Bioactivity Guided Study for the Isolation and Identification of Antidiabetic Compounds from Edible Seaweed—Ulva reticulata

Managing diabetes is challenging due to the complex physiology of the disease and the numerous complications associated with it. As part of the ongoing search for antidiabetic chemicals, marine algae have been demonstrated to be an excellent source due to their medicinal properties. In this study, Ulva reticulata extracts were investigated for their anti-diabetic effect by examining its inhibitory effects on α-amylase, α-glucosidase, and DPP-IV and antioxidant (DPPH) potential in vitro and its purified fraction using animal models. Among the various solvents used, the Methanolic extract of Ulva reticulata (MEUR) displayed the highest antidiabetic activity in both in vitro and in vivo; it showed no cytotoxicity and hence was subjected to bioassay-guided chromatographic separation. Among the seven isolated fractions (F1 to F7), the F4 (chloroform) fraction exhibited substantial total phenolic content (65.19 μg mL−1) and total flavonoid content (20.33 μg mL−1), which showed the promising inhibition against α-amylase (71.67%) and α-glucosidase (38.01%). Active fraction (F4) was further purified using column chromatography, subjected to thin-layer chromatography (TLC), and characterized by spectroscopy techniques. Upon structural elucidation, five distinct compounds, namely, Nonane, Hexadecanoic acid, 1-dodecanol, Cyclodecane methyl, and phenol, phenol, 3,5-bis(1,1-dimethylethyl) were identified. The antidiabetic mechanism of active fraction (F4) was further investigated using various in vitro and in vivo models. The results displayed that in in vitro both 1 and 24 h in vitro cultures, the active fraction (F4) at a concentration of 100 μg mL−1 demonstrated maximum glucose-induced insulin secretion at 4 mM (0.357 and 0.582 μg mL−1) and 20 mM (0.848 and 1.032 μg mL−1). The active fraction (F4) reduces blood glucose levels in normoglycaemic animals and produces effects similar to that of standard acarbose. Active fraction (F4) also demonstrated outstanding hypoglycaemic activity in hyperglycemic animals at a dose of 10 mg/kg B.wt. In the STZ-induced diabetic rat model, the active fraction (F4) showed a (61%) reduction in blood glucose level when compared to the standard drug glibenclamide (68%). The results indicate that the marine algae Ulva reticulata is a promising candidate for managing diabetes by inhibiting carbohydrate metabolizing enzymes and promoting insulin secretion.


Introduction
Diabetes mellitus (DM) is the most common metabolic disorder affecting various body organs and is associated with other complications such as coronary heart disease, stroke, liver damage, nephropathy, retinopathy, and peripheral neuropathy [1]. Since DM has become more prevalent in recent years, it has significantly impacted individual health [2]. The disease is characterized by hyperglycemia due to abnormalities in insulin production or action or a combined effect of both [3]. The reduction of postprandial hyperglycemia All the extracts were compared with standards acarbose, Diprotin A, and BHT, where n = 3 for each sample with S.E.M. The mean values were analyzed using two-way ANOVA (Tukey's multiple comparisons test). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05 considered as significant. Absorbance was measured at 540, 405, and 517 nm, respectively. All the extracts were compared with standards acarbose, Diprotin A, and BHT, where n = 3 for each sample with S.E.M. The mean values were analyzed using two-way ANOVA (Tukey's multiple comparisons test). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05 considered as significant.

Measurement of Cell Viability and In Vitro Hemolytic Activity of MEUR
The toxicological evaluation of MEUR was assessed by measuring the cell viability and hemolytic activity using mouse macrophage cells (J774) and human red blood cells, respectively. The results showed that a lower concentration of 250 µg/mL MEUR showed maximum viability (99%) for 24 h (Figure 2a). Similarly, MEUR did not show any hemolysis or erythrocyte membrane damage against human red blood cells at all the tested concentrations. There was only 6.92% cell lysis at 1000 µg/mL, and at a concentration of 250 µg/mL, MEUR showed 2.81% lysis against human red blood cells (Figure 2b).

Measurement of Cell Viability and In Vitro Hemolytic Activity of MEUR
The toxicological evaluation of MEUR was assessed by measuring the cell viability and hemolytic activity using mouse macrophage cells (J774) and human red blood cells, respectively. The results showed that a lower concentration of 250 μg/mL MEUR showed maximum viability (99%) for 24 h (Figure 2a). Similarly, MEUR did not show any hemolysis or erythrocyte membrane damage against human red blood cells at all the tested concentrations. There was only 6.92% cell lysis at 1000 μg/mL, and at a concentration of 250 μg/mL, MEUR showed 2.81% lysis against human red blood cells (Figure 2b).

