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Effect of Glycosin alkaloid from Rhizophora apiculata in non-insulin dependent diabetic rats and its mechanism of action: In vivo and in silico studies.

ABSTRACT

Background and aim: Diabetes mellitus is a complex multifactorial disorder that remains a great challenging task in the clinical practice. Rhizophora apiculata from Indian medicinal mangrove is widely used to treat inflammation, wound healing and diabetes. Bioassay guided fractionation was followed to isolate Glycosin from the ethanolic extract of R. apiculata. The antidiabetic effect of Glycosin in diabetic rats was investigated and determined their possible mechanism of action.

Methods: Diabetes was induced in adult Wistar rats by a single intraperitoneai injection of streptozotocin and nicotinamide. Based on the oral glucose tolerance test, Glycosin (50mg/kg b.wt.) was orally administrated to diabetic rats for a period of 45 days. In different intervals, blood glucose and body weight were recorded. After 45 days, blood samples were collected to determine serum lipid profile, level of plasma insulin, hemoglobin, liver, and kidney functions using the appropriate tests. In addition the levels of carbohydrate metabolic enzymes in the liver homogenate were also measured. To determine the molecular mechanism of action, we followed the molecular docking of Glycosin in its possible targets, dipeptidyl peptidase-IV (DPP-IV), Peroxisome proliferator-activated receptor gamma (PPAR[gamma]), phosphorylated insulin receptor, and protein tyrosine phosphatase 1B (PTP-1B).

Results: Glycosin treatment significantly (p < 0.01) reduced the blood-glucose level, increased the body weight, increase hemoglobin, high-density lipoprotein and insulin level, protein, in addition the activity of hexokinase when compared to untreated rats. Decreased activities of liver function enzymes as well as level of urea, and creatinine were observed in Glycosin treated rats. Docking simulation confirmed that Glycosin interacted with DPP-IV, Insulin receptor and PTP-1B and PPAR[gamma] with more affinity and binding energy.

Conclusion: Glycosin acts as antihyperglycemic agent, associated with antihyperlipidemic and possibility function as a ligand for proteins that are targets for antidiabetes drugs.

Keywords:

Diabetes mellitus

Docking

Glycosin

NMR

Mangrove

PPAR[gamma]

Introduction

Diabetes mellitus is a complex multifactorial disease that involves severe insulin dysfunction, conjunction with gross abnormalities in glucose homeostasis and lipid-protein metabolism. Over 90% of patients with diabetes have type 2 diabetes; the rest have type 1 diabetes (Attele et al. 2002). Non-insulin dependent diabetes is characterized by high blood glucose in the connection with insulin resistance and relative insulin deficiency. In India, 38 million individuals got the type 2 diabetes, a frequency that make it third place in the world after by China and United States of America (Kaveeshwar and Cornwall 2014). The modern life style and fast foods are critical factors for inducing obesity that enhances the insulin resistance (Klein et al. 2007). Several drugs are available in the market as anti-diabetes, however one of the key side effect caused by the modern anti-diabetic drugs is hyperglycemia but increasing weight gain which further contribute to the string of non-insulin dependent diabetes. Therefore, while these drugs are useful over the short-term, they are not the best treatment for the long-term health of type 2 diabetic patients (Chan et al. 2012). The most attractive circumstances would be the development of new anti-diabetic drugs without the side effect of promoting weight gain. Therefore, it is highly recommended to find new anti-diabetic molecules that stimulate glucose uptake by fat or muscle cells but distinct than thiazolidinediones or insulin mode of action. Also, ideally these novel drugs would not induce obesity or other side effects like brain damage, swelling, erythema, hepatotoxicity, and gastrointestinal disturbances (Sheng and Yang 2008; Patil et al. 2011).

In this regard, numerous plant extracts and their isolates have been assessed and affirmed in animal models for diabetes that made herbal remedies could serve as alternative treatments, as well as offer resources to search for new anti-diabetic molecules (Baldea et al. 2010). Extracts of mangroves and associated plants are being used as a traditional medicine and proved their hypoglycemic effect on animal models (Alikunhi et al. 2012; Gurudeeban et al. 2012). R. apiculata Blume (family: Rhizophoraceae) is a mangrove plant traditionally used to treat astringent, looseness of the bowels, torment and irritation in the Southeast coast of India (Kaliamurthi et al. 2014). Studies reported, that this plant has anticholinesterase, anti-plasmodial, anti-viral, anti-oxidant, free radical scavenging, anti-microbial, and anti-nociceptive activities (Sur et al. 2004; Vijayavel et al. 2006; Loo et al. 2008; Suganthy et al. 2009; Satyavani et al. 2015a). In search of the active molecules in mangrove extracts, different metabolites have been detected in R. apiculata such as benzoquinone, campesterol, cinnamate, and lupeol, sitosterol, stigmasterol, diethyl phthalate, epoxy purine,1,2 epoxy-hexobarbital, bromobimane, 1,3-cydohexadiene, oxyphenylon, [alpha]-isomethyl ionone and diazene also reported (Nebula et al. 2013; Satyavani et al. 2015b). Moreover, the phytochemical studies reported a huge amount of total alkaloids present in the ethanolic extract which exhibited significant a-glucosidase inhibition in the uncompetitive mode of action. The alkaloid includes sanguinarine, muscimol, glycosin, ergotamine, ergobasine, mescaline, pilosine and nordihydrocapsaicin were identified from the potential extract and its fraction (Selvaraj et al. 2015). In another hand computational studies reported the alkaloids of this plant act as an agonist for PPAR[gamma] receptor (Selvaraj et al. 2014). The antihyperglycemic and anti-hyperlipidemic effect of ethanolic extract and its dichloromethane fraction were tested on diabetic animal models, and showed that the dichloromethane fraction exhibit potential antidiabetic activity (Gurudeeban et al. 2015). In the continuation of previous studies, we isolated Glycosin from the active dichloromethane fraction of R. apiculata and examined its antidiabetic effect in non-insulin dependent diabetes animal models and determined their mechanism of action using molecular docking analysis.

Methods and methods

Chemicals and drug

Streptozotocin (STZ) and nicotinamide were purchased from Sigma, St. Louis, USA. Chemicals and solvents used were of analytical grade (Hi-media, Mumbai, India). The diagnostic kits for triglycerides, total cholesterol, and HDL-C were obtained from Asritha Diatech, Hyderabad, India.

