Glycosaminoglycan derived from field cricket and its inhibition 1 activity of diabetes based on anti-oxidative action 2 3

Glycosaminoglycan derived from field cricket and its inhibition 1 activity of diabetes based on anti-oxidative action 2 3 Mi Young Ahn1, Ban Ji Kim1, Ha Jeong Kim1, Jang Mi Jin 2, Hyung Joo 4 Yoon1, Jae Sam Hwang1 and Byung Mu Lee3 5 1Department of Agricultural Biology, National Academy of Agricultural Science, RDA, 6 Wanju-Gun 55365, South Korea; amy@korea.kr (MYA); kimbj0826@naver.com (BJK.); 7 musical2058@daum.net (HJK); yoonhj@korea.kr (HJY); jshwang@korea.kr (JSH) 8 2Korean Basic Science Institute, Ochang 863-883, South Korea; wlsjm03@gmail.com 9 3Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Suwon, 440-746, 10 South Korea; bmlee@skku.edu 11 Running title: Anti-oxidative effect of cricket glycan in db mice 12 13 Prepared for Nutrients 14


Introduction
The custom of eating of insects has come to be spread worldwide.Edible crickets acquired from industrial rearing systems are an alternative food increasingly used in countries including the Netherlands and several others.Gryllus bimaculatus (field cricket, Gb) water extract has been used in Oriental medicine as a crude drug for an antifebrile and lowering agent for high blood pressure.Recently, this extracts from this cricket, have the lowering activity of blood ethanol metabolite concentrations by enhancing the liver mitochondrial alcohol and acetaldehyde dehydrogenases [1] and anti-obesity effect through the inhibition of adipose tissue accumulation in high fat diet rats [2].Recently, we found a significant report on showing good result effects by consuming crickets: edible cricket consumption on gut microbiota in healthy adults [3].We are concerned about the type-2 diabetes is related with oxidative stress and influenced vascular disease such as a chronic disease, atherosclerosis, hypertension and nephropathy [4].
The antioxidant and anti-inflammatory activities of enzymatic hydrolysates and peptide fractions from selected heat-treated edible insects including cricket have been ascertained [5] with exception of other carbohydrate components of field cricket.Indeed, all human cells were coated with an array of glycoproteins, glycolipids and polysaccharides named glycocalyx, the surface proteoglycan/glycoprotein layer [6].Therefore, glycans can appeal distinct properties as biomarker targets [7]; especially N-glycans are also ubiquitous in nature providing structural and functional stability to N-linked glycoprotein, with flexibility [8].
In the overview of a total insect component survey concept, as one of the functional components, a mucin polysaccharide, glycosaminoglycan as an ingredient, is required to standardize and to manufacture not from a versatile natural source but from the same conditions of insect rearing.The distinctive role of Gb glycosaminoglycan has been reported as an anti-inflammatory effect in arthritis induced rat model [9], anti-obesity [10] and antilipidemic [11] effects in a high-fat diet.Nowadays, some glycosaminoglycans so retained anti-oxidant activity robust enough for that they scavenge free radicals thus repaired cellular oxidative damages [12].
We commonly use db/db mouse model to conduct research in this experiment on type 2 diabetes mellitus and its comorbidities including obesity and hypertension [13].Hence, there are some reports that a kind of glycosaminoglycan, heparin sulfate, plays a crucial role in proliferation, development and maturation of beta-cells thereby contributing to normal glucose hemostasis [14] and mouse  cell survival in the autoimmune type 1 diabetes [15].
Here, this G. bimaculatus glycosaminoglycan with anti-oxidant activities endeavored to contribute to anti-diabetic activities thus to repair cellular oxidative damage in protein carbonyl level and anti-oxidant enzyme induction ratios after treatment in db/db mice.
As a comparable insect glycosaminoglycan sample, following to a previous report, the dung beetle (Catharsius molossus, Ca) GAG with marked anti-aging activity by reducing oxidative damage in aged rats [16], was used as a positive control.In this work, we also characterized purified N-glycans throughout deglycosylation of GbG and analysis by MS and MS/MS (MALDI TOF MS).In this study, we found that field cricket revealed elements constituting glycosaminoglycan and displayed anti-oxidative activity for use as an antidiabetic agent with reducing cellular oxidative damages.

