Research Article120572-Amylase and 120572-Glucosidase Inhibitory Saponins fromPolyscias fruticosa Leaves

Tran Thi Hong Hanh Nguyen Hai Dang and Nguyen Tien Dat

Institute of Marine Biochemistry Vietnam Academy of Science and Technology 18-Hoang Quoc Viet Cau GiayHanoi 100000 Vietnam

Correspondence should be addressed to Nguyen Tien Dat ngtiendimbcvastvn

Received 21 February 2016 Accepted 17 April 2016

Academic Editor Nick Kalogeropoulos

Copyright copy 2016 TranThi Hong Hanh 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

Three bisdesmosidic saponins 3-O-[120573-D-glucopyranosyl-(1rarr4)-120573-D-glucuronopyranosyl] oleanolic acid 28-O-120573-D-glucopyran-osyl ester (1) polyscioside D (2) and 3-O-120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyloleanolic acid 28-O-120573-D-glucopyranosyl-(1rarr2)-120573-D-galactopyranosyl ester (3) were isolated from a methanol extract of Polysciasfruticosa (L) Harms leaves Compound 1 was obtained as a main constituent and compound 3 was reported for the first time andnamed as polyscioside I Saponin 1 inhibited porcine pancreas 120572-amylase and yeast 120572-glucosidase activities while 2 and 3 wereinactive Synergistic inhibitory effect on 120572-amylase was observed from the combination of low concentrations of 1 and acarboseThe findings suggest the use of P fruticosa and itsmajor saponin 1 for the prevention and treatment of diabetes and its complications

1 Introduction

Polyscias fruticosa (L) Harms (synonyms Nothopanax fru-ticosus (L) Miq and Panax fruticosus L) belonging to thefamily Araliaceae is widely used in Vietnam as a tonicagent for the treatment of ischemia and inflammation andto increase blood flow in the brain The leaves of thisplant are also eaten as a salad [1] Previous studies showedthat P fruticosa leaf extracts exhibited antipyretic anti-inflammatory analgesic and molluscicidal properties [2 3]and antidiabetic activity [4] A few reports on the chemicalcomposition of this plant indicated the presence of polyacety-lene [5] and saponins [6] Diabetes is a group of metabolicdiseases characterized by chronic hyperglycemia resultingfrom deficiency in insulin secretion or action Recent reportsrevealed that a high postprandial plasma glucose level ismoreharmful than fasting blood glucoseTherefore it is importantto control the postprandial blood glucose level to reducecomplications and mortality One therapeutic approach fortreating diabetes is to decrease postprandial glycemia byinhibiting enzymes responsible for carbohydrate hydrolysissuch as 120572-glucosidase and 120572-amylase [7]

Our search for antidiabetic agents of natural origin iden-tified a methanol extract of P fruticosa leaves that showedsignificant inhibitory activity against 120572-glucosidase and120572-amylase A phytochemical investigation of the methanolextract of P fruticosa leaves led to the isolation of threebisdesmosidic saponins (Figure 1) Their structures wereelucidated as 3-O-[120573-D-glucopyranosyl-(1rarr4)-120573-D-glucu-ronopyranosyl] oleanolic acid 28-O-120573-D-glucopyranosylester (1) [6 8] 3-O-120573-D-glucopyranosyl-(1rarr2)-[120573-D-gluco-pyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl oleanolic acid28-O-120573-D-glucopyranosyl ester or polyscioside D (2) [6]and 3-O-120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl oleanolic acid 28-O-120573-D-glucopyranosyl-(1rarr2)-120573-D-galactopyranosyl ester (3) new-ly named polyscioside I All compounds were evaluated fortheir inhibitory activity against porcine pancreas 120572-amylaseand yeast 120572-glucosidase

2 Experimental

21 General Experimental Procedures Optical rotation valueswere recorded a JASCO P-2000 digital polarimeter (JASCO

Hindawi Publishing CorporationJournal of ChemistryVolume 2016 Article ID 2082946 5 pageshttpdxdoiorg10115520162082946

2 Journal of Chemistry

O

12

34

56

7

89

10

1112

13

1415

16

1718

1920

21

22

23 24

25 26

27

30

28

29

O

OHO

O

HO O

O

OHHOHO

HO

O

OHHOHO

HO

O

OHHO

O

HO O

O

OHHOHO

HO

O

OHO

OH OH

O

OHHOHO

HO

O

OHHO

HO

HO

S1 S2

S3

S4

O

O

O

12

34

56

7

89

10

1112

13

1415

16

1718

1920

21

22

23 24

25 26

27

30

28

29

O

OHO

O

HO O

O

OHHOHO

HO

O

OHHOHO

HOO

OHO

OH OH

O

OHHOHO

HO

R1O

OR2

6998400998400

6998400998400

6998400998400

1998400998400

1998400998400

3998400998400

1998400998400

3998400998400

3998400998400

49984004998400

39984003998400

69984006998400

19984001998400

1998400998400998400998400

3998400998400998400998400

3998400998400998400

6998400998400998400998400

6998400998400998400

1998400998400998400

4998400998400998400

6998400998400998400998400998400

3998400998400998400998400998400

1998400998400998400998400998400

4998400

3998400

6998400

1998400

1998400998400998400998400

3998400998400998400998400

6998400998400998400 3998400998400998400

6998400998400998400

4998400998400998400

69984009984009984009984009984001998400998400998400

1998400998400998400998400998400

4998400

3998400

6998400

1998400

1 R1 = S1 R2 = S3

2 R1 = S2 R2 = S33 R1 = S2 R2 = S4

3

Figure 1 Structure of compounds 1ndash3 and key HMBC correlations of 3

Tokyo Japan) NMR experiments were carried out on aBruker AM500 FT-NMR spectrometer (Bruker Rheinstet-ten Germany) using tetramethylsilane (TMS) as internalstandard The HR-ESI-MS was recorded on API Q-STARPULSAR I of Applied Biosystem The bioassay absorbancewas read by xMark Microplate Spectrophotometer reader(Bio-Rad Laboratories USA)

22 Plant Material The leaves of Polyscias fruticosa werecollected in TienHaiThai Binh in July 2011 and identified byProfessor Tran Huy Thai Institute of Ecology and BiologicalResources Vietnam Academy of Science and TechnologyThe voucher specimens were deposited at the herbarium ofthe Institute of Ecology and Biological Resources

23 Extraction and Isolation The air-dried and powdered Pfruticosa leaves (20 kg) were extracted with methanol (5 L times3 times) at room temperature The combined extracts wereconcentrated to give 300 g of crude extract which was thenresuspended inwater (15 L) and successively partitionedwithhexane and ethyl acetate (each 05 L times 3 times) to obtain 75

and 92 g of hexane and ethyl acetate residues respectivelyThe water residue was filtered through a Diaion HP-20column eluted by 0 25 and 100 methanol in waterThe 100 methanol-eluted fraction was diluted with water(1 1 vv) and kept overnight at 4∘C Compound 1 as whitecrystals (500mg) was obtained by filtering The filtrate wasconcentrated and chromatographed on a silica gel columneluted with a gradient of 0 50 and 100 methanol inethyl acetate to afford three fractions F1ndashF3 Fraction F2 wasfractionated on a silica gel column eluted with chloroform-methanol-water (50 10 1 vv) to afford compound 2 (10mg)Compound 3 (205mg) was purified from F3 by a RP-18column (methanol-water 1 1 vv)

Polyscioside I (3)White powder mp gt300∘C [120572]D24 = +124

(c 01 MeOH) 1H NMR (500MHz DMSO-d6) 120575 067 (3H

br s H-26) 073 (3H br s H-24) 085 (6H br s H-25 29)087 (3H br s H-30) 097 (3H br s H-23) 106 (3H br sH-27) 301 (1H m H-3) 360 (1H m H-2101584010158401015840) 425 (1H 119889J = 70Hz H-110158401015840) 429 (1H 119889 J = 65Hz H-11015840) 462 (1H 119889J = 75Hz H-110158401015840101584010158401015840) 448 (1H 119889 J = 70Hz H-11015840101584010158401015840) 514 (1Hm H-11) 530 (1H 119889 J = 80Hz H-1101584010158401015840) 13C NMR (125MHz

Journal of Chemistry 3

DMSO-d6) see Table 1 HR-ESI-MS (positive)119898119911 12816127

[M + H]+ (calcd 12816116 for C60H97O29)

Acid Hydrolysis of 3 Compound 3 (10mg) was heated in2N HCl (5mL) at 80∘C for 2 h and then the solution wasextracted with ethyl acetate (2mL times 3) The sugar productsin the aqueous layer were identified as glucose (Rf 065)galactose (Rf 060) and glucuronic acid (Rf 012) in compar-ison with standards by silica gel thin layer chromatography(developedwith acetone-methanol-water 10 9 1 and sprayedby 10 sulfuric acid solution containing 2 vanillin) Afterpreparative thin layer chromatography the optical rotation ofeach sugar fraction was checkedThe positive optical rotationvalues led to the assignment of D-glucuronic acid D-glucoseand D-galactose [9]

24 Assay for 120572-Glucosidase Inhibition The yeast 120572-glu-cosidase (G0660 Sigma-Aldrich) enzyme inhibition assaywas performed by the digestion of p-nitrophenyl-120572-D-glucopyranoside [10] The sample solution (2 120583L dissolved inDMSO) and 05UmL 120572-glucosidase (40 120583L) were mixed in120120583L of 01M phosphate buffer (pH 70) After 5min prein-cubation 5mM p-nitrophenyl-120572-D-glucopyranoside solu-tion (40 120583L) was added and the solution was incubated at37∘C for 30min The absorbance of released 4-nitrophenolwasmeasured at 405 nmby using amicroplate reader (Molec-ular Devices CA) Acarbose was used as positive control(IC506504 120583gmL)

25 Assay for 120572-Amylase Inhibition The porcine pancreas 120572-amylase (A3176 Sigma-Aldrich) enzyme inhibitory activitywas measured using the method reported by Ashok Kumaret al [11] with slight modifications Substrate was pre-pared by dissolving 2-chloro-4-nitrophenyl-120572-D-maltotrioside (93834 Sigma-Aldrich) in phosphate buffer (pH 70)Thesample (2 120583L dissolved in DMSO) and 05 unitmL 120572-amylase(50120583L) were mixed in 100 120583L of 01M phosphate buffer (pH70) After 5min preincubation substrate solution (50120583L)wasadded and the solution was incubated at 37∘C for 15minTheabsorbances were measured at 405 nm Acarbose was used aspositive control (IC

50576120583gmL)