Effect of MEUR on STZ-Induced Diabetic Rats
We examined the hypoglycaemic effect of MEUR (250 mg kg −1 B.wt.) on STZ-induced diabetic rats (Table 1). Fasting blood glucose (FBG) level was measured at regular intervals (0, 7, 14, 21 & 28 days) in normal and diabetic rats treated with MEUR and standard antidiabetic drug glibenclamide (0.25 mg kg −1 B.wt.). The FBG level of untreated rats (diabetic control) remained significantly increasing (˃329 mg/dL) throughout the study. Oral administration of MEUR (46.21%) and glibenclamide (73.05%) in treated rats showed a notable reduction in the blood glucose level compared to the untreated diabetic rats.
The body weight of diabetic rats was reduced compared to normal rats; however, after administration of MEUR and glibenclamide for 28 days, their body weight increased compared to the untreated diabetic rats (Figure 3). There was also a change in serum parameters, including cholesterol, triglycerides, urea, and liver markers (ALT & AST) in STZ-induced diabetic rats (Table 2). After the administration of MEUR and glibenclamide, there was a significant reduction in the amount of cholesterol, triglycerides, ALT, and AST compared to untreated diabetic rats. Other parameters such as total protein, urea, albumin, and globulin were similar to normal control. Histopathology analysis reveals an extensive alteration in the morphology of the kidney, liver, and pancreas of diabetic rats

Effect of MEUR on STZ-Induced Diabetic Rats
We examined the hypoglycaemic effect of MEUR (250 mg kg −1 B.wt.) on STZ-induced diabetic rats (Table 1). Fasting blood glucose (FBG) level was measured at regular intervals (0, 7, 14, 21 & 28 days) in normal and diabetic rats treated with MEUR and standard antidiabetic drug glibenclamide (0.25 mg kg −1 B.wt.). The FBG level of untreated rats (diabetic control) remained significantly increasing (>329 mg/dL) throughout the study. Oral administration of MEUR (46.21%) and glibenclamide (73.05%) in treated rats showed a notable reduction in the blood glucose level compared to the untreated diabetic rats. The body weight of diabetic rats was reduced compared to normal rats; however, after administration of MEUR and glibenclamide for 28 days, their body weight increased compared to the untreated diabetic rats (Figure 3). There was also a change in serum parameters, including cholesterol, triglycerides, urea, and liver markers (ALT & AST) in STZ-induced diabetic rats (Table 2). After the administration of MEUR and glibenclamide, there was a significant reduction in the amount of cholesterol, triglycerides, ALT, and AST compared to untreated diabetic rats. Other parameters such as total protein, urea, albumin, and globulin were similar to normal control. Histopathology analysis reveals an extensive alteration in the morphology of the kidney, liver, and pancreas of diabetic rats ( Figure 4). The pathological changes observed in diabetic rats were retained to normal after administering MEUR and glibenclamide for 28 days.
( Figure 4). The pathological changes observed in diabetic rats were retained to normal after administering MEUR and glibenclamide for 28 days.
Among the 5 extracts, the methanolic extract of Ulva reticulata (MEUR), exhibiting the highest antidiabetic activity in both in vitro and in vivo studies, was subjected to chromatographic separation using a silica gel column (60-120 mesh).   The mean values were analyzed using two-way ANOVA (Tukey's multiple comparisons test). * p < 0.05 considered as significant.    Among the 5 extracts, the methanolic extract of Ulva reticulata (MEUR), exhibiting the highest antidiabetic activity in both in vitro and in vivo studies, was subjected to chromatographic separation using a silica gel column (60-120 mesh).

Determination of Total Phenols and Flavonoids
The total phenolic and flavonoid content of chloroform fraction (F4) of MEUR was estimated by using gallic acid (GA) and quercetin (Q) as standard (Figure 5b,c). The results showed that the total phenolic content (65.19 μg mL −1 ) was higher than the total flavonoid content (20.33 μg mL −1 ).

Purification of Compounds from the Active Fraction (F4)
The active fraction (F4) was further separated using a silica gel column (60-120 mesh) and subjected to thin-layer chromatography (TLC). Among the various solvents, petroleum ether: ethyl acetate in varying ratios yielded five individual compounds. The com- Figure 5. In vitro α-amylase, α-glucosidase inhibitory activity, and determination of total phenols and flavonoids of isolated fractions from MEUR. (a) In vitro α-amylase, α-glucosidase, (b) total phenols, and (c) flavonoids of various fractions of MEUR. Absorbance was measured at 540 nm (α-amylase) and 405 nm (α-glucosidase). Mean values were analyzed using two-way ANOVA (Tukey's multiple comparisons test). **** p < 0.0001 considered as significant.

