Biological Activities of Sargassum Algae Mediated ZnO and Co Doped ZnO Nanoparticles as Enhanced Antioxidant and Anti-Diabetic Agents

Brown macroalgae (BMG) were used as carriers for ZnO (ZnO/BMG) and cobalt-doped ZnO (Co-ZnO/BMG) via facile microwave-assisted hydrothermal synthesis. The multifunctional structures of synthesized composites were evaluated as enhanced antioxidant and anti-diabetic agents based on the synergistic effects of ZnO, Co-ZnO, and BMG. BMG substrate incorporation and cobalt doping notably enhanced the bioactivity of the synthesized ZnO nanoparticles. As an antioxidant, the Co-ZnO/BMG composite exhibited highly effective scavenging properties for the common free reactive oxygen radicals (DPPH [89.6 ± 1.5%], nitric oxide [90.2 ± 1.3%], ABTS [87.7 ± 1.8%], and O2●− [46.7 ± 1.9%]) as compared to ascorbic acid. Additionally, its anti-diabetic activity was enhanced significantly and strongly inhibited essential oxidative enzymes (porcine α-amylase (90.6 ± 1.5%), crude α-amylase (84.3 ± 1.8%), pancreatic α-glucosidase (95.7 ± 1.4%), crude intestinal α-glucosidase (93.4 ± 1.8%), and amyloglucosidase (96.2 ± 1.4%)). Co-ZnO/BMG inhibitory activity was higher than that of miglitol, and in some cases, higher than or close to that of acarbose. Therefore, the synthetic Co-ZnO/BMG composite can be used as a commercial anti-diabetic and antioxidant agent, considering the cost and adverse side effects of current drugs. The results also demonstrate the impact of cobalt doping and BMG integration on the biological activity of ZnO.


Introduction
The recent unexpected increase in the number of patients with diabetes worldwide represents a critical health issue, as an increase to 366 million patients by 2030 is predicted. Diabetes is currently the seventh leading cause of death [1][2][3]. This clinical pancreatic syndrome is classified into two main types: (A) diabetes mellitus type 1 (T1-DM) and (B) diabetes mellitus type 2 (T2-DM). The latter is widely distributed and is likely to comprise 90% of diabetes cases by 2030 [1,4]. T2-DM is a serious metabolic disorder that results in a significant generation of reactive oxygen species (ROS) or oxidizing radicals at abnormal concentrations and an increase in blood glucose levels (post-prandial hyperglycemia) [5,6]. Post-prandial hyperglycemia is the underlying reason for several clinical complications Therefore, the application of brown macroalgal microstructures as a substrate for pure or modified ZnO is expected to result in innovative multifunctional composites with significantly enhanced biomedical and biological applications. To the best of our knowledge, the biological activity and biomedical applications of synthetic ZnO nanoparticles under microwave irradiation, as well as the elemental doping of ZnO with brown macroalgae (Sargassum species), have not yet been fully investigated. Therefore, this study is the first to investigate the biological activities of pure ZnO and Co-doped ZnO (Co-ZnO) nanoparticles and their composites with brown macroalgae as potential antioxidant and anti-diabetic agents. We investigated the scavenging efficiencies of the synergetic forms of the synthetic structures for common oxidative radicals and their inhibitory effects on essential enzymes.

