Characterisation of Galloylated Cyanogenic Glucosides and ...

Research Article

Received: 17 August 2010; Revised: 13 December 2010; Accepted: 14 December 2010 Published online in Wiley Online Library: 14 April 2011

(wileyonlinelibrary.com) DOI 10.1002/pca.1312

516

Characterisation of Galloylated CyanogenicGlucosides and Hydrolysable Tanninsfrom Leaves of Phyllagathis rotundifoliaby LC‐ESI‐MS/MSHooi Poay Tan,a,b* Sui Kiong Lingb and Cheng Hock Chuaha

ABSTRACT:Introduction – Phyllagathis rotundifolia (Jack) Bl. (Melastomataceae) is a creeping herb found in Peninsular Malaysia andSumatra. Traditionally, a decoction of the leaves is used in the treatment of malaria, fever and stomach ache.Objective – To provide ESI‐MSn data which are applicable for chemical fingerprinting of P. rotundifolia to obviate laboriousisolation and purification steps.Methodology – The mass spectral data for the compounds isolated from the leaves of P. rotundifolia were obtained by liquidchromatography–electrospray ionisation tandem mass spectrometry.Results – The MS fragmentation patterns were obtained for galloylated cyanogenic glucosides based on prunasin (prunasin6′‐O‐gallate 1, prunasin 2′,6′‐di‐O‐gallate 2, prunasin 3′,6′‐di‐O‐gallate 3, prunasin 4′,6′‐di‐O‐gallate 4, prunasin 2′,3′,6′‐tri‐O‐gallate 5, prunasin 3′,4′,6′‐tri‐O‐gallate 6 and prunasin 2′,3′,4′,6′‐tetra‐O‐gallate 7), gallotannins (6‐O‐galloyl‐D‐glucose 8, 3,6‐di‐O‐galloyl‐D‐glucose 9, 1,2,3‐tri‐O‐galloyl‐β‐D‐glucose 10, 1,4,6‐tri‐O‐galloyl‐β‐D‐glucose 11, 3,4,6‐tri‐O‐galloyl‐D‐glucose 12,1,2,3,6‐tetra‐O‐galloyl‐β‐D‐glucose 13 and 1,2,3,4,6‐penta‐O‐galloyl‐β‐D‐glucose 14), ellagitannins [6‐O‐galloyl‐2,3‐O‐(S)‐hexahydroxy‐diphenoyl‐D‐glucose 15, praecoxin B 16 and pterocarinin C 17], ellagic acid derivatives (3′‐O‐methyl‐3,4‐methylenedioxyellagic acid 4′‐O‐β‐D‐glucopyranoside 18 and 3,3′,4‐tri‐O‐methylellagic acid 4′‐O‐β‐D‐glucopyranoside 19) andgallic acid 20 that were isolated from the leaves of P. rotundifolia.Conclusion – The ESI‐MSn technique facilitates identification of galloylated cyanogenic glucosides, hydrolysable tanninsand ellagic acid derivatives that were isolated from the leaves of P. rotundifolia. It yields MSn spectra that are useful foridentification of these compounds in complex samples and permit more complete fingerprinting of plant materials.Copyright © 2011 John Wiley & Sons, Ltd.

Supporting information can be found in the online version of this article.

Keywords: Phyllagathis rotundifolia; Melastomataceae; galloylated cyanogenic glucosides; hydrolysable tannins; ellagic acidderivative; liquid chromatography–mass spectrometry

* Correspondence to: Hooi Poay Tan, Medicinal Plants Division, Forest ResearchInstitute Malaysia, 52109Kepong, Selangor, Malaysia. E–mail: [email protected]

a Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia

b Medicinal Plants Division, Forest Research Institute Malaysia, 52109 Kepong,Selangor, Malaysia

IntroductionPhyllagathis rotundifolia (Jack) Bl. from the family ofMelastomataceae is a creeping herb found in PeninsularMalaysia and Sumatra. It has heart‐shaped leaves with darkgreen upper surface and reddish lower surface, short stem andpink or magenta flowers (Henderson, 1954; Ridley, 1922). Intraditional medicine, a decoction of the leaves is used for thetreatment of malaria, fever and stomachache as well as inparturition and as tonic (Burkill, 1966).