Determination of Total Phenols and Flavonoids
The total phenolic and flavonoid content of chloroform fraction (F4) of MEUR was estimated by using gallic acid (GA) and quercetin (Q) as standard (Figure 5b,c). The results showed that the total phenolic content (65.19 µg mL −1 ) was higher than the total flavonoid content (20.33 µg mL −1 ).

Purification of Compounds from the Active Fraction (F4)
The active fraction (F4) was further separated using a silica gel column (60-120 mesh) and subjected to thin-layer chromatography (TLC). Among the various solvents, petroleum ether: ethyl acetate in varying ratios yielded five individual compounds. The compounds were purified and characterized using spectrometry using GC-MS, HR-MS, and NMR ( 13 C and 1 H NMR).

In Vitro Insulin Secretion Studies
The isolated pancreatic islets treated with the test compounds (nonane, hexadecanoic acid, phenol, phenol, 3,5-bis(1,1-dimethylethyl)) and an active fraction (F4) for a period of 1 and 24 h showed significant insulin secretion in both normal (4 mM) and diabetic (20 mM) condition (Figure 11a-d). The two compounds (1-dodecanol, cyclodecane methyl) did not show any effect against pancreatic islets. The results were compared with standard drugs acarbose and glibenclamide (Figure 11e

In Vivo Antidiabetic Mechanism of Active Fraction (F4)
Based on the in vitro results obtained from the active fraction (F4) against enzyme inhibition (α-amylase and α-glucosidase) and insulin secretion studies, the fraction F4 was further tested for its hypoglycaemic effect in normoglycaemic animals, glucose-loaded hyperglycemic animals, and STZ-induced diabetic animals. The results obtained from normal, glucose-loaded hyperglycemic, and diabetic rats are shown in Tables 3-5. In normoglycaemic, the active fraction (F4) controlled the blood glucose level and showed a similar result as standard acarbose ( Table 3). The active fraction (F4) showed a remarkable hypoglycaemic effect at 10 mg/kg B.wt. concentration in glucose-loaded hyperglycemic animals. After 60 min administration of test samples, the blood glucose level was reduced gradually for both active fraction (F4) and acarbose (Table 4). In STZ-induced diabetic rats, the active fraction (F4) showed a 61% reduction in blood glucose level compared to the standard drug glibenclamide (68%). The biochemical parameters of active fraction (F4) were similar to that of the standard drug glibenclamide ( Table 5). The serum insulin level of the diabetic control was significantly low when compared to the control. After 14 days of treatment, serum insulin levels of active fraction (1.09 µg L −1 ) and glibenclamide (1.12 µg L −1 ) treated rats were near to normal rats when compared to diabetic rats ( Figure 12). The glucose levels were analyzed at 30, 60, 120, and 240 min in normal rats. Acarbose and glibenclamide served as positive control. Each value is expressed as mean ± S.E.M. (n = 6). The glucose levels were analyzed at 30, 60, 120, and 240 min in rats loaded with 2 g/kg B.wt of glucose. Acarbose served as a positive control. Each value is expressed as mean ± S.E.M. (n = 6). The effect of active fraction (F4) on fasting blood glucose and serum parameters of STZ-induced diabetic rats. Glibenclamide served as a positive control. Each value is expressed as mean ± S.E.M. (n = 6).

Discussion
The treatment of diabetes with natural products has been an integral part of traditional medical systems for centuries [31]. In natural resources, phytochemicals with diverse structures belonging to different chemical classes, such as flavonoids, phenols, tannins, alkaloids, terpenoids, steroids, saponins, and polysaccharides are present, which exhibit various bioactive properties [32]. Numerous research findings demonstrate that these natural bioactive compounds can prevent and treat diabetes and obesity by focusing on multiple targets, such as inhibiting carbohydrate-digesting enzymes, targeting activities to improve insulin resistance, insulin secretion, etc. Seaweeds are a potential source of novel compounds exhibiting various bioactivities that could be used in the quest for effective anti-diabetic treatments. An in vitro enzyme inhibitory study was conducted to evaluate the anti-diabetic potential of Sargassum polycystum and Sargassum wightii, which showed significant inhibitory effects against α-amylase and α-glucosidase and DPP-IV [33]. There were substantial antioxidant activities reported in various seaweed extracts [34][35][36]. Identifying novel antidiabetic compounds with high biomedical value can be accomplished by isolating and characterizing the bioactive compounds and investigating marine algae.
Inhibiting major carbohydrate-digesting enzymes like α-amylase and α-glucosidase and incretin-inhibiting enzymes results in a decrease in glucose absorption rate and, as a result, helps control postprandial hyperglycemia [37]. As part of our previous studies, we

