Structural characterization and hypoglycemic activity of Trichosanthes peel polysaccharide
Tianle Chen1,Min Zhang1,Jinglei Li,Maheen Mahwish Surhio,Beibei Li,Ming Ye*
College of Biotechnology and Food Engineering,Hefei University of Technology,Hefei230009,China
a r t i c l e i n f o
Article history:
Received4August2015 Received in revid form
29January2016
Accepted8February2016 Available online17February2016
Keywords:
Trichosanthes peel Polysaccharide
Purification
Structure
Hypoglycemic activity a b s t r a c t
Trichosanthes peel polysaccharide(TPP)was obtained from the aqueous extract of Trichosanthes peel by alcohol precipitation,deproteinization and decoloration.TPP was then parated and purified by Sephadex G-100column to obtain the homogeneous component TPP-1(1.2Â105Da).The compositions in monosaccharides were D-arabino(Ara),D-manno(Man),D-gluco(Glc)and D-galacto(Gal)with a molar ratio of1.00:3.27:4.26:6.01.Its backbone was compod of1,4,6-Gal p,1,4-Gal p,1,3,6-Man p and 1,4-Man p,while the branches comprid of1,3-Ara f,1-Ara f and1-Glc p.Diabetic mice experiments showed that the blood gluco levels in hyperglycemia mice reduced by22.47%,15.38%and12.72%after administration of high,medium and low dos of TPP-1,respectively.Compared with the negative control group,the contents of insulin and total superoxide dismuta of the hyperglycemia mice in groups treated with different dos of TPP-1were incread significantly,while the contents of biochemical indexes including malondialdehyde,creatinine,triglyceride,total cholesterol low density lipoprotein cholesterin and blood urea nitrogen were decread in different degrees.The results suggested that TPP-1possd strong hypoglycemic activity on streptozotocin-induced model mice.
孟姜女哭长城的故事
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1.Introduction
Diabetes mellitus(DM),a common endocrine dia,usually manifests as the disorder of blood gluco,fat and protein levels of the patients,and induces some complications including vascular lesions and diabetic neuropathy(Clements,1979).The prevalence of DM has incread substantially over the past years being the third most prevalent dia,which is following by cardiovascular system dias and cancer.It may po great threat to human health and life in the developed countries(Wang et al.,2013).DM is mainly caud by the metabolic disorder,and prents as hyper-glycemia,dyslipidemia and oxygen free radical metabolism enzyme defects(Kesavulu,Giri,Kameswara,&Apparao,2000;Nawata, Sohmiya,Kawaguchi,Nishiki,&Kato,2004;Scoppola,Montecchi, Menzinger,&Lala,2001).Sulfonylureas,biguanides and thiazoli-dinediones applied as oral anti-diabetic medicines have the ability to increa the reduction rate of blood sugar level,but behave poorly in regulating the gluco tolerance and dyslipidemia (Tahrani,Piya,Kennedy,&Barnett,2010).Moreover,the drugs may also produce toxicity and side effects if taken for a long time (Hu,Liu,Ni,&Lu,2014).Therefore,it is necessary to explore and develop
other natural and effective hypoglycemic drugs for the prevention and treatment of DM.
Plant polysaccharides contain many monosaccaride composi-tions with various structures and biological activities(Ding,Zhu,& Gao,2012).Polysaccharides isolated from Ophiopogon japonicas and Porphyra yezoensis have been proven to exert hypoglycemic and hypolipidemic effects,respectively(Chen et al.,2011;Qian,Zhou,& Ma,2014).Therefore,plant polysaccharides can be explored as a kind of natural medicines and new functional food.
Trichosanthes kirilowii Maxin belongs to Trichosanthes genus of cucurbitaceous,a perennial vine.Trichosanthes peel is the ripe pericarp of T.kirilowii Maxin and rich in oils,organic acid,poly-saccharide,flavones and protein(Qian,Dan Liu,&Peng,2010).It was reported that Trichosanthes peel was widely ud in traditional Chine medicine to treat dias of cerebrovascular,cardiovas-cular,and respiration systems due to the abilities to clear heat and dissipate phlegm,regulate theflow of vital energy and relieve chest stuffiness(Chen,Huang,&Wang,2006).However,there is no report with regard to the structure and hypoglycemic activity of Trichosanthes peel polysaccharide.