2.1.Preparation of field cricket glycosaminoglycan
Field cricket (Gryllus bimaculatus) was supplied from a cricket farm located in Hwasung, South Korea.These insects were freeze-dried, in the Department of Agricultural Biology, National Academy of Agricultural Science (NAAS), South Korea.Dried dung beetle (C. molossus) was purchased at a local market in China by Chinese phamacognosist and posted to NAAS.
These each insect glycosaminoglycan was purified by previous reported method [17] using removal of fat by ethanol and acetone, protein enzymatic hydrolysis by putting Alcalase (Sigma Aldrich, St. Louis, Mo., USA), protein precipitation by trichloroacetic acid (5%), impure materials cleansing with detergent, cetylpyridinium chloride (5%), dissolving of nonglycosaminoglycan with 2.5 M NaCl.We added five volumes of ethanol followed by centrifugation at 8000 g for 30 min.Thus the precipitate was then dissolved in water and then dialyzed against 100 volumes of water and freeze-dried.We loaded crude field cricket or

Neutral monosugar composition of digested N-glycan on the basis of GC-MS analysis
To induce the trimethylsilylation of samples, 10 mg of isolated Gb N-glycan in 100 µL was hydrolyzed by 1N HCl for 10 minute period, then dried with N2 gas.Accordingly, the evaporated samples were added to 200 µL pyridine, and 120 µL N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (Sigma Co., USA), reacted for 30 min at 65°C, and then injected into the GC-MS.In order to confirm the target components, we made a TMS mono, di-carbohydrate standard mixture, and further blended the dried standard mixture with a derivative sample of the TMS standard mixture.

Body weight and blood glucose detection
Body weights were measured per every week.Blood glucose levels were recorded weekly with glucose stick by using a blood glucose Nocodingone detector (Theragenetex.Co., Sungnam, Korea).Serum glucose levels were recorded by means of an auto-analyzer on the last day of the treatment schedule, after one-month of treatment was recorded using an autoanalyzer.Metformin was used as a positive control drug to retain an antidiabetic effect [18].

Organ and Adipose Tissue Weights
Absolute and relative weights (organ-to-body ratio) were measured for adrenal glands, kidneys, heart, liver, lung, spleen, stomach and pancreas.Abdominal fat to-body weight ratio was also determined.These measurements were made after sacrifice at the end of the onemonth treatment period.
These parameters were evaluated by means of an auto analyzer (Hitachi 7060 Automatic Clinical Analyzer, Tokyo, Japan).

Oxidative protein damage
Liver homogenate and blood were centrifuged (8000 g for 30 min).Supernatants were used to determine of carbonyl content.Protein oxidative stress was evaluated by measuring protein carbonyl content in the blood.Carbonyl content was determined with an enzyme-linked immunosorbent assay (ELISA) by means of OxiSelect™ protein carbonyl ELISA kit (Cell Biolabs, Inc., San Diego, CA, USA) according to the manufacturer's protocol.Catalase, CAT activity (U/mg protein) was measured on the basis of CAT-mediated decomposition of H2O2 [19].

Liver homogenate in preparation for oxidative enzyme detection
Five groups (DB-Hetero, DB-Homo, CaG5, GbG5, Metformin10) of liver tissues were homogenized on ice in a 10-fold volume lysis buffer PRO-PREP™ protein extraction solution (iNtRON, Busan, Korea).The supernatant of each liver homogenate after centrifugation (800 g, 10 min) was assayed for catalase, glutathione peroxidase, glutathione s-transferase and superoxide dismutase activities [20] using OxiSelect™ ELISA kit (Cell BioLabs, InC., San Diego, CA, USA) according to the assay manual.

Endothelial Nitric oxide synthase, laminin and VEGF on diabetic endothelial cells
Vasorelaxation and growth factor were also measured in adult diabetic type2 microvascular endothelial cells (D-HMVECs) were obtained from type 2 diabetics patients (Clonetics™, diabetic type II, Lonza CC-2928, Cambrex,Walkersville, MD, USA).Cells were grown in an endothelial cell basal medium (EBM)-2 with EGM-2 singlequots (Cambrex) at 37•C in an atmosphere containing 5% CO2.Cells were pretreated with 0.2 mg/ml of either GAG or Pravastatin were incubated prior to the determination of endothelial nitric oxide synthase (eNOS) [21] and Vascular endothelial growth factor (VEGF) [22] (Quantikine, R&D Systems, Inc., Minneapolis, MN, USA) as according to the manufacturers' instructions.The laminin level in endothelial cells was measured using Quantimatrix™ human laminin ELISA kits (Millipore, Billerica, MA, USA) in HMVEC cells or in upper mentioned hepatocytes according to the manufacturer's instructions.Positive controls were Paravastatin (CJ Healthcare Co., Seoul Korea) and Chitosan (Sigma Co., USA).The excised organs were included kidneys, heart, liver, lung, spleen, stomach, pancreas, testis and adipose tissue to be fixed 10% neutral formalin.After paraffin embedding, they were stained with hematoxylin and eosin, and Toluidine blue O, examined by light microscopy (Leica CTR6000, Hesse, Germany), and photographed.Adipocyte density (cells/mm 2 ) was determined in treated and control tissue by toluidine blue O stain (original magnification, x400).