Characterization of the Synthetic Structures
The obtained XRD patterns of the brown macroalgae incorporated as a substrate in addition to the ZnO/BMG and Co-ZnO/BMG composites are presented in Figure 1. The observed pattern of the algae exhibits the broad peak typical of amorphous organic materials (2theta angle of approximately 22 • ) in addition to several peaks possibly related to impurities and salts ( Figure 1A Figure 1B). The considerable deviation in the reported position of the ZnO identification peak compared to the JCPDS cards, in addition to the remarkable detection of the broad peak of the algal substrate, confirmed successful integration of the supported ZnO and the organic formwork of the algae. The observed pattern of Co-ZnO/BMG demonstrated a notable reduction in the intensities of the difficult-to-detect peaks of ZnO in the pattern of ZnO/BMG, in addition to a considerable deviation of these peaks to lower 2theta angles ( Figure 1C). Such structural observations, in addition to the lack of detection of clear peaks related to the formation of cobalt oxide, indicate the existence of cobalt as a doping element within the structure of ZnO rather than as a separated oxide phase [33]. The average crystallite size of the synthesized ZnO phase in ZnO/BMG was 51.7 nm, whereas that of Co-ZnO/BMG was 44.3 nm. Such a reduction in the crystallite size reflects the impact of the doped Co element, as it can hinder the progressive growth of ZnO crystals by forming Co-O-Zn surface chemical groups, in addition to significantly inhibiting the agglomeration potential of the synthetic crystals [34]. Previous findings were also based on the FT-IR spectra of the synthetic materials compared to the obtained spectrum of brown macroalgae ( Figure 2). The detected broad band with an area from 3200 to 3600 cm −1 implies the stretching vibration of N-H and O- Previous findings were also based on the FT-IR spectra of the synthetic materials compared to the obtained spectrum of brown macroalgae ( Figure 2). The detected broad band with an area from 3200 to 3600 cm −1 implies the stretching vibration of N-H and O-H bonds within the alginate, lipid, protein, and cellulose compounds of the algae structure ( Figure 2A) [35,36]. Additionally, other organic chemical groups were clearly identified from the spectrum of BMG, such as aliphatic -CH 2 -(2920 cm −1 ), asymmetrical carboxylate groups (1643 cm −1 ), symmetrical carboxylate groups (1447 cm −1 ), sulfate esters within the fucoidan structure (1256 cm −1 ), sulfonic acids (SO 3 ) (1161 cm −1 ), alcoholic groups (1064 and 1021 cm −1 ), and mannuronic groups of alginate structure (863 cm −1 ) (Figure 2A) [36][37][38]. The recognized spectra of ZnO/BMG and Co-ZnO/BMG demonstrated slight deviations in the positions of the previously identified bands of the algae. Moreover, new reduced bands at approximately 460 cm −1 were detected, implying O-Zn-O stretching ( Figure 2B,C) [21,39]. This finding indicates a clear interaction or formation of complexes between the chemical groups of the algal chemical structure and the supported ZnO nanoparticles [21,40]. The chemical structures identified based on the reported chemical groups significantly matched the elemental compositions according to the EDX spectra. The EDX spectrum of ZnO/BMG confirmed the presence of Zn (24.56%), O (40.9%), and ZnO and C mixture (34.54%) as the main components of the algae. The EDX spectrum of Co-ZnO/BMG confirmed the presence of Zn (22.16%), O (39.4%), and Co (3.2%) in the composition of Co-doped ZnO and C (35.24%) as the main composition of the algae. The XPS spectrum of the synthesized Co-ZnO/BMG particles was investigated to confirm the doping process, integration reactions, and oxidation states ( Figure 3). The full scan spectrum confirmed the elemental composition of the composite, which contained N, C, Co, Zn, and O ( Figure 3A). The three clear peaks in the O1s spectra indicate surface oxygen (532.12 eV), O-Zn (530.96 eV), and O-Co (530.27 eV) ( Figure 3B) [26]. The recognized three spectral peaks of C1s identify the bonds of C=O (287.58 eV), C-O (285.78 eV), and C-C (284.3 eV) ( Figure 3C) [41]. The two clear peaks observed in the N1s spectra imply the bonds of C-N (399.47 eV) and N-Zn (397.39 eV) ( Figure 3D). The spectra of zinc show the presence of Zn with two binding energies at 1021.5 eV (Zn 2p3/2) and 1044.6 eV (Zn 2p1/2) ( Figure 3E) [42]. Moreover, the clear peaks at 780.6 eV (785.3 eV satellite peak) and 796.46 eV (802.67 eV satellites peak) identify the state of the doped cobalt as Co 2p3/2 and Co 2p1/2, respectively ( Figure 3F) [43]. The chemical structures identified based on the reported chemical groups significantly matched the elemental compositions according to the EDX spectra. The EDX spectrum of ZnO/BMG confirmed the presence of Zn (24.56%), O (40.9%), and ZnO and C mixture (34.54%) as the main components of the algae. The EDX spectrum of Co-ZnO/BMG confirmed the presence of Zn (22.16%), O (39.4%), and Co (3.2%) in the composition of Co-doped ZnO and C (35.24%) as the main composition of the algae. The XPS spectrum of the synthesized Co-ZnO/BMG particles was investigated to confirm the doping process, integration reactions, and oxidation states ( Figure 3). The full scan spectrum confirmed the elemental composition of the composite, which contained N, C, Co, Zn, and O ( Figure 3A). The three clear peaks in the O1s spectra indicate surface oxygen (532.12 eV), O-Zn (530.96 eV), and O-Co (530.27 eV) ( Figure 3B) [26]. The recognized three spectral peaks of C1s identify the bonds of C=O (287.58 eV), C-O (285.78 eV), and C-C (284.3 eV) ( Figure 3C) [41]. The two clear peaks observed in the N1s spectra imply the bonds of C-N (399.47 eV) and N-Zn (397.39 eV) ( Figure 3D). The spectra of zinc show the presence of Zn with two binding energies at 1021.5 eV (Zn 2p 3/2 ) and 1044.6 eV (Zn 2p 1/2 ) ( Figure 3E) [42]. Moreover, the clear peaks at 780.6 eV (785.3 eV satellite peak) and 796.46 eV (802.67 eV satellites peak) identify the state of the doped cobalt as Co 2p 3/2 and Co 2p 1/2 , respectively ( Figure 3F) [43]. SEM and HRTEM analyses reflected the successful decoration of algae with synthetic ZnO and Co-ZnO nanoparticles ( Figure 4). The algal particles used as substrates appeared as massive compacted particles without definite outlines, which are common for natural particles in both the SEM ( Figure 4A) and HRTEM ( Figure 4B) images. Regarding the synthetic ZnO/BMG and Co-ZnO/BMG structures, the surface of the massive algal particles in the SEM images appeared to be highly decorated or ornamented with nanoparticles of semi-spherical forms related to the loaded ZnO ( Figure 4C,D) and Co-ZnO grains ( Figure 4E,F). The HRTEM images also confirmed the SEM observations and indicated the significant impedance of the synthetic ZnO ( Figure 4D) and Co-ZnO grains ( Figure 4F) as nanoparticles within the matrix of the incorporated particles of brown macroalgae as a substrate. The determined surface areas of BMG, ZnO/BMG, and Co-ZnO/BMG particles were 5.4, 11.3, and 10.4 m 2 /g, respectively.  SEM and HRTEM analyses reflected the successful decoration of algae with synthetic ZnO and Co-ZnO nanoparticles ( Figure 4). The algal particles used as substrates appeared as massive compacted particles without definite outlines, which are common for natural particles in both the SEM ( Figure 4A) and HRTEM ( Figure 4B) images. Regarding the synthetic ZnO/BMG and Co-ZnO/BMG structures, the surface of the massive algal particles in the SEM images appeared to be highly decorated or ornamented with nanoparticles of semi-spherical forms related to the loaded ZnO ( Figure 4C,D) and Co-ZnO grains (Figure 4E,F). The HRTEM images also confirmed the SEM observations and indicated the significant impedance of the synthetic ZnO ( Figure 4D) and Co-ZnO grains ( Figure 4F) as nanoparticles within the matrix of the incorporated particles of brown macroalgae as a substrate. The determined surface areas of BMG, ZnO/BMG, and Co-ZnO/BMG particles were 5.4, 11.3, and 10.4 m 2 /g, respectively.