Plant material

Fresh leaves of R. apiculata were collected from Kodiyampalayam coastal village, located in Nagapattinam district, Tamil Nadu, India during December 2009. The specimen identified and deposited in the herbarium of Centre of Advanced Study in Marine Biology Annamalai University, India (Voucher no. AUCASMB 10/2010). The leaves were cleaned, cut into pieces (0.5 1.5 x 1 x 0.2 [cm.sup.3]), shade dried and powdered.

Isolation and identification of active compound

1.5 kg of dried and coarsely powdered leaf material of R. apiculata was defatted with petroleum ether (3000 ml) at room tem perature for 24 h. After removing the solvent, the residue was extracted and soaked in 3000 ml of ethanol (80%) for 72 h with irregular shaking. The extract was filtered through Buchner funnel and concentrated using vacuum rotary evaporator at 40[degrees]C. At the end 65 g of crude ethanol extract was obtained. Then, the residue was extracted with 1 N HCl (750 ml) which was then neutralized sodium hydroxide and the precipitate was partitioned between equal amounts of water and dichloromethane (DCM) layers. DCM part was concentrated to obtain the extract and 45 g of this extract was chromatographed on a silica gel column (Merck 10-200 mesh, 750 g 2.5 x 40 [cm.sup.2]) and successively eluted with stepwise gradient of hexane: ethyl acetate solvent system (5%, 25%, 50%, 75% and 100%). 20 fractions were collected and finally five fractions were obtained based on thin layer chromatography (TLC) profiles. A light yellow color precipitate was obtained in fraction 3 eluted with 75% ethyl acetate and 25% hexane solvent system. The precipitate was washed with ethyl acetate-methanol (8:64) mixture. The crystalline solid material obtained was, spotted on a precoated silica gel (60 F254) s 0.25 mm thick TLC plate (Merck) and run in ethyl acetate: methanol (1:8) solvent system to obtain single spot. To characterize the purified material, the single spot was subjected to nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and mass spectral analysis. FT-IR spectra of isolated substance recorded on the Nicolet Avatar 660 FT-IR spectroscopy (Nicolet, USA) using KBr pellets. To obtain a good signal to noise ratio, 256 scans of isolated fraction 5 was taken in the range of 400-4000 [cm.sup.-1] and the resolution kept as 4 [cm.sup.-1]. Part of the fraction was obtained immediately dissolved in deuterium oxide (D20) for structural analysis by NMR spectra. The mass spectrophotometer (E1MS, 125 MHz) was used in characterizing the isolated fraction.

Structural characterization of Glycosin

IR: the absorption peak at 3427 [cm.sup.-1], 2926[cm.sup.-1], 2864[cm.sup.-1], 1633 [cm.sup.-1] and 1440 [cm.sup.-1]. MS (m/z): 251, 250 (100), 249, 222, 221, 220, 207, 193, 179. [sup.1]H NMR ([D.sub.2]O, 400 MHz): [[delta].sub.H] 3.60 (3H, s, N-C[H.sub.3]), 4.36 (2H, s, -C[H.sub.2]Ph), 7.37 (1H, dd, 7= 8.6, 1.3 Hz, H-8'), 7.39 (s, 5H, Ph), 7.54 (1H, t, J=7.8Hz, H-6'), 7.78 (1H, ddd, J=8.6, 7.8, 1.3Hz, H-7'), 8.35 (1H, dd J = 7.8,1.3 Hz, H-5'); [sup.13]C NMR ([D.sub.2]O, 100 MHz): [delta]C162.0 (C-2), 128.3 (C-3), 168.5 (C-4), 128.4 (C-5), 125.7 (C-6), 134.6 (C-7), 114.3 (C-8), 149.6 (C-9), 119.2 (C-10), 34.2 (N-C[H.sub.2]), 42.5 (C[H.sub.2]), 134.2 (C-1'), 128.5 (C-2'), 128.3 (C-3'), 127.4 (C-4'), 128.4 (C-5'), 128.1 (C-6'). The spectral characterization of Glycosin was shown in Supplementary material 1-4. The chemical name of the purified compound Glycosin is (syn. Arborine; Glycosine; Arborin) 2-benzyl-l-methylquinazoline-4-one.

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Structure of Glycosin

HPLC purity analysis of Glycosin

The identification of Glycosin in reverse phase HPLC (Shimadzu, Japan) with photodiode array detector (SPD-M30A), experiments was based on the retention time of external standard. HPLC analysis carried out in Sepak C18 column (Phenomenex, 5 [micro]m, 225, 4.6 mm, Merck) at room temperature. Methanol: acetonitrile: water system was used as mobile phase in gradient mode as follows: 15:38:47 in 0-22 min, 45:38:17 in 22-65 min. The flow rate of the mobile phase was 1.0 ml [min.sup.-1]. The sample was dissolved in mobile phase and filtered before injection. The solvents were degassed under vacuum by sonication at 5 min and filtered through nylon membrane (0.45 [micro]m). The data was processed by Empower software. The concentration of the Glycosin was calculated using calibration curves. The ranges of calibration curve were 0.04-0.13 mg/ml for Glycosin. The linear relationship was obtained correlating the concentration of Glycosin to the correspondent peak area. For peak purity analysis, spectra in the range of 210-400 nm were recorded at a frequency of 1 Hz. Threshold was calculated employing the noise and solvent angles. Reference spectra of Glycosin/Arborine standard were recorded in the Empower 2 software library for identification purposes. The spectra search improves the identification of compounds in complex matrices since different substances have identical retention times. Glycosin was identified in R. apiculata derived extract chromatogram through the comparison of peak apex spectrum against the results of reference standard solution recorded previously in the software library. The peak height of Glycosin showed in Supplementary material 5. The peak purity analysis provided by diode array detectors is essential to ensure reliability and accuracy of the chromatographic measurements of analytes in complex matrices. In the present work, the Glycosin peak was found pure since the purity angles was lower than the threshold angles and the threshold curves do not intersect the purity curves. The chromatographic method shows linearity over the range evaluated and the correlation coefficients for Glycosin was 0.9976. The concentration (mean [+ or -] standard deviation for n = 6) of Glycosin in the R. apiculata extracted was 9.58 [+ or -] 0.52 [micro]g/mg.