Statistical Analysis
Means and standard errors of parameters were determined for each group through the analysis of variance (ANOVA).Student's t-test was applied to determine significant differences between control and treated groups.A p value of less than 0.05 was considered statistically significant.

Yield of field cricket glycosaminoglycan
From 1 kg of each dried insect, the yield of freeze-dried GAG powder was about 1.52 g for CaG and 3.4 g for GbG  2A).The abdominal fat weight (g) of the treated GAG group over an one month, showed significant differences from that of the DB-Hetero (normal db mice) group but was no significantly difference from that of the DB-Homo group: DB-Hetero, 0.50±0.23;DB-Homo, 2.72±0.90;CaG5, 2.72±0.59;GbG5, 2.79±0.43;Metformin 10, 2.79±0.37.

Effects of CaG5 on blood glucose level
The blood glucose levels on heterozygous (normal) db mice were about 182.30 mg/dL.The glucose levels on homozygous db mice were shown to be increased with aging, proved from 486. treatment until the fourth week did not turn out to have antidiabetic effect on blood glucose level.

Decrease in oxidative damages
Protein carbonyl concentrations in blood was shown quite decrease along with treatment with insect glycosaminoglycans.Decreased levels (nmol/mg protein) were found in DB-Hetero, 8.39±0.24nmol/mg protein; DB-Homo, 7.25±0.17nmol/mg protein; CaG5, CON, p <0.05); and Metformin10, 6.74±0.71mg/dL, respectively (Fig. 3A).However, we cannot find statically significant differences in the case of hepatocyte carbonyl contents between the control and treatment groups of db mice (Figure 3A and 3D).As for blood cellular oxidative damage, protein oxidative damage was also reduced (CaG5, 81.5; GbG5, 81.5% and Metformin10, 93.0%) by these GaGs based on blood neutrophil carbonyl content.

Oxidative enzyme (catalase, GPx, GST, SOD) quantitation
We also found Figure 3B showed the Catalase, GPx and GST increase levels of db mice treated with GbG for a month.Catalase activities (mg protein/min) in db mice blood after one month of GAG treatment were as follows: db mice (CON), 32.18±1.4B).

The manufacture of the insect N-glycan fraction
The N-glycan (N-glycan) is to be refined from the cricket (G.bimaculatus) origin insect glycosaminoglycan and the concentration of the sodium chloride used in the ion exchange chromatography process in the elution is 0 M (Figure 5A).By confirming whether it was the low molecular weight or the degree of purity to confirm performed SAX (strong aion exchange process, Phenomex, 250x 10mm) -HPLC, and 232 nm after glycan lyase (the heparinase I,II,III, and the chondroitin ABCase) processing amid the single Peak was detected or purity was confirmed in Figure 5A.As this figure showed SAX-HPLC result, final low molecular weight chromatogram of cricket glycosaminoglycan noted from, the major SAX-HPLC peak glycosaminoglycan of insect, exists as one.After pooling this single peak, by heparinase II treatment, heparin disaccharide IV-H and I-S standard fragments were detected throughout MADI-TOF mass data program in this peak.

Identification of N-glycan derived from GbG
We have already noted for the monosaccharide composition of GbG [11].N-glycan of CbG was identified as Hex6, Hex5GlcNAc2, Hex6GlcNAc2, Hex7~10GlcNAc2 and m/z 1905.7 of Hex9GlcNAc2 by mass/mass spectroscopy (Fig. 5B and Fig. 5C).We found that the neutral mono-sugar of N-glycan derived from GbG, to be mainly hexose: L(+) rhamnose 81.10%,

Adipocyte density amid pathological observations
Pancreas tissues were repaired by treated GAG or metformin compared non-treated Homo-db mice at hematoxylin and eosin stain samples.Adipocyte density (cells/mm 2 ) was decreased in treated cardiac tissues compared to control by toluidine blue O stain, but in other organ tissues, toluidine stain deposit number was not shown to decreasing discrepancy as shown in Figure 6.

