Hypoglycemic polysaccharides from Auricularia auricula and Auricularia polytricha inhibit oxidative stress,NF-κB signaling and proinflammatory cytokine production in streptozotocin-induced diabetic mice

Hun Xing,Dongxio Sun-Wterhouse,c,∗,Chun Cui,b,∗

aSchool of Food Science and Engineering,South China University of Technology,Guangzhou,510640,Guangdong,China

bGuangdong Weiwei Biotechnology Co.,LTD,Guangzhou,510640,Guangdong,China

cSchool of Chemical Sciences,The University of Auckland,Private Bag 92019,Auckland,New Zealand

Keywords:

Polysaccharides

Auricularia auricula

Auricularia polytricha

Hypoglycemic effect

Oxidative stress

ABSTRACT

Auricularia auricula(AA)and Auricularia polytricha(AP)are popular edible fungi.This study successfully produced hypoglycemic polysaccharides from un-smashed or smashing and sieving(through a 10-mesh sieve)AA and AP(termed as AAP/AAP-10 and APP/APP-10)via scalable processes(water extraction,ethanolic precipitation and deproteinization).This is the first report to compare the effectiveness of AAP and APP in combating streptozotocin-induced oxidative stress and diabetes-related changes in mice(body weight,fasting blood glucose,serum insulin,proinflammatory mediator and cytokines,oxidative stress-related products,antioxidant enzymes).APP and AAP with different molecular weights and monosaccharide molar ratios could be therapeutic options for diabetes with a low dose(100mg/kg/day)likely working better.At the same dose,APP generally performed more effective than AAP,and AAP-10/APP-10 seemed slightly more beneficial than AAP/APP.One mechanism underlying these antidiabetic functions might involve the NF-κB and associated signalling pathways.AP is cheaper than AA,thereby representing a favorable source of functional polysaccharides.

1.Introduction

Diabetes mellitus (DM) is a complex metabolic disease characterized by insulin resistance or deficiency, hyperglycemia anddyslipidemia. The number of diabetic patients is estimated to reach366 million or more by 2030 based on International Diabetes Federation reports [1]. Oxidative stress and inflammation have beenconsidered as common risk factors associated with the pathogenesis of diabetes, as the total antioxidant defense could be lowered owing to dyslipidemia and hyperglycemia in prediabetes [2]. Whenthe reactive oxygen/nitrogen species (ROS/RNS) overwhelm theantioxidative defense system in an organism, the pathologicalstate of oxidative stress takes place, causing oxidative modification of antioxidant enzymes and oxidative stress-related markers:increases ofMDA (malondialdehyde, a product of lipid peroxidationreflecting the degree of tissue oxidation), activation of aldose reductase (AR), formation of advanced glycation end products (AGEs, asource of ROS formed increasingly under hyperglycemic conditionsand in the diabetic vasculature), generation of methylglyoxal (MG,a highly reactive dicarbonyl compound and a major precursor ofAGEs), and reduction of superoxide dismutase (SOD), catalase (CAT)and glutathione S-transferase (GST) [3,4]. Inflammatory response ismediated by the action of cytokines such as tumor necrosis factor-α(TNF-α), interferon-γ (IFN-γ) and interleukin-6 (IL-6), and nuclearfactor kappa-light-chain-enhancer of activated B cells (NF-κB, acritical proinflammatory mediator and transcription factor) plays aprotective role under oxidative stress while monitoring the expression of genes encoding cytokines [5,6].

Recently,antidiabetic and antioxidant polysaccharides obtained from natural sources have gained increasing attention,such as opuntia[7],dendrobium[8],ginseng[9],seaweed(likeGracilaria lemaneiformis)[10],and okra[11].Culinary-medicinal mushrooms have been frequently used in traditional medicine or included in the diet as therapeutic substances against a wide range of diseases.Their derived products including dietary supplement have high market value worldwide(annual value above$5 billion)[12].Auriculariaspp.mushrooms are one of the most important cultivated mushrooms in the world.Auricularia auricula(AA)andAuricularia polytricha(AP)are both popular therapeutic wood-ear fungi,but differ in taste,morphology and selling price.AA grows in temperate zones whilst AP is a tropical species.Recent studies reported that the polysaccharides from AA and AP have a variety of beneficial effects,including antioxidant activity,anticoagulant activity,inflammatory response-attenuating,hypoglycemic and anti-cancer properties[13–15].

