulin resistance and pancreatic β-cell dysfunction, resulting in hyperglycemia [5] .
Interestingly, it was found that there is a direct link between T2D and obesity. Obesity leads to increased cytokines production, fat disposition in body tissues and mitochondrial dysfunction which results in insulin resistance and pancreatic β-cell apoptosis [6].
The current treatment for T2D is the usage of oral hypoglycemic. On the other hand, insulin replacement therapy is the main treatment for patients with type 1 diabetes [7]. However, major undesirable effects of such drugs are the main cause for an urgent need for alternative therapies that may have no side-effects [8].
As a consequence there is a growing interest in phytomedicine for using plant extracts which are more safe, easily available, more affordable and have less incidence of adverse effects in comparison to synthetic antihyperglycemic drugs [9].
A bene cial approach for management of diabetes is to reduce the post-prandial hyperglycemia and to prevent lipids digestion and absorption. This is unswervingly associated with the preclusion of obesity and obesity-related diseases [10].
Exploring natural inhibitors of hydrolyzing enzymes in carbohydrates and lipids digestion can offer an attractive combinatorial therapeutic strategy for the management/prevention of postprandial hyperglycemia and obesity.
Moreover, DM appears to be an oxidative stress-related endocrine disorder and using antioxidants may be bene cial in its prophylaxis and treatment [11]. Aforementioned studies reported that in T2D the oxidative stress is increased due to chronic hyperglycemia leading to manufacture of reactive oxygen species (ROS) in β-cells, thereby depletes the antioxidant mechanism activity with enhancement of free radical generation [12]. Consequently, medicinal plants rich in antioxidant phytoconstituents can shield β-cells from reactive oxygen species (ROS) and can avoid diabetes induced by ROS [13].
Increasing evidence suggests that plant phenolic offer potential therapeutic antioxidant activities which are related to their capacity to scavenging a wide range of ROS [14].
So, looking for a relevant source of plant phenolics as potential therapeutic agents which can be used for decreasing blood glucose levels and lipids for dual control of diabetes and obesity via digestive enzyme inhibition, speci cally pancreatic α-amylase, intestinal β-glucosidase and pancreatic lipase [15], along with its ability to boost the body antioxidant system become an absolute necessity.
One of the famous plants genera which include phenolic-rich species is the genus Ammannia (Lythraceae). This genus is known to embrace species capable of synthesizing and accumulating high percent of phenolics [16]. Ammannia baccifera L. subsp. aegyptiaca (Willd.) Koehne Waste (AE) is an annual herb distributed in tropical regions, in Egypt, it was recorded in Nile and Oasis geographical regions. It grows as a summer weed in rice elds, ditches and swamps [17]. It is often treated as a synonym, variety of subspecies of Ammannia baccifera L., eg. [18]. However, AE is an accepted species separated from the other three species known from Egypt by its sessile or subsessile owers in compact axillary cymes. The type of this species was collected from Damietta, Egypt [17]. In folk medicine, its fruit has been mixed with ginger and this juice was useful in fever treatment [19]. Providing the available current literature is concerned, the phenolic pro le and biological activities of AE have never been investigated. However, other species has been reported to have antidiabetic activity [20].
This study aimed to subject the owering whole plant ethanol extract (AEEE) of AE to an intensive phytochemical investigation of its phenolic constituents and to investigate the in vitro antioxidant activity of AEEE and the isolated constituents. Along with evaluation of their inhibitory effects and antidiabetic activity against the extra pancreatic digestive enzymes and to use molecular docking analysis to demarcate their inhibition toward digestive enzymes. Furthermore, evaluation of the possible antidiabetic activity of the owering whole plant ethanol extract AEEE in streptozotocin (STZ) induced diabetic rats in order to assess the dual antidiabetic and anti-obesity effects of AEEE. This provide a related data for further study and its utilization as an adjuvant therapy in the management of T2D diabetes and obesity.
Being a weed, which mainly threatens water channels and rice elds, this study has focused on the exploitation and utilization of this plants in the pharmaceutical eld.