The aims of this study were to analysis the structure of the polysaccharide from Trichosanthes peel(TPP-1)and to evaluate its hypoglycemic activity.
*Corresponding author.Tel.:þ8655162919368.
E-mail address:(M.Ye). 1The authors contributed equally to this
work.Contents lists available at ScienceDirect
LWT-Food Science and Technology journal ho mep age:/lo
cate/lwt
dx.doi/10.1016/j.lwt.2016.02.024
0023-6438/©2016Elvier Ltd.All rights rerved.
LWT-Food Science and Technology70(2016)55e62
2.Materials and methods
2.1.Materials and reagents
Trichosanthes peel(T.kirilowii Maxin)was provided by Lushi Ecological Agricultural Technology Co.,Ltd(Anhui).
Sephadex G-100column was purchad from Sigma Chemical Co.(Zhengzhou,China).ELISA kits for the analysis of insulin(INS), triglyceride(TG),total cholesterol(TCH),low density lipoprotein cholesterin(LDLC),creatinine(Cr)and blood urea nitrogen(BUN) were purchad from Yansheng Biological Technology Co.,Ltd. (Shanghai,China).Total superoxide dismuta(T-SOD)kit and malondialdehyde(MDA)kit were purchad from Nanjing Jian-cheng Technology Co.,Ltd.(Nanjing,China).Sinocare glucometer and test paper were purchad from Changsha Sinocare,Inc. (Changsha,China).The reagents were of analytical grade unless otherwi specified.
2.2.Experimental animals
Sixty Kunming mice with the body weight of20±2g were purchad from Experimental Animal Center of Anhui Medical University,Hefei,China.All animals'treatments were strictly in accordance with the N
ational Institutes of Health Guide for the Care and U of Laboratory Animals.The animals were kept at23±2 C with the humidity of55±5%,and cultured in a14h:10h light e dark cycle.
2.3.Preparation of TPP-1
Trichosanthes peel was dried to a constant weight at the temper-ature of40e50 C in an oven,and grounded to80e100m m particles. The polysaccharide was extracted according to the method of Khodaei and Karboune(2013).Trichosanthes peel was suspended in NaOH alkaline solution,containing0.02M NaBH4,and the mixture were incubated at60 C for24h.The supernatants were recovered after centrifugation(4,000g,10min)followed byfiltration.The recovered polysaccharide solution was neutralized to pH7.0with 0.1M acetic acid,and mixed with three volumes of95%ethanol to obtain precipitate.Then,the precipitate was dialyzed in14,000Da dialysis bag against tap water and distilled water for48h succes-sively.Crude polysaccharides(TPP)were obtained after freeze-drying for24h.The crude polysaccharide was fractionated by Sephadex G-100chromatographic column.The major polysaccharide peak eluted by distilled water was collected and freeze-dried,and ud for further structure analysis and animal experiments.
The chromatography conditions of Sephadex G-100 (1.6cmÂ60cm)were as follows:the amount of lo
ading sample was20mg;eluent was double distilled water at aflow rate of 0.4mL minÀ1,and4mL of the solution was collected in each tube.
2.4.Purity and molecular weight(M W)determination
Ultraviolet(UV)absorption spectra of TPP-1was recorded with a Agilent Cary5000spectrophotometer(Agilent Technologies Co. USA).High performance liquid gel permeation chromatography (HPGPC)was further ud to test the purity of TPP-1and to calculate its molecular weight.The Waters-2414HPLC host(Waters Co.USA)that was equipped with Waters-1515parallax shading monitor was ud.Double distilled water was ud as eluent at the flow rate of0.5mL minÀ1with the Ultrahydrogel™2000analytical column temperature of35 C.The purity of the sample was determined according to the shape and distribution of the eluting peak.When the eluting peak was single,symmetric and narrow, with the feature of homogeneous distribution,it proved that TPP-1was a homogeneous polysaccharide.To obtain the molecular weight of TPP-1,standard T-ries dextrans were also eluted through the column,and a calibration curve was plotted.The mo-lecular weight of TPP-1was calculated according to the calibration curve established by standard T-ries dextrans(M W:4400,9900, 21,400,43,500,124,000,196,000,401,000Da).
