in vitro antidiabetic effects of isolated triterpene...

Research ArticleIn Vitro Antidiabetic Effects of Isolated Triterpene GlycosideFraction from Gymnema sylvestre

Rashmi S Shenoy 12 K V Harish Prashanth23 and H K Manonmani 12

1Department of Food Protectants and Infestation Control CSIR-Central Food Technological Research Institute Mysore 570020 India2Academy of Scientific and Innovative Research CSIR-Central Food Technological Research Institute Mysore 570020 India3Department of Biochemistry CSIR-Central Food Technological Research Institute Mysore 570020 India

Correspondence should be addressed to H K Manonmani manonmanicftriresin

Received 29 March 2018 Revised 30 May 2018 Accepted 10 June 2018 Published 8 August 2018

Academic Editor Hilal Zaid

Copyright copy 2018 Rashmi S Shenoy et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A triterpene glycoside (TG) fraction isolated and purified from ethanolic extract ofGymnema sylvestre (EEGS) was investigated forblood glucose control benefit using in vitromethodsThe HPLC purified active fraction TG was characterized using FTIR LC-MSand NMR The purified fraction (TG) exhibited effective inhibition of yeast 120572-glucosidase sucrase maltase and pancreatic 120572-amylase with IC

50values 316 plusmn 005 120583gmL 7407 plusmn 051 569 plusmn 002 and 117 plusmn 024 120583gmL respectively compared to control TG

was characterized to be a mixture of triterpene glycosides gymnemic acids I IV and VII and gymnemagenin In vitro studies wereperformedusingmouse pancreatic120573-cell lines (MIN6) TGdid not exhibit any toxic effects on120573-cell viability and showedprotectionagainst H

2O2induced ROS generation There was up to 134-fold increase in glucose stimulated insulin secretion (plt005) in a

dose-dependent manner relative to standard antidiabetic drug glibenclamide Also there was further one-fold enhancement inthe expression of GLUT2 compared to commercial standard DAG (deacylgymnemic acid) Thus the present study highlights theeffective isolation and therapeutic potential of TG making it a functional food ingredient and a safe nutraceutical candidate formanagement of diabetes

1 Introduction

Type II diabetes is one of the most widespread metabolicdisorders in the world characterized by hyperglycemia andhyperlipidemia About 190 million people are affected bydiabetes and the number is projected to rise to 642 mil-lion by 2040 [1] It is known that the onset of diabetesis related to many classic risk factors including oxidativestress [2] The complication associated with diabetes willbe enhanced due to the involvement of reactive oxygenspecies (ROS) [3] The ROS contributes to oxidative stressand in turn damages pancreatic islets and reduces insulinsecretion Growing evidences show that 120573-cells are centralto the development of type 2 diabetes [4] Therefore thefunctional defects in 120573-cells are often accompanied by areduction in expression of glucose transporter-2 (GLUT2)levels further characterized by increased apoptosis of 120573-cells[5 6] Therefore therapies for improving the 120573-cell function

have become the potential new strategy to control hyper-glycemia [7] Studies demonstrate that supplementation withnatural antioxidant products has shown improvement ofhyperglycemic status by reducing the oxidative stress [8]Also glucose generation and absorption in the intestine playa vital role in hyperglycemia management [9] Inhibition of120572-glucosidase and 120572-amylase which digest dietary starch intoglucose was studied as a method for controlling blood sugarlevels [10] Although several drugs for type II diabetes arein vogue they have shown side effects such as liver toxicityand gastrointestinal adversity [11] Thus the quest for safeeconomical and pharmacologically active molecules withmultifunctional attributes towards diabetes management isnecessary This can be achieved by screening natural sourcessuch as medicinal plants Moreover there is considerableinterest in the application of traditional plants due to theirnatural origin easily cultivable with minimum or no sideeffects

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 7154702 12 pageshttpsdoiorg10115520187154702

2 Evidence-Based Complementary and Alternative Medicine

Gymnema sylvestre (GS) a woody vine-like climbingplant is well known in Indian traditional medicine ldquoAyurv-edardquo It grows in the tropical forest of central and south-ern India [12] The leaves are used mostly as antiviraldiuretic antiallergic hypoglycemic hypolipidemic antibi-otic antianalgesic and antirheumatic agents [13] The leavesof GS contain triterpenoidal saponins belonging to oleananeand dammarane classes [13] Twenty different saponins andglycosides have been reported in Gymnema sylvestre [14]Several studies suggest that gymnemic acids may act asantidiabetic by promoting regeneration of islet cells andincrease insulin secretion [15] Also gymnemic acids havebeen reported to inhibit glucose absorption from the intestineand utilize glucose by enhancing the activities of enzymes ininsulin-dependent pathways [15]

The current focus of our study was to isolate and char-acterize bioactive agents (s) from Gymnema sylvestre andevaluate in vitro antidiabetic effects to initiate a search fornutraceutical pharmacophores towards inhibition of pancre-atic 120572-amylase and 120572-glucosidase with improvement in betacell function

2 Materials and Methods

21 Chemicals 120572-Amylase from porcine pancreas 120572-glucos-idase from Saccharomyces cerevisiae para-nitrophenyl-glu-copyranoside (pNPG) soluble starch and 3-45-(dimeth-ylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT)were procured from SRL chemicals (Bangalore India)Rat intestinal acetone powder (source of 120572-glucosidase)acarbose Anti-GLUT2 antibody anti-insulin antibody 2101584071015840-dichlorodihydrofluorescein diacetate (DCFH-DA) acridineorange (AO) and ethidium bromide (EtBr) were from SigmaChemical Co (St Louis MO USA) Glucose oxidase kitwas purchased from AGAPE diagnostics (India) ELISA kitswere purchased from Cytoglow Co (India) Analytical andHPLC grade solvents were from E Merck (India) The driedpowder of Gymnema sylvestre was obtained from NikhilaKarnataka Central Ayurvedic Pharmacy (Mysore India)Mouse pancreatic 120573-cell line (MIN6) was purchased fromNCCS (Pune India) Dulbeccorsquos minimal essential medium(DMEM) Fetal Bovine Serum (FBS) and antimycoticsolution were purchased from Himedia chemicals (India)All other chemicals used in the study were of analytical gradeand purchased from Himedia chemicals (India)

22 Preparation of Gymnema sylvestre (GS) Extracts GSpowder was extracted using different solvent systems likemethanol ethanol acetone ethyl acetate chloroform andwater respectively The GS powder along with respectivesolvents were left for 3 h refluxing and the resulting infusionswere filtered The filtrates were concentrated solvents wereevaporated using rotary evaporator (Cole-Parmer SB 1100Shanghai China) at 45∘C under reduced pressure and thenlyophilized The greenish-black powder thus obtained wasstored at 4∘C until used for further assays

23 120572-Glucosidase Inhibitory Assay Extracts of GS werescreened for 120572-glucosidase inhibitory activity according to

the method described in [16] The source of 120572-glucosidaseenzyme was from Saccharomyces cerevisiae The substratesolution pNPG (2mgmL)was prepared in 50mMphosphatebuffer pH 68 10 120583L of 120572-glucosidase (112 UmL) waspreincubated with 10 120583L of known concentrations of GS(extracted from different solvents) for 10 min pNPG (10 120583L)was added to start the reaction followed by incubation at37∘C for 20 min The reaction was stopped by adding 2 mLof 01 M Na

2CO3 The 120572-glucosidase activity was determined

by measuring the yellow-colored para-nitrophenol releasedfrom pNPG at 405 nm The results were expressed aspercentage of the blank control