3 Results and Discussion

Compound 3 was obtained as a white crystal and itshigh-resolution electrospray ionization mass spectrometryspectrum revealed an ion peak at 119898119911 12816127 [M + H]+corresponding to the molecular formula C

60H96O29 The

1H-NMR spectrum of 1 showed seven methyl groups at 120575H067 (3H br s H-26) 073 (3H br s H-24) 085 (6H brs H-25 29) 087 (3H br s H-30) 097 (3H br s H-23)and 106 (3H br s H-27) and five glycosidic anomericprotons at 120575H 425 (1H 119889 J = 70Hz H-110158401015840) 429 (1H 119889 J =65Hz H-11015840) 448 (1H 119889 J = 70Hz H-11015840101584010158401015840) 530 (1H 119889 J =80Hz H-1101584010158401015840) and 462 (1H 119889 J = 75Hz H-110158401015840101584010158401015840) The largecoupling constants of all anomeric protons indicated the120573-configuration of the sugars 13C-NMR and DEPT spectraindicated the presence of an olean triterpene skeleton with

Table 1 13C-NMR data of compounds 1ndash3measured in DMSO-1198896

C 1 2 3 C 1 2 31 381 381 381 3-O-GluA2 254 229 254 11015840 1050 1036 10363 879 884 883 21015840 735 793 7954 387 386 386 31015840 754 754 7535 550 550 550 41015840 829 823 8236 177 177 177 51015840 753 751 7517 322 323 327 61015840 1720 1718 17158 400 407 400 41015840-O-Glu9 470 470 470 110158401015840 1038 1033 103310 362 362 362 210158401015840 734 735 73611 229 229 229 310158401015840 763 762 76312 1216 1217 1215 410158401015840 700 699 69913 1434 1434 1435 510158401015840 770 770 77014 412 412 411 610158401015840 611 611 61015 271 277 279 21015840-O-Glu16 225 225 219 11015840101584010158401015840 mdash 1031 103217 459 459 458 21015840101584010158401015840 mdash 735 73618 407 407 407 31015840101584010158401015840 mdash 761 76019 455 455 455 41015840101584010158401015840 mdash 701 70020 302 303 302 51015840101584010158401015840 mdash 768 76721 332 332 332 61015840101584010158401015840 mdash 609 61022 316 316 323 28-O-GlcGal23 276 277 277 1101584010158401015840 941 941 91924 166 161 167 2101584010158401015840 723 723 76925 151 151 151 3101584010158401015840 766 766 74126 164 169 167 4101584010158401015840 695 695 70727 254 255 255 5101584010158401015840 776 777 76828 1752 1752 1752 6101584010158401015840 607 607 61829 327 327 327 2101584010158401015840-O-Glu30 233 233 234 110158401015840101584010158401015840 mdash mdash 1023

210158401015840101584010158401015840 mdash mdash 750310158401015840101584010158401015840 mdash mdash 770410158401015840101584010158401015840 mdash mdash 694510158401015840101584010158401015840 mdash mdash 775610158401015840101584010158401015840 mdash mdash 605

seven CH3 ten CH

2 five CH and 8 C signals (Table 1)

The aglycone was identified as oleanolic acid a commontriterpene of saponins in the genus Polyscias [6] The 120575Cvalues of C-3 at 120575C 883 and C-28 at 120575C 1752 suggested thatcompound 3 was a bisdesmosidic saponin with sugar unitsattached to these positions In addition five sugar moietieswere recognized by five anomeric methine groups at 120575C 1036(C-11015840) 1033 (C-110158401015840) 919 (C-1101584010158401015840) 1032 (C-11015840101584010158401015840) and 1023(C-110158401015840101584010158401015840) These data are similar to those of compound 2

4 Journal of Chemistry

except for the addition of a sugar moiety and the upfield shiftof the anomeric signal at 120575C from 941 to 919 Acid hydrolysisof compound 3 allowed the identification of D-glucuronicacid D-glucose and D-galactose Extensive analysis of theHMBC spectrum of compound 3 allowed the position ofthe sugar units to be determined The couplings from H-11015840(120575H 429) to C-3 (120575C 883) indicated that the glucuronic andglucosyl moieties attached to C-3 The correlations fromH-110158401015840 (120575H 425) to C-41015840 (120575C 823) and between H-41015840101584010158401015840 (120575H448) and C-21015840 (120575C 795) confirmed that the two glucosylunits were attached to C-21015840 andC-41015840 respectively Indeed the13C-NMR data of this fragment coincided well with the 120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl of compound 2 (Table 1) The HMBCcorrelations from H-1101584010158401015840 (120575H 531) to C-28 (120575C 1752) andthe anomeric C-1101584010158401015840 resonance shifted upfield at 120575C 919suggesting the galactose unit attached to C-28 The HMBCcouplings from H-110158401015840101584010158401015840 (120575H 462) to C-2101584010158401015840 (120575C 769) and fromH-2101584010158401015840 (120575H 360) to C-110158401015840101584010158401015840 (120575C 1023) and the COSY crosspeakbetween H-1101584010158401015840 and H-2101584010158401015840 indicated that the glucose unitattached to C-2101584010158401015840 Thus compound 3 was elucidated as3-O-120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl oleanolic acid 28-O-120573-D-glucopyranosyl-(1rarr2)-120573-D-galactopyranosyl ester namedpolyscioside I

The inhibitory effects of the isolated compounds againstporcine pancreas 120572-amylase and yeast 120572-glucosidase wereevaluated in comparison with the antidiabetic acarbose(Table 2) Compound 1 potently inhibited 120572-amylase and 120572-glucosidase with IC

50values of 271 and 4405 120583gmL respec-

tively In contrast compounds 2 and 3 did not show any effecton both enzymes Dou et al reported that the glucuronic acidunit at C-3 and glucosidic moiety at C-28 of the aglyconewere the functional groups essential for 120572-amylase and 120572-glucosidase inhibition the addition or replacement of glucoseby another sugar unit might thus abolish the inhibitoryactivity [12] In oral sucrose tolerance tests several oleanolicacid glycosides showed significant antihyperglycemic activityin the oral sucrose tolerance in rats [13 14] However the 21015840-O-120573-D-glucopyranosyl unit attached to the glucuronic acidmoiety was assumed to markedly reduce the hypoglycemiceffect [15] Consistent with this in our study compounds2 and 3 possessing a 21015840-O-120573-D-glucopyranosyl unit wereinactive regarding 120572-amylase and 120572-glucosidase inhibitionPrevious studies showed that P fruticosa leaf extracts stronglyinhibited rat intestinal 120572-glucosidase [16] and exhibitedhypoglycemic and antihyperglycemic activity in normal andstreptozotocin-diabetic albino rats [4] Our results suggestthat the observed hypoglycemic activity might be related tothe inhibition of 120572-amylase and 120572-glucosidase enzymes

It was of interest to establish whether compound 1and acarbose interact synergistically or additively in theinhibition of 120572-glucosidase and 120572-amylase Therefore theassay was performed in solutions containing these agentsalone or in a mixture at different concentrations The resultsshowed that the combination of compound 1 and acarboseproduced a synergistic inhibitory effect on porcine pancreas120572-amylase (Figure 2) but had no effect on yeast 120572-glucosidase(data not shown) Interestingly higher synergistic inhibition

Table 2 Inhibitory effects (IC50

plusmn SD 120583gmL) of 1ndash3 for 120572-amylaseand 120572-glucosidase

Compounds 120572-Amylase 120572-Glucosidase1 271 plusmn 205 4405 plusmn 1272 mdash mdash3 mdash mdashAcarbose 576 plusmn 412 6504 plusmn 334

was found from the combination of low concentrations ofindividual agents At 10 120583gmL compound 1 and acarbosealone exhibited only 15 and 42 inhibition respectivelyHowever their combination increased the inhibition up to328 At a higher concentration of 10 120583gmL the percentageof 120572-amylase inhibition of the mixture was equal to thesum of compound 1 and acarbose suggesting that theyproduced additive inhibition A slight increase in inhibitionwas obtained from the mixture in comparison with thatachieved by the individual agents at 100120583gmL These find-ings indicate that saponin 1 produced synergistic effects onporcine pancreas 120572-amylase inhibition when combined witha low concentration of acarbose

Acarbose is a potent 120572-glucosidase inhibitor and has beenused in the treatment of type 2 diabetes It can also beused in combination with other agents such as sulfonylureaand metformin in patients with diabetes However thelong-term use and high-dose administration of this drugcause severe side effects such as diarrhea flatulence andabdominal or stomach pain [17ndash19] Our study showed thatthe combination of P fruticosa saponin 1 with acarbose at alow concentration producedmore synergistic inhibition thaneither drug alone suggesting that theymight provide a signif-icant clinical benefit in delaying postprandial hyperglycemiaand diminishing the adverse effects of acarbose

4 Conclusion

Three bisdesmosidic saponins including two known 3-O-[120573-D-glucopyranosyl-(1rarr4)-120573-D-glucuronopyranosyl] oleano-lic acid 28-O-120573-D-glucopyranosyl ester (1) and 3-O-120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl oleanolic acid 28-O-120573-D-glucopyrano-syl ester (or polyscioside D 2) and one new 3-O-120573-D-glu-copyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glu-curonopyranosyl oleanolic acid 28-O-120573-D-glucopyranosyl-(1rarr2)-120573-D-galactopyranosyl ester (named polyscioside I3) were isolated from the leaves of P fruticosa Compound 1showed inhibitory effects against porcine pancreas 120572-amylaseand yeast 120572-glucosidase activities and produced a synergisticeffect with acarbose in 120572-amylase inhibition Further studyis needed to clarify the mechanism of enzyme inhibition aswell as the hypoglycemic properties of saponin 1

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Journal of Chemistry 5

1 10 100

in

hibi

tion

0102030405060708090

(120583gmL)

Compound 1Acarbose

Mix

lowast

lowast

lowast

Figure 2 The percentage inhibition by combination of 1 andacarbose on porcine pancreatic 120572-amylase Results are expressed asmeans plusmn SD 119899 = 3 lowast119901 lt 005 compared with acarbose alone

Acknowledgments

This work is supported in part by the Vietnam Academyof Science and Technology (VASTDLT0614-15) and theNational Foundation for Science and Technology Develop-ment (NAFOSTED 10401-201154) The authors thank theInstitute of Chemistry (VAST) for the NMR measurementsand Dr ML Bourguet-Kondracki (Musee National drsquoHistoireNaturelle Paris France) for providing HRMS experiment

References

[1] V C Vo Dictionary of Vietnamese Medicinal Plants vol 1Medical Publisher Hanoi Vietnam 2012