Although there are several studies on the hypoglycemic effects of polysaccharides from AA and AP,very few studies compared directly the polysaccharides obtained from AA and AP through the same industrially scalable process,in particular,their antidiabetic effects on streptozotocin(STZ)-induced diabetic mice,and their working mechanisms and the involved proinflammatory mediator and cytokines,antioxidant enzymes,and other oxidative stressrelated markers.Accordingly,this study investigated these aspects and aimed to obtain functional polysaccharides from AA or AP with a greater hypoglycemic effect and market potential.Two variables affecting the hypoglycemic and antioxidative actions,a smashingsieving pretreatment of initial mushroom raw material and the dose for administration in mice,were also examined.

2.Material and methods

2.1.Materials and chemicals

AA and AP,which met the Jiangshan Wanli Quality Internal Control Standard,were gifted from Jiangshan Wanli Traditional Medicine Co.Ltd.(Jiangshan,Zhejiang,China;Batch No.WLHME170601).The moisture contents of AA and AP were 11.1% and 12.2%,respectively.AA or AP was smashed and screened using different meshes(0,3,10,20,40,60,80 and 100-mesh).Based on the concentrations and yields of the obtained polysaccharide samples(Fig.S1),a 10-mesh sieve was selected(by which higher polysaccharide yields were achieved for both AA and AP).The AA-or AP-derived powder after smashing and sieving through a 10-mesh sieve was termed as “AA-10＇＇or “AP-10＇＇.Then the AA,AP,AA-10 and AP-10 samples were stored at−40◦C before use.Rhamnose,xylose,fucose,mannose,glucose and galactose standards were of analytical grade and purchased from Sigma Aldrich Chemical Co.(St Louis,USA).

2.2.Extraction and purification of polysaccharides

The extraction of polysaccharides from AA,AA-10,AP,and AP-10 were carried out following the method established in our laboratory[16]with some modifications.In brie fly,10g of AA,AP,AA-10,and AP-10 were mixed with 300mL of distilled water,respectively,at 105◦C for 2.5h,and this extraction step was repeated twice for each sample.Each of the obtained extracts was filtrated,and the supernatant was concentrated to 1/5 of the original volumeviaevaporation at 55◦C using a RE-52A rotary evaporator(Shanghai Yarong Biochemical Equipment Factory, Shanghai, China).Then the concentrated solution was treated by adding ethanol(final ethanol concentration:80%,V/V)and allowing the mixture to stand for 8h at 4◦C to get the precipitates.The precipitates were deproteinated through mixing with distilled water and then the Sevag reagent(chloroform:n-butanol=4:1,V/V), before vigorous homogenization and centrifugation at 8000gand 4◦C for 30min using a GL-21M refrigerated centrifuge(Chang sha Xiangzhi Centrifuge Instrument Co.Ltd.,Changsha,China).Deproteinization was repeated until no absorption was detected at 280nm.The deproteinated precipitates were lyophilized using a Scientz-18N freeze dryer(Ningbo Scientz Biotechnology CO.,LTD.,Ningbo,China)to yield the corresponding polysaccharide samples(termed as“AAP”,“APP”,“AAP-10”,and“APP-10”,respectively).