Plants in the family of Melastomataceae produce distinctoligomeric tannins (Calderon et al., 2003; Cronquist, 1981;Tan et al., 2010) or ellagic acid and its derivatives such as 3,3′‐di‐O‐methylellagic acid, 3,3′,4‐tri‐O‐methylellagic acid and 3′‐O‐methyl‐3,4‐methylenedioxyellagic acid which can be utilised aschemotaxonomic characteristics or definitive systematic markers(Hillis and Yazaki, 1973; Lowry, 1968; Yoshida et al., 2000). In thisstudy, we reported the fragmentation patterns of galloylatedcyanogenic glucosides, gallotannins, ellagitannins and ellagicacid derivatives by electrospray ionisation tandem mass

Copyright © 2011 John

spectrometry (ESI‐MSn) that can be used for fingerprinting ofplants collected from different localities as well as in theassessment of chemical changes in response to environmentalfactors.

Experimental

Solvents and chemicals

All reagent and solvents used were of analytical and HPLC grades. Allsolvents were purchased from Merck, Germany.

Phytochem. Anal. 2011, 22, 516–525Wiley & Sons, Ltd.

R1 R2

R2

R3 R4 R58: H H H H G9: H H G H G

10: G G G H H11: G H H G G12: H H G G G13: G G G H G14: G G G G G

O OR1OH

CNOR2

OR3

OR4

R1 R2 R3 R41: H H H G2: G H H G3: H G H G4: H H G G5: G G H G6: H G G G7:: G G G G

O1 OR1

OR2R3O

R4O

OR5 O1

HO OH

OH

OHHO

HO

OR1OR2O

OR3

O

C OCO

R1 R315: H H G16: H G G17: G G G

R1 R2

18: - CH2 -

19: CH3 CH3

O O

OHHO

HO

OH

O

O

O

O

H3COOR2

OR1

HO

HO

HO

COOH

20

OH

OH

OH

CO

G =

1

1'

2'3' 6

6

1 1'

3'3

1

6

1

47

1'

4'7'

14 1" 4"

Figure 1. Chemical structures (1–20) isolated from the leaves of Phyllagathis rotundifolia.

Characterisation of Galloylated Cyanogenic Glucosides and Tannins

Plant materials

The leaves of P. rotundifolia were collected from Pasoh Forest Reserve,Negeri Sembilan and the specimens (A624–A633) were identified anddeposited in the specimen collection of Genetic Laboratory in ForestResearch Institute Malaysia (FRIM).

51

Extraction, fractionation and purification

Dried and ground leaves of P. rotundifolia (330 g dry weight) wereextracted five times using methanol at room temperature, which yielded60.8 g methanol extract after removal of solvent by rotary evaporator at40 °C. Then, the methanol extract was suspended in water and extractedconsecutively with hexane and ethyl acetate. Each extract wasevaporated under reduced pressure at 40 °C and yielded a hexaneextract (10.5 g), ethyl acetate extract (11.1 g) and water extract (36.0 g).The ethyl acetate and water extracts were fractionated and purified by acombination of column chromatography utilising MCI gel CHP 20P(Supelco, Bellefonte, PA, USA), Chromatorex ODS (100‐200 mesh, FujiSilysia Chemical Ltd, Japan), Sephadex LH‐20 (Amersham Biosciences,UK), silica gel (230–400 mesh, Merck, Germany) and Toyopearl HW‐40F(Tosoh Corporation, Japan). TLC was performed on precoated silica gel60 F254 plates (0.2 mm thick, Merck, Germany) using the purificationprocess with solvent systems of chloroform:methanol:water (8:2:0.1;7:3:0.5; 6:4:1; 5:5:1.5 v/v) or benzene:ethyl formate:formic acid (1:7:1 v/v).Spots were visualised by UV at 254 and 365 nm and developed byspraying with 10% sulphuric acid followed by heating or 2% ethanoliciron chloride reagent. The ethyl acetate extract yielded prunasin 6′‐O‐gallate (1, 41.4 mg), prunasin 2′,6′‐di‐O‐gallate (2, 8.6 mg), prunasin 3′,6′‐di‐O‐gallate (3, 21.6 mg), prunasin 4′,6′‐di‐O‐gallate (4, 8.5 mg),prunasin 2′,3′,6′‐tri‐O‐gallate (5, 34.6 mg), prunasin 3′,4′,6′‐tri‐O‐gallate(6, 24.9 mg), prunasin 2′,3′,4′,6′‐tetra‐O‐gallate (7, 33.8 mg), 1,2,3‐tri‐O‐