Results
Total phenolic and avonoid contents Phenolic content of AEEE was determined as 380.5±3.88 mg GAE/g, where R 2 = 0.9985, and the equation for standard curve is Y = 0.0242X+ 0.0211. In addition, the total avonoid content in the AEEE was evaluated as 190±2.31 mg CE/g, where R 2 = 0.9989, and the equation for standard curve is y = 0.0048x + 0.0091. So AEEE was found to be a source of wide range of potent polyphenolic constituents.
Identi cation of polyphenols from AEEE Concentrated aqueous ethanol extract (3:1) AEEE was prepared from a fresh aerial part samples of AE. AEEE was fractionated over column chromatography over Sephadex LH 20, using water/methanol mixtures of decreasing polarities, followed by column fractionation over MCI gel of the 30 and 50% aqueous methanol Sephadex LH 20 column fractions afforded pure samples of compounds (1)(2)(3)(4)(5). The known compounds (2)(3)(4)(5) showed chromatographic, UV absorption and hydrolytic data identical with those reported for kaempfeol 3-O-rutinoside 2 [21], quercetin 3-O-rutinoside 3 [22], tellimagranidine-I 4 [16] and 2,3-α,β-digalloy glucose 5 [23], respectively. Compound 1, a yellow amorphous powder, showed chromatographic properties (dark purple spot on paper chromatogram under UV light, turning reddish orange when fumed with ammonia vapor, moderate migration in aqueous and organic solvents). UV spectral analysis of 1 in methanol and on addition of shift reagents [24,25] con rmed the presence of a avonol moiety with a free hydroxyl at 4′-position (stable MeONa spectrum in UV) and a substituted hydroxyl at the 7-position (showed no shift in UV with NaOAc). Acid hydrolysis of 1 yielded glucose (cochromatography), myricetin and gallic acid (co-chromatography and 1  (m) and 4.28 (d, J=12 Hz), assignable to two non-equivalent methylene glucose protons. The anomeric proton was found resonating at d ppm 5.28 as a doublet (J=8 Hz), thus proving a b-con guration of the sugar moiety. In this spectrum, the galloyl moiety exhibited the resonance of its two equivalent H-2′′′′and H-6′′′′ protons as a singlet at d ppm 6.98. The chemical shifts of the remaining glucose protons were found resonating in the region between δ ppm 3.95 and 3.3, hidden by H 2 O proton signals, while the myricetin proton resonances were located at δ 7.48 (s, H-2′, H-6′), 6.16 (IH, d, J=2 Hz, H-6), 6.39 (IH, d, J=2  Hz, H-8), thus con rming the structure of intermediate 1a as myricetin 3-O-b-(6′′-O-galloylglucoside) to the H-6 and H-8 of this moiety [26]. The 13 C NMR analysis established the structure of 1. It exhibited twelve glucose carbon signals. The two β-glucose anomers were detected at δ ppm 99.2 and 99.8, while the methylene C-6′′′ glucose carbon with a free hydroxyl and methylene glucose C-6′′ resonate up eld at δ ppm 60.2 and 63.2 respectively. Acylation by gallic acid resulted in de-shielding of the second resonance. The remaining glucose carbons were found resonating at δ ppm closely related to those reported to avonols 3,7-di-O-glycoside [27]. The presence of only one galloyl moiety in 1 followed from the single carboxyl carbon resonance at δ 166.5 and from the recognized characteristic pattern of the remaining galloyl carbons. Substitution at the 3-and 7-positions of myricetin was tracked from the relative up-eld shifts of C-3 and C-7 signals to δ 134.4 and 161.6, respectively. Down eld shifts of the signals corresponding oand p-carbons in comparison with the corresponding signals of myricetin was detected [27]. Furthermore, 4 C 1 conformer of the two glucose moieties was con rmed from the measured chemical The molecular docking analysis was performed by using MOE 2015 Software to predict the possible binding mechanism of MGGG with three targeted enzymes. In this study, interactions between the docking sites of enzymes and the phenolic molecule and some relevant parameters were applied for molecular docking analysis.
All of the molecular docking results regarding interactions between the molecule of MGGG, reference standards and digestive enzymes binding are summarized in (Table 1) and Fig. 2. In order to prove the reliability of the docking process, the native ligand derived from the X-ray crystallographic structures was redocked into the active sites of α-amylase as shown in Fig. 2 (A1),The binding mode of MGGG exhibited an energy binding of -8.80 kcal/mol and the complex was stabilized by four hydrogen bonds, one polar hydrogen bond with carbonyl group in chromone nucleus formed (green dash lines) with His305 residue of the binding pocket of α-amylase with a distance of 2.55 Å, while hydroxyl groups in B-ring and galloylted glucose moieties formed another three acidic and basic hydrogen bonds with Asp300, Glu233 and Lys200 with distance of 2.11,2.41 and 2.15 Å respectively. The binding mode of the commercial drug Acarbose which used as a positive control exhibited an energy binding of -7.23 kcal/mol and formed three hydrogen bonds, one acidic hydrogen bond with hydroxyl group in 4,6-dideoxyo-α-D-glucopyranosyl moiety (green dash lines) with Asp 300 residue of the binding pocket of the α-amylase with distance of 2.17 Å, while hydroxyl groups in α-D-glucopyranose moiety formed another two basic hydrogen bonds with Lys200 with distance of 2.16, 2.23 Å Fig. 2 (A2).