Nitric Oxide Scavenging
The extensive generation of reactive oxygen, especially during aerobic respiration and electron transportation, causes notable oxidative stress and numerous degenerative diseases [44]. Among these reactive species, the gaseous free nitric oxide radical (NOR) and its irregular generation may be associated with DNA fragmentation, cell cytotoxicity, neuronal cell death, and cell damage [6,45,46]. Metal oxides commonly used as antioxidant agents have demonstrated significant NOR scavenging properties. The scavenging activity of the synthetic ZnO nanoparticle against NOR was notably enhanced after cobalt doping and after the application of BMG particles as a substrate ( Figure 5A). The estimated scavenging percentage of BMG, commercial ZnO (C.ZnO), free synthetic ZnO (S.ZnO), and Co-ZnO for NIR were 22.2 ± 1.3%, 35.7 ± 1.5%, 36.8 ± 1.4%, and 54.7 ± 1.7%, respectively ( Figure 5A). Therefore, the cobalt doping process increased the scavenging activity of S. ZnO by approximately 17.9%, which is 19% higher than that of C. ZnO and matches the previously reported results in the literature ( Figure 5A) [25,47]. This was attributed to the reported surface electrons of the Co-ZnO nanoparticles, which effectively pair with the lone pairs of hydroxyl radicals [26,47]. Electron-supplying capacity is an essential controlling factor that significantly affects the antioxidant properties of metal oxides [48]. Therefore, the incorporation of Co into the synthetic ZnO structure increased the generation efficiency of surface free electrons, which improved the free radical scavenging performance.
The incorporation of the BMG substrate forming ZnO/BMG and Co-ZnO/BMG composites resulted in remarkable enhancement in the NOR scavenging activity to 57.2 ± 1.4% and 90.2 ± 1.3%, respectively ( Figure 5A). The reported activities of ZnO/BMG and Co-ZnO/BMG were significantly higher than those of free ZnO and Co-ZnO, respectively. The detected enhancement effect of the BMG substrate was attributed to its positive impact on the exposure and interactive interfaces of the loaded ZnO nanoparticles, in

Nitric Oxide Scavenging
The extensive generation of reactive oxygen, especially during aerobic respiration and electron transportation, causes notable oxidative stress and numerous degenerative diseases [44]. Among these reactive species, the gaseous free nitric oxide radical (NOR) and its irregular generation may be associated with DNA fragmentation, cell cytotoxicity, neuronal cell death, and cell damage [6,45,46]. Metal oxides commonly used as antioxidant agents have demonstrated significant NOR scavenging properties. The scavenging activity of the synthetic ZnO nanoparticle against NOR was notably enhanced after cobalt doping and after the application of BMG particles as a substrate ( Figure 5A). The estimated scavenging percentage of BMG, commercial ZnO (C.ZnO), free synthetic ZnO (S.ZnO), and Co-ZnO for NIR were 22.2 ± 1.3%, 35.7 ± 1.5%, 36.8 ± 1.4%, and 54.7 ± 1.7%, respectively ( Figure 5A). Therefore, the cobalt doping process increased the scavenging activity of S. ZnO by approximately 17.9%, which is 19% higher than that of C. ZnO and matches the previously reported results in the literature ( Figure 5A) [25,47]. This was attributed to the reported surface electrons of the Co-ZnO nanoparticles, which effectively pair with the lone pairs of hydroxyl radicals [26,47]. Electron-supplying capacity is an essential controlling factor that significantly affects the antioxidant properties of metal oxides [48]. Therefore, the incorporation of Co into the synthetic ZnO structure increased the generation efficiency of surface free electrons, which improved the free radical scavenging performance.
The incorporation of the BMG substrate forming ZnO/BMG and Co-ZnO/BMG composites resulted in remarkable enhancement in the NOR scavenging activity to 57.2 ± 1.4% and 90.2 ± 1.3%, respectively ( Figure 5A). The reported activities of ZnO/BMG and Co-ZnO/BMG were significantly higher than those of free ZnO and Co-ZnO, respectively. The detected enhancement effect of the BMG substrate was attributed to its positive impact on the exposure and interactive interfaces of the loaded ZnO nanoparticles, in addition to its reduction effect on the agglomeration affinities between the supported particles [21].
Moreover, the chemical compounds of brown macroalgae exhibit notable antioxidant activity, which increases the interaction rates with free active oxidizing species. Therefore, the synthetic Co-ZnO/BMG composite can be used as a more effective antioxidant against NOR compared to the separated phases of ZnO, Co-ZnO, and BMG as well as the ascorbic acid control (21.6 ± 1.3%) ( Figure 5A).