2.5.Analysis of monosaccharides
2mL of2M Trifluoroacetic acid(TFA)was added into TPP-1 (5mg)powder and the solution was hydrolyzed at121 C for4h. The subquent steps were carried out according to the method of Ye et al.(2011).NaBH4(40mg)was added and restored overnight under the room temperature.4mL of acetic anhydride and3mL of pyridine were added to the dried hydrolyzate and incubated at 110 C for1h.The acetylated derivative was loaded into gas chromatography-mass spectrometry(GC e MS)(Shimadzu Co. Japan).Conditions of GC e MS:quartz capillary column HP-5 (30mÂ0.25mmÂ0.25m m);temperature programming was lected for column temperature which incread from50 C to 250 C at the rate of10 C minÀ1;temperature of the injection port was260 C while theflow rate of He was1mL minÀ1;ion source:EI, 70eV;the molecular weight range:35e650.
2.6.Periodate oxidation and smith degradation
The method of Linker,Evans,and Impallomeni(2001)was referred with slight amendments.TPP-1(12.5mg)was dissolved in 25mL of15mM NaIO4,and then the solution was placed in dark.An aliquot of this solution(1mL)was taken out every6h and diluted 100times with distilled water.The absorbance of the solution was detected at223nm by the UV spectrophotometer.When it was stable at223nm,1mL of reaction liquid was drawn out and titrated with0.5mM NaOH to measure the production of formic acid.
The purpo of Smith degradation is to reduce periodate oxidation products to polyol with boron hydrides,and evaluate the linkage types and order of the polysaccharide components.Ac-cording to the method of Rout,Mondal,Chakraborty,and Islam (2008), 1.0mL of ethylene glycol was added into the poly-saccharide solution after periodate oxidation.The solution was shaken and then placed for3h,The mixture was dialyzed with distilled water for48h and concentrated by vacuum.NaBH4(80mg) was added and later restored for24h under the room temperature. Then,0.1M acetic acid was added to the solution for neutralization and dialyzed for48h with distilled water,and evaporated to dry-ness under the decompression.2mL of2.0M TFA was added and aled in a tube,and hydrolyzed at120 C for2h.Then5mL of methanol was added and evaporated to dryness again by decom-pression.The above procedures were repeated twice to eliminate TFA.The sample was acetylated according to the method described in the ction2.5and then GC e MS analysis was carried out.
2.7.Methylation analysis
自虐的诗The method of Needs and Selvendran(1993)was referred.TPP-1 (28.0mg)was taken and added into0.1mL of water.5mL of DMSO and3A molecular sieve(5mg)were added andfiltrated withfilter paper into the reactionflask.NaOH(80mg)powder was added into the solution and N2was injected int
o theflask.Under the room temperature,it was treated by ultrasonication for30min to obtain the solution.Later,1.0mL of98%CH3I was added and N2was injected into theflask.Ultrasonic treatment was carried out in20 C for1h to expel the residual CH3I.The procedures above were repeated for three times.The reaction was terminated by adding2mL of water and the solution was neutralized by25%acetic acid.Then,it was
T.Chen et al./LWT-Food Science and Technology70(2016)55e62 56
dialyzed for48h with distilled water and freeze dried.Detected by FT-IR,TPP-1was fully methylated if there was no absorption peak in 3400cmÀ1.2mL of2M TFA was added and hydrolyzed at120 C for 3h.The sample was acetylated bad on the method described in the ction2.5,and then GC e MS analysis was carried out.