Modeling of MGGG binding mode against α-amylase
The X-ray crystal structure and enzyme kinetics studies have revealed that the active site of α -amylase is characterized by the presence of three important residues namely Asp197, Glu233, and Asp300 [28]. It was reported that among the amino acid residues of α-amylase interacting with myricetin (an inhibitor of α-amylase) were Trp59, Lys200, Leu162, Val163, Ala198, and Glu233 [28]. A previous study found that αamylase inhibitors such as curcumin, interacted with the amino acid residues Lys200, Glu233, and Asp300, while rutin and quercetin interacted with the amino acid residues Try59, His305, Thr163, and Lys200 [29]. Therefore, the docking results con rmed MGGG had similarities with acarbose as they interacted with several key amino acids residues Asp300 and Lys200 of α-amylase, suggesting that those two amino acid residues may play an important role in the inhibitory activities of the two compounds.
Modeling of MGGG binding mode on β-glucosidase β-glucosidase active site of MGGG is characterized by the presence Trp425, Asn426 and Phosphate ion. A previous study found that quercetin xed in the binding pocket of β-glucosidase mainly through hydrogen bonds and hydrophobic interactions between the complexes. The residues involved in H-bonds were mainly polar amino acids, such as Trp442, Gln57 and Asn426 [30]. The molecular docking illustrated that MGGG bind strongly to β-glucosidase catalytic site with an energy binding of -8.98 kcal/mol. Hydroxyl groups in β-D-glucopyranose moiety formed one polar hydrogen bond with Gln165 (green dash lines) of the active pocket of β-glucosidase with a distance of 2.00, 2.40 Å and two strong metal ion interactions with PO 4 group attached (black dash lines) with Trp425 and Asn426 residues Fig. 2 (B1). In addition, MGGG and Acarbose Fig. 2 (B2) can form metal ion interactions with β-glucosidases via some common residues, namely Trp425 and Asn426. It can be speculated that these amino acids are extraordinarily critical to the binding of the two ligands with glucosidase enzyme, suggesting that MGGG is a good hypoglycemic agent. The magnitude of the binding energy indicates that MGGG showed the strongest interaction with β-glucosidase, con rming the in vitro data.
Modeling of MGGG binding mode on pancreatic lipase MGGG was able to bind to the active site of pancreatic lipase. The values of the binding free energy was-7.01 kcal/mol. β-D-glucopyranose moiety formed one polar hydrogen bond with Asn385 with a distance of 2.19 Å, while galloylglucose moiety formed another acidic hydrogen bond with Thr355 with a distance of 2.02 Å Fig. 2 (C1). Moreover, the binding mode of the reference Orlistat exhibited an energy binding of -5.62 kcal/mol with a carbonyl group in formamide moiety formed one polar hydrogen bond with Tyr389 with a distance of 2.31 Å. While other carbonyl group formed another basic hydrogen bond with Lys387 with distance of 2.30 Å and hydrophobic aliphatic tail show H-arene interaction with Phe410 Fig. 2 (C2). it was reported that rutin and isorhamnetin-3-O-rutinoside interacted with the amino acid residues Asn 320 and Asn 176 of pancreatic lipase, respectively [31]. For all tested digestive enzymes, mapping surface technique was carried out to show MGGG occupying the active pocket of these enzymes Fig. 3. lower than Trolox (the positive control), which had an IC 50 of 28.0±14.31µg/mL, further endorsing the potent antioxidant activity of new MGGG.