[2] B M Bernard N Pakianathan and M C Divakar ldquoOnthe antipyretic anti-inflammatory analgesic and molluscicidalproperties of Polyscias fruticosa (L) Harmsrdquo Ancient Science ofLife vol 17 pp 313ndash319 1998

[3] G Asumeng Koffuor A Boye S Kyei et al ldquoAwuku Anti-asthmatic property and possible mode of activity of an ethanolleaf extract of Polyscias fruticoserdquo Pharmaceutical Biology vol8 pp 1ndash10 2015

[4] M C Divakar and M B Bensita ldquoScreening of various leafextracts of Polyscias fruticosa Harms for their antidiabeticactivityrdquo Indian Journal of Natural Products vol 14 pp 24ndash281998

[5] J Lutomski T C Luan and T T Hoa ldquoPolyacetylenes inthe Araliaceae family Part IV The antibacterial and antifungalactivities of two main polyacetylenes from Panax vietnamensisHa et Grushv and Polyscias fruticosa (L) Harmsrdquo HerbaPolonica vol 38 pp 137ndash140 1992

[6] V D Huan S Yamamura K Ohtani et al ldquoOleanane saponinsfrom Polyscias fruticosardquo Phytochemistry vol 47 no 3 pp 451ndash457 1998

[7] P M de Souza P M de Sales L A Simeoni E C Silva DSilveira and P de Oliveira Magalhaes ldquoInhibitory activity of 120572-amylase and 120572-glucosidase by plant extracts from the Braziliancerradordquo Planta Medica vol 78 no 4 pp 393ndash399 2012

[8] C Borel M P Gupta and K Hostettmann ldquoMolluscicidalsaponins from Swartzia simplexrdquo Phytochemistry vol 26 no10 pp 2685ndash2689 1987

[9] L Voutquenne-Nazabadioko R Gevrenova N Borie et alldquoTriterpenoid saponins from the roots ofGypsophila trichotomaWenderrdquo Phytochemistry vol 90 pp 114ndash127 2013

[10] H H T Tran M C Nguyen H T Le et al ldquoInhibitors of 120572-glucosidase and 120572-amylase from Cyperus rotundusrdquo Pharma-ceutical Biology vol 52 no 1 pp 74ndash77 2014

[11] B S Ashok Kumar K Lakshman K N Jayaveea et alldquoAntidiabetic antihyperlipidemic and antioxidant activities ofmethanolic extract of Amaranthus viridis Linn in alloxaninduced diabetic ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 1-2 pp 75ndash79 2012

[12] F Dou M Xi J Wang et al ldquoalpha-Glucosidase and alpha-amylase inhibitory activities of saponins from traditional Chi-nese medicines in the treatment of diabetes mellitusrdquo DiePharmazie vol 68 no 4 pp 300ndash304 2013

[13] H Matsuda Y Li T Murakami N Matsumura J Yama-hara and M Yoshikawa ldquoAntidiabetic principles of naturalmedicines III Structure-related inhibitory activity and actionmode of oleanolic acid glycosides on hypoglycemic activityrdquoChemical and Pharmaceutical Bulletin vol 46 no 9 pp 1399ndash1403 1998

[14] M Yoshikawa T Murakami E Harada N Murakami JYamahara andHMatsuda ldquoBioactive saponins and glycosidesVII On the hypoglycemic principles from the root cortex ofAralia elata SEEM structure related hypoglycemic activity ofoleanolic acid oligoglycosiderdquo Chemical and PharmaceuticalBulletin vol 44 no 10 pp 1923ndash1927 1996

[15] M Yoshikawa T Murakami A Kishi T Kageura and HMatsuda ldquoMedicinal flowers III Marigold (1) hypoglycemicgastric emptying inhibitory and gastroprotective principles andnew oleanane-type triterpene oligoglycosides calendasaponinsA B C and D from egyptian Calendula officinalisrdquo Chemicaland Pharmaceutical Bulletin vol 49 no 7 pp 863ndash870 2001

[16] T T Mai N N Thu P G Tien and N Van Chuyen ldquoAlpha-glucosidase inhibitory and antioxidant activities of Vietnameseedible plants and their relationships with polyphenol contentsrdquoJournal of Nutritional Science and Vitaminology vol 53 no 3pp 267ndash276 2007

[17] M Rodier J L Richard L Monnier and J Mirouze ldquoEffect oflong term Acarbose (Bay g 5421) therapy on metabolic controlof non insulin dependent (type II) diabetes mellitusrdquoDiabete ampMetabolisme vol 14 no 1 pp 12ndash14 1988

[18] A J Scheen ldquoClinical efficacy of acarbose in diabetes mellitus acritical review of controlled trialsrdquo Diabetes amp Metabolism vol24 no 4 pp 311ndash320 1998

[19] S Akkarachiyasit P Charoenlertkul S Yibchok-Anun andS Adisakwattana ldquoInhibitory activities of cyanidin and itsglycosides and synergistic effect with acarbose against intestinal120572-glucosidase and pancreatic 120572-amylaserdquo International Journalof Molecular Sciences vol 11 no 9 pp 3387ndash3396 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

2 Journal of Chemistry

O

12

34

56

7

89

10

1112

13

1415

16

1718

1920

21

22

23 24

25 26

27

30

28

29

O

OHO

O

HO O

O

OHHOHO

HO

O

OHHOHO

HO

O

OHHO

O

HO O

O

OHHOHO

HO

O

OHO

OH OH

O

OHHOHO

HO

O

OHHO

HO

HO

S1 S2

S3

S4

O

O

O

12

34

56

7

89

10

1112

13

1415

16

1718

1920

21

22

23 24

25 26

27

30

28

29

O

OHO

O

HO O

O

OHHOHO

HO

O

OHHOHO

HOO

OHO

OH OH

O

OHHOHO

HO

R1O

OR2

6998400998400

6998400998400

6998400998400

1998400998400

1998400998400

3998400998400

1998400998400

3998400998400

3998400998400

49984004998400

39984003998400

69984006998400

19984001998400

1998400998400998400998400

3998400998400998400998400

3998400998400998400

6998400998400998400998400

6998400998400998400

1998400998400998400

4998400998400998400

6998400998400998400998400998400

3998400998400998400998400998400

1998400998400998400998400998400

4998400

3998400

6998400

1998400

1998400998400998400998400

3998400998400998400998400

6998400998400998400 3998400998400998400

6998400998400998400

4998400998400998400

69984009984009984009984009984001998400998400998400

1998400998400998400998400998400

4998400

3998400

6998400

1998400

1 R1 = S1 R2 = S3

2 R1 = S2 R2 = S33 R1 = S2 R2 = S4

3

Figure 1 Structure of compounds 1ndash3 and key HMBC correlations of 3

Tokyo Japan) NMR experiments were carried out on aBruker AM500 FT-NMR spectrometer (Bruker Rheinstet-ten Germany) using tetramethylsilane (TMS) as internalstandard The HR-ESI-MS was recorded on API Q-STARPULSAR I of Applied Biosystem The bioassay absorbancewas read by xMark Microplate Spectrophotometer reader(Bio-Rad Laboratories USA)

22 Plant Material The leaves of Polyscias fruticosa werecollected in TienHaiThai Binh in July 2011 and identified byProfessor Tran Huy Thai Institute of Ecology and BiologicalResources Vietnam Academy of Science and TechnologyThe voucher specimens were deposited at the herbarium ofthe Institute of Ecology and Biological Resources

23 Extraction and Isolation The air-dried and powdered Pfruticosa leaves (20 kg) were extracted with methanol (5 L times3 times) at room temperature The combined extracts wereconcentrated to give 300 g of crude extract which was thenresuspended inwater (15 L) and successively partitionedwithhexane and ethyl acetate (each 05 L times 3 times) to obtain 75

and 92 g of hexane and ethyl acetate residues respectivelyThe water residue was filtered through a Diaion HP-20column eluted by 0 25 and 100 methanol in waterThe 100 methanol-eluted fraction was diluted with water(1 1 vv) and kept overnight at 4∘C Compound 1 as whitecrystals (500mg) was obtained by filtering The filtrate wasconcentrated and chromatographed on a silica gel columneluted with a gradient of 0 50 and 100 methanol inethyl acetate to afford three fractions F1ndashF3 Fraction F2 wasfractionated on a silica gel column eluted with chloroform-methanol-water (50 10 1 vv) to afford compound 2 (10mg)Compound 3 (205mg) was purified from F3 by a RP-18column (methanol-water 1 1 vv)

Polyscioside I (3)White powder mp gt300∘C [120572]D24 = +124

(c 01 MeOH) 1H NMR (500MHz DMSO-d6) 120575 067 (3H

br s H-26) 073 (3H br s H-24) 085 (6H br s H-25 29)087 (3H br s H-30) 097 (3H br s H-23) 106 (3H br sH-27) 301 (1H m H-3) 360 (1H m H-2101584010158401015840) 425 (1H 119889J = 70Hz H-110158401015840) 429 (1H 119889 J = 65Hz H-11015840) 462 (1H 119889J = 75Hz H-110158401015840101584010158401015840) 448 (1H 119889 J = 70Hz H-11015840101584010158401015840) 514 (1Hm H-11) 530 (1H 119889 J = 80Hz H-1101584010158401015840) 13C NMR (125MHz

Journal of Chemistry 3

DMSO-d6) see Table 1 HR-ESI-MS (positive)119898119911 12816127

[M + H]+ (calcd 12816116 for C60H97O29)

Acid Hydrolysis of 3 Compound 3 (10mg) was heated in2N HCl (5mL) at 80∘C for 2 h and then the solution wasextracted with ethyl acetate (2mL times 3) The sugar productsin the aqueous layer were identified as glucose (Rf 065)galactose (Rf 060) and glucuronic acid (Rf 012) in compar-ison with standards by silica gel thin layer chromatography(developedwith acetone-methanol-water 10 9 1 and sprayedby 10 sulfuric acid solution containing 2 vanillin) Afterpreparative thin layer chromatography the optical rotation ofeach sugar fraction was checkedThe positive optical rotationvalues led to the assignment of D-glucuronic acid D-glucoseand D-galactose [9]

24 Assay for 120572-Glucosidase Inhibition The yeast 120572-glu-cosidase (G0660 Sigma-Aldrich) enzyme inhibition assaywas performed by the digestion of p-nitrophenyl-120572-D-glucopyranoside [10] The sample solution (2 120583L dissolved inDMSO) and 05UmL 120572-glucosidase (40 120583L) were mixed in120120583L of 01M phosphate buffer (pH 70) After 5min prein-cubation 5mM p-nitrophenyl-120572-D-glucopyranoside solu-tion (40 120583L) was added and the solution was incubated at37∘C for 30min The absorbance of released 4-nitrophenolwasmeasured at 405 nmby using amicroplate reader (Molec-ular Devices CA) Acarbose was used as positive control(IC506504 120583gmL)