Phytochem. Anal. 2011, 22, 516–525 Copyright © 2011 John

galloyl‐β‐D‐glucose (10, 7.0 mg), 1,4,6‐tri‐O‐galloyl‐β‐D‐glucose(11, 32.5 mg), 3,4,6‐tri‐O‐galloyl‐D‐glucose (12, 23.1 mg), 1,2,3,6‐tetra‐O‐galloyl‐β‐D‐glucose (13, 27.0 mg), 1,2,3,4,6‐penta‐O‐galloyl‐β‐D‐glucose (14, 21.3 mg), pterocarinin C (17, 55.5 mg) and gallic acid (20,31.6 mg). Compound 6‐O‐galloyl‐D‐glucose (8, 13.9 mg) was obtainedfrom the water extract. Several compounds such as 3, 6‐di‐O‐galloyl‐D‐glucose (9, 19.2 mg), 6‐O‐galloyl‐2,3‐O‐(S)‐hexahydroxy‐diphenoyl‐D‐glucose (15, 331.8 mg) and praecoxin B (16, 157.3 mg) were present inboth ethyl acetate and water extracts. A mixture of granular crystalcontaining compounds 18 (3′‐O‐methyl‐3,4‐methylenedioxyellagic acid4′‐O‐β‐D‐glucopyranoside) and 19 (3,3′,4‐tri‐O‐methylellagic acid 4′‐O‐β‐D‐glucopyranoside) was obtained when the ethyl acetate and waterextracts were each subjected to MCI gel CHP 20P column chromatog-raphy with a solvent system of methanol (50–100%) in water. These twocompounds (18, 8.1 mg; 19, 6.5 mg) were purified by preparative HPLC,using a Waters 600 HPLC system (Milford, MA, USA) equipped with aphotodiode array (PDA) detector. A Symmetry C18 reversed‐phase semi‐preparative column (7.8 i.d. × 300 mm, 7 μm) was used. The mobilephase consisted of a linear gradient of acetonitrile in water from 20 to30% over 25 min at a flow‐rate of 2.88 mL/min.

Sample preparation

The crude extract of P. rotundifolia was prepared at a concentration of2.0 mg/mL in methanol. Prior to injection, an adequate volume waspassed through a 0.22 μm polytetrafluoroethylene (PTFE) filter. Analiquot of 2 μl was injected for LC‐MS analysis.

For isolated compounds (1–20), each compound was prepared in astock solution of 1 mg/mL of 50% methanol. A serial dilution was donefor each stock solution to give a final concentration of 80 ppb. All thesolutions were filtered through 0.22 μm polytetrafluoroethylene (PTFE)filters and aliquots of 10 μL were injected for ESI‐MSn analysis.

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7

Table

1.Massspectral

data

ofcompo

unds

isolated

from

theleaves

ofP.

rotund

ifolia

No.

Com

poun

dRe

tention

time(m

in)

Elem

ental

compo

sitio

n[M

–H]–

Mass

accuracy

(ppm

)

MS2

MS3

1Prun

asin

6′‐O‐gallate

55.61

C21H21NO10

446.10

913

−0.3

125(2),16

9(22

),22

3(2),24

1(1),25

3(2),27

0(1),29

5(1),3

13(100

),35

9(2),4

19(5)

125(7),15

1(3),16

9(10

0),19

3(2),21

1(4),

223(15

),23

3(2),2

41(8),25

3(8),2

95(9)