Reducing power assay
The reducing power of phenolic compounds implicates its antioxidant activity. In the reducing power assay, the more antioxidant compounds convert the oxidation form of iron (Fe +3 ) in ferric chloride to ferrous (Fe +2 ). Fig. 4 showed that AEEE, MGGG and the isolated compounds (2-5) had concentrationdependent reducing power. MGGG showed the highest activity with a comparable reducing power to standard quercetin.
In-vitro enzyme assays In the current study, AEEE and its isolated compounds exhibited anti-α-amylase, βglucosidase, and pancreatic lipase activities ( Table 2).

Effect of AEEE on body weight
Treatment with AEEE at 500 mg/kg body weight significantly inhibited the reduction in the body weight induced by STZ compared to DC, Fig. 5.

Effect of AEEE on liver and kidney functions' markers
No signi cant changes in the liver and kidney functional tests were observed. After two weeks treatment diabetic groups treated with (250 or 500 mg/kg) AEEE alleviated the hepatocellular caused by STZ by reduction in the levels of AST and ALT [32]. In addition, they reversed the high creatinine and urea levels. Moreover, administration of extract preserved the values of serum AST, ALT, urea and creatinine, thus illustrating its non-toxic nature, as shown in Fig. 6. AEEE showed insigni cant changes in both liver and kidney functional tests with irrelevant alterations in all parameters under the present investigation.

Effect of AEEE on serum blood glucose, insulin and α-amylase
The serum glucose level of STZ-induced diabetic rats in groups IV and V remarkably decreased after administration of 250 and 500 mg/kg of AEEE, with increased insulin levels and reduced α amylase activity on comparison to the DC group.  Fig. 8.

Effect of AEEE on oxidative stress markers of pancreas
Remarkable reduction in antioxidant enzyme activity (SOD) and a signi cant increase in TBARS manufacture in pancreatic tissues of DC group versus NC group Fig. 9. AEEE treated diabetic groups increased SOD activity and signi cantly suppressed the formation of TBARS on comparison to DC group. The percentage change from diabetic groups for the 250 and 500 mg/kg doses were; 71.6, 42.78, 80.9 and 47.45, respectively. Furthermore, there was a signi cant difference between AEEE treated diabetic groups and glibenaclamide group (VI) in oxidative stress parameters Fig. 9.

Effect of AEEE on pancreas histopathological examination
Pancreas histopathological examination of NC, AE500-NC and AE250-NC groups showed normal picture Fig. 10 (A, b and C). Islets of Langerhans appears as circular shapes with normal cell lining, while the exocrine components and the interlobular duct surrounded with the supporting tissue appeared well organized and with normal morphology Fig. 10 (A and C). Image morphometry showed that the mean islets area in non-diabetic rats was (145.21 ± 3.49) mm 2 , whereas for AE500-NC and AE250-NC groups were (175.67 ± 1.85) and (167.89 ± 1.34) mm 2 , respectively. On the other hand, histopathological examination of diabetic rats showed the acinar cells with up normal morphology around the islets. The cells of islets were in degenerative form with asymmetrical vacuoles with intra islets hemorrhage, reduced size of islet cells and number of β-cells Fig. 10 (D and E). The diabetic rats mean islets area was (110.65 ± 9.41) mm 2 which in comparison with normal rats appeared smaller. Pancreas sections of AE500-DC and AE250-DC groups were microscopically investigated, suggesting the protection of the islets due to recovery of Langerhans' islets size with β-cells repair Fig. 10 (F and G). This regeneration of the β-cell was more obvious at higher dose, whereas with the mean of islets area for AE500-DC and AE250-DC groups were (185.33 ± 10.41) and (180.12 ± 6.52) mm 2 , respectively.