2.8.Partial acid hydrolysis analysis
According to the method of Tong et al.(2009),8mL of0.5M TFA was added into TPP-1(80mg).The sample was aled in a tube and hydrolyzed at90 C for4h.8mL of methanol were added to the solution for co-evaporation to eliminate TFA and then the solution was dialyzed in distilled water for48h.The solutions in and out of the bag were parately concentrated and4mL of ethanol was add
ed into each solution.The precipitate for the inner component and the supernate for the outer component were collected and methylation was carried out twice for the both of the components, respectively.Then,the methylated products of them were fully hydrolyzed and acetylized into the corresponding alditol acetates. The products were analyzed by GC e MS according to the method described in the ction2.5.
2.9.FT-IR analysis
According to the method of Ye et al.(2011),TPP-1was ground with KBr(1:100)and presd into tablets,and measured with a Fourier transform infrared spectrometer(Thermo Nicolet Co.USA) between4000and400cmÀ1.
2.10.NMR analysis
According to the method of Nep and Conway(2011)，TPP-1 (50mg)was put in a5mm NMR tube and dissolved in1.0mL of 99.96%D2O.1H NMR and13C NMR analysis were carried out on a Bruker Avance AV-500nuclear magnetic resonance spectrometer (Bruker Co.Germany).The resonance frequencies were at 500.13MHz for1H NMR and125.76MHz for13C NMR,and the test temperature was27 C.Chemical shifts were given in ppm.
2.11.Establishment and grouping of hyperglycemia model mice and determination of relating indexes
Mice were adaptively fed for7d and later no food but only water was offered for12h.Intraperitoneal injection(dosage: 140mg kgÀ1)was conducted to mice with streptozotocin(STZ) water solution(2%).The mice were intraperitoneally injected and normally fed for3concutive days.Then,the mice were fasted for 12h.The blood gluco(blood was drawn from the tip of tail)was measured by Sinocare glucometer(Sinocare,Inc.China)and the mice could be ud as hyperglycemia model mice if the blood gluco(FPG)level was higher than11.1mmol LÀ1(Ye et al.,2012).
Ten normal mice were lected as the normal control group and gavaged with normal saline(NS).Hyperglycaemia model mice were divided intofive groups(10mice per group)including the negative control group(10mL kgÀ1NS),TPP-1low-do group(50mL kgÀ1), TPP-1medium-do group(100mL kgÀ1),TPP-1high-do group (200mL kgÀ1)and the positive group(200mL kgÀ1metformin hydrochloride).They were gavaged once per day for30concutive days.During the experiment,the mice got free access to food and water.After the gavage treatment ended,the eyeballs of mice were picked out to obtain the sample blood.Later,the sample blood was centrifuged at3000rpm for5min and the rum was obtained. Contents of INS,Cr,BUN,TG,TC and LDLC in the s
erum were determined by iMark ELISA(Bio-Rad USA)according to enzyme-linked immunosorbent assay.T-SOD activity in the rum was assayed by hydroxylamine method,and MDA contents in the rum was measured by thiobarbituric acid method.
2.12.Statistical analysis
Software DPS v6.55was ud to compare the two samples,and T-test for variance analysis was carried out.All data obtained from each group of the tested mice were expresd as mean value±standard deviation.Differences were considered statisti-cally significant when p<0.05and very significant when p<0.01.
3.Results and discussion
3.1.Purity and molecular weight of Trichosanthes peel polysaccharide
The yield of the total polysaccharide obtained from Trichosanthes peel was1.91%.TPP was parated and purified through Sephadex G-100column chromatography(Fig.1),obtaining two elution peaks which were named as TPP-1and TPP-2,respectively.Peak shape of the former was in symmetry and the distribution of molecular weight of polysaccharide was relatively centralized.Thus,
TPP-1was collected for further structural analysis and animal experiments. There was no absorption peak between260and280nm in the UV spectrogram of TPP-1(Fig.2A),indicating that TPP-1did not contain protein or nucleic acid(Qiu,Ma,Ye,Yuan,&Wu,2013).