2.3.Determination of molecular weight,neutral monosaccharide composition and uronic acid content

The molecular weight(MW)of polysaccharides in AAP,AAP-10,APP,and APP-10 was determined using the method by Wu et al.[15]with some modifications.Brie fly,the analysis of MW was performed using a gel permeation chromatography(GPC;Hitachi Ltd.,Tokyo,Japan)system equipped with a G-5000 PWXL(Tosho TSKGEL,Japan)at 50◦C and a RI detector(Bischoff,Hitachi Ltd.,Tokyo,Japan),using degassed and filtered double distillated water as the eluting solution at a flow rate of 0.5mg/min.Commercial standards(MW 7200,16230,35600,74300,170000,535000,1580000 and 2754000)were applied for calibration.

The monosaccharide composition(MC)was analyzed by using HPLC method according to Xu et al.[17].AAP,AAP-10,APP,and APP-10 were hydrolyzed with trifluoroacetic acid(1mol/L)at 100◦C for 12h.The residual acid was removed under a stream of N2in a 60◦C water-bath after adding methanol and taking to dryness three times,then distilled water(1mL)was added to dissolve the residue.The hydrolysates were analyzed by HPLC on a Waters HPLC system(Waters Technologies Palo,Alto,CA,USA)equipped with a differential detector(2414),using an Agilent-ZORBAX NH2 column(4.6mm×250mm,5μm,Agilent technology co.,LTD,USA).The flow rate was 1mL/min and the mobile phase was acetonitrile-water(65:35,V/V).The injection volume of monosaccharide standards and the hydrolysates was 20μL.The temperature of column and optical unit were both set at 35◦C.Identification of the monosaccharides in the hydrolysates was carried out by comparing their retention times and absorption with those of standards under the same HPLC conditions.

The total uronic acid content was determined colorimetrically according to the method reported by Sun-Waterhouse et al.[18]by using galacturonic acid as standard.

2.4.Fourier transform infrared spectroscopy analysis

Fourier transform infrared(FTIR)spectra of the freeze-dried AAP,AAP-10,APP,and APP-10 were acquired under the conditions reported by Xiang et al.[19].All the FTIR spectra were obtained in the range of 4000 and 500cm−1with a spectral resolution of 4cm−1.The obtained spectra were normalized using OMNIC 8.0(Thermo Fisher Nicolet,America)for further analysis.

2.5.Animal trial

Sprague-Dawley mice(male,aged4–5weeks,weighing(210±9.4)g)were obtained from the Experimental Animal Center of Jinan University(Guangdong,China),and maintained with free access to food and water in a controlled animal room(12h light/dark cycle,(23±2)◦C,humidity(50±5%)).All experiments were performed in accordance with the internationally accepted principles and guidelines for the care and use of laboratory animals.The maintenance of mice and induction of diabetes using STZ were performed as described by Liu et al.[11].The mice were divided into 10 groups(12 mice per group):the normal mice as the nor-mal control(NC group);the STZ-induced diabetic group without the administration of any test polysaccharide sample(MC group);the STZ-induced diabetic mice with a low dose APP,AAP,APP-10 or AAP-10 treatment only(termed as “APPL”,“AAPL”,“APP-10L”,and“AAP-10L”,respectively,with a dose of 100 mg/kg body weight/dayviaoral administration using a lavage needle);the STZ-induced diabetic mice with a relatively high dose APP,AAP,APP-10 or AAP-10 treatment only(termed as “APPH”,“AAPH”,“APP-10H”,and “AAP-10H”,respectively,with at a dose of 300 mg/kg body weight/dayviaoral administration using a lavage needle).After 4-week treatment,all the mice were fasted for 12h,and anesthetized with chloral hydrate(400mg/kg·body weight).Their blood samples were first collected from the orbital sinus,and centrifuged immediately for 5min at 8000gand 4◦C using the above-described refrigerated centrifuge to obtain sera for biochemical analyses.Then,the mice were sacrificed by cervical dislocation,and after necropsy,the liver samples were collected and stored at−80◦C for further use.

Table 1Molecular weight and monosaccharide composition of the polysaccharides.