Discussion
Nowadays searching for natural compounds that have both antidiabetic and antioxidant activities, with fewer side effects, is still challenging. Polyphenols, in particular avonoids, are suggested as better therapeutic agents in the management of free radical mediated diseases particularly diabetes mellitus and its chronic complications due to their potent antioxidant activity which has been demonstrated by both in vitro and animal models studies [33]. They are hydroxylated phenolic substances and the hydroxyl group mediate their antioxidant effects by scavenging free radicals by chelating metal ions [34].
Flavonols the class of MGGG is a sub class of avonoids, are effective as antioxidant and antidiabetic agents mostly depending on their chemical structure. Both, con guration and the total number of hydroxyl groups increased both activities and substantially regulate the mechanisms of radical scavenging [35] and antidiabetic effects. Thus, the total of hydroxyl groups, hydroxyl con guration, the catechol structure in the B ring, C-2-C-3 double bond, and C-4 ketonic functional group are the essential features in the manifestation of bioactivity of avonoids especially for antidiabetic effect [36]. Myrecetin signi cantly improves insulin resistance besides antioxidant, anti-in ammatory, aldose reductase inhibitory actions [37].
AE is an annual herb that grows as a summer weed in rice elds. Despite the reported antidiabetic activity of other Ammannia species, there is no data in the literature on phytoconstituents, antidiabetic activity and, its mechanism of action. In the current study, phytochemical investigation of AEEE resulted in the isolation of unique acylated avonoid, myricetin 3-O-β-4 C 1 -(6′′-O-galloyl glucopyranosid) 7-O-β-4 C 1glucopyranoside, along with four additional known phenolics. Signi cant antioxidant potential of AEEE, polyphenols 2-5, and highest antioxidant potential exhibited by MGGG are recorded with IC5 0 2.37±0.56, 2.01±0.23 and 158.13±2.82 µg/mL in comparison to standard against DPPH, ORAC and ferrous reducing assay, respectively. Several reports showed that the radical scavenging capacities increased with an increase in the number of phenolic hydroxyl groups; this was observed for the three classes of isolated compounds: avonoids, gallotannins and ellagitannins [38]. Moreover, the antioxidant activity increased as the number of galloyl units increased; however, it is not affected by their position and not in uenced by the presence of a hemiacetal hydroxyl and the 4,6-O-HHDP groups. Also, the presence of two adjacent phenolic hydroxyl groups on the galloyl moiety is signi cant and this justi ed the higher antioxidant capacity of tellimagrandin-I compared to nilocitin and the potential of MGGG, Flavonoids with potent antioxidant activity were shown to be effective in management of diabetes [39].
Pancreatic amylase and intestinal glucosidase are crucial enzymes involved in glucose formation [40]. βglucosidases catalyze the breakdown of alkyl and aryl-β-glycosides , disaccharides and short chain oligosaccharides with dual activities of hydrolysis and transglycosylation [41]. Therefore, the study of βglucosidase enzyme inhibitors is important for treatment of type 2 diabetes [42]. However, the effect of inhibitors on β-glucosidase was illustrated barely. Phenolic and avonoids content in roselle were responsible for the inhibitory activity α-/β-glucosidase. Pancreatic α-amylase catalyses the rst step in the starch breakdown. Suppression of intestinal α-amylase activity hinders the starch and oligosaccharides breakdown to monosaccharides before absorption, this results profound control of T2D.
Acarbose an oral hypoglycemic agent, is used for the inhibition of α-amylase [43]. Flavonoids as myricetin, the core of the new isolate MGGG, luteolin, quercetin were potent inhibitors. In fact, pancreatic lipase plays a key role for triglyceride absorption in the small intestine. [44]. Thereby, the hindrance of triglycerides absorption by lipase inhibition is a main route for avoiding obesity and management of T2D.
The commercial drug Orlistat strongly inhibits the activity of pancreatic lipase [45]. Pancreatic lipase inhibitory activity has been attributed to various types of phytochemicals, such as saponins, polyphenols and terpenes [46].