排骨糯米饭
The results of HPGPC showed that the elution peak of TPP-1was a single symmetric peak with a relatively narrow distribution, suggesting that TPP-1had reached the chromatographic grade. Thus,TPP-1was identified to be a homogeneous polysaccharide. M W of TPP-1was calculated to be1.2Â105Da according to the equation of the standard curve(log M W¼595þ93.50TÀ11.45T2þ0.68T3,T reprented elution time) and retention time(16.225min)of the elution peak(Fig.2B).
3.2.TPP-1monosaccharide components
TPP-1was acetylated after being fully hydrolyzed by TFA.The total ion chromatogram of GC e MS analysis showed four peaks (Fig.3A)with retention times of20.906min,21.079min,21.216min and21.406min.The peaks reprented the hexa-acetate of D-0102030405060708090100110
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Fig.1.Chromatogram of the crude polysaccharide(TPP)extracted from Trichosanthes peel on Sephadex G-100column eluted double distilled water at aflow rate of 0.4mL minÀ1.According to the order of the elution time,two elution peaks were named as TPP-1and TPP-2,respectively.
T.Chen et al./LWT-Food Science and Technology70(2016)55e6257
arabitol,D-mannitol,D-glucitol and D-galactitol,respectively.Analysis by area normalization method showed that the molar ratio was about 1.0:3.27:4.26:6.01.The results showed that TPP-1was compod of D -arabino (Ara),D -manno (Man),D -gluco (Glc)and D -galacto (Gal).
3.3.Structural characterization of TPP-1
In the periodate oxidation reaction,0.642mol periodic acid was consumed and 0.291mol formic acid was produced by each mole of anhydrogluco unit.The ratio of periodic acid consumption to formic acid production was about 2.2:1.0.The production of formic acid suggested that 1/6or 1/glucosidic bond was prent.The fact that the consumption of periodate was more than two times the productio
想象猫咪n of formic acid indicated that there might prent 1/2,1/4,1,2/6or 1,4/6glucosidic bonds,which only consumed periodic acid but not produced formic acid.
GC e MS analysis for Smith degradation products showed the existence of mannitol,glucitol and a small quantity of glycerinum.The generation of the hexitols suggested that TPP-1might contain
1/3,1,3/6,1,2/3and 1,2/4glucosidic bonds,which could hardly be oxidized by periodic acid.The generation of glycerinum suggested that TPP-1might contain 1/and 1/6glucosidic bonds.Methylation products of TPP-1(Table 1)were mainly compod of 2,5-Me 2-Ara,2,3,5-Me 3-Ara,2,4-Me 2-Man,2,3,6-Me 3-Man,2,3,4,6-Me 4-Glc,2,3-Me 2-Gal and 2,3,4-Me 3-Gal with a molar ratio of about 0.80:0.60:1.10:2.20:4.17:4.34:2.01,which was in accor-dance with the result of GC e MS analysis of the monosaccharide components.The existence of 2,5-Me 2-Ara,2,3,5-Me 3-Ara,2,4-Me 2-Man,2,3,6-Me 3-Man,2,3,4,6-Me 4-Glc,2,3-Me 2-Gal and 2,3,4-Me 3-Gal indicated that TPP-1was compod of 1,3-Ara f ,1-Ara f ,1,3,6-Man p ,1,4-Man p ,1-Glc p ,1,4,6-Gal p and 1,6-Gal p .
The two components inside and outside the dialysis bag (named as TPP-1a and TPP-1b,respectively)were collected for methylation.The methylation products of TPP-1a consisted of 2,3-Me 2-Gal,2,3,4-Me 3-Gal,2,4-Me 2-Man and 2,3,6-Me 3-Man,which suggested that the backbone of
TPP-1was compod of 1,4,6-Gal p ,1,6-Gal p ,1,3,6-Man p and 1,4-Man p ,with a molar ratio of 4.25:1.91:1.14:2.17(Table 1),while the methylation products of TPP-1b contained 2,5-Me 2-Ara,2,3,5-Me 3-Ara and 2,3,4,6-Me 4-Glc,suggesting that the branches of TPP-1consisted of 1,3-Ara f ,1-Ara f and 1-Glc p with a molar ratio of 0.90:0.70:4.12.