Discussion
The treatment of diabetes with natural products has been an integral part of traditional medical systems for centuries [31]. In natural resources, phytochemicals with diverse structures belonging to different chemical classes, such as flavonoids, phenols, tannins, alkaloids, terpenoids, steroids, saponins, and polysaccharides are present, which exhibit various bioactive properties [32]. Numerous research findings demonstrate that these natural bioactive compounds can prevent and treat diabetes and obesity by focusing on multiple targets, such as inhibiting carbohydrate-digesting enzymes, targeting activities to improve insulin resistance, insulin secretion, etc. Seaweeds are a potential source of novel compounds exhibiting various bioactivities that could be used in the quest for effective anti-diabetic treatments. An in vitro enzyme inhibitory study was conducted to evaluate the anti-diabetic potential of Sargassum polycystum and Sargassum wightii, which showed significant inhibitory effects against α-amylase and α-glucosidase and DPP-IV [33]. There were substantial antioxidant activities reported in various seaweed extracts [34][35][36]. Identifying novel antidiabetic compounds with high biomedical value can be accomplished by isolating and characterizing the bioactive compounds and investigating marine algae.
Inhibiting major carbohydrate-digesting enzymes like α-amylase and α-glucosidase and incretin-inhibiting enzymes results in a decrease in glucose absorption rate and, as a result, helps control postprandial hyperglycemia [37]. As part of our previous studies, we have conducted various in vitro and in vivo tests on different marine seaweeds that target this primary mechanism of enzyme inhibition [24,25,33,[38][39][40].
In this study, Ulva reticulata extracts were investigated for their inhibitory effects on α-amylase, α-glucosidase, and DPP-IV and antioxidant (DPPH) potential using in vitro assays. Among the various extracts tested, the methanolic extract of U. reticulata (MEUR) displayed a significant α-amylase (61%), α-glucosidase (97%), and DPP-IV (44%) inhibitory activity among the five extracts. This could be because the seaweed may contain a high concentration of polar bioactive chemicals soluble in highly polar solvents such as methanol. Methanol has been reported to be more efficient in extracting polyphenols with lower molecular weights [41,42]. These findings suggest that methanol is the most effective solvent for extracting bioactive chemicals from Ulva reticulata.
Seaweed exhibiting potential antidiabetic activity must be evaluated for its efficacy and toxicity to avoid potential dangers such as unwanted side effects, overdose, and toxicity. Measures of cell viability and hemolytic activity of potentially bioactive compounds are essential for developing new drug treatments [43,44]. Furthermore, the measurement of cell viability and in vitro hemolytic activity of MEUR revealed 99% cell viability after 24 h of treatment at 250 µg mL −1 and showed no signs of hemolysis or erythrocyte membrane damage. Even at a higher concentration (1000 µg mL −1 ), the MEUR showed significantly less (6.92%) cell lysis, and in the presence of (250 g mL −1 ) MEUR, only 2.81% of human red blood cells were lysed. The extracts have a low to hemolytic impact on human erythrocytes. The obtained results from the study suggest that the MEUR is non-toxic and safe.
Research in animal models is crucial in discovering innovative and effective treatments for chronic diseases like diabetes [45]. The oral administration of MEUR on STZ-induced diabetic rats demonstrated a significant reduction in blood glucose levels (46.21%) compared with glibenclamide (73.05%). Diabetic rats had reduced body weight compared to normal rats, but after administering MEUR and glibenclamide for 28 days, their body weight significantly increased. MEUR-and Glibenclamide-treated rats reduce triglycerides and total cholesterol compared to the untreated, which inhibits hypercholesterolemia and decreases the risk of atherosclerosis [46]. Treated extracts and the standard group also showed a gradual reduction in ALT and AST. This marked the hepatoprotective effect of MEUR. Other parameters like total protein, urea, albumin, and globulin were similar to normal control. In our earlier studies, we have reported the antidiabetic activity of methanolic extract of Chaetomorpha antennina in S.T.Z-induced rats model, with results suggesting that at a period of 28 days (250 mg kg −1 B.wt) concentration reduced the fasting blood glucose level to 39.97% and that of positive control glibenclamide (0.25 mg kg −1 B.wt) was 73.05%, respectively [24].
In our study, MEUR, which exhibited the highest antihyperglycemic activity, was selected for Bioactivity-guided isolation of active compounds responsible for antidiabetic action, and seven fractions were separated (F1-F7). Among the fractions, F4 (chloroform) exhibited considerable α-amylase (71.67%) and α-glucosidase (38.01%) inhibition. Additionally, the fractions were found to be rich in total phenolic (65.19 µg mL −1 ) and total flavonoid content (20.33 µg mL −1 ). The α-amylase and α-glucosidase inhibition is attributed to the presence of phenols and flavonoids. The enzyme inhibition is due to phenols and flavonoids directly binding to enzyme amino acid residues (AARs), preventing substrate binding, or interacting with AARs around the active site, preventing substrate binding [47][48][49].
In vitro α-amylase and α-glucosidase inhibition study of isolated compounds and its active fraction (F4) was carried out, revealing active fraction (F4) showed the α-amylase (71.67%) and α-glucosidase (38.01%) inhibition at a concentration of 100 µg mL −1 as compared to the individual identified compounds. Carbohydrate-digesting enzymes αamylase and α-glucosidase are associated with postprandial hyperglycemia; as a result, inhibition of these major enzymes helps reduce glucose release and absorption in the small intestine [50].
Insulin secretagogues lower blood glucose by promoting insulin secretion, boosting insulin levels in the blood, and thereby managing diabetes [51]. Multiple research attempts have been carried out over the last three decades to produce an insulin-secreting beta cell line that maintains normal insulin secretion control, but only a few have been successful [52]. In the present study, non-toxic concentrations of nonane, hexadecanoic acid, phenol, phenol, 3,5-bis(1,1-dimethylethyl), and the active fraction (F4) stimulated concentration-dependent insulin release from isolated mouse pancreatic islets, for a period of 1, and 24 h showed significant insulin secretion in both normal (4 mM) and diabetic (20 mM) condition. Active fraction (F4) at a concentration of 100 µg mL −1 showed maximum glucose-induced insulin secretion at 4 mM (0.357 and 0.582 µg L −1 ) and 20 mM (0.848 and 1.032 µg L −1 ) concentration in both 1 and 24 h in vitro culture. The results of the insulin secretagogue activity were compared with standard drugs acarbose and glibenclamide, where maximum glucose-induced insulin secretion was seen in Active fraction (F4) and glibenclamide, and comparatively, acarbose showed less insulin secretion. Moreover, similar results were also previously reported with fucoidan extract of the seaweed Fucus vesiculosus, stimulating insulin secretion [51].
The in vivo antidiabetic mechanism of active fraction (F4) was further investigated using various Male albino wistar rat models. The results demonstrated that the active fraction (F4) reduces blood glucose levels in normoglycaemic animals and produces effects similar to that of standard acarbose. Active fraction (F4) demonstrated outstanding hypoglycaemic activity in hyperglycemic animals at a dose of 10 mg/kg B.wt. In the STZ-induced diabetic rat model, the active fraction (F4) showed (61%) reduction in blood glucose level when compared to the standard drug glibenclamide (68%). Significant increase in serum insulin was also observed compared to the diabetic control. Our findings suggest that among the various solvents used, the methanolic extract of Ulva reticulata (MEUR) displayed the highest antidiabetic activity in both in vitro and in vivo; it showed no cytotoxicity and hence was subjected to bioassay-guided chromatographic separation. Among the seven isolated fractions (F1 to F7), the F4 (chloroform) fraction exhibited substantial total phenolic content (65.19 µg mL −1 ) and total flavonoid content (20.33 µg mL −1 ), which showed demonstrated the promising inhibition against α-amylase (71.67%) and α-glucosidase (38.01%). Active fraction (F4) was further purified and characterized. Upon structural elucidation, five distinct compounds, namely nonane, hexadecanoic acid, 1-dodecanol, cyclodecane methyl, and phenol, 3,5-bis(1,1-dimethylethyl) were identified. Active fraction (F4) at a concentration of 100 µg mL −1 showed maximum glucose-induced insulin secretion at 4 mM (0.357 and 0.582 µg L −1 ) and 20 mM (0.848 and 1.032 µg L −1 ) concentration in both 1 and 24 h in vitro culture, and also exhibited promising antidiabetic activity in various in vivo models. The study's findings strongly imply that Ulva reticulata has the potential to help manage diabetes.