The in vitro enzymatic inhibition results showed that AEEE and all isolates 1-5 possessed antidiabetic and anti-obesity activity, based on inhibition of pancreatic lipase, β-glucosidase and α-amylase. Apart from compound 3 [47], AEEE and all isolates were rst reported. Moreover, signi cant difference existed between AEEE and its isolates as shown in Table (2). It was reported that acylated avonoids, the class of the new isolate MGGG and its core (myricetin), have been previously reported to have strong antidiabetic activity [37,48]. MGGG exhibited the highest % of inhibition in all three enzyme assays with antidiabetic and anti-obesity properties higher than the reference standards Acarbose and Orlistate geometric complementation and excellent mapping which increase its inhibitory activity and was involved in various type of interactions with the active site residues of the target enzymes. In addition, in all tested enzymes MGGG showed RMSD value lower than 2.00 Å which con rm its occupancy in the original site of crystal ligand and ensure the validation of molecular docking study. From the molecular structural point of view, actually, presence of multiple polar hydroxyl groups is very important for the stabilization of MGGG at the catalytic sites of the tested digestive enzymes due to the availability of the electron donating groups (-OH) forming electronegative and electron cloud system inside the pocket that can do tted stabilized interactions with polar and charged amino acids. This justify the higher inhibitory potential of MGGG compared to the used standard drugs towards digestive enzymes. In addition, docking results revealed that hydroxyl groups of the B-ring, galloylglucose and β-D-glucopyranose moieties are vital for its inhibition power and were the most critical groups for the stabilities in the active sites among the tested enzymes.
In fact, the interaction forces of the hydrogen bond are considered to play a critical role in stabilizing the ligand-enzyme complex in order to exert catalytic reaction, which depend mainly on the number of hydrogen bonds, distance between hydrogen bonds donor/acceptor and attached amino acids in targeted pocket, DG free energy score and RMSD value should be lower than 2 Å. Therefore, in terms of binding energy and binding a nity and compared with the standards Orlistat and acarbose, MGGG showed higher binding free energies for all tested digestive enzymes, with the highest inhibitory activity against β-glucosidase (DG= -8.98 kcal mol) and the lowest inhibitory activity against pancreatic lipase (DG= -7.01 kcal mol). This is due to the polar nature of MGGG and the presence of some polar and nonpolar amino acids in the critical pocket of pancreatic lipase, so the region of non-polar amino acids is not fully occupied. The glucose level was signi cantly reduced and the insulin level was elevated after administration of AEEE as compared to DC group. The histopathological results further confirmed this, which demonstrate that the structural integrity of islets of Langerhans was recovered. Furthermore, they decreased α-amylase activity. Phenolics are reported to decrease the activity of digestive enzymes. [49,50] and this is full agreement with the in vitro assay and molecular docking results.
In this investigation, the abnormal serum lipid profile was established in diabetic rats. This result agrees with Pepato et al. and Sharma et al. [51,52]. This abnormal serum lipid profile was inverted after incorporation of AEEE with both doses. Hence, the extract could be helpful in re ning lipid metabolism which will in turn aid in the protection against different diabetic complications.
AEEE treatment of diabetic groups signi cantly increased SOD activity and signi cantly inhibited the creation of TBARS when matched with the DC group. This may be attributed to its high phenolic and avonoids and in agreement with the high in vitro antioxidant potential of both the extract and new isolate using DPPH, ORAC and ferrous reducing assays. Furthermore, AEEE was considered safe as they decreased the levels of AST, ALT, creatinine and urea compared to DC. The biochemical ndings agree with histopathological modi cations of β-cells of pancreas. Such histopathological modi cations were decreases by incorporation of AEEE extract at both doses. The current study agrees with previous studies on antidiabetic and anti-obesity effects of herbal extracts [53,54]. Preparation of AEEE Aerial parts of AE (2.5 kg) were extracted by being re uxed with EtOH/H 2 O (3:1, 3 times, each with 3 L, for 8 h, under re ux). The solvent was removed under reduced pressure at 50 °C to yield dark brown amorphous material (150 g).