Experimental animals

Male, albino Wistar rats, weighing 200-250 g were procured from the Central Animal House Facility, Rajah Muthiah Medical College and Hospital (RMMC & H), Annamalai University utilized in this study. Animals were fed on pellet diet (Hindustan Pvt. Ltd., India) and water ad libitum. The experimental studies were conducted in accordance with the National Institute of Health's guidelines for laboratory animals and approved by the Institutional Animal Ethical Committee of RMMC&H, Annamalai University (Prop, no.: 922, Ref. no,160/1999/CPCSEA).

Determination of lethal dose 50 (LD50) of Glycosin

The acute oral toxicity study was performed according to OECD-423 guidelines (Wilhelm and Maibach 2008). Animals were fasted for 4 h with free access to water only. Animals were randomly distributed (number of normal-fed: 3 rats and number of Glycosintreated: 9 rats) into one control group (group 1), and three treated groups, containing three rats in each group. Rats were treated orally with three different doses of 50, 300, and 2000 (mg/kg b.wt.) of Glycosin respectively. 0.5% carboxymethyl cellulose in phosphate buffer saline (pH 7.4) used as a vehicle. The control group received vehicle alone. The animals were observed for first 72 h and up to seven days for any signs of behavioral changes, toxicity effect, mortality and body weight. The number of rats died within 24 h was noted, and their [LD.sub.50] of the Glycosin was calculated (Aliu and Nwude 1982). No lethality was observed up to 2g/kg of body weight of the animal. Therefore, the effective dose fixation study was carried with 1 /10th, 1 /20th and 1 /40th of 2000 mg/kg of the dose of Glycosin.

Induction of diabetes mellitus

Non-insulin-dependent diabetes mellitus (NIDDM) was induced in Wistar rats that had been fasted overnight by a single-dose injection of STZ (60 mg/kg b.wt. I.P.) followed by the injection with (I.P., 120 mg/kg b.wt.) nicotinamide 15 min later. NIDDM-ensured rats were applied glucose tolerance test, fasting insulin resistance index (FIRI) and the results were compared to that of the control group and NIDDM controls rats. Experimental rats were fed with 5% glucose orally in order to avoid the hypoglycemia during the injection of STZ. FIRI was calculated via blood sampling from ocular sinus and measurement of fasting insulin and glucose levels (Duncan et al. 1995). The results of the glucose tolerance test showed that fasting blood glucose level for the control group and NIDDM rats was significantly different at 30 min and 2 h after glucose injection. FIRI also revealed a significant increase in diabetic rats compared to the control group.

Effective dose fixation study

About 36 rats (6 normal and 30 diabetics) were fasted overnight with free access to water. Initial blood glucose of each rat was measured with One-Touch Select Simple (Johnson & Johnson Pvt. Ltd. Mumbai, India). Each animal was administered with glucose (2g/kg) orally by means of gastric intubation, excludes control group. They were divided into six groups (n = 6) as normal control, diabetic control, positive control (metformin 100 mg/kg b.wt.), and the remaining groups were fed with the Glycosin (50, 100 and 200 mg/kg b.wt.). Blood glucose was measured at 0th, 60th, and 120th min intervals. From the data obtained, the effective dose was fixed as 50 mg/kg b.wt. for the following studies.

Effect of Glycosin on diabetic rats in prolonged treatment

Glycosin (50 mg/kg) or metformin (100 mg/kg) was administered every day orally using the intragastric tube for 45 days. 0.5% carboxymethyl cellulose in phosphate buffer saline (pH 7.4) was used as a vehicle. 35 rats were divided into five groups (7 normal and 28 diabetic rats): control rats were treated either with vehicle (1 ml) or Glycosin 50 mg/kg, Diabetic rats were vehicle (1 ml), Glycosin (50 mg/kg) treated rats or metformin (100 mg/kg) treated rats. The bodyweight of rats were measured on the first and last day of the experiment. Blood samples were collected on 0th, 7th, 15th, 30th, and 45th days in the morning at 7.30 a.m. from each rat to determine the fasting blood-glucose levels that was estimated by the glucose oxidase--peroxidase method (Trinder 1969). Serum cholesterol, triglycerides, and HDL-C were also evaluated in normal and diabetic rats by an auto-analyzer using diagnostic kits according to the manufacturer's instruction (Asritha Diatech, Hyderabad, India).

At the end of the 45th days, the rats were anesthetized with intramuscular ketamine (24 mg/kg/body) injection and sacrificed by cervical decapitation. Blood was collected with anticoagulant tubes (heparin--35 units/ml of blood) and also in tubes without anticoagulant for plasma and serum separation. Haemoglobin, glycosylated hemoglobin (HbA1c) level was estimated in the serum samples of experimental rats (Drabkin and Austin 1932; Larsen et al. 1990). The plasma insulin level was determined by using ELISA kit (Linco Research, Saint-Charles, MO, USA) (Csont 2013). The level of serum liver marker (ALT, AST, ALP, ACP and bilirubin), serum urea, uric acid and creatinine were determined (Ghodkar 1994; Karmen et al. 1955; Kind and King 1954; Malloy and Evelyn 1937). In addition, the protein content of the supernatant was determined (Lowry et al. 1951).

Estimation of carbohydrate metabolic enzymes

The liver tissues of experimental rats were dissected out and washed with ice-cold saline immediately. Then, 1 g of fresh liver was chopped and homogenized in ice-cold sucrose (15 ml, 250 mM) for 2 min, centrifuged at 10,000g for 30 min. The supernatant was used as the enzymatic source to estimate hexokinase (EC2.7.1.1), Glucose-6-phosphatase (EC 3.1.3.9), and Fructose-1,6-biphosphatase (EC3.1.3.11) levels as described by Cori and Cori (1952), Crane and Sols (1953) and Shibib et al. (1993).