Collection of Seaweeds
Fresh seaweed, U. reticulata, was collected from intertidal and subtidal regions of Karunagappalli (Latitude 9 • 3 16" N; Longitude 76 • 32 7" E) Kollam, Kerala (India) in November 2012. It grows attached to rocky substrates, and after it gets mature, thalli easily detach and become free living vegetative algae. Mature thalli have irregular shapes, are light to dark green in color, and range in size from a few centimeters to approximately a meter. (Figure 13). The collected seaweed was identified and authenticated by Dr. P. Kaladharan, Principal Scientist and Scientist In-Charge, Calicut Regional Center of Central Marine Fisheries Research Institute, Kerala (India). A voucher specimen (VMBL-06) was deposited in the Marine Biotechnology and Bioproducts Laboratory, Vellore Institute of Technology. detach and become free living vegetative algae. Mature thalli have irregular shapes, are light to dark green in color, and range in size from a few centimeters to approximately a meter. (Figure 13). The collected seaweed was identified and authenticated by Dr. P. Kaladharan, Principal Scientist and Scientist In-Charge, Calicut Regional Center of Central Marine Fisheries Research Institute, Kerala (India). A voucher specimen (VMBL-06) was deposited in the Marine Biotechnology and Bioproducts Laboratory, Vellore Institute of Technology.