Porcine Pancreatic α-Amylase Inhibition Assay
The anti-diabetic activities of BMG, S.ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG were assessed in synergetic studies to determine their inhibitory effects on α-amylase. The α-amylase enzyme is an essential and dominant digestive enzyme that causes significant breakdown of long chains of carbohydrate polymers, such as starch, into simple forms, such as maltose, which also immediately transforms into glucose species [6]. Therefore, the synthetic structures that display significant and rapid inhibition against the α-amylase enzyme will cause a considerable decline in the breakdown rates of the complex carbohydrates and, in turn, slow the absorption of dietary starches and limit spikes in blood sugar levels [54]. Therefore, post-prandial hyperglycemia in diabetes can

DPPH Radical Scavenging
DPPH scavenging by ZnO/BMG and Co-ZnO/BMG was tracked in synergetic tests and compared to that of BMG, C.ZnO, S.ZnO, and Co-ZnO. These marked behaviors matched the properties observed during the scavenging reactions of NOR ( Figure 5B). The DPPH scavenging percentage determined using Co-ZnO/BMG was 89.6 ± 1.5% which was clearly higher than the recognized efficiency using ZnO/BMG (66.4 ± 1.8%) by approximately 23.2% ( Figure 5B). Based on the recognized activities by BMG (36.7 ± 1.66%), C.ZnO (41.7 ± 1.6%), S.ZnO (43.2 ± 1.2%), and Co-ZnO (70.3 ± 1.1%), the cobalt doping modification in combination with the incorporation of the BMG substrate strongly enhanced the scavenging activity of ZnO for the DPPH radical which also is higher than the activity of ascorbic acid standard (76.3 ± 1.3%) ( Figure 5B). The scavenging of the DPPH radical by the surfaces of synthetic metal oxides occurs via electron (e − )/proton (H + ) transfer reactions towards the organic framework structure of the DPPH radical [49,50]. The doped cobalt ions within the ZnO crystals induce the generation rate of surface electrons and charge separation efficiency, which enhances the activity of Co-ZnO as compared to ZnO. In addition, the negatively charged groups of the BMG chemical compounds might accelerate the charge separation reactions and enhance the effect of the algal substrate on the interactive interfaces between the DPPH radical and bioactive ZnO structures [51].

ABTS Radical Scavenging
ABTS scavenging tests have been used as an effective indicator of the antioxidant potential of synthetic materials, especially composites or hybrid structures, based on the actual decline in the concentration of the diffused ABTS cation radical (ABTS •+ ). Synthetic structures exhibiting effective hydrogen-donating antioxidant mechanisms can be used as scavengers of ABTS •+ radicals. Therefore, the synthetic metal oxides either as pure forms or modified products by doping reactions or hybridization by the suitable substrate are highly endorsed as scavengers for ABTS •+ . The detected ABTS •+ scavenging percentage of BMG (33.4 ± 1.8%), C.ZnO (36.4 ± 1.4%), S.ZnO (38.3 ± 1.3%), Co-ZnO (65.4 ± 1.8%), and ZnO/BMG (60.3 ± 1.77%) was lower than the value detected for the ascorbic acid control (75.4 ± 1.1%) ( Figure 5C). Impeding the Co-doped ZnO nanoparticles into the matrix of the BMG (Co-ZnO/BMG) resulted in a significant enhancement in its scavenging activity for ABTS •+ radical to 87.7 ± 1.8%, i.e., higher than the recognized activity of ascorbic acid ( Figure 5C).

Superoxide Radical Scavenging
The superoxide anion (O 2 •− ) is a reactive oxygen radical generated within the cellular organelles such as mitochondria transforms immediately into other oxidizing species such as hydroxyl radicals ( • OH) and H 2 O 2 . The uncontrolled and non-regulated generation of O 2 •− and its transformed products cause severe pathophysiological conditions in addition to numerous hazardous health risks and degenerative diseases, including the cellular destruction of DNA, RNA, and protein [52,53]. Generally, the biological system of the human body has natural defenses against the diffused O 2 •− as well as its transformed products and their oxidative stresses to maintain safe physiological homeostasis. However, various diseases have adverse effects on the biological responses in human organs during the generation of defense functions, depending on the type or the quantities required to control radical levels. Therefore, the effective scavenging of O 2 •− by the innovative and biocompatible multifunctional structures has been strongly recommended. The determined BMG scavenging percentage for O 2 •− (13.4 ± 1.8%), C.ZnO (8.7 ± 1.2%), S.ZnO (12.7 ± 1.5%), and ZnO/BMG (16.8 ± 1.3%) were lower than the reported scavenging efficiency of the ascorbic acid control (17.3 ± 1.3%) ( Figure 5D). However, the cobalt-doped ZnO (Co-ZnO) (22.4 ± 1.4%) and its integrated products with BMG (Co-ZnO/BMG) (46.7 ± 1.9%) showed significantly higher activity than the commercially used ascorbic acid ( Figure 5D).