2Prun

asin

2′,6′‐d

i‐O‐

gallate

130.59

C28H25NO14

598.11

981

−0.7

169(2),295

(1),29

7(1),313

(3),40

5(2),428

(4),

446(10

0),4

65(12),5

54(0.2),58

0(1)

125(1),16

9(15

),22

3(1),24

1(0.4),25

3(2),

270(0.2),29

5(1),31

3(10

0),35

9(0.3),

419(1)

3Prun

asin

3′,6′‐d

i‐O‐

gallate

116.66

C28H25NO14

598.11

981

−0.7

169(3),29

5(1),29

7(2),31

3(6),32

1(0.4),40

5(2),42

8(16

),44

6(10

0),4

65(19),5

54(0.4)

125(2),16

9(20

),22

3(1),24

1(0.4),25

3(3),

270(0.3),29

5(1),31

3(10

0),35

9(0.4),

419(1)

4Prun

asin

4′,6′‐d

i‐O‐

gallate

120.16

C28H25NO14

598.11

957

+1.1

169(5),21

1(1),29

5(3),29

7(6),31

3(11

),40

5(1),42

8(47

),44

6(10

0),4

65(56),5

54(0.8)

125(2),16

9(20

),22

3(1),21

1(0.4),24

1(0.4),25

3(2),29

5(1),31

3(10

0),35

9(1),

419(2)

5Prun

asin

2′,3′,6′‐tri‐O‐

gallate

187.36

C35H29NO18

750.13

000

−1.6

279(1),383

(1),42

8(1),446

(1),44

9(6),465

(3),

553(3),5

80(43),5

98(100

),61

7(3)

169(2),23

5(0.4),29

5(1),29

7(2),31

3(6),

405(1),42

8(21

),44

6(45

),46

5(0.3),55

4(1)

6Prun

asin

3′,4′,6′‐tri‐O‐

gallate

211.54

C35H29NO18

750.13

068

−0.7

383(1),41

0(1),42

8(3),44

6(1),44

9(2),46

5(0.4),55

3(1),5

80(1),59

8(10

0),6

17(4)

169(2),23

5(0.2),29

5(1),29

7(1),31

3(4),

405(2),428

(14),4

46(100

),46

5(16

),55

4(0.4)

7Prun

asin

2′,3′,4′,6′‐tetra‐

O‐gallate

244.15

C42H33NO22

902.14

069

−1.6

429(0.3),44

7(0.4),44

9(0.3),56

4(1),58

0(2),

598(3),6

17(1),73

2(20

),75

0(10

0),7

69(1)

295(1),383

(1),42

8(2),447

(2),44

9(5),465

(7),55

3(2),580

(38),598

(95),617

(100

)8

6‐O‐Galloyl‐ D‐glucose

5.70

,7.50

C13H16O10

331.06

747

+1.2

117(0.2),12

5(0.2),15

1(0.2),16

9(5),19

3(2),

211(1),2

41(1),27

1(10

0),2

95(0.2),31

3(1)

125(0.3),169

(11),193

(0.6),21

1(10

0),223

(0.2),23

5(0.2),2

53(1)

93,

6‐di‐O‐Galloyl‐ D‐

glucose

26.23,

26.99

C20H20O14

483.07

839

+0.7

169(2),193

(3),21

1(2),271

(6),31

3(4),331

(4),

405(0.6),4

23(100

),43

9(1),4

65(2)

169(4),19

3(12

),21

1(26

),21

7(1),23

5(2),

253(10

),27

1(88

),32

9(0.3),37

8(0.4),

405(10

0)10

1,2,3‐tri‐O

‐Galloyl‐β‐ D‐

glucose

32.80

C27H24O18

635.08

862

−0.6

169(2),235

(0.4),27

1(0.4),295

(2),31

3(3),405

(0.4),42

1(6),4

65(100

),48

3(23

),61

7(1)

125(4),16

9(41

),19

3(4),23

5(8),29

5(31

),31

3(10

0),32

1(3),37

7(3),42

1(82

),44

7(5)