Preparation of Seaweed Extracts
Collected seaweeds were cleaned, holdfasts were removed, shade dried, and finely powdered. The powdered seaweed (25 g) was extracted with various solvents (250 mL) based on polarity (petroleum ether, benzene, ethyl acetate, acetone, and methanol) using the Soxhlet apparatus for 24 h. Each filtrate was concentrated to dryness under reduced pressure using a rotary evaporator (model-PBU-6, Superfit Continental Pvt. Ltd., Mumbai, India). The samples were lyophilized using a freeze dryer (Penguin Classic Plus, Lark Innovation Fine Technology, Chennai, India) and stored in a refrigerator at 2-8 °C for further use in subsequent experiments.

In Vitro α-Amylase and α-Glucosidase Inhibition Study
The α-amylase and α-glucosidase inhibitory activity of the extracts (250-1000 μg mL −1 ) were determined as described earlier by [40]. Acarbose (250-1000 μg mL −1 ) was used as a positive control. The tests were performed in triplicates, and the inhibitory activity was calculated as percentage inhibition using the formula.

Preparation of Seaweed Extracts
Collected seaweeds were cleaned, holdfasts were removed, shade dried, and finely powdered. The powdered seaweed (25 g) was extracted with various solvents (250 mL) based on polarity (petroleum ether, benzene, ethyl acetate, acetone, and methanol) using the Soxhlet apparatus for 24 h. Each filtrate was concentrated to dryness under reduced pressure using a rotary evaporator (model-PBU-6, Superfit Continental Pvt. Ltd., Mumbai, India). The samples were lyophilized using a freeze dryer (Penguin Classic Plus, Lark Innovation Fine Technology, Chennai, India) and stored in a refrigerator at 2-8 • C for further use in subsequent experiments.

In Vitro α-Amylase and α-Glucosidase Inhibition Study
The α-amylase and α-glucosidase inhibitory activity of the extracts (250-1000 µg mL −1 ) were determined as described earlier by [40]. Acarbose (250-1000 µg mL −1 ) was used as a positive control. The tests were performed in triplicates, and the inhibitory activity was calculated as percentage inhibition using the formula. DPP-IV inhibitory activity was determined according to the method by [53]. Seaweed extracts of various concentrations (2.5, 10, 40, and 80 µg mL −1 ) were prepared in Tris-HCl buffer (50 mM, pH 7.5). The assay was performed according to the standardized procedure of Diprotin A (0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 µg mL −1 ) as standard. The tests were performed in triplicates, and the percentage of DPP-IV inhibition was calculated as follows: % Inhibition = [(Abs control − Abs* samples )/Abs control ] × 100

Free Radical Scavenging Activity (DPPH)
Free radical scavenging activity was determined according to the method of [54]. Seaweed extracts of various concentrations (250-1000 µg mL −1 ) were prepared. Butylated hydroxytoluene (BHT) was used as a positive control. The tests were performed in triplicates. Scavenging activity was expressed as percentage inhibition using the following formula.

In Vitro Hemolytic Activity
The hemolytic activity of the crude extracts (250-1000 µg mL −1 ) was evaluated as described by [55].

Experimental Animal
Male albino Wistar rats between 2 and 3 months of age, weighing 180-280 g, were used for the study. Animals were maintained in the animal house, Center for Biomedical Research, VIT, Vellore. Rats were housed in polypropylene cages, maintained under standard temperature conditions (22 ± 2 • C) on a 12 h light and dark cycle. They were fed with a standard rat pellet diet and water ad libitum. Animals were maintained as the principles and guidelines of the Institutional Animal Ethical Committee (IAEC) following the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines on animal care. All animal experiments were approved by the IAEC, VIT/IAEC/10th/14 March/No.26.