2.6.Biochemical analysis

The glucose level in the collected tail blood was determined using a portable electronic blood glucose meter.The collected liver sample was homogenised and then centrifuged(3000g,4◦C;for 10min)to obtain the supernatant for the analyses of the oxidative indices,inflammatory factors and antioxidant enzymes using the corresponding commercial assay kits.Oxidative indices included AR,ROS,AGEs,MG and MDA.inflammatory factors included IL-6,IFN-γ,TNF-α,and NF-κB in the liver,along with the serum insulin.Antioxidant enzymes of interest included SOD,CAT,and GST.

2.7.Statistical analysis

All data were expressed as “mean±standard deviation(SD)”of at least three independent measurements.One-way analysis of variance was performed using Minitab 17(Minitab Inc.,State College,PA,USA),and Tukey’s test at a confidence interval of 95% was used to determine the significance of the difference(P＜0.05)between data.One-way analysis of variance was used to evaluate the data.

3.Results and discussion

3.1.MW,MC and uronic acid content of AAPs and APPs

The MW values of AAP and AAP-10 were 9–10 times as high as those of APP and APP-10(Table 1),indicating the size difference for the polysaccharides in the different types of mushrooms.The difference in the MW values between AAP and AAP-10,or between APP and APP-10,was likely caused by the additional sample treatments including smashing and sieving. The smashing step led to the disruption or even damage of the cell walls of mushroom,and the sieving allowed the particles with a reduced size to be collected for subsequent extraction. As a result, a more effective extraction of the polysaccharides from mushrooms was facilitated.The higher MW of the polysaccharides from the smashed samples(AAP-10 and APP-10),as compared to those for their un-smashed samples(AAP and APP),was possibly due to the release of the high-MW polysaccharides originated from the mushroom cell walls.NaturalAuricularia auriculawas previously found to contain acidic heteropolysaccharides of 30×104–50×104Da[20].Another study showed that a polysaccharide fromAuricularia auriculawith a MW of 2.77×104Da was obtained by microwave-assisted extraction[21].Auricularia polytrichawas reported to have a low MW polysaccharide(MW:2.8×104Da)[22].But a salt-soluble polysaccharide fromAuricularia polytrichawas found to have a MW of about 9.30×105Da[23],while the polysaccharides obtained fromAuricularia polytrichaby high-speed counter current chromatography were of 162,259 and 483kDa[24].

All the four polysaccharide-containing samples in this study(AAP,AAP-10,APP and APP-10)consisted of fucose,glucose,galactose,xylose,rhamnose and mannose but with different molar ratios of these monosaccharides(Table 1).Previously,a polysaccharide obtained fromAuricularia auricula viamicrowave-assisted extraction was found to contain glucose,galactose,mannose,arabinose and rhamnose at the molar ratio of 37.53:1:4.32:0.93:0.91[21].In terms ofAuricularia polytricha,mannose,galactose and glucose were reported in molar ratios of 4.2:2.3:1.1,3.3:1.7:0.4,3.5:2.1:0.6 for the polysaccharide[22].Furthermore,both AAPs and APPs had relatively high uronic acid contents,with the former being much higher than the latter(Table 1).These results indicated that AAP and APP were both acidic polysaccharides,which was in accordance with the previous reports[20,22].Sun-Waterhouse et al.[18,25]found that a polysaccharide fraction with a higher uronic acid content(including those from onion and apple)likely exhibited a higherin vitroantioxidant activity.

3.2.FTIR spectra of AAP,AAP-10,APP and APP-10

FTIR spectroscopy was used to examine further compositional and structural features of the obtained polysaccharides.The FTIR spectra of AAP,AAP-10,APP and APP-10 resembled with a little difference in certain signals(Fig.1).The FTIR spectra of these samples all had a broad and intense signal peak around 3600–3200cm−1(indicating the O-H stretching vibration of hydroxyl group),and a weak absorption around 3000–2800cm−1(indicating a C-H stretching vibration)[21,26].The presence of the signal bands around 1630–1400cm−1in all the four polysaccharide samples indicated the occurrence of deprotonated-COO−groups in the polysaccharide structures of these samples(which agreed with their high uronic acid contents).The C-O group in uronic acid can exhibit stretching vibrations at 1625 cm-1(in the form of deprotonated-COO−group)and/or 1740–1760cm−1(in the form of protonated carboxylic acid-COOH group)[27].