Estimation of total phenolic and avonoid contents
Folin-Ciocalteu reagent was used for measuring phenolic content and estimated as gallic acid equivalents (GAE) per g of sample. Aluminium chloride (AlCl 3 ) used for measuring total avonoid content (colorimetric assay and estimated as catechin equivalents (CE) per g of sample using [55].

Molecular Modelling
The docking analysis was performed by using MOE 2015 software. The binding sites were generated from the co-crystallized ligands, within crystal protein (PDB codes: 2QV4 -2ZOX -2OXE). To prepare the protein for the docking experiments, water molecules were removed. The crystallographic disorders and un lled valence atoms were corrected, using protein report and utility and clean protein options. The protein geometry was corrected by applying CHARMM and MMFF94 force elds. The rigidity of binding site was obtained by applying xed atom constraint. The active site essential amino acids were de ned and prepared for docking process. The structures of tested compounds (ligands) were imported as MDL-SD le format. The 3D structures of the ligands were prepared for docking by rst protonated, then their energy was minimized by applying 0.05 RMSD kcal/mol using CHARMM force eld. The Molecular docking processes process was carried out using CDOCKER protocol. The receptor was held rigid while the ligands were allowed to be exible during the re nement each molecule was allowed to produce ten different interaction poses with the protein. The docking scores (-CDOCKER interaction energy) of the best-tted poses with the active sites at the tested enzymes were recorded. The output from of MOE was further analyzed with Discovery Studio 2.5 software. These processes were used to predict the proposed binding mode, a nity, preferred orientation of each docking pose and binding free energy (∆G) of the tested compound with pancreatic α-amylase, intestinal β-glucosidase and pancreatic lipase [56].
In-vitro studies DPPH assay The assay was carried out for AEEE and isolated phenolics according to Brand-Williams et al. [57].
Oxygen radical absorbance capacity (ORAC assay) The antioxidant assay was applied on AEEE and isolated phenolics [58].

Reducing power assay
The assay was carried out on AEEE and isolated phenolics [59].

α-Amylase inhibition
The assay was implemented in accordance with [53,60]. The percentage of inhibition can be estimated using the following equation.

β-Glucosidase inhibition
The assay was done in conformity with [61,62] and using the same formula for amylase.

Pancreatic lipase inhibition
Determination of % inhibition of pancreatic lipase was calculated as prescribed by Hegazi [53] and using the same formula for amylase.

Experimental animals
Male Sprague-Dawley rats (170-220 g) were acquired from the National Research Centre (NRC, Giza, Egypt). Animals were acclimatized in our animal facility for one week before the experiment. Animals had total access to standard laboratory food pellets and water ad libitum under temperature-controlled conditions and 12 h light-dark cycles. The animal experiments were conducted according to the international regulations of the usage and welfare of laboratory animals and were approved by the Ethics Committee of the National Research Centre, Cairo, Egypt, Protocol number 49/261 (2019).

Acute oral toxicity
The acute oral toxicity of AEEE was adpoted in male Sprague-Dawley rats according to OECD guideline No.423 (OECD, 2001). Based on a pilot study in our laboratories, limit test was performed. Animals were fasted overnight and the extract was administered orally using gastric feeding needle at a dose of 2000 mg/kg (10 mL/kg dosing volume) [63].

Induction of diabetes
Induction of diabetes was done by a single intraperitoneal injection of streptozotocin (STZ) solution dissolved in freshly prepared citrate buffer (0.1 mol/L, pH 4.5) at a dosage of 60 mg/kg. After 72 h tail vein blood was collected to determine fasting blood glucose level colorimetrically (Diamond Diagnostics, Cairo, Egypt). Glucose levels over 200 mg/dL were considered diabetic and included in the study.