Experimental Design
Twenty-four rats were divided into four equal groups (n = 6) as follows: (1) Normal control: Rats fed with normal food and water (2) Diabetic control: Rats were made diabetic by a single intraperitoneal injection of streptozotocin (STZ) at a concentration of 45 mg kg −1 body weight. (3) Diabetic rats treated with glibenclamide (Positive control): Rats were made diabetic by STZ (45 mg kg −1 body weight) and treated orally with standard antidiabetic drug glibenclamide (0.25 mg kg −1 body weight) once daily for 28 days. (4) Diabetic rats treated with methanolic extract of U. reticulata (MEUR): Rats were made diabetic by STZ (45 mg kg −1 body weight) and treated orally with methanolic extract of U. reticulata (250 mg kg −1 body weight) once daily for 28 days.
After 3 days of STZ-injection, fasting blood glucose (FBG) values above 250 mg dL −1 were considered diabetic. The treatment started on the third day, and diabetic animals were considered for further study and continued for 28 days. FBG levels were measured with a One Touch Select simple TM glucometer, and body weights were checked in regular intervals (0, 7, 14, 21, and 28 days) during the experimental period. At the end of the experiment, the animals were made to fast overnight, and the blood was collected. The collected blood was incubated for 15-30 min at room temperature, the serum was separated by centrifugation (3000 rpm), and the collected serum was used for various biochemical parameters using standard kits (Span Diagnostics Ltd., Surat, India). The animals were sacrificed on the 28th day, and organs, kidneys, liver, and pancreas were collected for histopathological studies.

Bioassay-Guided Fractionation and Isolation of Active Compounds from MEUR
Methanolic extract of Ulva reticulata (MEUR), which showed the highest antidiabetic activity in both in vitro and in vivo, was subjected to chromatographic separation using a silica gel column (60-120 mesh). Initially, the powdered seaweed material (200 g) was extracted using Methanol, and the extract was evaporated to dryness under a vacuum using a rotary evaporator (Super Fit-Rotavap, model-PBU-6, India). The methanolic extract (10 g) was further subjected to column chromatography. MEUR loaded onto silica gel column (60-120 mesh) were eluted with various solvents, starting with less polarity solvent in the sequence, such as hexane (F1), benzene (F2), Dichloromethane (F3), chloroform (F4), ethyl acetate (F5), methanol (F6), and finally, with water (F7). Further, each fraction was assayed for in vitro antidiabetic activity (α-amylase and α-glucosidase). Finally, the active fraction (F4) was subjected to TLC, and the individual compounds were separated using column chromatography (60-120 mesh). Later these discrete compounds were identified and characterized based on spectroscopic methods, including NMR (C-13, H-NMR), GC-MS, and HR-MS spectrometry.

Determination of Total Phenols and Flavonoids
The concentration of total phenolic present in the active fraction was determined using Folin-Ciocalteu's reagent [56]. Briefly, 100 µL of active fraction (F4) of MEUR and 500 µL of Folin-Ciocalteu's reagent, and 1 mL of Na 2 CO 3 (20%) were mixed and incubated at room temperature for 90 min. The absorbance was measured at 760 nm. Results were expressed as µg gallic acid equivalents per mg of extract (µg GAE/mg). Similarly, the concentration of total flavonoids in the active fraction was determined according to the aluminum chloride colorimetric method [57]. Briefly, the active fraction (F4) 100 µL was mixed with 95% alcohol, 10% aluminum chloride hexahydrate, 0.1 mL of 1 M potassium acetate, and 2.8 mL of deionized water. After incubation for 40 min at room temperature, the absorbance was measured at 415 nm. Results were expressed as µg quercetin equivalents per mg of extract (µg QE/mg).

In Vitro α-Amylase and α-Glucosidase Inhibition Study of Isolated Compounds and Their Active Fraction (F4)
The α-amylase and α-glucosidase inhibitory activity was determined as described by [58]. Purified compounds and the active fraction (F4) of varying concentrations (25-100 µg/mL) were used for the assays, and acarbose was used as a positive control. The experiments were performed in triplicates, and the inhibitory activity was calculated as percentage inhibition using the formula described previously.

In Vitro Insulin Secretion Studies Using Isolated Pancreatic Islets
Pancreatic islets were isolated from adult male Wistar rats by using the standard collagenase digestion method [59]. Islet cells having viability greater than 90% were chosen for the studies. Insulin secretion study was determined in both 1 and 24 h time intervals to evaluate the effect of isolated compounds and its active fraction against pancreatic islet cells. Therefore, the isolated islets (150 cells/mL medium) were incubated with 5% CO 2 at 37 • C in a humidified incubator. Test samples of varying concentrations (25-100 µg/mL) were treated with normal (4 mM glucose) and diabetic (20 mM) conditions, respectively. After incubation, cells were centrifuged at 1500× g for 15 min at 4 • C. The supernatant obtained was subjected to measure insulin secretion according to the manufacturer's instruction (Mercodia ultrasensitive rat insulin ELISA, Uppsala, Sweden).