Fig.1.FTIR spectra of AAP,AAP-10,APP and APP-10.

Slight differences among the AAP,AAP-10,APP and APP-10 were detected in the weak signals at 1022–1066 and 890cm−1(which were assigned to the C–O–H bending and C–O–C stretching vibration of the glycosidic linkages including β-(1–4)linkages in polysaccharides)[27].While both had a weak absorption at 889cm−1,AAP had a weak absorption at 1067cm−1.This difference might result from the differences between AA and AP in glutamic acid content[28],crystalline/amorphous proportion[29]and polymerization degree.The presence of 1067 and 2967cm−1in AAP(instead of 1035 and 2973cm−1in APP)suggested that compared to APP,AAP had a slightly higher polymerization degree and higher amount of glutamic acid moieties(such glutamic acid moieties might result in an amide linkage that contributed to the signal at 2967cm−1).AA was reported to have much higher glutamic acid(5.53% dry weight)than AP(0.03% dry weight)[28].Also,a slight difference in the region of 1022–1067cm−1between AAP and AAP-10,with essentially no difference between APP and APP-10,suggesting that the smashing followed by sieving might affected AA to a greater extent(compared with AP),and during the subsequent extraction,loss or change of glutamic acid/derived amide residues and reduction of polymerization might take place in AAP-10(compared with AAP).

3.3.The anti-diabetic effects of AAP,AAP-10,APP and APP-10 inthe STZ-induced diabetic mice

After STZ injection,the fasting blood glucose(FBG)level in the MC group was significantly(P＜0.05)higher than that of the NC group(Fig.2A),indicating that the diabetic mouse model was successfully established.STZ is a nitric oxide(NO)donor and can cause the generation of superoxide anions,hydrogen peroxide and hydroxyl radicals,increase of FBG level,and partial or complete,damage to islet and hepatic cells and their mitochondria[30].

After the four-week treatment,the FBG level of the MC group remained remarkably higher than that of the NC group,whilst the FBG levels of all the APP-or AAP-treated groups were significantly(P＜0.05)reduced compared with the MC group(Fig.2A).These results were in accordance with the previous findings[31].The FBG levels for the treatment groups increased in this order:AAP-10L～=APPL＜APP-10L＜APP-10H ～=AAP-10H＜AAPL ～=AAPH ～=APPH,indicating that a low dose of AAP or APP generally worked better than a high dose to reduce the FBG level. At a low dose,APP samples were more effective to decrease the FBG level,while the smashing followed by sieving led to a greater FBG-lowering effect of AAP-10 L compared to APP-10L.APPs and AAPs reversed the FBG level in the STZ-induced diabetic mice probably through their contributions to the repairing of the damaged β-cells in the pancreatic islets,the promotion of insulin synthesis(also see the results of serum insulin in Section 3.4),and the reduction of the level of plasma glucose.

Changes in body weight before and after the induction of diabetes by STZ and the 4-week treatment with AAPs or APPs are summarized in Fig.2B.No statistically significant differences(P＞0.05)were detected in the initial body weight between the ten experimental groups of mice prior to the injection of STZ.During the trial period,the body weights of the NC group increased steadily.An insignificant or marginal increase(P＞0.05)of body weight was observed in the STZ-treated mice,which agreed to the previous findings[7].After the AAP/APP administration period,the body weights of the MC group were higher than those of APP-/AAP-treated groups,indicating that the AAPs or APPs could facilitate a weight loss in the diabetic mice.At either dose,AAP-10 and APP-10 seemed to be slightly less effective in weight loss compared to AAP and APP.