24 Preparative HPLC of Ethanolic Extract of Gymnemasylvestre (EEGS) 200 g of dried plant material was extractedusing 1 L absolute ethanol under refluxing conditions for3h The extracted volume was concentrated using rotaryevaporator (Cole-Parmer SB 1100 Shanghai China) at 45∘Cat high pressure and brought down to 500 mL This wasfurther concentrated to sim5 g wet weight and a stock of 1mgmL was prepared using HPLC grade methanol solutionThe stock solution was filtered using nylon 6 membraneconsisting of six multiple filter papers with a pore size of 045120583m degassed by ultrasonic treatment before use PreparativeHPLC (gradient mode) was carried out using ODSC-18 silicacolumn (25 cm X 212 mm 12 120583m) The mobile phase wasprepared using two solvent systems solvent A water (HPLCGRADE) containing 1 acetic acid solvent B acetonitrilecontaining 1 acetic acidThemobile phasewas filtered usingnylon 6 multiple filter membranes having a pore size of 045120583m The conditions for HPLC [0 min solvent B (5 ) 5 min(8 ) 7 min (12 ) 12 min (18 ) 17 min (22) 24 min (35) 26min (100) 30min (100) 32min (5) 35min stop]were set constant flow rate was maintained at 5 mLminThedetector was set at 254 nm and fractions were collected Thefractions were made solvent free using a rotary evaporatorfreeze-dried and then resuspended in known amount ofphosphate buffer (50 mM pH 68) and were analyzed forpercentage 120572-glucosidase inhibitory activity

25Thin Layer Chromatography (TLC) andHigh PerformanceTLC (HPTLC) The purity of the HPLC purified activefraction was tested by thin layer chromatography Silica gel-G was taken as stationary medium and coated over glassslides The mobile phase used was tolueneethylacetateaceticacid (571 vv) [17] Deacylgymnemic acid (DAG) was usedas a standard The purity of the active fraction was checkedby HPTLC HPTLC system equipped with a Linomat IVsample applicator a twin-chamber tank a model III TLCscanner and CATS 40 integration software was employedAluminum-backed layers (10times 10 cm) of silica gelGF254wereused as absorbent The mobile phase used was chloroformand methanol (82 vv) The spotted plates were developedup to 80 mm under chamber saturation conditions After air-drying the solvent the plates were scanned at 205 nm [18]

26 Characterization by FTIR LC-MS and NMR The iso-lated active fraction was characterized by spectroscopicmethods FTIR (Bruker USA) spectra were recorded in the

Evidence-Based Complementary and Alternative Medicine 3

range of 4000-400 cmminus1 by blending samples with KBr LC-MS (Shimadzu Japan) was used to detect the molecularweights of the bioactive agents (s) detected in the activefraction The separation was carried out using C18 column(150 x 46 mm ID 5120583) column temperature maintained at40∘C Mobile phase consisted of methanol and water bothcontaining methanol and water (8020) at a flow rate of 1mLmin over 25minutesThe electrospray ionisationwas car-ried out in both positive and negativemode 1Hand 13CNMRspectra were recorded in DMSO-d

6on a Bruker Avance

USA 400 MHz spectrometer (Bruker Karlsruhe Germany)Assignments of 13C signals were based on respective chemicalshift values

27 120572-Amylase Inhibitory Assay This assay was carried outusing amodified procedure described in [19] Briefly a total of100 120583L of different concentration of TG (250 ngndash500 120583g mL)was placed in a tube and 100 120583L of 50 mM sodium phosphatebuffer (pH 68) containing 120572-amylase solution (15 mgmL)was addedThis solutionwas preincubated at 25∘C for 20minafter which 100 120583L of 1 soluble starch solution in 50 mMsodium phosphate buffer (pH 68) was added and incubatedat 25∘C for 10 min The reaction was stopped by adding500 120583L of dinitrosalicylic acid (DNS) reagent followed byincubation in boilingwater for 5min and then cooled to roomtemperature The resulting mixture was diluted with 5 mLdistilledwater and the absorbancewasmeasured at 540 nmAcontrol was prepared using the same procedure replacing theextract with distilled waterThe 120572-amylase inhibitory activitywas calculated as percentage inhibition Concentrations ofextracts resulting in 50 inhibition of enzyme activity (IC

50)

were determined graphically

28 Rat Intestinal 120572-Glucosidase Inhibitory Activity (Sucraseand Maltase Inhibitory Activities) The assay was based onthe modified method described [20] 100 mg of rat intestinalacetone powder was dissolved in 3 mL of NaCl (09 )solution and centrifuged at 12000 g for 30 min and usedfor the assay The enzyme solution (30 120583L) was incubatedwith 40 120583L of sucrose (400 mM) for sucrase assay Variousconcentrations of the extract were taken and made up tofinal volume of 100 120583l with 01 M phosphate buffer pH 68The reaction mixture was incubated at 37∘C for 30 min Thereaction was then stopped by suspending mixtures in boilingwater for 10 min The concentrations of glucose releasedfrom the reaction mixtures were determined by glucoseoxidase method with absorbance at a wavelength of 505 nmConcentrations of extracts resulting in 50 inhibition ofenzyme activity (IC

50) were determined graphically

Similarly maltase assay was also carried out using thesame protocol except that sucrose was replaced with 10 120583Lmaltose (86 mM)

29 Cell Culture MIN6 cells were maintained under 5CO2 37∘C in DMEM media supplemented with 10 FBS

2 mM L-glutamine 100 UmL of penicillin and 01 mgmLstreptomycin

291 Cell Viability Assay 3-4 5-(Dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) assay was based on

the protocol described [21] MIN6 cells were seeded at adensity of 2 times 104 cellswell in 96-well plates for 24 h Thenthe cells were treated with various concentrations of TG andEEGS (20ndash650 120583g) and incubated for 24 h After the requiredperiod of treatment MTT solution [10 120583L 5 mgmL MTT inphosphate-buffered saline (PBS)] was added with continuedincubation for 3 h After that DMSO was added and againincubated for 30 min in the dark The cell viability was thenrecorded at 570 nm

292 Intracellular ROS The quantification of the cellu-lar antioxidant activity was determined according to themethod described in [22] To measure ROS levels 2101584071015840-dichlorodihydrofluorescein diacetate (DCFH-DA) was usedThe cells were seeded in a 96-well microtiter plate at a densityof 5 X104 cellswell After 24 h the cells were treated with 50and 100120583g concentration of TG and EEGS and were furtherincubated for 24 h at 5 CO

2 37∘C Then 25 120583M DCFH-

DA was added and incubation was continued further for 1h The intracellular oxidation of DCFH-DA in the wells wasmeasured using fluorescence microplate reader at excitation(488 nm) and emission (525 nm)

293 Fluorescence Microscopy of MIN6 Cells MIN6 cellswere seeded at a density of 1times105 cellsmL on poly-L-Lysinecoated cover slips in 6 well plates containing DMEM for 24h H2O2treated cells were used as control to show oxidative

stress Subsequently the cells were treated with 50 and 100120583g concentrations of TG devoid of H

2O2for 24 h Cells

were stained by double dye method using acridine orange(AO) and ethidium bromide (11) The images were then cap-tured using a fluorescence microscope (Nikon Tokyo Japan)[23]

294 Glucose Stimulated Insulin Secretion Assay DMEM(Serum-free) without glucose supplemented with 2 mM L-glutamine and 25 mMHEPES pH 74 was used in this studyGlucose solution was prepared at different concentrations (13 5 8 and 25 mM) and incubated at 37∘C before use MIN6cells were seeded at a density of 1X106 in 96-well plates for24 h Subsequently the cells were treated with 50 and 100 120583gconcentrations of TG Prior to the insulin secretion assay thecells were starved in Krebs-Ringer solution containing 01bovine serum albumin (BSA) with 1 mM glucose for 1h andthe wells were washed twice with the same buffer The cellswere later incubated with Krebs-Ringer solution containing3 8 15 and 25 mM glucose 10 mM glibenclamide and 8mM glucose 30 mM KCl and 8 mM glucose for 1 h) Insulinsecreted into the medium was measured using an ELISA kit[23]