Porcine Pancreatic α-Amylase Inhibition Assay
The anti-diabetic activities of BMG, S.ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG were assessed in synergetic studies to determine their inhibitory effects on α-amylase. The α-amylase enzyme is an essential and dominant digestive enzyme that causes significant breakdown of long chains of carbohydrate polymers, such as starch, into simple forms, such as maltose, which also immediately transforms into glucose species [6]. Therefore, the synthetic structures that display significant and rapid inhibition against the α-amylase enzyme will cause a considerable decline in the breakdown rates of the complex carbohydrates and, in turn, slow the absorption of dietary starches and limit spikes in blood sugar levels [54]. Therefore, post-prandial hyperglycemia in diabetes can be safely and significantly controlled [24]. The α-amylase enzyme inhibition percentage by Co-ZnO (78.5 ± 1.7%) and Co-ZnO/BMG (90.6 ± 1.5%) demonstrates remarkable anti-diabetic activity as compared to the commercial drugs used as standards (acarbose (75.2 ± 1.7%) and miglitol (18.3 ± 1.4%)) ( Figure 6A). In addition, the prepared S.ZnO (44.8 ± 1.5%) and ZnO/BMG structures (61.9 ± 1.8%) exhibited enhanced inhibition effects on the αamylase enzyme as compared to BMG (38.6 ± 1.8%), C.ZnO (40.3 ± 1.6%), and miglitol (18.3 ± 1.4%) ( Figure 6A). The reported enhancement in the anti-diabetic activity of the prepared ZnO was attributed to cobalt-doping functionalization and the incorporated BMG substrate. The BMG substrate effectively reduced the agglomeration potential of synthetic ZnO and Co-ZnO on its surface, which enhanced the exposure and interactive interfaces of their particles with enzymes [14,47,54,55]. The agglomeration and aggregation of metal oxides adversely affect their biological activities and inhibit oxidative enzymes [47]. Based on the inhibitory activity of Co-ZnO and Co-ZnO/BMG on the porcine pancreatic α-amylase enzyme, they can be recommended as enhanced, low-cost, effective, and safe anti-diabetic agents compared with expensive commercial drugs that commonly lead to side effects [56,57].

Murine Pancreatic α-Amylase Inhibition
The inhibitory activities of BMG, S.ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG on murine pancreatic α-amylase enzyme were assessed to determine the suitability of these products to inhibit the activity of metabolically crude active enzymes. Inhibition tests were performed using a synergetic design, considering the impact of cobalt doping and the incorporated BMG substrate. The synthetic Co-ZnO and Co-ZnO/BMG inhibited the activity of α-amylase enzyme (crude active enzyme) by 68.7 ± 1.9% and 84.3 ± 1.8%, respectively ( Figure 6B). This demonstrates their higher anti-diabetic activities as compared to acarbose (61.4 ± 1.5%) and miglitol (11.2 ± 1.6%), commonly used commercial drugs and controls ( Figure 6B). Additionally, the prepared S.ZnO (38.5 ± 1.87%) and ZnO/BMG structures (54.4 ± 1.9%) exhibited enhanced inhibition effects on the α-amylase enzyme compared to BMG (29.2 ± 1.1%), C.ZnO (9.8 ± 1.1%), and miglitol (18.3 ± 1.4%) ( Figure  6B). These experimental findings reflect the significant potential of Co-ZnO and Co-ZnO/BMG as enhanced and effective anti-diabetic agents against the commercial α-amylase enzyme and the metabolically crude active enzyme.

Murine Pancreatic α-Amylase Inhibition
The inhibitory activities of BMG, S.ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG on murine pancreatic α-amylase enzyme were assessed to determine the suitability of these products to inhibit the activity of metabolically crude active enzymes. Inhibition tests were performed using a synergetic design, considering the impact of cobalt doping and the incorporated BMG substrate. The synthetic Co-ZnO and Co-ZnO/BMG inhibited the activity of α-amylase enzyme (crude active enzyme) by 68.7 ± 1.9% and 84.3 ± 1.8%, respectively ( Figure 6B). This demonstrates their higher anti-diabetic activities as compared to acarbose (61.4 ± 1.5%) and miglitol (11.2 ± 1.6%), commonly used commercial drugs and controls ( Figure 6B). Additionally, the prepared S.ZnO (38.5 ± 1.87%) and ZnO/BMG structures (54.4 ± 1.9%) exhibited enhanced inhibition effects on the α-amylase enzyme compared to BMG (29.2 ± 1.1%), C.ZnO (9.8 ± 1.1%), and miglitol (18.3 ± 1.4%) ( Figure 6B). These experimental findings reflect the significant potential of Co-ZnO and Co-ZnO/BMG as enhanced and effective anti-diabetic agents against the commercial α-amylase enzyme and the metabolically crude active enzyme.

Pancreatic α-Glucosidase Inhibition
The inhibitory effects of BMG, S.ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG on α-Glucosidase enzyme were followed to control its level as an essential and highly effective enzyme during the metabolic reactions of dietary starch and carbohydrates. Therefore, the development of effective inhibition gents against the α-Glucosidase enzyme can regulate and control the absorption rates of glucose compounds into the blood and significantly decrease hyperglycemia [16]. The inhibition activities of Co-ZnO (82.6 ± 1.6%) and Co-ZnO/BMG (95.7 ± 1.4%) against the pancreatic α-Glucosidase enzyme suggest promising anti-diabetic potential of the Co-ZnO/BMG compared with that of miglitol (90.2 ± 1.3%) and the separate phase of ZnO and BMG investigated in this study ( Figure 7A). Moreover, the detected inhibition percentage is close to the determined effect of acarbose (96.4 ± 1.4%) ( Figure 7A). The assessed S.ZnO (70.8 ± 1.5%) and ZnO/BMG structures (81.4 ± 1.3%) exhibited enhanced inhibition effects compared to BMG (34.3 ± 1.6%) and C.ZnO (68.7 ± 1.4%) ( Figure 7A). These previously reported behaviors agree with the results presented in the literature regarding the synergetic effect of transition metal doping processes and the application of an effective substrate on the bioactivity of ZnO, either as an antioxidant or an anti-diabetic agent.