Histopathological studies

The liver was dissected out, washed with ice-cold saline immediately to remove blood, and stored in 10% formalin to avoid decomposition. Then the tissues were washed in 70% alcohol to remove the excess picric acid for three days and dehydrated in alcohol. The tissues cleared using xylene, infiltrated with molten paraffin at 58[degrees]C and finally embedded in paraffin. 5 [micro]m thick sections of the tissues obtained by rotary microtome and stained us ing Ehrlich's hematoxylin with eosin. The tissues were examined under the light microscope. The tissues were examined to assess the hepatic stetosis (HS), lobular inflammation (LI) and liver cell injury (LCI). Briefly, the LCI was graded 0-2 according to ballooning regeneration (0--none; 1--few ballon cells; 2--many cells are prominent ballooning). The HS characterized by vacuolated hepatocytes. These three scores were added and the sum was used as a histopathological scores. This assessment was carried out by three independent researchers that were unaware of the experimental conditions.

In silico docking studies of bioactive alkaloid Glycosin

Preparation of receptors

The 3D valid structure of antidiabetic target proteins such as the Dipeptidyl peptidase-4 receptor (PDB ID: 2R1P), Phosphorylated Insulin Receptor (PDB ID: 1IR3), Peroxisome proliferator activated receptor gamma (PDB ID: 1ZGY) and Protein Tyrosine Phosphatase 1 (PDB ID: 2F70), were obtained from Protein Data Bank. LigPlot tools were used to confirm the non-peptidic and the reversible character of the DPP-IV, PTP-1B, PPAR[gamma] and insulin receptor ligands present in each protein complex by discarding one mutation in their amino acid sequences (Wallace et al. 1995).

Preparation of ligands

The 3D structure of Glycosin was retrieved from the PubChem database. Sdf file was converted into .pdb file by Open Babel. The optimized ligands were docked using Autodock 4.0.

Molecular docking

The MGL (Molecular Graphics Laboratory) tools of AutoDock (4.0) version were used to create PDBQT files from traditional PDB files. Receptor file was prepared with the addition of polar hydrogen, Kollman charges and solvation parameters. The pre-calculated grid maps at the size were set at 60, 60, and 60 [Angstrom] (x, y, and z) to include all the amino acid residues that present in the receptor. The spacing between grid points was 0.575 angstroms. The Lamarckian genetic algorithm (LGA) chosen to confirm the maximum of 10 conformers was considered to the docking process with the population size of 150 individuals. The complete docking process performed in Intel CORETM i5, 64 bit Operating System and 4GB RAM in Lenovo Win 7 PC.

Statistical analysis

The values were presented as mean [+ or -] standard deviation. The data analyzed using ANOVA and means were compared with Dunnett's multiple range tests using the Graph pad instat software.

Results

Effective dose fixation of Clycosin on experimental rats

Antihyperglycemic activity of the effective dose of Glycosin was evaluated on experimental rats using the oral glucose tolerance test (OGTT) and results are shown in Table 1. Among the three different doses used, the dose of 50mg/kg showed most prominent glucose lowering potential at the time interval of 2nd h following 100 and 200 mg/kg of Glycosin administration, reducing the blood glucose level by 25% as compared with the control group. At 2nd h the effect of the Glycosin was clearer with the same low dose of 50 mg/kg reducing the elevated glucose level. Overall, it observed that the response elicited was not dose- dependent, and 50 mg/kg dose was the most effective, displaying a consistent blood glucose lowering effect in diabetic rats. Hence, further studies were carried out with Glycosin at the dosage of 50 mg/kg.

Antihyperglycemic activity of Glycosin

The present study was completed using 25 rats and 10 rats died during the execution of the study. Table 1 represents the level of blood glucose in the control, diabetic, Glycosin treated, and metformin treated rats. The level of blood glucose was significantly (p < 0.01) decreased in Glycosin treated rats compared to the untreated diabetic rats. Table 2 summarize the effects on body weight and changes in the level of hemoglobin, glycosylated hemoglobin, plasma insulin, plasma protein, liver and kidney function test in experimental rats. In the untreated diabetic rats, there was a significant decrease in the level of hemoglobin and body weight (p < 0.05) compared to control rats. There was a significant increase in the level of hemoglobin, body weight (p < 0.01) and a decrease in glycosylated hemoglobin in the Glycosin treated rats than compared to the untreated diabetic rats.

In the untreated diabetic rats after 45 days, there was a significant decrease in plasma insulin when compared to normal rats. Oral administration of Glycosin and metformin increased plasma insulin drastically when compared to diabetic control. The untreated diabetic rats showed a considerable increase in the serum TG, TC and decrease in HDL level when compared with normal. The rat treated with Glycosin and metformin had a reduced TG, TC and increased HDL level when compared with the untreated diabetic rats. There were no significant changes observed in the normal rats treated with Glycosin when compared to control rats.

In diabetic rats, significant (p<0.05) increases were observed in the AST, ALT, ALP, ACP, urea, creatinine and bilirubin levels compared to normal control rats. Glycosin significantly decreased the level of AST, ALT, ALP, ACP, urea, uric acid, creatinine, bilirubin, and increased the level of protein when compared to diabetic control. There is no significant difference between the Glycosin and metformin treated rats.

Effect of Glycosin on carbohydrate metabolic enzymes

The activity of hexokinase is remarkably decreased while glucose-6-phosphatase and fructose 1, 6-bisphosphatase are significantly (p < 0.05) elevated in diabetic control compared with normal rats. Oral administration of Glycosin and metformin increased the hexokinase activity and decreased the activities of glucose 6-phosphatase and fructose 1,6-bisphosphatase compared to diabetic control rats. There was no statistical significance in parameters estimated in control and control treated with Glycosin (Table 3).

Histopathological studies

The normal control rats and Glycosin, treated diabetic rats (p < 0.05) showed absence of lobular inflammation and liver cell injury compared to diabetic rats. The histopathological mean scores of Glycosin treated rats were considerably lower (0.63) compared to metformin treated diabetic rats (1.44). However, there was no significant difference between Glycosin and metformin treated diabetic rats. In the case of diabetic control, the HS, LI and LCI scores was significantly higher (Table 4). The liver tissues of diabetic rats showed periportal necrosis of the hepatocytes near the portal areas, dilated and congested portal vessels as well as areas of inflammatory cell infiltration. Oral administration of Glycosin made the appearance of hepatic lobules. In addition, the central vein and sinusoids were normal and no inflammation, fatty changes or fibrosis was seen in Glycosin treated group.