295 Measurement of GLUT 2 Levels The surface translo-cation of GLUT2 was measured according to the protocolsdescribed [24] The cells were treated with TG at 50 and 100120583gmL for 24 h After treatment the target GLUT 2 level inthe cell lysate was measured using ELISA kit The primaryantibody specific to the target protein GLUT2 was addedand allowed to bind to their target epitopes The secondaryantibody tagged with HRP which was specific to the primary


TGAC

Fraction III (TG)acarbose comparison0

20

40

60

Inhi

bitio

n

(a)

TGDAG

(b) (c)

Triterpeneglycoside

(d)

Figure 1 120572-glucosidase inhibition and identification of the active fraction TG (a) shows the120572-glucosidase inhibition of the active fractionIII (TG) relative to the positive control acarbose (AC) Values are expressed asmeanplusmn SD (b) represents the TLC pattern of the active fractiontriterpene glycoside (TG labelled as C) and the available commercial standard deacylgymnemic acid (DAC labelled as GA) (c) representsthe HPTLC pattern of triterpene glycoside (TG) The active fraction was checked for purity using TLC and HPTLC TLC spot indicated thepresence of gymnemic acid and HPTLC displayed rf value of 056 (d) depicts FTIR spectrum of TG The characteristic FTIR peaks of TGwith ester link 1710 cm-1 revealed presence of triterpene glycoside

antibody was added and the detection of GLUT2 was doneusing TMB-H

2O2substrate at 450 nm

210 Statistical Analysis Results were represented as mean plusmnSD Two-way ANOVA was employed to carry out statisticalanalysis using GraphPad prism version 50The experimentswere conducted in triplicate A confidence limit of plt005wasconsidered statistically significant

3 Results

31 Extraction and Isolation of Bioactive Agents (s) from Gym-nema sylvestre Dried leaf powder of GS (Gymnema sylvestre)was extracted using different solvents methanol ethanolacetone ethyl acetate chloroform and water All the solventextracts were subjected to 120572-glucosidase inhibitory activityEthanolic extract showed maximum inhibition of 972 (Table 1)This fraction was therefore chosen for further stud-ies The ethanolic extract of Gymnema sylvestre (EEGS) wassubjected to chromatographic fractionation using preparativegradient HPLC system The four peaks (fractions) obtainedwere collected made solvent free and resuspended in known

Table 1 120572-glucosidase inhibition for different solvent extracts ofGS

Solvents 120572-glucosidase InhibitionMethanol 522 plusmn 050Ethanol 9724 plusmn 024Acetone 8034 plusmn 063Ethyl acetate 71 plusmn 005Chloroform 7524 plusmn 040Water -Known amount of GS powder was extracted using different solvent sys-temsThe experiments were done in triplicate and average values weredetermined Values are expressed as meanplusmn SD

amount of phosphate buffer (50mM pH 68) 120572-Glucosidaseinhibitory activity was assayed for all the collected fractionsFraction III showed the highest inhibitory activity of 533compared to standard acarbose (positive control) whichexhibited 26 inhibition against 120572-glucosidase (Figure 1(a))Theother fractions showed lesser inhibitory activities rangingfrom 30 to 40 and hence fraction III was used for further


(a)

(b)

Figure 2 NMR spectrum of the active fraction TG (a) 1H-NMR (b) 13C-NMR

studies In the subsequent investigation triterpene glycosideswere identified in the active fraction III after evaluationand characterization by TLC HPTLC FTIR NMR and LC-MS This triterpene glycoside active fraction was termed asTG The presence of gymnemic acid was indicated in TG byTLC (Figure 1(b)) when spotted along with the commercialstandard deacylgymnemic acid (DAG) We also observed asingle peak when TG was subjected to HPTLC with rf value056 (Figure 1(c))

FTIR spectrum showed the presence of bands at wave-lengths 3335 2930 and 1710 cmminus1 corresponding to OHCH and C=O functional groups The characteristic FTIRpeaks of TG with ester link 1710 cmminus1 (Figure 1(d)) revealedpresence of typical gymnemic acid To further elucidate thestructure TG was subjected to NMR assignments (Figure 2)Table 2 gives the details of NMR chemical shifts assigned forTG1H NMR spectrum of TG was observed in DMSOndashd

6

solvent The characteristic protons were found in theirexpected range (Figure 2) Further the spectroscopic anal-yses employing chemical carbon (13C) shifts assignments(Table 2) were in good agreement with theMS negativemodedata (Figure 3(a)) The spectrum suggested the presence of

a tigloyl group at C-21 (Table 2) and acetyl group (172 ppmndashC=O amp 23 ppm ndashCH

3) at C-28 suggesting Gymnemic acid

I Saponins are classified as monodesmosides with a singlesaccharide chain generally linked to C-3 depending on thenumber of sugars present As usual 77-76 ppm will appearfor gymnemic acids due to glycosylation shifts But we found70 ppm for C-3 triterpenoids in their free form (sapogenins)indicating the presence of gymnemagenin The presence ofthis compound was again reconfirmed with MS data inpositive mode (Figure 3(b)) The sugar moiety was foundto be 3-O-120573-D-glucuronic acid (175ppm ndashC=O) Moreoverthe pattern gave a peak at NMR sugar region (103 ppm)and they appeared to consist of glucuronic acid alone andno other types of sugar moieties were present Comparisonin relative assignments revealed the presence of a mixtureof gymnemagenin and gymnemic acids I IV and VII andthey differ only in presenceabsence of acetyl and tigloylgroups respectively These results are in good agreementwith literature data with plusmn 1-2 ppm observed shifts whichmight be due to complex interactions in the above mixture ofsaponins in TG Based on results obtained from 1H 13C andFTIR analyses the structure of TG was proposed as shown inFigure 1(d)


Table 2 13C NMR spectral data for gymnemagenin and respective gymnemic acids (120575 in DMSO-d6)

Position Gymnemagenin (120575) Gymnemic acid I (120575) Gymnemic acid IV (120575) Gymnemic acid VII (120575)C-1 386 ++ ++ 386C-2 272 268 268 272C-3 706 773 765 765C-4 423 429 423 423C-5 491 -- -- 491C-6 177 176 176 177C-7 322 -- -- 322C-8 402 ++ ++ 402C-9 467 466 466 467C-10 362 ++ ++ 362C-11 249 245 236 249C-12 1215 1248 1222 1215C-13 1418 -- -- 1418C-14 419 -- -- 419C-15 342 356 356 342C-16 673 -- -- 673C-17 460 460 464 460C-18 420 -- -- 420C-19 464 -- -- 464C-20 342 360 360 342C-21 770 784 784 770C-22 737 725 725 737C-23 706 623 623 706C-24 134 146 144 134C-25 162 163 163 162C-26 170 171 171 170C-27 272 273 273 272C-28 600 630 600 600C-29 318 295 292 318C-30 197 216 212 197

Acetyl 1 - 1726Acetyl 2 ndash 225

Tigloyl 1rsquo - 1709Tigloyl 2rsquo - 1297Tigloyl 3rsquo - 1391Tigloyl 4rsquo - 14 4Tigloyl 5rsquo -126

3-0-120573-D-glucopyranosiduronic acidC1 - 1039C2 - 740C3 - 766C4 - 724C5 - 752C6 - 1759

++ overlapped signals -- not assigned


Table 3 IC50values for porcine 120572-amylase yeast 120572-glucosidase and mammalian 120572-glucosidase inhibitory potential of TG

Enzymes IC50values for TG (120583gmL) IC

50values for acarbose (120583gmL)