Pancreatic α-Glucosidase Inhibition
The inhibitory effects of BMG, S.ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG on α-Glucosidase enzyme were followed to control its level as an essential and highly effective enzyme during the metabolic reactions of dietary starch and carbohydrates. Therefore, the development of effective inhibition gents against the α-Glucosidase enzyme can regulate and control the absorption rates of glucose compounds into the blood and significantly decrease hyperglycemia [16]. The inhibition activities of Co-ZnO (82.6 ± 1.6%) and Co-ZnO/BMG (95.7 ± 1.4%) against the pancreatic α-Glucosidase enzyme suggest promising anti-diabetic potential of the Co-ZnO/BMG compared with that of miglitol (90.2 ± 1.3%) and the separate phase of ZnO and BMG investigated in this study ( Figure  7A). Moreover, the detected inhibition percentage is close to the determined effect of acarbose (96.4 ± 1.4%) ( Figure 7A). The assessed S.ZnO (70.8 ± 1.5%) and ZnO/BMG structures (81.4 ± 1.3%) exhibited enhanced inhibition effects compared to BMG (34.3 ± 1.6%) and C.ZnO (68.7 ± 1.4%) ( Figure 7A). These previously reported behaviors agree with the results presented in the literature regarding the synergetic effect of transition metal doping processes and the application of an effective substrate on the bioactivity of ZnO, either as an antioxidant or an anti-diabetic agent.

Murine Intestinal α-Glucosidase Inhibition
The inhibitory effects of BMG, S.ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG on the active crude intestinal α-Glucosidase enzyme were evaluated in addition to the previously tested commercial forms or more realistic investigations. The recognized results show notable inhibition influences of both Co-ZnO (80.2 ± 1.6%) and Co-ZnO/BMG (93.4 ± 1.8%) on the crude intestinal α-Glucosidase enzyme ( Figure 7B). The activity of Co-ZnO/BMG was significantly higher than the determined effects of miglitol (88.6 ± 1.4%) and close to the determined activity of acarbose (94.6 ± 1.34%) drug ( Figure 7B). The synthetic S.ZnO (65.6 ± 1.2%) and ZnO/BMG structures (78.8 ± 1.3%) exhibited enhanced inhibition effects as compared to BMG (30.6 ± 1.2%) and C.ZnO (58.7 ± 1.4%) ( Figure 7B). The previously determined activities of synthetic ZnO and its composite with BMG (ZnO/BMG) were adequate based on the reported adverse side effects of common drugs as well as their high production costs. Generally, ZnO nanoparticles ex-

Murine Intestinal α-Glucosidase Inhibition
The inhibitory effects of BMG, S.ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG on the active crude intestinal α-Glucosidase enzyme were evaluated in addition to the previously tested commercial forms or more realistic investigations. The recognized results show notable inhibition influences of both Co-ZnO (80.2 ± 1.6%) and Co-ZnO/BMG (93.4 ± 1.8%) on the crude intestinal α-Glucosidase enzyme ( Figure 7B). The activity of Co-ZnO/BMG was significantly higher than the determined effects of miglitol (88.6 ± 1.4%) and close to the determined activity of acarbose (94.6 ± 1.34%) drug ( Figure 7B). The synthetic S.ZnO (65.6 ± 1.2%) and ZnO/BMG structures (78.8 ± 1.3%) exhibited enhanced inhibition effects as compared to BMG (30.6 ± 1.2%) and C.ZnO (58.7 ± 1.4%) ( Figure 7B). The previously determined activities of synthetic ZnO and its composite with BMG (ZnO/BMG) were adequate based on the reported adverse side effects of common drugs as well as their high production costs. Generally, ZnO nanoparticles exhibit promising anti-diabetic activity and contribute to considerably reducing blood glucose levels. Moreover, synthetic ZnO nanoparticles demonstrated a remarkable enhancement of insulin receptors, glucokinase activity, glucokinase genes, and serum insulin [58].

Amyloglucosidase Inhibition
The amyloglucosidase enzyme is also very effective during the breakdown of complex carbohydrates as well as the absorption of the obtained simple compounds. Therefore, inhibition of amyloglucosidases by safe, effective, and low-cost synthetic compounds significantly reduces the transformation performance of complex sugar compounds [59]. The measured amyloglucosidase inhibition percentage using BMG, C.ZnO, S.ZnO, ZnO/BMG, Co-ZnO, and Co-ZnO/BMG was 51.3 ± 1.3%, 65.4 ± 1.3%, 68.3 ± 1.8%, 80.3 ± 1.2%, 86.4 ± 1.2%, and 96.2 ± 1.4%, respectively ( Figure 8). However, all the investigated structures (BMG, S.ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG) exhibited considerable inhibition effects on the amyloglucosidase enzyme, but only the Co-ZnO/BMG multifunctional composite had higher activity than that of miglitol (88.3 ± 1.8%) and acarbose (95.6 ± 1.7%) (Figure 8). Therefore, a multifunctional composite that involves doping ZnO with cobalt over a BMG substrate (Co-ZnO/BMG) is recommended as an enhanced anti-diabetic agent that is characterized by low production cost, high biocompatibility, few side effects, and strong inhibition of common oxidative enzymes.