In silico docking interaction of Glycosin with target receptors

Glycosin has a good solubility, oral bioavailability and possess Lipinski's drug-like molecules (Table 5). Therefore, Glycosin was used in docking simulation with four different receptors: DPP-IV, Insulin receptor tyrosine kinase, PPAR[gamma] and PTP-1B. Docking simulation of Glycosin with target proteins generated six clusters of conformers using a root mean square difference (RMSD) tolerance of 2.0 [Angstrom] on the amino acid residues ARG; TRP; TYR; THR; GLU; HIS; SER in the target proteins. The binding energies of Glycosin with target proteins were shown in Supplementary material 6. Table 6 indicates the binding energy, intermolecular energy and hydrogen bond donor and acceptor of three different receptors and selected ligands. The result revealed Glycosin showed the foremost interaction with PPAR[gamma], DPP-IV receptor, insulin receptor, and PTP-1B, respectively (Fig. 1).

Discussion

Number of mangrove plant extracts with hypoglycemic activity have been evaluated and confirmed to be effective in animal models (Morada et al. 2011; Gurudeeban et al. 2012). In order to explore specific molecule and maximize the therapeutic benefits, efforts should be geared toward searching and discovery of novel anti-diabetic agent. Initially, we studied [alpha]-glucosidase inhibitory effect of leave extracts of R. apiculata and Glycosin was identified from the effective fraction of R. apiculata. Therefore, the present study Glycosin was isolated from R. apiculata and evaluated for possible treatment of diabetes mellitus. Earlier, Chatterjee and Majumdar (1953) reported the chemical constitution and synthesis of Glycosin alkaloid from Glycosmis pentaphylla. After that, Glycosin was found to have a growth inhibitory effect on the larvae of Drosophila melanogaster (Ahmad et al. 1996).

The 1H-NMR range demonstrated the frequency of two singlets at 3.60 and 4.36 due to the N-C[H.sub.3] and benzylic methylene protons, respectively. A singlet at 7.37 was assigned to the five protons of the isolated phenyl nucleus. A doublet of doublet at 7.39 which was partly masked by the phenyl nucleus signal and a triplet centered at 7.54 (J = 7.8 Hz) were referred to the protons at C-8 and C-6, respectively. Proton resonances of H-7 occurred as a doublet of doublet of doublet at 7.78 (J = 8.6, 7.8 and 1.3 Hz). Another doublet of doublet at 8.35 (J = 7.8 and 1.3 Hz) was assigned to the deshielded fragrant proton in the peri-position to the carbonyl group of a quinazoline system. A complete assignment for the [sup.1]H NMR chemical shifts of Glycosin was made by comparison with the data of similar compounds reported by Ahmad et al. (1996). The [sup.13]C NMR spectrum showed the presence of 16 carbons with four quaternary carbons at 168.0, 162.1, 149.5 and 119.1. The range likewise demonstrated the frequency of one methylene carbon which was confirmed. Complete assignments for the [sup.13]C NMR compound arrangements of Glycosin were made by comparison with the data of similar compounds for both the heterocyclic and the isolated phenyl part of the molecule (Chatterjee and Majumdar 1953; Dreyer and Brenner 1980; Ahmad et al. 1996).

In the present study, we used STZ/nicotinamide diabetic rat, with abnormal glucose tolerance and FIR1 to confirm non-insulin-dependent diabetes mellitus in rats. The elevated blood-glucose level results from the process of hepatic glycogenolysis, gluconeogenesis and insufficient uptake of glucose by the cells (Hijmans et al. 2014). Glycosin treatment reduces the blood-glucose level and increases hemoglobin, indicates the activation of liver homeostasis. During diabetes condition, the excess glucose present in the blood reacts with hemoglobin to form HbAlC, which results in the increases of non-enzymatic glycosylation to develop diabetes linked vascular complications (Kondeti et al. 2010). In the current study, the untreated diabetic rats showed a higher level of HbAlC compared to normal rats, indicating their poor glycemic control. In Glycosin treated rats, the level of HbAlC significantly decreased and total hemoglobin increased, which might be the result of an improvement in the glucose metabolism. From the result of lipid profile, it has been observed that Glycosin exhibited a significant andhyperlipidemic effect on diabetic rats compared to untreated diabetic rats. The result indicated that the presence of potent antidiabetic active constituent which produced an antihyperglycemic effect (Sharma et al. 2010; Sanyal et al. 2010). The hypo-insulinemia observed in diabetic control is gradually intensified during the experimental period. A significant increase in the plasma insulin levels of the Glycosin treated rats compared to the diabetic group may indicate the regeneration of the [beta]-cells, which is possibly due to the active constituent present in the extract, induces the pancreas cells with the capacity to regenerate (Dusane and Joshi 2013).

The level of hexokinase and glucose 6-bisphosphatase is indirectly proportional to each other in such action of hexokinase decreases in diabetic rats. The treatment of Glycosin increases the level of hexokinase by improving the glycolytic metabolic pathway. Increased glucose-6-phosphatase activity in diabetic rats indicates formation of NADPH and enhances the lipogenesis (Ananda et al. 2012). Fructose-1,6-bisphosphatase is upregulated in [beta]-cell lines exposed to high levels of fat. In the untreated diabetic rats, the increased level of triglycerides and total cholesterol enhances the activity of fructose-1,6-bisphosphatases, and it becomes normalized when the treated with Glycosin. Creatinine and urea are the potential indicators to demonstrate the function of the kidney. Increased urea production in diabetic rats might be the catabolic action of both liver and plasma proteins. Glycosin treatment has appreciably normalized the content of protein and urea. In response to STZ treatment, creatinine was increased in the serum, suggesting an impairment of kidney functions (Hassan et al. 2009). Glycosin clearly shows an improvement in kidney functions.

Likewise, oral administration of Glycosin showed a significant decrease in these enzyme levels. Aspartate transaminase and alanine transaminase (AST and ALT) are important liver marker enzymes, which are increased during diabetes. However, after treatments with the Glycosin level of these enzymes were reduced. In the diabetic control, elevated level of transaminase that is active in the absence of insulin because of the availability of amino acids in the blood stream is responsible for the high rate of gluconeogenesis and ketogenesis metabolism (Rui 2014). In the diabetic rats, the rate of these enzymes is increased in the blood circulation, which depicted the failure in the function of both kidney and liver. Althnaian et al. (2013) observed the mitochondrial damage and cellular membrane integrity in experimental rats through an elevated level of AST in the plasma. Glycosin treatment restored the function of liver and kidney.