120572-glucosidase (yeast source) 316 plusmn 005 614 plusmn 005rat Intestinal 120572-glucosidase (sucrase) 7407 plusmn 051lowast 4430 plusmn 073lowast

rat Intestinal 120572-glucosidase (maltase) 569 plusmn 002 0059 plusmn 005porcine 120572-amylase 117 plusmn 024 018 plusmn 002At different concentrations of TG IC50 values for yeast 120572-glucosidase sucrase maltase and porcine 120572 amylase were studied Values are expressed as meanplusmn SDof three independent experiments made in duplicatelowastIndicating the significant levels at plt005

Gymnemic acid VII (666849)

Gymnemic acid IV (764950)

Gymnemic acid I (806987)

(a)

Gymnemagenin(50672)

(b)

Figure 3 (a) LC chromatogram of TG ESI -ve mode (b) LC chromatogram of TG ESI +ve mode

In ESI -ve mode the molecular weights were observedto be 765 665 and 805 respectively This indicated that TGcontained gymnemic acids I (805) IV (665) and VII (765)all of which have similar triterpene backbone but differ inthe positioning of functional groups Under ESI +ve modepeaks corresponding to triterpene backbone were observedThis indicated the presence of gymnemagenin a triterpenoidaglycone of gymnemic acid with a molecular weight of 507(mz) (Figures 3(a) and 3(b))These results correlate very wellwith the NMR patterns obtained

32 Enzyme InhibitoryActivities When subjected to differentconcentrations of TG the inhibition of 120572-glucosidase wasobserved to be concentration dependent This activity washigher compared to acarbose at their respective similar con-centrations (Table 3) TG showed 120572-glucosidase inhibitory

activity with an IC50value of 316 plusmn 005 120583gmL Similarly 120572-

amylase sucrase and maltase enzyme activities were inhib-ited by TG (Table 3) with IC

50values of 117 plusmn 024 120583gmL

7407 plusmn 511 120583gmL and 569 plusmn 002 120583gmL respectively

33 In Vitro Activity of TG Using MIN6 Cell (Pancreatic 120573-Cell) Lines To examine whether the ethanolic extract ofGymnema sylvestre (EEGS) and its active fraction (TG)induced any cytotoxic effects MTT assay was performedWhen extracts were used at concentrations ranging from 20to 650 120583gmL the viability retained up to 90 (plt005) evenat highest concentration studied (650 120583g) (Figure 4(a)) Thisdemonstrated the nontoxic effects of TG

To further demonstrate that TG did not induce anycytotoxic effects MIN6 cells were treated with TG andstained using differential stainingmethod usingAO andEtBr


EEGSTG

lowast lowast

0

50

100

150

Cel

l via

bilit

y

g

g

g

g

g

g

Concentration (g)

(a)

lowast

lowast

lowast

500

0

200

0

350

0

100

00

Hyd

roge

n pe

roxi

de

Con

trol

unt

reat

ed ce

lls

Concentration (g)

Control untreated cellsHydrogen peroxide(250 M) EEGS

TG

0

1000

2000

3000

RFU

(Rel

ativ

e fluo

resc

ence

uni

ts)

(b)

(c) (d) (e)

Figure 4 Effects of TG on120573-cells viability (a)MTTwas performed to study the cytotoxic effects of TGMIN6 cells were treated with differentconcentrations of TG and the cell viability was studied (b) To study the ROS effect of GS extracts on MIN6 cells treatment was carried outat concentrations 20 35 50 and 100 120583g levels H

2O2was used as positive control Fluorescent microscopy images showing the effect of TG

on MIN6 cells where (c) shows positive control (hydrogen peroxide) (d) shows TG treated cells (50 120583g) and (e) shows TG treated cells(100 120583g) Green regions indicate uptake of acridine orange showing live 120573 cells Orange-red color displays apoptosisdead cells All valueswere represented as meanplusmn SD Experiments were performed in triplicate Analysis was performed using two-way ANOVA lowastRepresentingsignificance at plt005

AOEtBr double staining combines the properties of twodyes to determine the type of cell death The micrographsclearly indicate noncytotoxic nature of TG (50 and 100 120583g)The cells appeared green and there was no decrease in cellnumber H

2O2(200 120583M) treated cells appeared orange-red

and they were agglomerated indicating cell death due totoxicity (Figures 4(c) 4(d) and 4(e))

The influence of TG on the generation of reactive oxygenspecies (ROS) was determined at 20 35 50 and 100 120583gmLconcentrations H

2O2(200 120583M) treatment was used as

control A normal control without any treatment was alsoused A significant reduction (plt005) in the formation ofROS was observed compared to H

2O2treated control at all

the concentrations studied (Figure 4(b)) But the increase

in sample concentration did not show any incrementalsignificance

34 Insulin Secretion by 120573-Cells The 120573-cells displayed dose-dependent response in insulin secretion when stimulatedwith increasing concentrations of glucose (3-25 mM) in thepresence of 50 and 100 120583g concentrations of TG TG at 50 120583glevel displayed 10-fold increase (plt005) in insulin secretionwhen cells were exposed to a lower concentration of glucose(3mM and 8mM) compared to the control The glucoseconcentrations 5 and 25 mM were considered as normo-glycemic and glucotoxic respectively But the cells incubatedwith normoglycemic glucose levels showed a little rise ininsulin levels (134-fold plt005) At glucotoxic conditions


(a)

lowastlowastlowast

lowastlowastlowast

controlTG

Std DAG

00

05

10

15

20

Fold

Incr

ease

(Rel

ativ

e uni

ts)

50 100controlConcentration (g)

(b)

Figure 5 Effect of TG on in vitro antidiabetic activity (a) MIN6 cells were treated with TG at 50 and 100120583g concentrations Deacylgymnemicacid (DAG) an available commercial standard fromGS and glibenclamide a commercial drug used for diabetes treatment were also used forcomparison (b) represents effect of TG on enhancement of GLUT2 levels Treatment with TG and standard DAG (from GS) was done at 50and 100 120583g concentrations All experiments were carried out in triplicate and results were represented as meanplusmn SD Analysis was performedusing two-way ANOVA lowastRepresenting significance level at plt005 lowastlowastlowastRepresenting significance level at plt0001

the response was quite lower than that of normoglycemicglucose levels yet significant compared to the control respec-tively as illustrated in Figure 5(a) (116-fold plt005) Whencells were treated with TG at 100 120583g at low concentrationsof glucose (3mM) there was 112-fold increase in insulinlevel (plt005) compared to control At normoglycemic andglucotoxic glucose levels the response of the cells was similarto TG at 50 120583g level in insulin secretion as shown in theFigure 5(a) The results obtained were comparable with thepositive controls (KCl and glibenclamide) used in this study

35 Effect of TG on GLUT2 We examined the effect ofTG on GLUT2 by quantitative ELISA using anti-GLUT2antibody tagged to HRP (Figure 5(b)) TG treated cellsdemonstrated better increase in GLUT-2 levels at 50 and 100120583g concentrations compared to commercial standard DAG(deacylgymnemic acid) Preincubation of cells at 50 120583g of TGshowed a slight increase in GLUT2 levels (05 fold) yet it wassignificant compared to control (plt001) When cells weretreated with TG at 100 120583g a single fold increase in GLUT2levels was observed when compared to control (plt0001)

4 Discussion

Pancreatic 120572-amylase breaks down complex carbohydratesinto oligosaccharides and disaccharides Further the intesti-nal 120572-glucosidases digest diet-derived oligosaccharides anddisaccharides into monosaccharides mainly glucose Inprevious reports the ethanolic extract of Gymnema mon-tanum leaves demonstrated appreciable 120572-glucosidase and 120572-amylase inhibitory activity comparable to that of acarbosea commercially available drug that inhibits 120572-glucosidase

to decrease postprandial hyperglycemia [25] In the presentstudy we have observed that isolated active fraction TGinhibited the activity of pancreatic 120572-amylase intestinal 120572-glucosidases (sucrase and maltase) and yeast 120572-glucosidasein a concentration-dependent manner as shown in theTable 3 The inhibition effect was significant (plt005) againstintestinal 120572-glucosidase (sucrase) with an IC