Preparation of Incorporated Brown Macroalgae (BMG)
The collected brown macroalgal (Sargassum) samples were washed thoroughly with distilled water for four cycles of approximately 15 min to ensure effective removal of the attached salts and associated debris. The washed biomass products were then dried gently in an oven for approximately 24 h at 50 • C to ensure complete dehydration. Afterward, the dehydrated products were ground into smaller pieces using a domestic handheld blender and then transferred into a ball mill (Planetary Ball Mill PM 400) to obtain microfractions of the algae. Then, the microfractions were sieved, and the obtained fractions within the range from 50 to 200 µm (95% < 180, 75% < 120, 50% < 100, and 25% < 60 µm) were used in the further experimental procedure.

Synthesis of ZnO and Co-ZnO Composites with BMG (ZnO/BMG and Co-ZnO/BMG)
The synthesis of ZnO and Co-doped ZnO, both as separated phases or as composites with BMG, was performed using the microwave hydrothermal-assessed precipitation method. Approximately 0.153 g of the main Zn precursor (zinc nitrate salt) was dissolved in 40 mL of distilled water in a Teflon autoclave as a separate synthesis mixture, while another synthesis system was prepared by co-dissolving the zinc-bearing salt (Zn(NO 3 ) 2 ·6H 2 O), and a cobalt-bearing salt (Co(NO 3 ) 2 ·6H 2 O) in a Teflon-lined autoclave. The dissolved quantities were adjusted to obtain 4 wt % of both ZnO and Co-ZnO, and the doped parent cobalt was 1 wt %. After the homogenization period reached 60 min, the systems were treated with ammonium hydroxide solution (1 M), which was added gently to separate the synthesis mixtures with regular detection of the solution pH until reaching a pH of 10.5. Subsequently, the two mixtures were stirred for an additional 120 min before being transferred to round-bottom flasks. The flasks were subjected to domestic microwave irradiation at an adjusted power of 700 W for 15 min. Then, the synthesized ZnO and Co-ZnO solid particles were extracted, washed using distilled water to get rid of the residual ammonium ions, and dried overnight at 60 • C. The synthesis of the ZnO/BMG and Co-ZnO/BMG composites involved the incorporation of algae (1 g) during the starting step of the production process. The ground fractions of the algae were dispersed and homogenized within the precursor solutions of zinc nitrate, and the complex zinc nitrate/cobalt nitrate in two individual synthesis mixtures, and they were mixed using a magnetic stirrer for 120 min. Subsequently, the two mixtures were subjected to the same synthesis procedures as those used for pure ZnO and Co-ZnO, including the synthesis pH (10.5), microwave irradiation (700 W; 15 min), washing, and drying stages (12 h; 60 • C). Finally, all products were kept in specific containers, labeled, and used in characterization and biological assay tests. The synthesis procedure is illustrated in Figure 9.

Characterization Techniques
The formed crystal phases and their structural properties were assessed based on the X-ray diffraction patterns of these materials obtained using PANalytical XRD diffractometer (Empyrean) with a Cu-Ká radiation source considering the measuring range between 5° and 80°. The chemical composition was assessed based on the EDX elemental composition using the energy-dispersive X-ray (EDX) technique and the X-ray photoelectron spectra, which were determined by ESCALAB MK II spectrometer with Al anode X-ray exciting source (Al Ká = 1487.6 eV). In addition, the essential chemical functional groups were identified from the Fourier-transform infrared (FT-IR) spectra of the studied

Characterization Techniques
The formed crystal phases and their structural properties were assessed based on the X-ray diffraction patterns of these materials obtained using PANalytical XRD diffractometer (Empyrean) with a Cu-Ká radiation source considering the measuring range between 5 • and 80 • . The chemical composition was assessed based on the EDX elemental composition using the energy-dispersive X-ray (EDX) technique and the X-ray photoelectron spectra, which were determined by ESCALAB MK II spectrometer with Al anode X-ray exciting source (Al Ká = 1487.6 eV). In addition, the essential chemical functional groups were identified from the Fourier-transform infrared (FT-IR) spectra of the studied materials, which were obtained using a Fourier Transform Infrared spectrometer (FTIR−8400S). The surface forms of the synthesized structures and their general morphologies were described based on their SEM images, which were obtained using a scanning electron microscope (Gemini-Zeiss, Ultra 55). The internal features and nature of the integration processes were evaluated based on the HRTEM images of the synthetic materials, which were obtained using a transmission electron microscope (JEOL-JEM, 2100). In addition to the porous properties, the texture of the surface areas was determined using a Beckman Coulter surface area analyzer (SA3100 type) based on the N 2 adsorption/desorption curves of the materials.

Scavenging of Nitric Oxide Radical (NOR)
The scavenger potentialities of ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG for NOR radicals were determined using an assay described by Kitture et al. [60]. All assessed materials were mixed separately with sodium nitroprusside (2 mL; 10 mM) in pre-prepared phosphate buffer solutions with pH 7.4 (500 µL). The obtained mixtures were incubated separately at 25 • C for 150 min. After incubation, the mixtures were mixed with 500 mL of sulphanilic acid (1 M) and then incubated for another short period (5 min). The mixture was then mixed with naphthyl ethylenediamine dihydrochloride (0.1% w/v, 1 mL) and incubated for 30 min. Finally, the absorbance of the tested mixtures was determined using a microplate reader at 540 nm and compared with that of the control solutions, and the results were used to calculate the scavenging percentage using Equation (1).

Scavenging of the DPPH Radical
The scavenging assay described by Robkhob et al. [6] was used to evaluate the potentialities of ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG as scavengers for the DPPH radical. The prepared materials (100 µg/mL; 20 µL) were mixed separately with 80 µL of methanolic solutions, which were supplemented with the tested DPPH radicals (100 µM) using specific 96 well plates. Then, the obtained mixtures were incubated separately for 20 min in the dark, and the absorbance of tested mixtures was determined by a microplate reader at 517 nm and compared to that of the control solutions and the results were used to calculate the scavenging percentage using Equation (2).