Experimental design
Male Sprague-Dawley were randomly divided into 6 groups, comprising six rats each as follows; Group I: Normal control rats (NC). Group II: Normal rats treated with AEEE (500 mg/kg) (AE 500-NC). Group III: Diabetic control (DC). Group IV: Diabetic rats treated with AEEE (250 mg/kg) (AE250-DC). Group V: Diabetic rats treated with AEEE (500 mg/kg) (AE500-DC). Group VI: Diabetic rats treated with standard drug glibenclamide (0.25 mg/kg). Groups I and III received only the vehicle (distilled water). Administration of different oral doses of AEEE started 72 h after STZ injection. This was done using an intragastric tube to the treated group daily till the experiment ended. Weight measurement was done at the beginning of the study and at the end of the 28 th day Doses were chosen based on previous literature [49].
Blood and tissue sampling FBG was measured 14 and 28 d after treatment. After the 28th day, blood samples were taken from the retro-orbital venous plexus under light ether anesthesia after overnight fasting. Pancreatic tissues were dissected. They were washed in ice-cold saline solution immediately. After that they were divided into two portions. One was homogenized in 0.1 mol/L potassium phosphate buffer (pH 7.4) using Tissue master TM125 (Omni International, USA). After centrifugation at 3000 r/min for 10 min, the clear supernatant was kept at −80 °C for biochemical assays. The second portion was placed in 10% formalin for histopathological investigation [64].

Assay of Biomarkers
Determination of liver and kidney functions markers Serum aspartate transaminase (AST), alanine transaminase (ALT), serum urea and creatinine level were measured as kidney function tests using kits provided by Spectrum Diagnostics Company, (Egypt). The operational processes were measured in accordance with the kit instructions.

Measurement of serum lipid pro le
Triacylglycerol (TAG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were assayed colorimetrically using (Reactivos GPL,Barcelona, Spain ). Low-density lipoprotein cholesterol (LDL-C) was calculated from TAG and HDL-C values according to Friedewald's formula [65]: Determination of oxidative stress markers in pancreatic tissue Superoxide dismutase activity (SOD) was estimated in accordance with Minami and Yoshikawa [66].

Histopathological investigation
Histopathologic examination was performed by light microscopy on pancreas specimen xed in 10% formalin. After xation, the samples were processed to obtain 5 µm thick para n sections followed by staining with hematoxilin and eosin (H & E) then observation under a Leica photomicroscope.

Image morphometry
The morphometric analysis was performed at the Pathology Department, National Research Center using the Leica Qwin 500 Image Analyzer (LEICA Imaging Systems Ltd., Cambridge, England) which consisted of Leica DM-LB microscope with JVC color video camera attached to a computer system Leica Q 500IW E stained slide. The results were expressed in (µm 2 ) with the mean of standard deviations (SD) [68].

Statistical Analysis
The results are expressed as mean of standard deviations (SD). The differences among the various groups were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test.
The level of signi cance was taken at p values ≤ 0.05. All analyses were done using the SPSS ver. 25.0 (IBM, Chicago, USA). GraphPad prism® software (version 6.00 for Windows) was implemented.  Molecular docking of MGGG with digestive enzymes. (A1) 2D interactions between MGGG and amino acid residues in the active site of α-amylase, (A2) 2D interactions between standard Acarbose and amino acid residues in the active site of α-amylase, (B1) 2D interactions between MGGG and amino acid residues in the active site of β-glucosidase, (B2) 2D interactions between standard Acarbose and amino acid residues in the active site of β-glucosidase, (C1) 2D interactions between MGGG and amino acid residues in the active site of pancreatic lipase, (C2) 2D interactions between standard rlistate and amino acid residues in the active site of pancreatic lipase. The green dashed lines stand for hydrogen bonds and the purple dashed lines stands for pi interactions.  Reducing power of AEEE and isolated compounds compared with quercetin as standard. Results are given as mean ± SD of three replicate analyses.

Figure 5
Page 28/32 AEEE effect on body weight during the experimental period (28 days). Results are stated as mean ± S.D. (n = 6). Results are considered signi cantly different at P < 0.05. (a) is statistically different from NC group; (b) is statistically different from DC group; (c) is statistically different from standard drug.

Figure 6
Effect of AEEE on serum liver and kidney function markers. Results are denoted by means for six rats ± SD in each group.