50value of

7407 plusmn 051 120583gmL relative to acarbose which showedIC50

value of 4430 plusmn 073 120583gmL We found promisinginhibition with IC

50value for TG (316 plusmn 005 120583gmL) against

yeast 120572-glucosidase compared to acarbose Similar effectswere observed in vivo where inhibition of 120572-glucosidaseand other intestinal enzymes such as maltase and sucraseoccurs in polysaccharides and oligosaccharides digestion[26] 120572-Amylase and 120572-glucosidase showed different inhibi-tion patterns probably due to structural differences relatedto the origins of the enzymes [27] Gymnemic acid (GA)isolated from GS not only inhibits glucose absorption in thesmall intestine [27] but also suppresses hyperglycemia andhyperinsulinemia in an oral glucose tolerance test [28] Theefficiency of GA in inhibiting glucose absorption in the smallintestine was found to be increased by a combined effectwith acarbose and voglibose [28] Thus we have attemptedto study antihyperglycemic effect induced by TG in vitrosince it seems to be little more feasible than that in vivo Theinhibition of these enzymes could be probably because ofthe synergistic action of triterpene glycosides present in theactive fraction TG as substantiated by in vitro antidiabeticstudies Bekzod Khakimov et al (2016) [29] have shown intheir study the mass spectral fragmentation patterns of 49saponin peaks detected from plant originating from eightdifferent triterpenoid aglycones with different MW Further


they have shown that continuously measured proton NMRdata during chromatography separation along with massspectrometry data revealed significant differences includingcontents of saponins types of aglycones and numbers ofsugar moieties attached to the aglycone The review [30] hasalso showed the results about the isolation and chemistry ofthe triterpenoids and their physicochemical characterizationwill be better studied using MS FTIR and NMR spectraldata

Previous study showed that the aqueous extract of GSdecreased blood glucose levels in rats and humans withoutimproving insulin sensitivity [31] An insulin secretagoguefunction was ascribed to GS following observations that itsadministration led to elevations in serum insulin levels inindividuals with type 2 diabetes [32] However to identifythe mode of action for glucose-lowering agents we havecarried out experiments in vitro for potential effects of GSextracts to stimulate insulin secretion by direct action at 120573-cells Pretreatment with (50minus100 120583g) of TG for 2 h triggered asignificant increase of glucose-induced insulin secretion in adose-dependentmannerThismay bemainly attributed to theeffect of TG on the insulin secretion enhancement capacityof MIN6 cells The ability of TG to further stimulate insulinsecretion at 15 mM glucose demonstrated that TG acts byenhancing glucose metabolism within the 120573-cells TG wasalso an efficient amplifier of insulin secretion thus assistingin sustained amplification of insulin secretion at 15 and 25mM glucose concentrations respectivelyThe ability of TG tostimulate insulin secretion at substimulatory glucose concen-trationswas comparable to glibenclamide a standard drug forstimulating insulin secretion used in our study GS has beenwidely utilized in Indian medicine (Ayurveda) for the treat-ment of diabetes and other disorders [33] GS extracts containvarious biologically active compounds that exert antidiabeticeffects [34] GS leaves contain numerous saponin compo-nents some of which may solubilize mammalian cell mem-brane proteins in much the same way as digitonin which wasused experimentally to permeabilize plasmamembranes [35]Themajor saponins present in the isolated active fraction TGshowed the stimulatory effect on insulin release from MIN6120573-cells at 50 and 100120583g accompanied by 90-95 cell viabilityas revealed by staining with 11 mixture of ethidium bromide(EtBr) and acridine orange (AO) In one of the studies novelisolate of GS termed OSA (named after Om Santal Adivasi)showed insulinotropic activities on 120573-cell lines and isolatedhuman islets in vitrohadbeneficial effects on glycemic controlin patients with type 2 diabetes [36] OSA at around 025mgmL stimulated insulin secretion in vitro without adverseeffects on cell viability Similarly in our study TG at 50120583gmLlevel stimulated insulin secretion in vitro without hostileeffects on cell viability Even at 650 120583gmL concentrationtested 90 of 120573-cells were viable These investigations sug-gested that membrane damaging components in TG or theirconcentrations might be much lower Differential stainingwith ethidium bromide and acridine orange was an estab-lished test of cell viability that paves the way for rapid visualreadout of compromised plasma membrane integrity In thisstudy increased dye uptake of acridine orange to give greenfluorescence indicated the nontoxic effects of TG on 120573-cells

The pancreatic islet includes the most vulnerable cellsfor the attack after ROS generation in human as it has lessantioxidant enzymes as compared to other organs [8] Thedamage of pancreatic islet leads to decrease in insulin secre-tion resulting in hyperglycemic conditions and regenerationof ROS In vitroGS alcoholic leaf extract showed antioxidantability by inhibiting 11-diphenyl-2-picrylhydrazyl (DPPH)and scavenging superoxide and hydrogen peroxide [37] Inthe present study H

2O2was chosen as knownmodel biologi-

cal ROS A significant reduction in the formation of ROS wasobserved compared to control at all the concentrations of TGstudied

Many medicinal plants are used in the traditionalmedicine to enhance the translocation of GLUT and thiscould lead to a new approach for treating type 2 diabetesGLUT2 is a facilitative glucose transporter located in theplasma membrane of pancreas liver intestinal kidney cellsand the hypothalamus areas [38] Due to its high capacityand low affinity GLUT2 transports dietary sugars glucosefructose and galactose in a broad range of physiologicalconcentrations GLUT2 has the highest capacity and thelowest affinity for glucose which allows glucose uptake inthe beta cells only when the glucose level is high and insulinsecretion is necessary [38] According to literature ethanolicextract of Catharanthus roseus enhanced GLUT2 levels instreptozotocin-induced diabetic Wistar rats In our studiesat 50 120583g concentration of TG there was 05-fold increaseof GLUT2 compared to control At 100120583g concentration theincrease was 1-fold (plt005) In a similar previous study con-ductedmethanolic leaf extract ofGymnema sylvestre (MLGS)demonstrated a significant and dose-dependent increase inglucose uptake in all the concentrations of MLGS studied[39] TG at 50 and 100 120583g elevated GLUT2 levels comparableto standardDAG (deacylgymnemic acid) Renewal of glucosetransport induction would therefore enhance the uptake ofglucose and thus help to combat hyperglycemic conditions

5 Conclusion

In conclusion the present study exhibited an evidence thatisolated active fraction triterpene glycoside TG from Gym-nema sylvestre could inhibit the activity of pancreatic 120572-amylase 120572-glucosidase sucrase and maltase Further it wasshown to enhance the GLUT2 protein levels and ameliorateimpaired insulin secretion of MIN6 cells However theunderlying molecular mechanism remains to be understoodThe isolated and characterized active fraction triterpeneglycoside (TG) gives insight into dietary antidiabetic benefitsby reducing antinutritional factors of saponins in Gymnemasylvestre

Abbreviations

GS Gymnema sylvestreEEGS Ethanolic extract of Gymnema sylvestre119901NPG Para-nitrophenyl-glucopyranosideTLC Thin layer chromatographyHPTLC High performance thin layer

chromatographyHPLC High performance liquid chromatography


FTIR Fourier transform infrared spectrumLC-MSMS Liquid chromatography mass

spectrometryNMR Nuclear magnetic resonanceTG Active fraction III mixture of triterpenoid

glycosidesMIN6 Pancreatic 120573-cell lines from mouseMTT 3-45-(Dimethylthiazol-2-yl)-25-diphenyl

tetrazolium bromideROS Reactive oxygen speciesDCFH-DA 2101584071015840-dichlorodihydrofluorescein

diacetate101584010158401015840101584010158401015840GLUT2 Glucose transporter 2

Data Availability

All the data used to support the findings of this study areincluded in the manuscript