Scavenging of ABTS Radical
The scavenging assay described by Dappula et al. [49] was used to evaluate the potentialities of ZnO, Co.ZnO, ZnO/BMG, and Co-ZnO/BMG as scavengers for ABTS radical. The ABTS solutions used during the tests were prepared by simple dissolution of the ABTS compound (44 mg) in deionized water (10 mL). These solutions were supplemented separately with potassium persulfate (3 mM) as an essential step to generate free cations (ABTS •+ ) of the ABTS radical. This reaction was conducted in the dark at 25 • C for up to 18 h. Then, the reaction mixtures were diluted with methanol at a ratio of 1:29 to obtain fresh ABTS •+ . Subsequently, the synthetic materials were mixed separately (100 µg/mL) with the previously prepared ABTS solutions (290 µL), and the mixtures were kept for 30 min. The absorbance of the tested mixtures was determined using a microplate reader at 734 nm and compared to that of the control solutions, and the results were used to calculate the scavenging percentage using Equation (3).

Scavenging of Superoxide Radical
The scavenging assay described by Robkhob et al. [6] was applied to evaluate the potentialities of ZnO, Co.ZnO, ZnO/BMG, and Co-ZnO/BMG as scavengers for superoxide radical (O •− ). The prepared materials (100 µL) were mixed separately with previously prepared mixtures of EDTA (200 µL; 12 mM), ethanol (200 µL), riboflavin (100 µL; 20 µg), and NBT (100 µL; 0.1 mg). The products of the previous step were supplemented separately with a phosphate buffer solution (3 mL) and exposed to a light source for approximately 5 min. Subsequently, the absorbance of the tested mixtures was determined using a microplate reader at 540 nm and compared with that of the control solutions, and the results were used to calculate the scavenging percentage using Equation (1).

Inhibition Assay of Porcine Pancreatic α-Amylase
The inhibition assay described by Robkhob et al. [6] was used to evaluate the potential of ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG as inhibition agents against the commonly tested commercial pancreatic α-amylase enzyme. The prepared materials were mixed separately at 100 µg/mL with the investigated α-amylase enzyme (50 µg/mL), and the obtained mixtures were subjected to an incubation process for 10 min at 37 • C. Afterward, the mixtures were supplemented with a starch substrate (1%), and the absorbance was determined by a microplate reader at 540 nm and compared with that of the control solutions, and the results were used to calculate the inhibition percentage using Equation (4).

Inhibition Assay of Crude Murine Pancreatic α-Amylase
This assay was performed to ensure the suitability of ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG to inhibit crude active enzymes in comparison with commercially available enzymes. Crude enzymes were extracted from the pancreas of a 10-week-old Swiss male mouse. The mouse was subjected to starvation for approximately 12 h. Then, the starved pancreas was excised and immersed carefully in a phosphate buffer solution containing protease inhibitors. Next, the cell-free supernatant was extracted by a fast centrifugation process (15 min; 10,000 rpm), and the mixture was diluted regularly until the detection of 0.4 by the microplate reader at a wavelength of 280 nm. Under these conditions, the pancreas can be used as a source of the active crude enzyme, and the inhibition assay of the synthetic materials was performed following the procedures described in Section 3.5.1.

Inhibition Assay of α-Glucosidase
The inhibition assay described Sanap et al. [61] was used to evaluate the potentialities of ZnO, Co.ZnO, ZnO/BMG, and Co-ZnO/BMG as inhibition agents against the commercial form of the α-Glucosidase enzyme. The prepared materials were mixed separately at 100 µg/mL with the investigated α-Glucosidase enzyme (100 µL; 0.1 unit/mL), and the resulting mixtures were incubated for 60 min at 37 • C. Subsequently, the mixtures were supplemented with pNPG (10 mL) before re-incubation for an additional 10 min. Then, the Na 2 CO 3 solution (0.1 M; 2 mL) was added to the mixtures to stop the reactions. Finally, the absorbance of the nitrophenol released from pNPG within the investigated mixtures was determined using a microplate reader at 420 nm and compared with that of the control solutions, and the results were used to calculate the inhibition percentage using Equation (5).

Inhibition Assay of Crude Murine Intestinal α-Glucosidase
The crude intestinal α-Glucosidase enzyme was isolated according to the previously reported procedure during the extraction of the crude α-amylase enzyme in Section 3.5.2. The inhibition assays of ZnO, Co.ZnO, ZnO/BMG, and Co-ZnO/BMG against the crude intestinal α-Glucosidase enzyme were performed following the same procedures described in Section 3.5.3 incorporating p-nitrophenyl-α-D-glucopyranoside as substrate.

Amyloglucosidase Inhibition Assay
The inhibition assay described by Lawande et al. [62] was used to evaluate the potential of ZnO, Co-ZnO, ZnO/BMG, and Co-ZnO/BMG as inhibitors of the amyloglucosidase enzyme. The prepared materials were mixed separately at 100 µg g/mL with the investigated amyloglucosidase enzyme (0.1 unit/mL), and the resulting mixtures were incubated for 10 min at 37 • C with 1% of starch substrate. Subsequently, the absorbance of the mixtures was determined using a microplate reader at 540 nm and compared with that of the control solutions, and the results were used to calculate the scavenging percentage using Equation (4).