Computational drug design approach is very useful, reasonable, minimize the cost and accelerate the discovery of new molecules to be considered for clinical studies unlike the traditional approach for drug discovery (Young 2009). The inhibition of DPPIV prevents the inactivation of gastric inhibitory polypeptide and glucagon-like peptide-1. Activation of GLP-1 and GIP can stimulate insulin secretion, which results in lowering of glucose levels and improvement of the glycemic control (Kushwaha et al. 2015). Insulin sensitivity to cells is attributed to phosphorylation of the insulin receptor, protein tyrosine phosphatase 1B, which dephosphorylates the tyrosine residues of IR proteins, is primarily responsible for insulin resistance in T2DM (Jung et al. 2013). Nuclear receptor protein PPAR-[gamma] plays a vital role in adipogenic pathway and secretion of adiponectin, lipid storage and glucose metabolism (Grygiel-Gorniak 2014). Glycosin is beneficial in the management of diabetes mellitus that could be through the enhancement of the activity of PPAR[gamma] by increasing insulin sensitivity to skeletal muscle and inhibiting hepatic gluconeogenesis. Therefore, the results of docking experiment indicate the identified alkaloids are able to bind PPAR[gamma], DPP-IV, PTP1B and insulin receptor and act as potential modulators through the inhibiting hepatic gluconeogenesis, activation of GLP-1 and ameliorating the insulin-dependent signaling pathway diabetes condition.

Conclusion

In conclusion, this study gives solid confirmation of the beneficial effect of Glycosin on non-insulin dependent diabetes mellitus, which were evaluated for the first time. Glycosin given at a dose of 50mg/kg (oral gavage) after streptozotocin that induced the destruction of insulin producing [beta]-cells improved antihyperglycemic condition. The beneficial changes in biochemical parameters including carbohydrate metabolic enzymes, lipid profile, serum marker enzymes and plasma insulin were also coupled with parallel lucrative changes in the histopathological appearance of liver tissue and computational studies on PPAR[gamma], DPP-IV, PTP-1B, and insulin receptor inhibitory action evidence.

In summary, our results strongly suggest potential clinical benefits of Glycosin usage to regulate hyperglycemic condition against non-insulin dependent diabetes mellitus and this protection is associated with antihyperlipidemic and inhibitory interaction of target receptor-Glycosin by molecular docking simulation.

Authors' contributions

S.G. and ICS. developed the concept and designed experiments. T.R. was research guide. S.G and ICS. performed collection, experimental studies of diabetes, S.G. performed molecular and in silico studies. T.R. provided chemicals, instrumental studies and advised on experimental part.

http://dx.doi.org/10.1016/j.phymed.2016.03.004

ARTICLE INFO

Article history:

Received 11 April 2015

Revised 19 February 2016

Accepted 10 March 2016

Abbreviations: ACP, acid phosphatase; ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate amino transferase; CMC, carboxyl methyl cellulose; CPCSEA, committee for the purpose of control and supervision of experiments on animals; DPP-IV, dipeptidyl peptidase-4 receptor; T2DM, type 2 diabetes mellitus; FDA, food and drug administration; I.P., intraperitoneai; NAD, nicotinamide; STZ, streptozotocin; S.D., standard deviation; PTP-1B, protein tyrosine phosphatase IB.

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and have no significant financial support for this work that could have influenced its outcome.

Acknowledgments

The authors are grateful to the authorities of Annamalai University for providing necessary support during the study period. The authors extend special thanks to Dr. S. Sengottuvelu, Nandha College of Pharmacy, Erode and Dr. Antony Muthu Prabu, Central Instrumentation Facility, Department of Chemistry, Annamalai University, for FTIR and NMR analysis. Authors are thankful to Sciencedit-DW for reviewing and editing the language of the manuscript.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/J.phymed.2016.03.004.

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Gurudeeban Selvaraj *, Satyavani Kaliamurthi Ramanathan Thirugnasambandan

Centre of Advanced Study in Marine Biology. Faculty of Marine Sciences. Annamalai University, Parangipettai 608502, Tamil Nadu, India

* Corresponding author. Tel.: +91 95430 98008 (mobile).

E-mail address: gurudeeb99@gmail.com (G. Selvaraj).

Table 1
Changes in the blood glucose level for effective dose fixation and
prolonged treatment of Glycosin in experimental rats.

OGTT test for effective dose fixation of Glycosin in the blood
glucose level (mg/dl)

Group        Normal control          Diabetic control

0th min      89 [+ or -] 7.74        251 [+ or -] 11.54
60th min     94 [+ or -] 9.98 (b)    545 [+ or -] 10.24
120th min    95 [+ or -] 8.75 (b)    530 [+ or -] 12.45

Group        Diabetic + Glycosin     Diabetic + Glycosin
             (50 mg/kg of b.wt.)     (100 mg/kg of b.wt.)

0th min      245 [+ or -] 10.65      248 [+ or -] 12.25
60th min     457 [+ or -] 10.35 (a)  385 [+ or -] 11.14 (a)
120th min    370 [+ or -] 10.16 (b)  345 [+ or -] 11.25 (a)

Group        Diabetic + Glycosin     Diabetica- Metformin
             (200 mg/kg of b.wt.)    (100 mg/kg of b.wt.)

0th min      247 [+ or -] 11.65      249 [+ or -] 10.18
60th min     415 [+ or -] 10.21 (a)  425 [+ or -] 09.57 (a)
120th min    390 [+ or -] 10.27 (a)  375 [+ or -] 10.56 (b)

Changes in the blood glucose level (mg/dl) of Glycosin treated normal
and diabetic rats

Group        Normal                  Control + Glycosin
             control                 (50 mg/kg of b.wt.)