. Effect in Normoglycemic Animals
Rats fed with normal food and water were made to fast overnight with water ad libitum. The control group received distilled water, and at the same time, positive control (acarbose & glibenclamide) and test samples at a concentration of 10 mg/kg B. wt. were administered using the exact vehicle. FBG levels of each rat were measured at 1 /2, 1, 2, and 4 h after the administration of samples.

Effect in Glucose-Loaded Hyperglycemic Animals
Rats that fasted overnight were administrated glucose (2 g/kg B.wt) after the oral administration of the test and positive control at a concentration of 10 mg/kg B.wt. FBG was measured just before and after the administration of the test samples.

Effect in STZ-Induced Diabetic Animals
Rats were made diabetic by a single intraperitoneal injection of streptozotocin (STZ) at a concentration of 45 mg/kg body weight. After 3 days of STZ injection, fasting blood glucose (FBG) values above 250 mg/dL were considered diabetic. The treatment started on the third day, and animals with diabetes were treated with test samples and the standard antidiabetic drug glibenclamide at a concentration of 10 mg/kg B.wt. FBG levels were measured with a One Touch Select simple TM glucometer, and body weights were checked in regular intervals (0, 7, and 14 days) during the experimental period. At the end of the experiment, the animals fasted overnight, and blood was collected. The collected blood was incubated for 15-30 min at room temperature, the serum was separated by centrifugation, and the collected serum was used for analyzing the insulin content (Mercodia ultrasensitive rat insulin ELISA, Uppsala, Sweden) and various other biochemical parameters using standard kits (Span Diagnostics Ltd., Surat, India).

Statistical Analysis
One-way analysis of variance (ANOVA) and two-way ANOVA followed by Tukey's multiple comparison tests and Dunn's multiple comparisons test was used to compare results. Graph-Pad Prism, Version 5, was used for all the statistical analysis. Values were expressed as mean ± SEM, and the level of statistical significance was taken at p < 0.05.

Conclusions
Diabetes is one of the most prevalent pathological conditions affecting healthy living globally and is accompanied by multiple side effects [60]. It is reported that Diabetes mellitus is frequent in both the elderly and the young population [61]. To effectively and efficiently lower postprandial glycemic levels, novel inhibitors with improved preclinical and clinical trial profiles should be found and produced from natural substances. Marine algae (seaweed) can be considered as one of the prospective sources, with highly bioactive unexplored compounds in the ongoing hunt for effective anti-diabetic medicines. Antidiabetic effectiveness profiles for a variety of marine algae have been previously reported [39,40,[62][63][64].
Findings from this study indicated that the extract of MEUR (Ulva reticulata) and its active fraction F4 examined could be a promising therapeutic agent with better therapeutic efficacy. The extracts and the active fraction exhibited potential anti-diabetic activity with strong inhibitory activity against α-amylase and α-glucosidase, DPP-IV, and antioxidant (DPPH) potential. Active fraction (F4) displayed a prominent in vivo antidiabetic activity due to the presence of five distinct compounds, namely, nonane, hexadecanoic acid, 1dodecanol, cyclodecane methyl, and phenol, 3,5-bis (1,1-dimethylethyl). The presence of phenols and flavonoids and the isolated compounds in the fractions also validate the antidiabetic action of the active fraction (F4). The in vivo antidiabetic mechanism of active fraction (F4) was investigated, and it was found to have a significant hypoglycemic impact in glucose-loaded, hyperglycemic rats and STZ-induced diabetic animals. Conclusively, our research findings suggest that Ulva reticulata and the bioactive chemicals extracted from it are potentially effective and that safe inhibitors of diabetes mellitus can be used to reduce postprandial hyperglycemia. writing-original draft, P.S.U., A.A. and K.S.; writing, reviewing and editing, P.S.U., A.A., K.S. and M.A.J. All authors have read and agreed to the published version of the manuscript.

Funding:
The authors wish to thank the Department of Biotechnology, Government of India, for financial support (Grant no. B.T./BioCARe/03/347/2010-11) and Vellore Institute of Technology for providing all necessary facilities.
Institutional Review Board Statement: Male albino Wistar rats (180-280 g) aged 2-3 months were obtained from the animal house, Center for Biomedical Research, Vellore Institute of Technology, Vellore. All procedures were as per the principles and guidelines of the IAEC, following CPCSEA guidelines on laboratory animals. All animal experiments were approved by the IAEC, VIT/IAEC/8th/23.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.