Discussion
Glycosaminoglycan can play a role in the curing increased blood glucose, hyperglycemia, and other complicated diseases.As an insect source, glycosaminoglycan extracted from the mucous membrane in the cricket inner cortex has been reported to have an antilipidemic effect on high fat diet rat [11], and blood-pressure-lowering [23] and anti-inflammatory effects on adjuvant-induced edema rats [9].The results of the present study displayed that, after, treatment with each glycosaminoglycan, sera BUN levels were decreased (CaG5, 68.5%; GbG5, 75.5% and Metformin10, 68.4%) compared to those in the DB-Homo group.
Furthermore, the levels of total albumin, alkaline phosphatase, ALT, AST, creatinine, glucose, and HDL-cholesterol were also decreased by these GAGs, demonstrating their anti-diabetic thus thereby complicating disease amelioration effects.
In fact, some oxygenic functional groups, including epoxy, hydroxyl, and carbonyl groups enable a chemical structure constituent to exert a potent free radical scavenging activities [24].
In addition to antioxidant activities, GbG displayed antidiabetic activities accompanied by no adverse effects found in in vitro and in vivo models.
We also found some instances of the anti-oxidant action of glycosaminoglycan with scavenging activities through free radical causing cellular oxidative damages [25].On the other hand, scavenger enzymes like SOD with cofactors, Cu or Zn in cardiovascular target sites promote increases of antioxidant defenses [26].Levels of anti-oxidative enzymes and the following activities of catalase, glutathione peroxidase, glutathione-s-transferase and SOD were also increased by this GbGs.Henceforth; hepatocellular oxidative stress by free radical damage could be scavenged with the help of these antioxidant enzymes.In case of db mice experiment, CbG5 appeared to have anti-oxidant activities that are increasing ones of catalase by 114.9%,GPX by 248.1%, GST by 117.6% as well as SOD by 125.7%.We found as for cellular oxidative damage, protein oxidative damage was also to be reduced (CaG5, 81.5; GbG5, 81.5% and Metformin10, 93.0%) by these GaGs on the basis of blood neutrophil carbonyl content.However, we recognize some down laying elements such as glycosaminoglycan and mucopolysaccharide: purified human GAGs have shown to reduce cell death, limit DNA fragmentation and thus protein oxidation, decreased OH• generation and lactate dehydrogenase activity, inhibited lipid peroxidation and improved endogenous antioxidant defenses [27].On the other hand, the mucopolysaccharidosis type II (MPS II), a lysosomal storage disorder could be caused by deficient enzyme iduronate-2-sulfatase that is responsible for the degradation of glycosaminoglycans dermatan and heparin sulfate, could be protected against lipid peroxidation while protein damage in these patients by enzyme replacement therapy [28].Furthermore, the proposed mechanism of action on cricket glycosaminoglycan in db mice can be summarized as figure 6B.This anti-oxidative action of cricket glycosaminoglycan in type 2 diabetic mice could be supported in the following role that GAG lowers molecular weight of heparin, increases SOD levels, and inhibits oxidative stress [29] and glycosaminoglycan (from Urechis unicinctus) significantly enhances liver SOD and GSH-Px activity [30].We leave those concerns for further human clinical researches to come.

Conclusions
These results from sero-biochemical, hepatocellular anti-oxidant assay in db mice data suggest cricket (G.bimaculatus) could be used as natural anti-diabetic agents and functional food.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 March 2019 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 March 2019 doi:10.20944/preprints201903.0136.v1 2.4. Animals
(diabetes) male db mice at 12-weeks of age, were purchased from Samtako Co. Ltd. (Osan, Korea).All procedures were accorded with NIH Guidelines for Care and Use of Laboratory Animals.All experiments were approved (approval number: NIAS 201605) by Laboratory Animals' Ethical Committee of the National Academy of Agricultural Science, Rural Development Administration, Republic of Korea.Mice were acclimated for 6 months under normal husbandry conditions.They were provided free access to normal diet (D10001, AIN-76A rodent diet, Research Diet Inc., New Brunswick, NJ, USA) and water ad libitum.These mice were allocated into two (negative and positive) control groups and two groups with each GAG treatment (11 mice per group).They were distributed according to similarity in weight

Posted: 12 March 2019 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 March 2019 doi:10.20944/preprints201903.0136.v1
5 mg/dL at the age of 12 weeks to 578.2 mg/dL at the age of 17 weeks.The second week mg/dL; and Metformin 10, 540.08±48.16mg/dL(Figure2B).Early GbG treatment in the second week showed the lowering effect on the blood glucose level in db mice but long GbG Preprints (www.preprints.org)| NOT PEER-REVIEWED |