25 Assay for 120572-Amylase Inhibition The porcine pancreas 120572-amylase (A3176 Sigma-Aldrich) enzyme inhibitory activitywas measured using the method reported by Ashok Kumaret al [11] with slight modifications Substrate was pre-pared by dissolving 2-chloro-4-nitrophenyl-120572-D-maltotrioside (93834 Sigma-Aldrich) in phosphate buffer (pH 70)Thesample (2 120583L dissolved in DMSO) and 05 unitmL 120572-amylase(50120583L) were mixed in 100 120583L of 01M phosphate buffer (pH70) After 5min preincubation substrate solution (50120583L)wasadded and the solution was incubated at 37∘C for 15minTheabsorbances were measured at 405 nm Acarbose was used aspositive control (IC

50576120583gmL)

3 Results and Discussion

Compound 3 was obtained as a white crystal and itshigh-resolution electrospray ionization mass spectrometryspectrum revealed an ion peak at 119898119911 12816127 [M + H]+corresponding to the molecular formula C

60H96O29 The

1H-NMR spectrum of 1 showed seven methyl groups at 120575H067 (3H br s H-26) 073 (3H br s H-24) 085 (6H brs H-25 29) 087 (3H br s H-30) 097 (3H br s H-23)and 106 (3H br s H-27) and five glycosidic anomericprotons at 120575H 425 (1H 119889 J = 70Hz H-110158401015840) 429 (1H 119889 J =65Hz H-11015840) 448 (1H 119889 J = 70Hz H-11015840101584010158401015840) 530 (1H 119889 J =80Hz H-1101584010158401015840) and 462 (1H 119889 J = 75Hz H-110158401015840101584010158401015840) The largecoupling constants of all anomeric protons indicated the120573-configuration of the sugars 13C-NMR and DEPT spectraindicated the presence of an olean triterpene skeleton with

Table 1 13C-NMR data of compounds 1ndash3measured in DMSO-1198896

C 1 2 3 C 1 2 31 381 381 381 3-O-GluA2 254 229 254 11015840 1050 1036 10363 879 884 883 21015840 735 793 7954 387 386 386 31015840 754 754 7535 550 550 550 41015840 829 823 8236 177 177 177 51015840 753 751 7517 322 323 327 61015840 1720 1718 17158 400 407 400 41015840-O-Glu9 470 470 470 110158401015840 1038 1033 103310 362 362 362 210158401015840 734 735 73611 229 229 229 310158401015840 763 762 76312 1216 1217 1215 410158401015840 700 699 69913 1434 1434 1435 510158401015840 770 770 77014 412 412 411 610158401015840 611 611 61015 271 277 279 21015840-O-Glu16 225 225 219 11015840101584010158401015840 mdash 1031 103217 459 459 458 21015840101584010158401015840 mdash 735 73618 407 407 407 31015840101584010158401015840 mdash 761 76019 455 455 455 41015840101584010158401015840 mdash 701 70020 302 303 302 51015840101584010158401015840 mdash 768 76721 332 332 332 61015840101584010158401015840 mdash 609 61022 316 316 323 28-O-GlcGal23 276 277 277 1101584010158401015840 941 941 91924 166 161 167 2101584010158401015840 723 723 76925 151 151 151 3101584010158401015840 766 766 74126 164 169 167 4101584010158401015840 695 695 70727 254 255 255 5101584010158401015840 776 777 76828 1752 1752 1752 6101584010158401015840 607 607 61829 327 327 327 2101584010158401015840-O-Glu30 233 233 234 110158401015840101584010158401015840 mdash mdash 1023

210158401015840101584010158401015840 mdash mdash 750310158401015840101584010158401015840 mdash mdash 770410158401015840101584010158401015840 mdash mdash 694510158401015840101584010158401015840 mdash mdash 775610158401015840101584010158401015840 mdash mdash 605

seven CH3 ten CH

2 five CH and 8 C signals (Table 1)

The aglycone was identified as oleanolic acid a commontriterpene of saponins in the genus Polyscias [6] The 120575Cvalues of C-3 at 120575C 883 and C-28 at 120575C 1752 suggested thatcompound 3 was a bisdesmosidic saponin with sugar unitsattached to these positions In addition five sugar moietieswere recognized by five anomeric methine groups at 120575C 1036(C-11015840) 1033 (C-110158401015840) 919 (C-1101584010158401015840) 1032 (C-11015840101584010158401015840) and 1023(C-110158401015840101584010158401015840) These data are similar to those of compound 2

4 Journal of Chemistry

except for the addition of a sugar moiety and the upfield shiftof the anomeric signal at 120575C from 941 to 919 Acid hydrolysisof compound 3 allowed the identification of D-glucuronicacid D-glucose and D-galactose Extensive analysis of theHMBC spectrum of compound 3 allowed the position ofthe sugar units to be determined The couplings from H-11015840(120575H 429) to C-3 (120575C 883) indicated that the glucuronic andglucosyl moieties attached to C-3 The correlations fromH-110158401015840 (120575H 425) to C-41015840 (120575C 823) and between H-41015840101584010158401015840 (120575H448) and C-21015840 (120575C 795) confirmed that the two glucosylunits were attached to C-21015840 andC-41015840 respectively Indeed the13C-NMR data of this fragment coincided well with the 120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl of compound 2 (Table 1) The HMBCcorrelations from H-1101584010158401015840 (120575H 531) to C-28 (120575C 1752) andthe anomeric C-1101584010158401015840 resonance shifted upfield at 120575C 919suggesting the galactose unit attached to C-28 The HMBCcouplings from H-110158401015840101584010158401015840 (120575H 462) to C-2101584010158401015840 (120575C 769) and fromH-2101584010158401015840 (120575H 360) to C-110158401015840101584010158401015840 (120575C 1023) and the COSY crosspeakbetween H-1101584010158401015840 and H-2101584010158401015840 indicated that the glucose unitattached to C-2101584010158401015840 Thus compound 3 was elucidated as3-O-120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl oleanolic acid 28-O-120573-D-glucopyranosyl-(1rarr2)-120573-D-galactopyranosyl ester namedpolyscioside I

The inhibitory effects of the isolated compounds againstporcine pancreas 120572-amylase and yeast 120572-glucosidase wereevaluated in comparison with the antidiabetic acarbose(Table 2) Compound 1 potently inhibited 120572-amylase and 120572-glucosidase with IC

50values of 271 and 4405 120583gmL respec-

tively In contrast compounds 2 and 3 did not show any effecton both enzymes Dou et al reported that the glucuronic acidunit at C-3 and glucosidic moiety at C-28 of the aglyconewere the functional groups essential for 120572-amylase and 120572-glucosidase inhibition the addition or replacement of glucoseby another sugar unit might thus abolish the inhibitoryactivity [12] In oral sucrose tolerance tests several oleanolicacid glycosides showed significant antihyperglycemic activityin the oral sucrose tolerance in rats [13 14] However the 21015840-O-120573-D-glucopyranosyl unit attached to the glucuronic acidmoiety was assumed to markedly reduce the hypoglycemiceffect [15] Consistent with this in our study compounds2 and 3 possessing a 21015840-O-120573-D-glucopyranosyl unit wereinactive regarding 120572-amylase and 120572-glucosidase inhibitionPrevious studies showed that P fruticosa leaf extracts stronglyinhibited rat intestinal 120572-glucosidase [16] and exhibitedhypoglycemic and antihyperglycemic activity in normal andstreptozotocin-diabetic albino rats [4] Our results suggestthat the observed hypoglycemic activity might be related tothe inhibition of 120572-amylase and 120572-glucosidase enzymes

It was of interest to establish whether compound 1and acarbose interact synergistically or additively in theinhibition of 120572-glucosidase and 120572-amylase Therefore theassay was performed in solutions containing these agentsalone or in a mixture at different concentrations The resultsshowed that the combination of compound 1 and acarboseproduced a synergistic inhibitory effect on porcine pancreas120572-amylase (Figure 2) but had no effect on yeast 120572-glucosidase(data not shown) Interestingly higher synergistic inhibition

Table 2 Inhibitory effects (IC50

plusmn SD 120583gmL) of 1ndash3 for 120572-amylaseand 120572-glucosidase

Compounds 120572-Amylase 120572-Glucosidase1 271 plusmn 205 4405 plusmn 1272 mdash mdash3 mdash mdashAcarbose 576 plusmn 412 6504 plusmn 334

was found from the combination of low concentrations ofindividual agents At 10 120583gmL compound 1 and acarbosealone exhibited only 15 and 42 inhibition respectivelyHowever their combination increased the inhibition up to328 At a higher concentration of 10 120583gmL the percentageof 120572-amylase inhibition of the mixture was equal to thesum of compound 1 and acarbose suggesting that theyproduced additive inhibition A slight increase in inhibitionwas obtained from the mixture in comparison with thatachieved by the individual agents at 100120583gmL These find-ings indicate that saponin 1 produced synergistic effects onporcine pancreas 120572-amylase inhibition when combined witha low concentration of acarbose

Acarbose is a potent 120572-glucosidase inhibitor and has beenused in the treatment of type 2 diabetes It can also beused in combination with other agents such as sulfonylureaand metformin in patients with diabetes However thelong-term use and high-dose administration of this drugcause severe side effects such as diarrhea flatulence andabdominal or stomach pain [17ndash19] Our study showed thatthe combination of P fruticosa saponin 1 with acarbose at alow concentration producedmore synergistic inhibition thaneither drug alone suggesting that theymight provide a signif-icant clinical benefit in delaying postprandial hyperglycemiaand diminishing the adverse effects of acarbose

4 Conclusion

Three bisdesmosidic saponins including two known 3-O-[120573-D-glucopyranosyl-(1rarr4)-120573-D-glucuronopyranosyl] oleano-lic acid 28-O-120573-D-glucopyranosyl ester (1) and 3-O-120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl oleanolic acid 28-O-120573-D-glucopyrano-syl ester (or polyscioside D 2) and one new 3-O-120573-D-glu-copyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glu-curonopyranosyl oleanolic acid 28-O-120573-D-glucopyranosyl-(1rarr2)-120573-D-galactopyranosyl ester (named polyscioside I3) were isolated from the leaves of P fruticosa Compound 1showed inhibitory effects against porcine pancreas 120572-amylaseand yeast 120572-glucosidase activities and produced a synergisticeffect with acarbose in 120572-amylase inhibition Further studyis needed to clarify the mechanism of enzyme inhibition aswell as the hypoglycemic properties of saponin 1