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors thank Director Central Food TechnologicalResearch Institute Mysore for providing the necessary facil-ities to carry out this work and CSIR-Delhi for providingCSIR-GATE fellowship for Rashmi S Shenoy

Supplementary Materials

The graphical representation depicts the gist of entiremanuscript In this work we have attempted to evaluatethe antidiabetic potential of Gymnema sylvestre The activefraction ldquoTGrdquo has been characterized and explored for itsantidiabetic effects The active fraction TG showed inhi-bition of the disaccharidase activity owing to its glucosecontrol benefits TG also improved insulin secretion andglucose transporter protein levels in beta cells in vitro(Supplementary Materials)

References

[1] T Kawai I Takei A Shimada et al ldquoEffects of olmesartanmedoxomil an angiotensin II type 1 receptor antagonist onplasma concentration of B-type natriuretic peptide in hyper-tensive patients with type 2 diabetes mellitus A preliminaryobservational open-label studyrdquo Clinical Drug Investigationvol 31 no 4 pp 237ndash245 2011

[2] H Sakuraba H Mizukami N Yagihashi R Wada C Hanyuand S Yagihashi ldquoReduced beta-cell mass and expression ofoxidative stress-related DNA damage in the islet of JapaneseType II diabetic patientsrdquoDiabetologia vol 45 no 1 pp 85ndash962002

[3] R Kakkar S V Mantha J Radhi K Prasad and J KalraldquoIncreased oxidative stress in rat liver and pancreas during pro-gression of streptozotocin-induced diabetesrdquo Clinical Sciencevol 94 no 6 pp 623ndash632 1998

[4] P Marchetti S Del Prato R Lupi and S Del Guerra ldquoThepancreatic beta-cell in human Type 2 diabetesrdquo NutritionMetabolism amp Cardiovascular Diseases vol 16 pp S3ndashS6 2006

[5] W-H Kim J W Lee Y H Suh et al ldquoAICAR potentiates ROSproduction induced by chronic high glucose Roles of AMPKin pancreatic 120573-cell apoptosisrdquo Cellular Signalling vol 19 no 4pp 791ndash805 2007

[6] S R Sampson E Bucris M Horovitz-Fried et al ldquoInsulinincreases H

2O2-induced pancreatic beta cell deathrdquo Apoptosis

vol 15 no 10 pp 1165ndash1176 2010[7] E Bonora ldquoProtection of pancreatic beta-cells Is it feasiblerdquo

Nutrition Metabolism amp Cardiovascular Diseases vol 18 no 1pp 74ndash83 2008

[8] R Robertson H Zhou T Zhang and J S Harmon ldquoChronicoxidative stress as a mechanism for glucose toxicity of the betacell in type 2 diabetesrdquoCell Biochemistry and Biophysics vol 48no 2-3 pp 139ndash146 2007

[9] W F Caspary ldquoPhysiology and pathophysiology of intestinalabsorptionrdquo American Journal of Clinical Nutrition vol 55 no1 pp 299Sndash308S 1992

[10] F Zhao YWatanabe H Nozawa A Daikonnya K Kondo andS Kitanaka ldquoPrenylflavonoids and phloroglucinol derivativesfrom hops (Humulus lupulus)rdquo Journal of Natural Products vol68 no 1 pp 43ndash49 2005

[11] N Tewari V Tiwari R Mishra et al ldquoSynthesis and bioevalua-tion of glycosyl ureas as120572-glucosidase inhibitors and their effecton mycobacteriumrdquo Bioorganic amp Medicinal Chemistry vol 11no 13 pp 2911ndash2922 2003

[12] S Najafi and S S Deokule ldquoStudies on Gymnema sylvestre- amedicinally important plant of the familyAsclepiadaceaerdquoTGSvol 9 pp 26ndash32 2011

[13] V A Khramov A A Spasov and M P Samokhina ldquoChemicalcomposition of dry extracts of Gymnema sylvestre leavesrdquoPharmaceutical Chemistry Journal vol 42 no 1 pp 29ndash31 2008

[14] P D Trivedi and K Pundarikakshudu ldquoA validated high perfor-mance thin-layer chromatographic method for the estimationof gymnemic acids through gymnemagenin in Gymnemasylvestre materials extracts and formulationsrdquo InternationalJournal of Applied Sciences vol 6 p 19 2008

[15] P V Kanetkar K S Laddha andM Y KamatGymnemic acidsA molecular perspective of its action on carbohydrate Posterpresented at the 16th ICFOST meet organized by CFTRI andDFRL India Mysore 2004

[16] Y-M Kim Y-K JeongM-HWangW-Y Lee andH-I RheeldquoInhibitory effect of pine extract on 120572-glucosidase activity andpostprandial hyperglycemiardquo Nutrition Journal vol 21 no 6pp 756ndash761 2005

[17] K Y Kim K A Nam H Kurihara and S M Kim ldquoPotent 120572-glucosidase inhibitors purified from the red alga Grateloupiaellipticardquo Phytochemistry vol 69 no 16 pp 2820ndash2825 2008

[18] P V Kanetkar R S Singhal K S Laddha and M Y KamatldquoExtraction and quantification of gymnemic acids throughgymnemagenin from callus cultures of Gymnema sylverstrerdquoPhytochemical Analysis vol 17 no 6 pp 409ndash413 2006

[19] Y-M KimM-HWang andH-I Rhee ldquoA novel120572-glucosidaseinhibitor from pine barkrdquo Carbohydrate Research vol 339 no3 pp 715ndash717 2004

[20] S Adisakwattana andB Chanathong ldquo120572-glucosidase inhibitoryactivity and lipid-lowering mechanisms of Moringa oleiferaleaf extractrdquo European Review for Medical and PharmacologicalSciences vol 15 no 7 pp 803ndash808 2011


[21] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[22] C-L Hsu B O-H Hong Y S Yu and G-C Yen ldquoAntioxidantand Anti-Inflammatory effects of Orthosiphon aristatus andits bioactive compoundsrdquo Journal of Agricultural and FoodChemistry vol 58 no 4 pp 2150ndash2156 2010

[23] K Cheng K Ho R Stokes C Scott and S M Lau ldquoHypoxia-inducible factor-1alpha regulates beta cell function in mouseand human isletsrdquoThe Journal of Clinical Investigation vol 120pp 2171ndash2183 2010

[24] S Kadan B Saad Y Sasson and H Zaid ldquoIn vitro evaluationof anti-diabetic activity and cytotoxicity of chemically analysedOcimum basilicum extractsrdquo Food Chemistry vol 196 pp1066ndash1074 2016

[25] A A F Sima and S Chakrabarti ldquoLong-term suppression ofpostprandial hyperglycaemia with acarbose retards the devel-opment of neuropathies in the BBW-ratrdquoDiabetologia vol 35no 4 pp 325ndash330 1992

[26] T Matsui T Tanaka S Tamura et al ldquo120572-glucosidase inhibitoryprofile of catechins and theaflavinsrdquo Journal of Agricultural andFood Chemistry vol 55 no 1 pp 99ndash105 2007

[27] M Yoshikawa N Nishida H Shimoda M Takada Y Kawa-hara and H Matsuda ldquoPolyphenol constituents from salaciaspecies Quantitative analysis of mangiferin with 120572-glucosidaseand aldose reductase inhibitory activitiesrdquoYakugakuZasshi vol121 no 5 pp 371ndash378 2001

[28] H Luo T Imoto and Y Hiji ldquoInhibitory effect of voglibose andgymnemic acid on maltose absorption in vivordquo World Journalof Gastroenterology vol 7 no 2 pp 270ndash274 2001