0th day      82 [+ or -] 10.02 (b)    81.8 [+ or -] 9.05
7th day      85 [+ or -] 9.56 (b)       87 [+ or -] 10.12 (b)
15th day     86 [+ or -] 10.01 (b)      88 [+ or -] 9.65 (b)
30th day     87 [+ or -] 6.35 (b)       87 [+ or -] 5.35 (b)
45th day     85 [+ or -] 7.25 (b)    89.33 [+ or -] 7.50 (b)

Group        Diabetic control        Diabetic + Glycosin
                                     (50 mg/kg of b.wt.)

0th day      362.5 [+ or -] 10.25    381.9 [+ or -] 8.75
7th day        370 [+ or -] 12.52      350 [+ or -] 11.35 (a)
15th day       400 [+ or -] 9.85       270 [+ or -] 10.21 (a)
30th day       410 [+ or -] 9.65       165 [+ or -] 9.15 (b)
45th day       425 [+ or -] 8.45     125.9 [+ or -] 8.65 (b)

Group        Diabetic + Metformin
             (100 mg/kg of b.wt.)

0th day      373.9 [+ or -] 9.35
7th day        365 [+ or -] 10.45 (a)
15th day       325 [+ or -] 10.25 (a)
30th day       185 [+ or -] 9.35 (b)
45th day       155 [+ or -] 10.25 (b)

Note: Values are mean [+ or -] SD for six animals each. Values not
sharing a common superscript differ significantly at * p < 0.05,
** p < 0.01 vs diabetic rats.

Table 2
Effect of Glycosin on body weight, hemoglobin, glycosylated
hemoglobin, lipid profile, liver and kidney function tests in the
normal and diabetic rats.

Parameters            Normal control            Control + Glycosin

                                                (50 mg/kg of b.wt.)

Body weight (g)
Initial               200.3 [+ or -] 0.90       204.2 [+ or -] 1.12
Final                 213.3 [+ or -] 0.88 (b)   180.1 [+ or -] 0.83 (b)

Biochemical parameters
Hemoglobin (g %)      14.27 [+ or -] 0.92 (b)   14.29 [+ or -] 0.89 (b)
Glycosylated           0.45 [+ or -] 0.01 (b)    0.46 [+ or -] 0.03 (b)
  hemoglobin (mg/g)
Plasma insulin        19.13 [+ or -] 0.55 (b)   19.02 [+ or -] 0.75 (b)
  (U/ml)
Protein (g/dl)         8.17 [+ or -] 0.05 (b)    8.16 [+ or -] 0.11 (b)

Lipid profile
TC (mg/dl)               85 [+ or -] 3.6 (b)       84 [+ or -] 3.3 (b)
TG (mg/dl)               82 [+ or -] 4.5 (b)       86 [+ or -] 4.1 (b)
HDL (mg/dl)              54 [+ or -] 2.6 (b)       51 [+ or -] 3.9 (b)

Liver function test
AST ([micro]g/dl)     41.38 [+ or -] 1.21 (b)   40.33 [+ or -] 1.15
ALT ([micro]g/dl)     40.32 [+ or -] 1.25 (b)   41.13 [+ or -] 1.25
ALP ([micro]g/dl)      12.1 [+ or -] 1.25 (b)   12.03 [+ or -] 1.25
ACP ([micro]g/dl)      9.82 [+ or -] 0.25 (b)    9.62 [+ or -] 0.27
Bilirubin (mg/dl)      0.78 [+ or -] 0.09 (b)    0.72 [+ or -] 0.08 (b)

Kidney function test
Urea (mg/dl)          15.98 [+ or -] 0.68 (b)   15.81 [+ or -] 0.61 (b)
Creatinine (mg/dl)     0.63 [+ or -] 0.07 (b)    0.62 [+ or -] 0.05 (b)
Uric acid (mg/dl)      0.96 [+ or -] 0.01 (b)    0.96 [+ or -] 0.02 (b)

Parameters            Diabetic control       Diabetic + Glycosin
                                             (50 mg/kg of b.wt.)

Body weight (g)
Initial               220.9 [+ or -] 2.6    220.8 [+ or -] 1.32
Final                 103.4 [+ or -] 0.60   155.2 [+ or -] 2.62 (b)

Biochemical parameters
Hemoglobin (g %)       8.04 [+ or -] 0.02   14.24 [+ or -] 0.89 (b)
Glycosylated           0.82 [+ or -] 0.08    0.55 [+ or -] 0.08 (b)
  hemoglobin (mg/g)
Plasma insulin         4.12 [+ or -] 1.05   14.65 [+ or -] 1.27 (b)
  (U/ml)
Protein (g/dl)         3.67 [+ or -] 0.12    6.45 [+ or -] 0.17 (b)

Lipid profile
TC (mg/dl)              175 [+ or -] 5.4       89 [+ or -] 4.9 (b)
TG (mg/dl)              164 [+ or -] 3.2       95 [+ or -] 2.2 (b)
HDL (mg/dl)              34 [+ or -] 2.5       59 [+ or -] 4.3 (a)

Liver function test
AST ([micro]g/dl)     86.65 [+ or -] 1.25   45.22 [+ or -] 1.19 (b)
ALT ([micro]g/dl)    112.65 [+ or -] 3.25   47.56 [+ or -] 2.25 (b)
ALP ([micro]g/dl)     42.35 [+ or -] 1.25    17.5 [+ or -] 1.25 (a)
ACP ([micro]g/dl)     28.28 [+ or -] 0.15   15.04 [+ or -] 0.21 (b)
Bilirubin (mg/dl)      5.38 [+ or -] 0.14    0.92 [+ or -] 0.07 (b)

Kidney function test
Urea (mg/dl)          36.10 [+ or -] 3.97   14.25 [+ or -] 1.26 (b)
Creatinine (mg/dl)     1.25 [+ or -] 0.3     0.98 [+ or -] 0.02 (b)
Uric acid (mg/dl)      1.67 [+ or -] 0.04    1.09 [+ or -] 0.05 (a)

Parameters            Diabetic + Metformin
                      (100 mg/kg of b.wt.)