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Journal of Chemistry 5

1 10 100

in

hibi

tion

0102030405060708090

(120583gmL)

Compound 1Acarbose

Mix

lowast

lowast

lowast

Figure 2 The percentage inhibition by combination of 1 andacarbose on porcine pancreatic 120572-amylase Results are expressed asmeans plusmn SD 119899 = 3 lowast119901 lt 005 compared with acarbose alone

Acknowledgments

This work is supported in part by the Vietnam Academyof Science and Technology (VASTDLT0614-15) and theNational Foundation for Science and Technology Develop-ment (NAFOSTED 10401-201154) The authors thank theInstitute of Chemistry (VAST) for the NMR measurementsand Dr ML Bourguet-Kondracki (Musee National drsquoHistoireNaturelle Paris France) for providing HRMS experiment

References

[1] V C Vo Dictionary of Vietnamese Medicinal Plants vol 1Medical Publisher Hanoi Vietnam 2012

[2] B M Bernard N Pakianathan and M C Divakar ldquoOnthe antipyretic anti-inflammatory analgesic and molluscicidalproperties of Polyscias fruticosa (L) Harmsrdquo Ancient Science ofLife vol 17 pp 313ndash319 1998

[3] G Asumeng Koffuor A Boye S Kyei et al ldquoAwuku Anti-asthmatic property and possible mode of activity of an ethanolleaf extract of Polyscias fruticoserdquo Pharmaceutical Biology vol8 pp 1ndash10 2015

[4] M C Divakar and M B Bensita ldquoScreening of various leafextracts of Polyscias fruticosa Harms for their antidiabeticactivityrdquo Indian Journal of Natural Products vol 14 pp 24ndash281998

[5] J Lutomski T C Luan and T T Hoa ldquoPolyacetylenes inthe Araliaceae family Part IV The antibacterial and antifungalactivities of two main polyacetylenes from Panax vietnamensisHa et Grushv and Polyscias fruticosa (L) Harmsrdquo HerbaPolonica vol 38 pp 137ndash140 1992

[6] V D Huan S Yamamura K Ohtani et al ldquoOleanane saponinsfrom Polyscias fruticosardquo Phytochemistry vol 47 no 3 pp 451ndash457 1998

[7] P M de Souza P M de Sales L A Simeoni E C Silva DSilveira and P de Oliveira Magalhaes ldquoInhibitory activity of 120572-amylase and 120572-glucosidase by plant extracts from the Braziliancerradordquo Planta Medica vol 78 no 4 pp 393ndash399 2012

[8] C Borel M P Gupta and K Hostettmann ldquoMolluscicidalsaponins from Swartzia simplexrdquo Phytochemistry vol 26 no10 pp 2685ndash2689 1987

[9] L Voutquenne-Nazabadioko R Gevrenova N Borie et alldquoTriterpenoid saponins from the roots ofGypsophila trichotomaWenderrdquo Phytochemistry vol 90 pp 114ndash127 2013

[10] H H T Tran M C Nguyen H T Le et al ldquoInhibitors of 120572-glucosidase and 120572-amylase from Cyperus rotundusrdquo Pharma-ceutical Biology vol 52 no 1 pp 74ndash77 2014

[11] B S Ashok Kumar K Lakshman K N Jayaveea et alldquoAntidiabetic antihyperlipidemic and antioxidant activities ofmethanolic extract of Amaranthus viridis Linn in alloxaninduced diabetic ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 1-2 pp 75ndash79 2012

[12] F Dou M Xi J Wang et al ldquoalpha-Glucosidase and alpha-amylase inhibitory activities of saponins from traditional Chi-nese medicines in the treatment of diabetes mellitusrdquo DiePharmazie vol 68 no 4 pp 300ndash304 2013

[13] H Matsuda Y Li T Murakami N Matsumura J Yama-hara and M Yoshikawa ldquoAntidiabetic principles of naturalmedicines III Structure-related inhibitory activity and actionmode of oleanolic acid glycosides on hypoglycemic activityrdquoChemical and Pharmaceutical Bulletin vol 46 no 9 pp 1399ndash1403 1998

[14] M Yoshikawa T Murakami E Harada N Murakami JYamahara andHMatsuda ldquoBioactive saponins and glycosidesVII On the hypoglycemic principles from the root cortex ofAralia elata SEEM structure related hypoglycemic activity ofoleanolic acid oligoglycosiderdquo Chemical and PharmaceuticalBulletin vol 44 no 10 pp 1923ndash1927 1996

[15] M Yoshikawa T Murakami A Kishi T Kageura and HMatsuda ldquoMedicinal flowers III Marigold (1) hypoglycemicgastric emptying inhibitory and gastroprotective principles andnew oleanane-type triterpene oligoglycosides calendasaponinsA B C and D from egyptian Calendula officinalisrdquo Chemicaland Pharmaceutical Bulletin vol 49 no 7 pp 863ndash870 2001

[16] T T Mai N N Thu P G Tien and N Van Chuyen ldquoAlpha-glucosidase inhibitory and antioxidant activities of Vietnameseedible plants and their relationships with polyphenol contentsrdquoJournal of Nutritional Science and Vitaminology vol 53 no 3pp 267ndash276 2007

[17] M Rodier J L Richard L Monnier and J Mirouze ldquoEffect oflong term Acarbose (Bay g 5421) therapy on metabolic controlof non insulin dependent (type II) diabetes mellitusrdquoDiabete ampMetabolisme vol 14 no 1 pp 12ndash14 1988

[18] A J Scheen ldquoClinical efficacy of acarbose in diabetes mellitus acritical review of controlled trialsrdquo Diabetes amp Metabolism vol24 no 4 pp 311ndash320 1998

[19] S Akkarachiyasit P Charoenlertkul S Yibchok-Anun andS Adisakwattana ldquoInhibitory activities of cyanidin and itsglycosides and synergistic effect with acarbose against intestinal120572-glucosidase and pancreatic 120572-amylaserdquo International Journalof Molecular Sciences vol 11 no 9 pp 3387ndash3396 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 3

DMSO-d6) see Table 1 HR-ESI-MS (positive)119898119911 12816127

[M + H]+ (calcd 12816116 for C60H97O29)

Acid Hydrolysis of 3 Compound 3 (10mg) was heated in2N HCl (5mL) at 80∘C for 2 h and then the solution wasextracted with ethyl acetate (2mL times 3) The sugar productsin the aqueous layer were identified as glucose (Rf 065)galactose (Rf 060) and glucuronic acid (Rf 012) in compar-ison with standards by silica gel thin layer chromatography(developedwith acetone-methanol-water 10 9 1 and sprayedby 10 sulfuric acid solution containing 2 vanillin) Afterpreparative thin layer chromatography the optical rotation ofeach sugar fraction was checkedThe positive optical rotationvalues led to the assignment of D-glucuronic acid D-glucoseand D-galactose [9]

24 Assay for 120572-Glucosidase Inhibition The yeast 120572-glu-cosidase (G0660 Sigma-Aldrich) enzyme inhibition assaywas performed by the digestion of p-nitrophenyl-120572-D-glucopyranoside [10] The sample solution (2 120583L dissolved inDMSO) and 05UmL 120572-glucosidase (40 120583L) were mixed in120120583L of 01M phosphate buffer (pH 70) After 5min prein-cubation 5mM p-nitrophenyl-120572-D-glucopyranoside solu-tion (40 120583L) was added and the solution was incubated at37∘C for 30min The absorbance of released 4-nitrophenolwasmeasured at 405 nmby using amicroplate reader (Molec-ular Devices CA) Acarbose was used as positive control(IC506504 120583gmL)

25 Assay for 120572-Amylase Inhibition The porcine pancreas 120572-amylase (A3176 Sigma-Aldrich) enzyme inhibitory activitywas measured using the method reported by Ashok Kumaret al [11] with slight modifications Substrate was pre-pared by dissolving 2-chloro-4-nitrophenyl-120572-D-maltotrioside (93834 Sigma-Aldrich) in phosphate buffer (pH 70)Thesample (2 120583L dissolved in DMSO) and 05 unitmL 120572-amylase(50120583L) were mixed in 100 120583L of 01M phosphate buffer (pH70) After 5min preincubation substrate solution (50120583L)wasadded and the solution was incubated at 37∘C for 15minTheabsorbances were measured at 405 nm Acarbose was used aspositive control (IC

50576120583gmL)

3 Results and Discussion

Compound 3 was obtained as a white crystal and itshigh-resolution electrospray ionization mass spectrometryspectrum revealed an ion peak at 119898119911 12816127 [M + H]+corresponding to the molecular formula C

60H96O29 The

1H-NMR spectrum of 1 showed seven methyl groups at 120575H067 (3H br s H-26) 073 (3H br s H-24) 085 (6H brs H-25 29) 087 (3H br s H-30) 097 (3H br s H-23)and 106 (3H br s H-27) and five glycosidic anomericprotons at 120575H 425 (1H 119889 J = 70Hz H-110158401015840) 429 (1H 119889 J =65Hz H-11015840) 448 (1H 119889 J = 70Hz H-11015840101584010158401015840) 530 (1H 119889 J =80Hz H-1101584010158401015840) and 462 (1H 119889 J = 75Hz H-110158401015840101584010158401015840) The largecoupling constants of all anomeric protons indicated the120573-configuration of the sugars 13C-NMR and DEPT spectraindicated the presence of an olean triterpene skeleton with

Table 1 13C-NMR data of compounds 1ndash3measured in DMSO-1198896

C 1 2 3 C 1 2 31 381 381 381 3-O-GluA2 254 229 254 11015840 1050 1036 10363 879 884 883 21015840 735 793 7954 387 386 386 31015840 754 754 7535 550 550 550 41015840 829 823 8236 177 177 177 51015840 753 751 7517 322 323 327 61015840 1720 1718 17158 400 407 400 41015840-O-Glu9 470 470 470 110158401015840 1038 1033 103310 362 362 362 210158401015840 734 735 73611 229 229 229 310158401015840 763 762 76312 1216 1217 1215 410158401015840 700 699 69913 1434 1434 1435 510158401015840 770 770 77014 412 412 411 610158401015840 611 611 61015 271 277 279 21015840-O-Glu16 225 225 219 11015840101584010158401015840 mdash 1031 103217 459 459 458 21015840101584010158401015840 mdash 735 73618 407 407 407 31015840101584010158401015840 mdash 761 76019 455 455 455 41015840101584010158401015840 mdash 701 70020 302 303 302 51015840101584010158401015840 mdash 768 76721 332 332 332 61015840101584010158401015840 mdash 609 61022 316 316 323 28-O-GlcGal23 276 277 277 1101584010158401015840 941 941 91924 166 161 167 2101584010158401015840 723 723 76925 151 151 151 3101584010158401015840 766 766 74126 164 169 167 4101584010158401015840 695 695 70727 254 255 255 5101584010158401015840 776 777 76828 1752 1752 1752 6101584010158401015840 607 607 61829 327 327 327 2101584010158401015840-O-Glu30 233 233 234 110158401015840101584010158401015840 mdash mdash 1023