3.4.Effects of AAPs and APPs on inflammatory cytokines

NF-κB plays a crucial role in various biological processes including immune responses,inflammation,stress responses,and cell growth,survival,development and apoptosis.inflammatory response is mediated by the action of cytokines such as TNF-α and IL-6.In this study,compared with the NC group,the MC group had significantly(P＜0.05)higher levels of proinflammatory mediator(NF-κB)and pro-inflammatory cytokines(IFN-γ,IL-6 and TNF-α),but a lower level of serum insulin(Table 2),which further indicated that the model group was successfully established.After administration,the levels of the cytokines and NF-κB of the APP/AAP-treated mice were reversed to the levels lower than those of the MC group(with some being comparable or even lower than those of the normal group),whilst the serum insulin levels of the APP-/AAP-treated mice were restored to the levels higher than those of the MC group.Both polysaccharides from AA and AP could favourably regulate the inflammatory factors.The effectiveness of APP in regulating the cytokines,NF-κB and serum insulin seemed slightly greater than that of AAP.Such effectiveness depended on the dose and type of polysaccharide from AA/AP sample and the absence/presence of smashing and sieving),and varied with the type of biomarker(cytokine,NF-κB or serum insulin).The smashed and sieved samples exhibited greater desirable effects on IL-6,INF-γ and serum insulin, although the smashing-sieving process caused an insignificant difference in regulating TNF-α,but opposite effects on NF-κB between APP and AAP.Acute STZ injection has been used to study oxidative damage on tissues,as STZ can inhibit partially or completely insulin secretion by the pancreas through the selective destruction of β-cells in the pancreatic islets,and induce the generation of a range of ROS including H2O2in islet cells over time,causing oxidative stress[32].The treatment with AAP or APP sample could downregulate the proinflammatory cytokines, inhibit NF-κB and increase serum insulin in the mice with STZ-induced diabetes.

3.5.Oxidative stress-modulating effects of AAP or APP administration in STZ-induced diabetic mice

Fig.2.Effect of AAP or APP administration on(A)fasting blood glucose(FBG)levels(“Initial”refer to “before diabetes induction by STZ”,and “Final”refer to “four weeks after the administration with AAPs or APPs”,respectively),and(B)body weights in STZ-induced diabetic mice(“Initial”,“After modelling”,and “After administration”refer to “before diabetes induction by STZ”,“one week after diabetes induction by STZ but before administration with AAPs/APPs”,and “four weeks after the administration with AAPs or APPs”,respectively).Different letters(a,b,c)or(A,B,C)on top of the columns indicate significant differences at P＜ 0.05 among the ten groups.

Table 2Effect of AAP or APP administration on the inflammatory cytokines in STZ-induced diabetic mice.

Table 3Oxidative stress-modulating effects of AAP or APP administration in STZ-induced diabetic mice.

Compared with the NC group, the MC group had higher contents of oxidative stress-related products(including ROS,AGEs,MDA and MG),a higher activity of an aldehyde-metabolizing enzyme(i.e.AR),and lower activities of endogenous antioxidative defense enzymes(including SOD and CAT),with an insignificant change(P＞0.05)in GST(Table 3).After the administration with AAP/APP sample,the levels of ROS,AGEs,MDA,MG,AR were lowered whilst the levels of antioxidant enzymes increased.In terms of ROS and MG reduction,in general,the APP samples were slightly more effective than the AAP samples subjected to the same production process,and a low dose of AAP/APP sample worked either better or the same compared with a high dose(although the dose-dependence was not obvious).In terms of AGEs and MDA,similar effects were found for the AAP and APP samples produced through the same process and applied at the same dose,except for AAPL(which exhibited the greatest suppression).In terms of AR,APP samples were generally more effective than AAP samples,and there were interplays between the applied dose of AAP/APP samples and the smashingsieving treatment.