In the FT-IR spectrogram (Fig.3B)of TPP-1,the wide and strong absorption peak at 3388cm À1resulted from the O e H stretching vibration (Qiu et al.,2013),and the weaker absorption peak at 2935cm À1was mainly contributed by the C e H (e CHOH,e CH 2e and e CH 3)stretching vibration (Zou,Zhang,Yao,Niu,&Gao,2010).The absorption peak at 1640cm À1was caud by the bending mode of bound water (Sun,Liu,Yang,&Kennedy,2010),and the ab-sorption peak at 1413cm À1was C e O stretching vibration (Su flet,Nicolescu,Popescu,&Chitanu,2011).The absorption peak at 1140cm À1was the characteristic peak of pyranoside and the weak small peak at 865cm À1indicated that TPP-1contained a -glucosidic bonds (Luo,He,Zhou,Fan,&Chun,2010).
Almost all the proton signals prented between d 3.00e 5.30in the 1H NMR spectrogram (Fig.4A),and the proton signals were typical polysaccharide signals.The signal peaks between 3.50and 4.50ppm were caud by protons on sugar rings (Qiu et al.,2013),therefore the signal peaks between 3.57and 4.14ppm were assigned to protons on sugar rings.The resonance signal peaks 溢组词语
at d >4.8ppm suggested that TPP-1contained a -con figuration of saccharide residues (Wang et al.,2011).The shifts of anomeric protons in TPP-1were all greater than the shift of 4.8ppm.Thus it could be concluded that TPP-1was linked by a -glucosidic bonds.In the 13C NMR spectrogram,d 90e 110ppm was the anomeric carbon area,and d 62e 85ppm was the absorption signal peak of C2~C5.TPP-1contained six types of anomeric carbon signals (Fig.4B).The signal peak at d 101.648ppm was assigned to the anomeric carbon of a -D-Gal p (Popov et al.,2011).The signal peaks at d 100.232ppm and d 97.649ppm were medium,and they were resulted from anomeric carbon of a -D-Man p and a -D-Glc p ,respectively (Patra et al.,2013;Wang et al.,2013).The signal peak at d 98.786ppm was the lowest and it was probably caud by anomeric carbon of a -D-Gal p (Sarkar et al.,2012).The resonance peaks at d 106.533ppm and d 107.135ppm were assigned to the anomeric carbon of a -L-Ara f (He et al.,2009;Kang et al.,2011).Furthermore,the signal peaks which appeared at d 82e 88ppm indicated that there were furan-gluco residues in TPP-1(Bushmarinov et al.,2004).Assignments of other carbons (C-1~C-6)were shown in Table 2(Dang et al.,2013).
According to the above results,we deduced that the backbone chain of TPP-1consisted of 1,6-Gal p ,1,3,6-Man p,1,4-Man p and 1,4,6-Gal p ,and the branches were compod of 1-Ara f ,1-Glc p and 1,3-Ara f ,and one of the possible repeat units of TPP-1which was as follows:
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Fig. 2.(A)Ultraviolet (UV)spectrum analysis of TPP-1.Ultraviolet spectrum was recorded with a Agilent Cary5000spectrophotometer in a range from 190to 400nm;(B)High performance liquid gel permeation chromatography (HPGPC)of TPP-1.The purity and molecular weight was measured by HPGPC with the mobile pha of double-distilled water at a flow rate of 0.5mL min À1and column temperature of 35 C.
T.Chen et al./LWT -Food Science and Technology 70(2016)55e 62
58
Fig.3.(A)Total ion chromatogram of acetylated derivatives of total hydrolyzate of TPP-1.TPP-1was hydrolyzed,acetylated and analyzed by gas chromatography-mass spectrometry (GC e MS);(B)Fourier transform infrared (FT-IR)spectrogram of TPP-1.Infrared (IR)absorption was measured with KBr pellet (mass ratio 1:100)by Fourier transform infrared spectrometer Nicolet 67in a range from 4000to 400cm À1.
T.Chen et al./LWT -Food Science and Technology 70(2016)55e 6259