210158401015840101584010158401015840 mdash mdash 750310158401015840101584010158401015840 mdash mdash 770410158401015840101584010158401015840 mdash mdash 694510158401015840101584010158401015840 mdash mdash 775610158401015840101584010158401015840 mdash mdash 605

seven CH3 ten CH

2 five CH and 8 C signals (Table 1)

The aglycone was identified as oleanolic acid a commontriterpene of saponins in the genus Polyscias [6] The 120575Cvalues of C-3 at 120575C 883 and C-28 at 120575C 1752 suggested thatcompound 3 was a bisdesmosidic saponin with sugar unitsattached to these positions In addition five sugar moietieswere recognized by five anomeric methine groups at 120575C 1036(C-11015840) 1033 (C-110158401015840) 919 (C-1101584010158401015840) 1032 (C-11015840101584010158401015840) and 1023(C-110158401015840101584010158401015840) These data are similar to those of compound 2

4 Journal of Chemistry

except for the addition of a sugar moiety and the upfield shiftof the anomeric signal at 120575C from 941 to 919 Acid hydrolysisof compound 3 allowed the identification of D-glucuronicacid D-glucose and D-galactose Extensive analysis of theHMBC spectrum of compound 3 allowed the position ofthe sugar units to be determined The couplings from H-11015840(120575H 429) to C-3 (120575C 883) indicated that the glucuronic andglucosyl moieties attached to C-3 The correlations fromH-110158401015840 (120575H 425) to C-41015840 (120575C 823) and between H-41015840101584010158401015840 (120575H448) and C-21015840 (120575C 795) confirmed that the two glucosylunits were attached to C-21015840 andC-41015840 respectively Indeed the13C-NMR data of this fragment coincided well with the 120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl of compound 2 (Table 1) The HMBCcorrelations from H-1101584010158401015840 (120575H 531) to C-28 (120575C 1752) andthe anomeric C-1101584010158401015840 resonance shifted upfield at 120575C 919suggesting the galactose unit attached to C-28 The HMBCcouplings from H-110158401015840101584010158401015840 (120575H 462) to C-2101584010158401015840 (120575C 769) and fromH-2101584010158401015840 (120575H 360) to C-110158401015840101584010158401015840 (120575C 1023) and the COSY crosspeakbetween H-1101584010158401015840 and H-2101584010158401015840 indicated that the glucose unitattached to C-2101584010158401015840 Thus compound 3 was elucidated as3-O-120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl oleanolic acid 28-O-120573-D-glucopyranosyl-(1rarr2)-120573-D-galactopyranosyl ester namedpolyscioside I

The inhibitory effects of the isolated compounds againstporcine pancreas 120572-amylase and yeast 120572-glucosidase wereevaluated in comparison with the antidiabetic acarbose(Table 2) Compound 1 potently inhibited 120572-amylase and 120572-glucosidase with IC

50values of 271 and 4405 120583gmL respec-

tively In contrast compounds 2 and 3 did not show any effecton both enzymes Dou et al reported that the glucuronic acidunit at C-3 and glucosidic moiety at C-28 of the aglyconewere the functional groups essential for 120572-amylase and 120572-glucosidase inhibition the addition or replacement of glucoseby another sugar unit might thus abolish the inhibitoryactivity [12] In oral sucrose tolerance tests several oleanolicacid glycosides showed significant antihyperglycemic activityin the oral sucrose tolerance in rats [13 14] However the 21015840-O-120573-D-glucopyranosyl unit attached to the glucuronic acidmoiety was assumed to markedly reduce the hypoglycemiceffect [15] Consistent with this in our study compounds2 and 3 possessing a 21015840-O-120573-D-glucopyranosyl unit wereinactive regarding 120572-amylase and 120572-glucosidase inhibitionPrevious studies showed that P fruticosa leaf extracts stronglyinhibited rat intestinal 120572-glucosidase [16] and exhibitedhypoglycemic and antihyperglycemic activity in normal andstreptozotocin-diabetic albino rats [4] Our results suggestthat the observed hypoglycemic activity might be related tothe inhibition of 120572-amylase and 120572-glucosidase enzymes

It was of interest to establish whether compound 1and acarbose interact synergistically or additively in theinhibition of 120572-glucosidase and 120572-amylase Therefore theassay was performed in solutions containing these agentsalone or in a mixture at different concentrations The resultsshowed that the combination of compound 1 and acarboseproduced a synergistic inhibitory effect on porcine pancreas120572-amylase (Figure 2) but had no effect on yeast 120572-glucosidase(data not shown) Interestingly higher synergistic inhibition

Table 2 Inhibitory effects (IC50

plusmn SD 120583gmL) of 1ndash3 for 120572-amylaseand 120572-glucosidase

Compounds 120572-Amylase 120572-Glucosidase1 271 plusmn 205 4405 plusmn 1272 mdash mdash3 mdash mdashAcarbose 576 plusmn 412 6504 plusmn 334

was found from the combination of low concentrations ofindividual agents At 10 120583gmL compound 1 and acarbosealone exhibited only 15 and 42 inhibition respectivelyHowever their combination increased the inhibition up to328 At a higher concentration of 10 120583gmL the percentageof 120572-amylase inhibition of the mixture was equal to thesum of compound 1 and acarbose suggesting that theyproduced additive inhibition A slight increase in inhibitionwas obtained from the mixture in comparison with thatachieved by the individual agents at 100120583gmL These find-ings indicate that saponin 1 produced synergistic effects onporcine pancreas 120572-amylase inhibition when combined witha low concentration of acarbose

Acarbose is a potent 120572-glucosidase inhibitor and has beenused in the treatment of type 2 diabetes It can also beused in combination with other agents such as sulfonylureaand metformin in patients with diabetes However thelong-term use and high-dose administration of this drugcause severe side effects such as diarrhea flatulence andabdominal or stomach pain [17ndash19] Our study showed thatthe combination of P fruticosa saponin 1 with acarbose at alow concentration producedmore synergistic inhibition thaneither drug alone suggesting that theymight provide a signif-icant clinical benefit in delaying postprandial hyperglycemiaand diminishing the adverse effects of acarbose

4 Conclusion

Three bisdesmosidic saponins including two known 3-O-[120573-D-glucopyranosyl-(1rarr4)-120573-D-glucuronopyranosyl] oleano-lic acid 28-O-120573-D-glucopyranosyl ester (1) and 3-O-120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl oleanolic acid 28-O-120573-D-glucopyrano-syl ester (or polyscioside D 2) and one new 3-O-120573-D-glu-copyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glu-curonopyranosyl oleanolic acid 28-O-120573-D-glucopyranosyl-(1rarr2)-120573-D-galactopyranosyl ester (named polyscioside I3) were isolated from the leaves of P fruticosa Compound 1showed inhibitory effects against porcine pancreas 120572-amylaseand yeast 120572-glucosidase activities and produced a synergisticeffect with acarbose in 120572-amylase inhibition Further studyis needed to clarify the mechanism of enzyme inhibition aswell as the hypoglycemic properties of saponin 1

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Journal of Chemistry 5

1 10 100

in

hibi

tion

0102030405060708090

(120583gmL)

Compound 1Acarbose

Mix

lowast

lowast

lowast

Figure 2 The percentage inhibition by combination of 1 andacarbose on porcine pancreatic 120572-amylase Results are expressed asmeans plusmn SD 119899 = 3 lowast119901 lt 005 compared with acarbose alone

Acknowledgments

This work is supported in part by the Vietnam Academyof Science and Technology (VASTDLT0614-15) and theNational Foundation for Science and Technology Develop-ment (NAFOSTED 10401-201154) The authors thank theInstitute of Chemistry (VAST) for the NMR measurementsand Dr ML Bourguet-Kondracki (Musee National drsquoHistoireNaturelle Paris France) for providing HRMS experiment

References

[1] V C Vo Dictionary of Vietnamese Medicinal Plants vol 1Medical Publisher Hanoi Vietnam 2012

[2] B M Bernard N Pakianathan and M C Divakar ldquoOnthe antipyretic anti-inflammatory analgesic and molluscicidalproperties of Polyscias fruticosa (L) Harmsrdquo Ancient Science ofLife vol 17 pp 313ndash319 1998

[3] G Asumeng Koffuor A Boye S Kyei et al ldquoAwuku Anti-asthmatic property and possible mode of activity of an ethanolleaf extract of Polyscias fruticoserdquo Pharmaceutical Biology vol8 pp 1ndash10 2015

[4] M C Divakar and M B Bensita ldquoScreening of various leafextracts of Polyscias fruticosa Harms for their antidiabeticactivityrdquo Indian Journal of Natural Products vol 14 pp 24ndash281998

[5] J Lutomski T C Luan and T T Hoa ldquoPolyacetylenes inthe Araliaceae family Part IV The antibacterial and antifungalactivities of two main polyacetylenes from Panax vietnamensisHa et Grushv and Polyscias fruticosa (L) Harmsrdquo HerbaPolonica vol 38 pp 137ndash140 1992

[6] V D Huan S Yamamura K Ohtani et al ldquoOleanane saponinsfrom Polyscias fruticosardquo Phytochemistry vol 47 no 3 pp 451ndash457 1998

[7] P M de Souza P M de Sales L A Simeoni E C Silva DSilveira and P de Oliveira Magalhaes ldquoInhibitory activity of 120572-amylase and 120572-glucosidase by plant extracts from the Braziliancerradordquo Planta Medica vol 78 no 4 pp 393ndash399 2012

[8] C Borel M P Gupta and K Hostettmann ldquoMolluscicidalsaponins from Swartzia simplexrdquo Phytochemistry vol 26 no10 pp 2685ndash2689 1987

[9] L Voutquenne-Nazabadioko R Gevrenova N Borie et alldquoTriterpenoid saponins from the roots ofGypsophila trichotomaWenderrdquo Phytochemistry vol 90 pp 114ndash127 2013