[29] B Khakimov L H Tseng M Godejohann S Bak and S BEngelsen ldquoScreening for triterpenoid saponins in plants usinghyphenated analytical platformsrdquo Molecules vol 21 no 12Article ID 21121614 2016

[30] G Di Fabio V Romanucci A De Marco and A ZarrellildquoTriterpenoids from Gymnema sylvestre and their pharmaco-logical activitiesrdquoMolecules vol 19 no 8 pp 10956ndash10981 2014

[31] M Tominaga M Kimura K Sugiyama et al ldquoEffects ofSeishin-renshi-in and Gymnema sylvestre on insulin resistancein streptozotocin-induced diabetic ratsrdquo Diabetes Research andClinical Practice vol 29 no 1 pp 11ndash17 1995

[32] K Baskaran B Kizar Ahamath K Radha Shanmugasundaramand E R B Shanmugasundaram ldquoAntidiabetic effect of aleaf extract from Gymnema sylvestre in non-insulin-dependentdiabetes mellitus patientsrdquo Journal of Ethnopharmacology vol30 no 3 pp 295ndash305 1990

[33] R Bradley E B Oberg C Calabrese and L J Standish ldquoAlgo-rithm for complementary and alternativemedicine practice andresearch in type 2 diabetesrdquo The Journal of Alternative andComplementary Medicine vol 13 no 1 pp 159ndash175 2007

[34] Y Sugihara H Nojima H Matsuda T Murakami MYoshikawa and I Kimura ldquoAntihyperglycemic effects of gym-nemic acid IV a compound derived from Gymnema sylvestreleaves in streptozotocin-diabeticmicerdquo Journal of AsianNaturalProducts Research vol 2 no 4 pp 321ndash327 2000

[35] A Varadi A Grant M McCormack et al ldquoIntracellular ATP-sensitive K+ channels in mouse pancreatic beta cells Against arole in organelle cation homeostasisrdquo Diabetologia vol 49 no7 pp 1567ndash1577 2006

[36] A Al-Romaiyan B Liu H Asare-Anane et al ldquoA novelGymnema sylvestre extract stimulates insulin secretion from

human islets in vivo and in vitrordquo Phytotherapy Research vol24 no 9 pp 1370ndash1376 2010

[37] S R Rachh H V Patel M T Hirpara M R RupareliyaA S Rachh and N M Bhargava ldquoIn vitro evaluation ofantioxidant activity of Gymnema sylvestrerdquo In vitro evaluationof antioxidant activity of Gymnema sylvestre vol 54 no 2 pp141ndash148 2009

[38] A Leturque E Brot-Laroche M Le Gall E Stolarczyk and VTobin ldquoThe role of GLUT2 in dietary sugar handlingrdquo Journalof Physiology and Biochemistry vol 61 no 4 pp 529ndash538 2005

[39] P M Kumar M V Venkataranganna K Manjunath GL Viswanatha and G Ashok ldquoMethanolic leaf extract ofGymnema sylvestre augments glucose uptake and amelioratesinsulin resistance by upregulating glucose transporter-4 perox-isome proliferator-activated receptor-gamma adiponectin andleptin levels in vitrordquo Journal of Intercultural Ethnopharmacol-ogy vol 5 no 2 pp 146ndash152 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


Gymnema sylvestre (GS) a woody vine-like climbingplant is well known in Indian traditional medicine ldquoAyurv-edardquo It grows in the tropical forest of central and south-ern India [12] The leaves are used mostly as antiviraldiuretic antiallergic hypoglycemic hypolipidemic antibi-otic antianalgesic and antirheumatic agents [13] The leavesof GS contain triterpenoidal saponins belonging to oleananeand dammarane classes [13] Twenty different saponins andglycosides have been reported in Gymnema sylvestre [14]Several studies suggest that gymnemic acids may act asantidiabetic by promoting regeneration of islet cells andincrease insulin secretion [15] Also gymnemic acids havebeen reported to inhibit glucose absorption from the intestineand utilize glucose by enhancing the activities of enzymes ininsulin-dependent pathways [15]

The current focus of our study was to isolate and char-acterize bioactive agents (s) from Gymnema sylvestre andevaluate in vitro antidiabetic effects to initiate a search fornutraceutical pharmacophores towards inhibition of pancre-atic 120572-amylase and 120572-glucosidase with improvement in betacell function

2 Materials and Methods

21 Chemicals 120572-Amylase from porcine pancreas 120572-glucos-idase from Saccharomyces cerevisiae para-nitrophenyl-glu-copyranoside (pNPG) soluble starch and 3-45-(dimeth-ylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT)were procured from SRL chemicals (Bangalore India)Rat intestinal acetone powder (source of 120572-glucosidase)acarbose Anti-GLUT2 antibody anti-insulin antibody 2101584071015840-dichlorodihydrofluorescein diacetate (DCFH-DA) acridineorange (AO) and ethidium bromide (EtBr) were from SigmaChemical Co (St Louis MO USA) Glucose oxidase kitwas purchased from AGAPE diagnostics (India) ELISA kitswere purchased from Cytoglow Co (India) Analytical andHPLC grade solvents were from E Merck (India) The driedpowder of Gymnema sylvestre was obtained from NikhilaKarnataka Central Ayurvedic Pharmacy (Mysore India)Mouse pancreatic 120573-cell line (MIN6) was purchased fromNCCS (Pune India) Dulbeccorsquos minimal essential medium(DMEM) Fetal Bovine Serum (FBS) and antimycoticsolution were purchased from Himedia chemicals (India)All other chemicals used in the study were of analytical gradeand purchased from Himedia chemicals (India)

22 Preparation of Gymnema sylvestre (GS) Extracts GSpowder was extracted using different solvent systems likemethanol ethanol acetone ethyl acetate chloroform andwater respectively The GS powder along with respectivesolvents were left for 3 h refluxing and the resulting infusionswere filtered The filtrates were concentrated solvents wereevaporated using rotary evaporator (Cole-Parmer SB 1100Shanghai China) at 45∘C under reduced pressure and thenlyophilized The greenish-black powder thus obtained wasstored at 4∘C until used for further assays

23 120572-Glucosidase Inhibitory Assay Extracts of GS werescreened for 120572-glucosidase inhibitory activity according to

the method described in [16] The source of 120572-glucosidaseenzyme was from Saccharomyces cerevisiae The substratesolution pNPG (2mgmL)was prepared in 50mMphosphatebuffer pH 68 10 120583L of 120572-glucosidase (112 UmL) waspreincubated with 10 120583L of known concentrations of GS(extracted from different solvents) for 10 min pNPG (10 120583L)was added to start the reaction followed by incubation at37∘C for 20 min The reaction was stopped by adding 2 mLof 01 M Na

2CO3 The 120572-glucosidase activity was determined

by measuring the yellow-colored para-nitrophenol releasedfrom pNPG at 405 nm The results were expressed aspercentage of the blank control

24 Preparative HPLC of Ethanolic Extract of Gymnemasylvestre (EEGS) 200 g of dried plant material was extractedusing 1 L absolute ethanol under refluxing conditions for3h The extracted volume was concentrated using rotaryevaporator (Cole-Parmer SB 1100 Shanghai China) at 45∘Cat high pressure and brought down to 500 mL This wasfurther concentrated to sim5 g wet weight and a stock of 1mgmL was prepared using HPLC grade methanol solutionThe stock solution was filtered using nylon 6 membraneconsisting of six multiple filter papers with a pore size of 045120583m degassed by ultrasonic treatment before use PreparativeHPLC (gradient mode) was carried out using ODSC-18 silicacolumn (25 cm X 212 mm 12 120583m) The mobile phase wasprepared using two solvent systems solvent A water (HPLCGRADE) containing 1 acetic acid solvent B acetonitrilecontaining 1 acetic acidThemobile phasewas filtered usingnylon 6 multiple filter membranes having a pore size of 045120583m The conditions for HPLC [0 min solvent B (5 ) 5 min(8 ) 7 min (12 ) 12 min (18 ) 17 min (22) 24 min (35) 26min (100) 30min (100) 32min (5) 35min stop]were set constant flow rate was maintained at 5 mLminThedetector was set at 254 nm and fractions were collected Thefractions were made solvent free using a rotary evaporatorfreeze-dried and then resuspended in known amount ofphosphate buffer (50 mM pH 68) and were analyzed forpercentage 120572-glucosidase inhibitory activity