Body weight (g)
Initial                212.8 [+ or -] 1.2
Final                  169.5 [+ or -] 0.60 (b)

Biochemical parameters
Hemoglobin (g %)       14.45 [+ or -] 0.90 (b)
Glycosylated            0.53 [+ or -] 0.04 (b)
  hemoglobin (mg/g)
Plasma insulin         15.25 [+ or -] 1.45 (b)
  (U/ml)
Protein (g/dl)          7.26 [+ or -] 0.15 (b)

Lipid profile
TC (mg/dl)                81 [+ or -] 1.4 (a)
TG (mg/dl)                86 [+ or -] 2.1 (b)
HDL (mg/dl)               57 [+ or -] 2.1 (b)

Liver function test
AST ([micro]g/dl)      43.65 [+ or -] 1.16 (b)
ALT ([micro]g/dl)       40.9 [+ or -] 1.25 (b)
ALP ([micro]g/dl)      13.01 [+ or -] 1.25 (a)
ACP ([micro]g/dl)      10.55 [+ or -] 0.51 (b)
Bilirubin (mg/dl)       0.83 [+ or -] 0.06 (b)

Kidney function test
Urea (mg/dl)           12.57 [+ or -] 1.78 (b)
Creatinine (mg/dl)      0.89 [+ or -] 0.09 (b)
Uric acid (mg/dl)       0.97 [+ or -] 0.01 (ns)

Note: Values are mean [+ or -] SD for six animals each. Values not
sharing a common superscript followed by DMRT. (ns) not significant
vs control.

(a) p < 0.05 by comparison with diabetic rats.

(b) p < 0.01 by comparison with diabetic rats.

Table 3
Effect of Glycosin on carbohydrate metabolic enzyme levels in the
normal and diabetic rats.

Enzymes               Normal control            Control + Glycosin
                                                (50 mg/kg of b.wt.)

Hexokinase (unit      0.252 [+ or -] 0.01 (a)   0.241 [+ or -] 0.01 (a)
  a/mg protein)
Glucose 6             0.186 [+ or -] 0.01 (a)   0.174 [+ or -] 0.03 (a)
  phosphatase (unit
  b/mg protein)
Fructose 1,6-bis-     0.421 [+ or -] 0.03 (b)   0.435 [+ or -] 0.05 (a)
  phosphatase (unit
  c/mg protein)

Enzymes               Diabetic control      Diabetica-Glycosin
                                            (50 mg/kg of b.wt.)

Hexokinase (unit      0.081 [+ or -] 0.02   0.233 [+ or -] 0.02 (a)
  a/mg protein)
Glucose 6             0.403 [+ or -] 0.01   0.204 [+ or -] 0.03 (a)
  phosphatase (unit
  b/mg protein)
Fructose 1,6-bis-     0.688 [+ or -] 0.02   0.459 [+ or -] 0.01 (b)
  phosphatase (unit
  c/mg protein)

Enzymes               Diabetic + Metformin
                      (100 mg/kg of b.wt.)

Hexokinase (unit      0.233 [+ or -] 0.01 (a)
  a/mg protein)
Glucose 6             0.203 [+ or -] 0.01 (a)
  phosphatase (unit
  b/mg protein)
Fructose 1,6-bis-     0.464 [+ or -] 0.02 (b)
  phosphatase (unit
  c/mg protein)

Note: Values are mean [+ or -] SD for six animals each. Values not
sharing a common superscript followed by DMRT.

Unit a: The enzyme activity was expressed as units of glucose
phosphorylated/min/mg protein.

Unit b: The enzyme activity was estimated as units of inorganic
phosphorus liberated/min/mg protein.

Unit c: The enzyme activity was expressed as units of inorganic
phosphorus liberated/h/mg protein.

(a) p < 0.05 by comparison with diabetic rats.

(b) p < 0.01 by comparison with diabetic rats.

Table 4
Histopathological scoring of liver tissues of experimental rats.

Parameters            Normal control     Control a- Glycosin
                                         (50 mg/kg of b.wt.)

                      Mean       Range   Mean         Range

Hepatic stetosis      0.31 (a)   0-1     0.30 (a)     0-1
  (HS)
Lobular               0          0       0            0
  inflammation (LI)
Liver cell injury     0          0       0            0
  (LCI)
Histopathological     0.31       0-1     0.30         0-1
  score

Parameters            Diabetic control    Diabetic + Glycosin
                                          (50 mg/kg of b.wt.)

                      Mean     Range      Mean        Range

Hepatic stetosis      1.28     1-1.61     1.12 (a)    0.31-1.27
  (HS)
Lobular               0.05     0.031      0           0
  inflammation (LI)
Liver cell injury     0.39     0-1        0.31 (a)    0-1
  (LCI)
Histopathological     1.72     0-2.61     1.43        0.31-2.27
  score

Parameters            Diabetic a- Metformin
                      (100 mg/kg of b.wt.)

                      Mean         Range

Hepatic stetosis      0.63 (a)     0-1.44
  (HS)
Lobular               0            0
  inflammation (LI)
Liver cell injury     0            0
  (LCI)
Histopathological     0.63         0-1.44
  score

(a) p < 0.05 vs diabetic control.

Table 5
ADMET profile of Glycosin.

Physiochemical parameters       Description

Molecular weight (g/mol)        250.30
logP                            2.47
tPSA                            34.89
HB donors                       0
HB acceptors                    3
HBD + HBA                       3
Lipinski violations             0
Solubility (mg/l)               10463.81
Oral bioavailability (VEBER)    Good
Phospholipidosis                Non-inducer
PPL_Friendly                    Yes

Table 6
Molecular docking interaction of Glycosin with target receptors.

Receptors   Binding energy   Total inter-molecular   No. of H bonds
            (kcal/mol)       energy (Kcal/mol)

DPPIV       -6.15            -6.36                   2

PTP-1       -6.35            -6.51                   3

IR          -5.91            -6.10                   1
PPAR        -7.68            -7.81                   2
[gamma]

Receptors   H bond       H bond     Ref. RMSD
            donor        acceptor

DPPIV       SER106:HG1   LIG1:0     111.91
            TRP157:HN1   L1G1:0

PTP-1       TYR176:HH    LIG 1:0    50.644
            GLU186:HN    L1G 1:0
            THR178: 0    LIG 1:N

IR          ARG1174:HN   LIG 1:0    44.91
PPAR        SER289:HG    LIG 1:0    39.83
[gamma]     HIS323:HE2   LIG 1:0
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Author:Selvaraj, Gurudeeban; Kaliamurthi, Satyavani; Thirugnasambandan, Ramanathan
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Jun 1, 2016
Words:8967
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