[10] H H T Tran M C Nguyen H T Le et al ldquoInhibitors of 120572-glucosidase and 120572-amylase from Cyperus rotundusrdquo Pharma-ceutical Biology vol 52 no 1 pp 74ndash77 2014

[11] B S Ashok Kumar K Lakshman K N Jayaveea et alldquoAntidiabetic antihyperlipidemic and antioxidant activities ofmethanolic extract of Amaranthus viridis Linn in alloxaninduced diabetic ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 1-2 pp 75ndash79 2012

[12] F Dou M Xi J Wang et al ldquoalpha-Glucosidase and alpha-amylase inhibitory activities of saponins from traditional Chi-nese medicines in the treatment of diabetes mellitusrdquo DiePharmazie vol 68 no 4 pp 300ndash304 2013

[13] H Matsuda Y Li T Murakami N Matsumura J Yama-hara and M Yoshikawa ldquoAntidiabetic principles of naturalmedicines III Structure-related inhibitory activity and actionmode of oleanolic acid glycosides on hypoglycemic activityrdquoChemical and Pharmaceutical Bulletin vol 46 no 9 pp 1399ndash1403 1998

[14] M Yoshikawa T Murakami E Harada N Murakami JYamahara andHMatsuda ldquoBioactive saponins and glycosidesVII On the hypoglycemic principles from the root cortex ofAralia elata SEEM structure related hypoglycemic activity ofoleanolic acid oligoglycosiderdquo Chemical and PharmaceuticalBulletin vol 44 no 10 pp 1923ndash1927 1996

[15] M Yoshikawa T Murakami A Kishi T Kageura and HMatsuda ldquoMedicinal flowers III Marigold (1) hypoglycemicgastric emptying inhibitory and gastroprotective principles andnew oleanane-type triterpene oligoglycosides calendasaponinsA B C and D from egyptian Calendula officinalisrdquo Chemicaland Pharmaceutical Bulletin vol 49 no 7 pp 863ndash870 2001

[16] T T Mai N N Thu P G Tien and N Van Chuyen ldquoAlpha-glucosidase inhibitory and antioxidant activities of Vietnameseedible plants and their relationships with polyphenol contentsrdquoJournal of Nutritional Science and Vitaminology vol 53 no 3pp 267ndash276 2007

[17] M Rodier J L Richard L Monnier and J Mirouze ldquoEffect oflong term Acarbose (Bay g 5421) therapy on metabolic controlof non insulin dependent (type II) diabetes mellitusrdquoDiabete ampMetabolisme vol 14 no 1 pp 12ndash14 1988

[18] A J Scheen ldquoClinical efficacy of acarbose in diabetes mellitus acritical review of controlled trialsrdquo Diabetes amp Metabolism vol24 no 4 pp 311ndash320 1998

[19] S Akkarachiyasit P Charoenlertkul S Yibchok-Anun andS Adisakwattana ldquoInhibitory activities of cyanidin and itsglycosides and synergistic effect with acarbose against intestinal120572-glucosidase and pancreatic 120572-amylaserdquo International Journalof Molecular Sciences vol 11 no 9 pp 3387ndash3396 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 Journal of Chemistry

except for the addition of a sugar moiety and the upfield shiftof the anomeric signal at 120575C from 941 to 919 Acid hydrolysisof compound 3 allowed the identification of D-glucuronicacid D-glucose and D-galactose Extensive analysis of theHMBC spectrum of compound 3 allowed the position ofthe sugar units to be determined The couplings from H-11015840(120575H 429) to C-3 (120575C 883) indicated that the glucuronic andglucosyl moieties attached to C-3 The correlations fromH-110158401015840 (120575H 425) to C-41015840 (120575C 823) and between H-41015840101584010158401015840 (120575H448) and C-21015840 (120575C 795) confirmed that the two glucosylunits were attached to C-21015840 andC-41015840 respectively Indeed the13C-NMR data of this fragment coincided well with the 120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl of compound 2 (Table 1) The HMBCcorrelations from H-1101584010158401015840 (120575H 531) to C-28 (120575C 1752) andthe anomeric C-1101584010158401015840 resonance shifted upfield at 120575C 919suggesting the galactose unit attached to C-28 The HMBCcouplings from H-110158401015840101584010158401015840 (120575H 462) to C-2101584010158401015840 (120575C 769) and fromH-2101584010158401015840 (120575H 360) to C-110158401015840101584010158401015840 (120575C 1023) and the COSY crosspeakbetween H-1101584010158401015840 and H-2101584010158401015840 indicated that the glucose unitattached to C-2101584010158401015840 Thus compound 3 was elucidated as3-O-120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl oleanolic acid 28-O-120573-D-glucopyranosyl-(1rarr2)-120573-D-galactopyranosyl ester namedpolyscioside I

The inhibitory effects of the isolated compounds againstporcine pancreas 120572-amylase and yeast 120572-glucosidase wereevaluated in comparison with the antidiabetic acarbose(Table 2) Compound 1 potently inhibited 120572-amylase and 120572-glucosidase with IC

50values of 271 and 4405 120583gmL respec-

tively In contrast compounds 2 and 3 did not show any effecton both enzymes Dou et al reported that the glucuronic acidunit at C-3 and glucosidic moiety at C-28 of the aglyconewere the functional groups essential for 120572-amylase and 120572-glucosidase inhibition the addition or replacement of glucoseby another sugar unit might thus abolish the inhibitoryactivity [12] In oral sucrose tolerance tests several oleanolicacid glycosides showed significant antihyperglycemic activityin the oral sucrose tolerance in rats [13 14] However the 21015840-O-120573-D-glucopyranosyl unit attached to the glucuronic acidmoiety was assumed to markedly reduce the hypoglycemiceffect [15] Consistent with this in our study compounds2 and 3 possessing a 21015840-O-120573-D-glucopyranosyl unit wereinactive regarding 120572-amylase and 120572-glucosidase inhibitionPrevious studies showed that P fruticosa leaf extracts stronglyinhibited rat intestinal 120572-glucosidase [16] and exhibitedhypoglycemic and antihyperglycemic activity in normal andstreptozotocin-diabetic albino rats [4] Our results suggestthat the observed hypoglycemic activity might be related tothe inhibition of 120572-amylase and 120572-glucosidase enzymes

It was of interest to establish whether compound 1and acarbose interact synergistically or additively in theinhibition of 120572-glucosidase and 120572-amylase Therefore theassay was performed in solutions containing these agentsalone or in a mixture at different concentrations The resultsshowed that the combination of compound 1 and acarboseproduced a synergistic inhibitory effect on porcine pancreas120572-amylase (Figure 2) but had no effect on yeast 120572-glucosidase(data not shown) Interestingly higher synergistic inhibition

Table 2 Inhibitory effects (IC50

plusmn SD 120583gmL) of 1ndash3 for 120572-amylaseand 120572-glucosidase

Compounds 120572-Amylase 120572-Glucosidase1 271 plusmn 205 4405 plusmn 1272 mdash mdash3 mdash mdashAcarbose 576 plusmn 412 6504 plusmn 334

was found from the combination of low concentrations ofindividual agents At 10 120583gmL compound 1 and acarbosealone exhibited only 15 and 42 inhibition respectivelyHowever their combination increased the inhibition up to328 At a higher concentration of 10 120583gmL the percentageof 120572-amylase inhibition of the mixture was equal to thesum of compound 1 and acarbose suggesting that theyproduced additive inhibition A slight increase in inhibitionwas obtained from the mixture in comparison with thatachieved by the individual agents at 100120583gmL These find-ings indicate that saponin 1 produced synergistic effects onporcine pancreas 120572-amylase inhibition when combined witha low concentration of acarbose

Acarbose is a potent 120572-glucosidase inhibitor and has beenused in the treatment of type 2 diabetes It can also beused in combination with other agents such as sulfonylureaand metformin in patients with diabetes However thelong-term use and high-dose administration of this drugcause severe side effects such as diarrhea flatulence andabdominal or stomach pain [17ndash19] Our study showed thatthe combination of P fruticosa saponin 1 with acarbose at alow concentration producedmore synergistic inhibition thaneither drug alone suggesting that theymight provide a signif-icant clinical benefit in delaying postprandial hyperglycemiaand diminishing the adverse effects of acarbose

4 Conclusion

Three bisdesmosidic saponins including two known 3-O-[120573-D-glucopyranosyl-(1rarr4)-120573-D-glucuronopyranosyl] oleano-lic acid 28-O-120573-D-glucopyranosyl ester (1) and 3-O-120573-D-glucopyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glucuronopyranosyl oleanolic acid 28-O-120573-D-glucopyrano-syl ester (or polyscioside D 2) and one new 3-O-120573-D-glu-copyranosyl-(1rarr2)-[120573-D-glucopyranosyl-(1rarr4)]-120573-D-glu-curonopyranosyl oleanolic acid 28-O-120573-D-glucopyranosyl-(1rarr2)-120573-D-galactopyranosyl ester (named polyscioside I3) were isolated from the leaves of P fruticosa Compound 1showed inhibitory effects against porcine pancreas 120572-amylaseand yeast 120572-glucosidase activities and produced a synergisticeffect with acarbose in 120572-amylase inhibition Further studyis needed to clarify the mechanism of enzyme inhibition aswell as the hypoglycemic properties of saponin 1

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Journal of Chemistry 5

1 10 100

in

hibi

tion

0102030405060708090

(120583gmL)

Compound 1Acarbose

Mix

lowast

lowast

lowast

Figure 2 The percentage inhibition by combination of 1 andacarbose on porcine pancreatic 120572-amylase Results are expressed asmeans plusmn SD 119899 = 3 lowast119901 lt 005 compared with acarbose alone

Acknowledgments

This work is supported in part by the Vietnam Academyof Science and Technology (VASTDLT0614-15) and theNational Foundation for Science and Technology Develop-ment (NAFOSTED 10401-201154) The authors thank theInstitute of Chemistry (VAST) for the NMR measurementsand Dr ML Bourguet-Kondracki (Musee National drsquoHistoireNaturelle Paris France) for providing HRMS experiment

References

[1] V C Vo Dictionary of Vietnamese Medicinal Plants vol 1Medical Publisher Hanoi Vietnam 2012

[2] B M Bernard N Pakianathan and M C Divakar ldquoOnthe antipyretic anti-inflammatory analgesic and molluscicidalproperties of Polyscias fruticosa (L) Harmsrdquo Ancient Science ofLife vol 17 pp 313ndash319 1998