25Thin Layer Chromatography (TLC) andHigh PerformanceTLC (HPTLC) The purity of the HPLC purified activefraction was tested by thin layer chromatography Silica gel-G was taken as stationary medium and coated over glassslides The mobile phase used was tolueneethylacetateaceticacid (571 vv) [17] Deacylgymnemic acid (DAG) was usedas a standard The purity of the active fraction was checkedby HPTLC HPTLC system equipped with a Linomat IVsample applicator a twin-chamber tank a model III TLCscanner and CATS 40 integration software was employedAluminum-backed layers (10times 10 cm) of silica gelGF254wereused as absorbent The mobile phase used was chloroformand methanol (82 vv) The spotted plates were developedup to 80 mm under chamber saturation conditions After air-drying the solvent the plates were scanned at 205 nm [18]

26 Characterization by FTIR LC-MS and NMR The iso-lated active fraction was characterized by spectroscopicmethods FTIR (Bruker USA) spectra were recorded in the



6on a Bruker Avance



50)










2 37∘C Then 25 120583M DCFH-




2O2for 24 h Cells





TGAC


20

40

60

Inhi

bitio

n

(a)

TGDAG

(b) (c)

Triterpeneglycoside

(d)





3 Results






(a)

(b)




6





















(a)

Gymnemagenin(50672)

(b)











EEGSTG

lowast lowast

0

50

100

150

Cel

l via

bilit

y

g

g

g

g

g

g

Concentration (g)

(a)

lowast

lowast

lowast

500

0

200

0

350

0

100

00

Hyd

roge

n pe

roxi

de

Con

trol

unt

reat

ed ce

lls

Concentration (g)


TG

0

1000

2000

3000

RFU

(Rel

ativ

e fluo

resc

ence

uni

ts)

(b)

(c) (d) (e)















(a)

lowastlowastlowast

lowastlowastlowast

controlTG

Std DAG

00

05

10

15

20

Fold

Incr

ease

(Rel

ativ

e uni

ts)


(b)




4 Discussion



50value of












5 Conclusion


Abbreviations









Data Availability




Acknowledgments




References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















6on a Bruker Avance



50)










2 37∘C Then 25 120583M DCFH-




2O2for 24 h Cells





TGAC


20

40

60

Inhi

bitio

n

(a)

TGDAG

(b) (c)

Triterpeneglycoside

(d)





3 Results






(a)

(b)




6





















(a)

Gymnemagenin(50672)

(b)











EEGSTG

lowast lowast

0

50

100

150

Cel

l via

bilit

y

g

g

g

g

g

g

Concentration (g)

(a)

lowast

lowast

lowast

500

0

200

0

350

0

100

00

Hyd

roge

n pe

roxi

de

Con

trol

unt

reat

ed ce

lls

Concentration (g)


TG

0

1000

2000

3000

RFU

(Rel

ativ

e fluo

resc

ence

uni

ts)

(b)

(c) (d) (e)















(a)

lowastlowastlowast

lowastlowastlowast

controlTG

Std DAG

00

05

10

15

20

Fold

Incr

ease

(Rel

ativ

e uni

ts)


(b)




4 Discussion



50value of












5 Conclusion


Abbreviations









Data Availability




Acknowledgments




References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















TGAC


20

40

60

Inhi

bitio

n

(a)

TGDAG

(b) (c)

Triterpeneglycoside

(d)





3 Results






(a)

(b)




6





















(a)

Gymnemagenin(50672)

(b)











EEGSTG

lowast lowast

0

50

100

150

Cel

l via

bilit

y

g

g

g

g

g

g

Concentration (g)

(a)

lowast

lowast

lowast

500

0

200

0

350

0

100

00

Hyd

roge

n pe

roxi

de

Con

trol

unt

reat

ed ce

lls

Concentration (g)


TG

0

1000

2000

3000

RFU

(Rel

ativ

e fluo

resc

ence

uni

ts)

(b)

(c) (d) (e)















(a)

lowastlowastlowast

lowastlowastlowast

controlTG

Std DAG

00

05

10

15

20

Fold

Incr

ease

(Rel

ativ

e uni

ts)


(b)




4 Discussion



50value of












5 Conclusion


Abbreviations









Data Availability




Acknowledgments




References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a)

(b)




6





















(a)

Gymnemagenin(50672)

(b)











EEGSTG

lowast lowast

0

50

100

150

Cel

l via

bilit

y

g

g

g

g

g

g

Concentration (g)

(a)

lowast

lowast

lowast

500

0

200

0

350

0

100

00

Hyd

roge

n pe

roxi

de

Con

trol

unt

reat

ed ce

lls

Concentration (g)


TG

0

1000

2000

3000

RFU

(Rel

ativ

e fluo

resc

ence

uni

ts)

(b)

(c) (d) (e)















(a)

lowastlowastlowast

lowastlowastlowast

controlTG

Std DAG

00

05

10

15

20

Fold

Incr

ease

(Rel

ativ

e uni

ts)


(b)




4 Discussion



50value of












5 Conclusion


Abbreviations









Data Availability




Acknowledgments




References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



































(a)

Gymnemagenin(50672)

(b)











EEGSTG

lowast lowast

0

50

100

150

Cel

l via

bilit

y

g

g

g

g

g

g

Concentration (g)

(a)

lowast

lowast

lowast

500

0

200

0

350

0

100

00

Hyd

roge

n pe

roxi

de

Con

trol

unt

reat

ed ce

lls

Concentration (g)


TG

0

1000

2000

3000

RFU

(Rel

ativ

e fluo

resc

ence

uni

ts)

(b)

(c) (d) (e)















(a)

lowastlowastlowast

lowastlowastlowast

controlTG

Std DAG

00

05

10

15

20

Fold

Incr

ease

(Rel

ativ

e uni

ts)


(b)




4 Discussion



50value of












5 Conclusion


Abbreviations









Data Availability




Acknowledgments




References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















EEGSTG

lowast lowast

0

50

100

150

Cel

l via

bilit

y

g

g

g

g

g

g

Concentration (g)

(a)

lowast

lowast

lowast

500

0

200

0

350

0

100

00

Hyd

roge

n pe

roxi

de

Con

trol

unt

reat

ed ce

lls

Concentration (g)


TG

0

1000

2000

3000

RFU

(Rel

ativ

e fluo

resc

ence

uni

ts)

(b)

(c) (d) (e)















(a)

lowastlowastlowast

lowastlowastlowast

controlTG

Std DAG

00

05

10

15

20

Fold

Incr

ease

(Rel

ativ

e uni

ts)


(b)




4 Discussion



50value of












5 Conclusion


Abbreviations









Data Availability




Acknowledgments




References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a)

lowastlowastlowast

lowastlowastlowast

controlTG

Std DAG

00

05

10

15

20

Fold

Incr

ease

(Rel

ativ

e uni

ts)


(b)




4 Discussion



50value of












5 Conclusion


Abbreviations









Data Availability




Acknowledgments




References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























5 Conclusion


Abbreviations









Data Availability




Acknowledgments




References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























Data Availability




Acknowledgments




References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















in vitro antidiabetic effects of isolated triterpene...

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