In terms of restoring SOD,AAP seemed more effective than APP,except for APP-10L(which restored the most).In terms of CAT,AAP samples and APP samples exhibited essentially the same restoring effect.In terms of GST,both AAP and APP samples could raise the GST level above that of the normal group,with the low dose of these polysaccharide samples working more effective than the high dose.The low dose of AAPL or APPL led to the highest GST level(34–36 U/mg prot).A previous study reported that a water-soluble polysaccharide(AAPI-a)obtained fromAuricularia auricular viaultrasonic-assisted extraction and purification by anion-exchange and gel-permeation chromatography exhibited antioxidant activities through decreasing significantly(P＜0.05) the MDA level while increasing SOD and glutathione(GSH)activities in D-galactoseinduced ageing mice[33].

In diabetic animals,free radicals accumulate rapidly,causing oxidative stress.The antioxidant status of tissues is closely associated with the etiology of diabetes,and the treatment with an antioxidant may alleviate diabetic complications[34].The abovementioned data support that all the AAP and APP samples exhibited a potential in alleviating STZ-induced oxidative stress and protecting the antioxidant enzymes.At the same dose,APP exhibited greater effectiveness in monitoring AR,ROS,AGEs and MG,greater or similar effectiveness in protecting antioxidant enzymes,but less effectiveness in regulating MDA compared to AAP.At the same dose,the polysaccharide samples from the smashed and sieved mushroom materials generally performed better than(if not,at least comparably to)those from the mushrooms without smashing and sieving in regulating these oxidative stress-related markers.

4.Conclusions

In this study,hypoglycemic polysaccharides were successfully produced from two types of commercially available edible mushroomsAuricularia auriculaandAuricularia polytricha(AA and AP,respectively)after water extraction,precipitation with ethanol(final ethanol concentration:80%V/V),and deproteinization.A portion of the initial mushroom materials(AA or AP)was pretreated by smashing and sieving(through a 10-mesh sieve),before being subjected to the polysaccharide preparation process to obtain“AAP-10”and “APP-10”polysaccharide samples.The AAP,APP,AAP-10 and APP-10 all contained heteropolysaccharides with different MWs(AAP and AAP-10＞APP and APP-10)and different molar ratios of neutral monosaccharides,fucose,glucose,galactose,xylose,rhamnose and mannose(AAPs and APPs were acidic polysaccharides, with AAP samples having much higher uronic acid contents than APP samples).

The extracted polysaccharide samples from AA and AP with and without the smashing-sieving treatment all exhibited a hypoglycemic effect and could alleviate STZ-induced oxidative stress and subsequent pathologic processes of diabetes.A low dose of these polysaccharides performed more effective than a high dose,and at the same dose,APP was generally more effective than AAP.One possible mechanism underlying these hypoglycemic and antidiabetic functions of APP and AAP might involve the NF-κB and other signalling pathways(as NF-κB functions at the crossroads of number of signalling pathways),as well as pro-inflammatory cytokines and endogenous antioxidant enzymes.Further studies should be conducted to understand the mechanisms underlying the oxidative stress-induced acceleration of the progression of diabetic complications.Moreover,there were interplays among the applied dose of AAP/APP samples,type of mushroom,and the smashing-sieving pretreatment inin vivoexperiments with mice.The smashing-sieving pretreatment of the mushrooms might be slightly beneficial,although considerable and consistent advantages of this pretreatment in monitoring the examined parameters were not found.Since AA is much more expensive than AP and APP seemed more effective than AAP at the same dose in combating oxidative stress-induced diabetes,AP might be a more desirable source of functional polysaccharides with hypoglycemic and antioxidative properties.More in-depth studies should be undertaken to elucidate the structure-function relationship for AAPs and APPs.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors are grateful for the financial support from National key Technologies R&D Program for 13th Five-year Plan(2016YFD0400803),National Natural Science Foundation of China(No.31201416)and Science and Technology Plan of Guangdong Province(2017ZD093).

Appendix A.Supplementary data

Supplementary material related to this article can be found,in the online version,at doi:10.1016/j.fshw.2020.06.001.