[3] G Asumeng Koffuor A Boye S Kyei et al ldquoAwuku Anti-asthmatic property and possible mode of activity of an ethanolleaf extract of Polyscias fruticoserdquo Pharmaceutical Biology vol8 pp 1ndash10 2015

[4] M C Divakar and M B Bensita ldquoScreening of various leafextracts of Polyscias fruticosa Harms for their antidiabeticactivityrdquo Indian Journal of Natural Products vol 14 pp 24ndash281998

[5] J Lutomski T C Luan and T T Hoa ldquoPolyacetylenes inthe Araliaceae family Part IV The antibacterial and antifungalactivities of two main polyacetylenes from Panax vietnamensisHa et Grushv and Polyscias fruticosa (L) Harmsrdquo HerbaPolonica vol 38 pp 137ndash140 1992

[6] V D Huan S Yamamura K Ohtani et al ldquoOleanane saponinsfrom Polyscias fruticosardquo Phytochemistry vol 47 no 3 pp 451ndash457 1998

[7] P M de Souza P M de Sales L A Simeoni E C Silva DSilveira and P de Oliveira Magalhaes ldquoInhibitory activity of 120572-amylase and 120572-glucosidase by plant extracts from the Braziliancerradordquo Planta Medica vol 78 no 4 pp 393ndash399 2012

[8] C Borel M P Gupta and K Hostettmann ldquoMolluscicidalsaponins from Swartzia simplexrdquo Phytochemistry vol 26 no10 pp 2685ndash2689 1987

[9] L Voutquenne-Nazabadioko R Gevrenova N Borie et alldquoTriterpenoid saponins from the roots ofGypsophila trichotomaWenderrdquo Phytochemistry vol 90 pp 114ndash127 2013

[10] H H T Tran M C Nguyen H T Le et al ldquoInhibitors of 120572-glucosidase and 120572-amylase from Cyperus rotundusrdquo Pharma-ceutical Biology vol 52 no 1 pp 74ndash77 2014

[11] B S Ashok Kumar K Lakshman K N Jayaveea et alldquoAntidiabetic antihyperlipidemic and antioxidant activities ofmethanolic extract of Amaranthus viridis Linn in alloxaninduced diabetic ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 1-2 pp 75ndash79 2012

[12] F Dou M Xi J Wang et al ldquoalpha-Glucosidase and alpha-amylase inhibitory activities of saponins from traditional Chi-nese medicines in the treatment of diabetes mellitusrdquo DiePharmazie vol 68 no 4 pp 300ndash304 2013

[13] H Matsuda Y Li T Murakami N Matsumura J Yama-hara and M Yoshikawa ldquoAntidiabetic principles of naturalmedicines III Structure-related inhibitory activity and actionmode of oleanolic acid glycosides on hypoglycemic activityrdquoChemical and Pharmaceutical Bulletin vol 46 no 9 pp 1399ndash1403 1998

[14] M Yoshikawa T Murakami E Harada N Murakami JYamahara andHMatsuda ldquoBioactive saponins and glycosidesVII On the hypoglycemic principles from the root cortex ofAralia elata SEEM structure related hypoglycemic activity ofoleanolic acid oligoglycosiderdquo Chemical and PharmaceuticalBulletin vol 44 no 10 pp 1923ndash1927 1996

[15] M Yoshikawa T Murakami A Kishi T Kageura and HMatsuda ldquoMedicinal flowers III Marigold (1) hypoglycemicgastric emptying inhibitory and gastroprotective principles andnew oleanane-type triterpene oligoglycosides calendasaponinsA B C and D from egyptian Calendula officinalisrdquo Chemicaland Pharmaceutical Bulletin vol 49 no 7 pp 863ndash870 2001

[16] T T Mai N N Thu P G Tien and N Van Chuyen ldquoAlpha-glucosidase inhibitory and antioxidant activities of Vietnameseedible plants and their relationships with polyphenol contentsrdquoJournal of Nutritional Science and Vitaminology vol 53 no 3pp 267ndash276 2007

[17] M Rodier J L Richard L Monnier and J Mirouze ldquoEffect oflong term Acarbose (Bay g 5421) therapy on metabolic controlof non insulin dependent (type II) diabetes mellitusrdquoDiabete ampMetabolisme vol 14 no 1 pp 12ndash14 1988

[18] A J Scheen ldquoClinical efficacy of acarbose in diabetes mellitus acritical review of controlled trialsrdquo Diabetes amp Metabolism vol24 no 4 pp 311ndash320 1998

[19] S Akkarachiyasit P Charoenlertkul S Yibchok-Anun andS Adisakwattana ldquoInhibitory activities of cyanidin and itsglycosides and synergistic effect with acarbose against intestinal120572-glucosidase and pancreatic 120572-amylaserdquo International Journalof Molecular Sciences vol 11 no 9 pp 3387ndash3396 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 5

1 10 100

in

hibi

tion

0102030405060708090

(120583gmL)

Compound 1Acarbose

Mix

lowast

lowast

lowast

Figure 2 The percentage inhibition by combination of 1 andacarbose on porcine pancreatic 120572-amylase Results are expressed asmeans plusmn SD 119899 = 3 lowast119901 lt 005 compared with acarbose alone

Acknowledgments

This work is supported in part by the Vietnam Academyof Science and Technology (VASTDLT0614-15) and theNational Foundation for Science and Technology Develop-ment (NAFOSTED 10401-201154) The authors thank theInstitute of Chemistry (VAST) for the NMR measurementsand Dr ML Bourguet-Kondracki (Musee National drsquoHistoireNaturelle Paris France) for providing HRMS experiment

References

[1] V C Vo Dictionary of Vietnamese Medicinal Plants vol 1Medical Publisher Hanoi Vietnam 2012

[2] B M Bernard N Pakianathan and M C Divakar ldquoOnthe antipyretic anti-inflammatory analgesic and molluscicidalproperties of Polyscias fruticosa (L) Harmsrdquo Ancient Science ofLife vol 17 pp 313ndash319 1998

[3] G Asumeng Koffuor A Boye S Kyei et al ldquoAwuku Anti-asthmatic property and possible mode of activity of an ethanolleaf extract of Polyscias fruticoserdquo Pharmaceutical Biology vol8 pp 1ndash10 2015

[4] M C Divakar and M B Bensita ldquoScreening of various leafextracts of Polyscias fruticosa Harms for their antidiabeticactivityrdquo Indian Journal of Natural Products vol 14 pp 24ndash281998

[5] J Lutomski T C Luan and T T Hoa ldquoPolyacetylenes inthe Araliaceae family Part IV The antibacterial and antifungalactivities of two main polyacetylenes from Panax vietnamensisHa et Grushv and Polyscias fruticosa (L) Harmsrdquo HerbaPolonica vol 38 pp 137ndash140 1992

[6] V D Huan S Yamamura K Ohtani et al ldquoOleanane saponinsfrom Polyscias fruticosardquo Phytochemistry vol 47 no 3 pp 451ndash457 1998

[7] P M de Souza P M de Sales L A Simeoni E C Silva DSilveira and P de Oliveira Magalhaes ldquoInhibitory activity of 120572-amylase and 120572-glucosidase by plant extracts from the Braziliancerradordquo Planta Medica vol 78 no 4 pp 393ndash399 2012

[8] C Borel M P Gupta and K Hostettmann ldquoMolluscicidalsaponins from Swartzia simplexrdquo Phytochemistry vol 26 no10 pp 2685ndash2689 1987

[9] L Voutquenne-Nazabadioko R Gevrenova N Borie et alldquoTriterpenoid saponins from the roots ofGypsophila trichotomaWenderrdquo Phytochemistry vol 90 pp 114ndash127 2013

[10] H H T Tran M C Nguyen H T Le et al ldquoInhibitors of 120572-glucosidase and 120572-amylase from Cyperus rotundusrdquo Pharma-ceutical Biology vol 52 no 1 pp 74ndash77 2014

[11] B S Ashok Kumar K Lakshman K N Jayaveea et alldquoAntidiabetic antihyperlipidemic and antioxidant activities ofmethanolic extract of Amaranthus viridis Linn in alloxaninduced diabetic ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 1-2 pp 75ndash79 2012

[12] F Dou M Xi J Wang et al ldquoalpha-Glucosidase and alpha-amylase inhibitory activities of saponins from traditional Chi-nese medicines in the treatment of diabetes mellitusrdquo DiePharmazie vol 68 no 4 pp 300ndash304 2013

[13] H Matsuda Y Li T Murakami N Matsumura J Yama-hara and M Yoshikawa ldquoAntidiabetic principles of naturalmedicines III Structure-related inhibitory activity and actionmode of oleanolic acid glycosides on hypoglycemic activityrdquoChemical and Pharmaceutical Bulletin vol 46 no 9 pp 1399ndash1403 1998

[14] M Yoshikawa T Murakami E Harada N Murakami JYamahara andHMatsuda ldquoBioactive saponins and glycosidesVII On the hypoglycemic principles from the root cortex ofAralia elata SEEM structure related hypoglycemic activity ofoleanolic acid oligoglycosiderdquo Chemical and PharmaceuticalBulletin vol 44 no 10 pp 1923ndash1927 1996

[15] M Yoshikawa T Murakami A Kishi T Kageura and HMatsuda ldquoMedicinal flowers III Marigold (1) hypoglycemicgastric emptying inhibitory and gastroprotective principles andnew oleanane-type triterpene oligoglycosides calendasaponinsA B C and D from egyptian Calendula officinalisrdquo Chemicaland Pharmaceutical Bulletin vol 49 no 7 pp 863ndash870 2001

[16] T T Mai N N Thu P G Tien and N Van Chuyen ldquoAlpha-glucosidase inhibitory and antioxidant activities of Vietnameseedible plants and their relationships with polyphenol contentsrdquoJournal of Nutritional Science and Vitaminology vol 53 no 3pp 267ndash276 2007

[17] M Rodier J L Richard L Monnier and J Mirouze ldquoEffect oflong term Acarbose (Bay g 5421) therapy on metabolic controlof non insulin dependent (type II) diabetes mellitusrdquoDiabete ampMetabolisme vol 14 no 1 pp 12ndash14 1988

[18] A J Scheen ldquoClinical efficacy of acarbose in diabetes mellitus acritical review of controlled trialsrdquo Diabetes amp Metabolism vol24 no 4 pp 311ndash320 1998

[19] S Akkarachiyasit P Charoenlertkul S Yibchok-Anun andS Adisakwattana ldquoInhibitory activities of cyanidin and itsglycosides and synergistic effect with acarbose against intestinal120572-glucosidase and pancreatic 120572-amylaserdquo International Journalof Molecular Sciences vol 11 no 9 pp 3387ndash3396 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of