111,4,6‐tri‐O

‐Galloyl‐β‐ D‐

glucose

35.14

C27H24O18

635.08

839

−0.9

169(5),22

1(7),27

1(5),29

5(5),31

3(13

),40

5(3),42

3(32

),46

5(10

0),4

83(82),6

17(12)

125(2),16

9(22

),19

3(3),23

5(9),25

3(1),

271(1),29

5(13

),31

3(10

0),40

5(2),44

7(7)

123,4,6‐tri‐O

‐Galloyl‐ D‐

glucose

28.95,

33.42

C27H24O18

635.08

815

−1.3

169(9),23

5(2),27

1(4),29

5(14

),31

3(9),40

5(5),42

3(30

),46

5(68

),48

3(10

0),6

17(11)

125(1),16

9(11

),19

3(8),21

1(4),24

1(3),

271(13

),31

3(16

),33

1(24

),42

3(10

0),

465(8)

131,2,3,6‐tetra‐O‐Galloyl‐β‐

D‐glucose

43.07

C34H28O22

787.09

864

−1.7

295(1),40

3(2),42

1(0.4),42

9(1),44

7(2),46

5(3),52

9(0.2),5

73(4),61

7(10

0),6

35(31)

169(6),23

5(5),27

7(5),29

5(10

),40

3(57

),42

1(12

),44

7(65

),46

5(80

),52

9(7),57

3(100

)14

1,2,3,4,6‐pe

nta‐O‐

Galloyl‐β‐ D‐glucose

67.70

C41H32O26

939.11

006

+0.9

329(0.4),4

39(0.4),44

7(0.2),5

15(0.2),59

9(1),

601(0.2),6

17(3),72

5(1),7

69(100

),78

7(8)

277(4),40

3(5),42

9(15

),44

7(20

),51

1(6),

555(7),57

3(4),59

9(47

),61

7(10

0),72

5(25)

T. Hooi Poay et al.

Phytochem. Anal. 2011, 22, 516–525Copyright © 2011 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/pca

518

Table

1.(Con

tinued)

No.

Com

poun

dRe

tention

time

(min)

Elem

ental

compo

sitio

n[M

–H]–

Mass

accuracy

(ppm

)

MS2

MS3

156‐O‐Galloyl‐2,3‐O‐(S)‐

hexahy

droxy‐

diph

enoy

l‐ D‐glucose

25.48,

27.94

C27H22O18

633.07

252

−1.3

229(1),24

9(1),25

7(1),27

5(19

),30

1(10

0),

319(0.4),4

81(3),5

89(0.3),61

5(1)

171(8),18

5(5),22

9(23

),25

7(10

0),27

3(15),2

84(3)

16PraecoxinB

29.65,

35.82

C34H26O22

785.08

375

−0.7

275(15

),30

1(10

0),31

9(3),40

5(2),42

3(32

),46

5(9),4

83(75),6

33(35),7

41(1),76

7(2)

171(30

),22

9(23

),25

7(10

0),2

73(16)

17Pterocarinin

C56

.65

C41H30O26

937.09

595

+0.7

275(1),3

01(17),3

13(1),46

5(12

),48

3(7),6

17(9),63

5(10

0),7

67(2),78

5(19

),91

9(0.4)

169(1),27

1(0.4),29

5(3),31

3(8),40

5(1),

423(3),46

5(10

0),48

3(64

4),59

1(0.3),

617(0.3)

183′‐O‐M

ethy

l‐3,4‐

methy

lene

dioxyellagic

acid

4′‐O‐β‐ D‐

glucop

yran

oside

76.21

C22H18O13

489.06

738

−0.2

327a,3

12(100

),28

3(0.2),

171(4),2

12(4),24

0(89

),25

6(5),2

84(100

)

193,3′,4‐tri‐O‐M

ethy

lellagic

acid

4′‐O‐β‐ D‐

glucop

yran

oside

91.08

C23H22O13

505.09

872

−0.1

343a,3

28(100

),31

3(0.1)

171(0.2),2

99(1),31

3(10

0)

20Gallic

acid

4.80

C7H6O5

169.01

428

+0.2

125(10

0)‐

a Rep

resented

theintensefrag

men

tiondu

eto

theremov

alof

glucose(m

/z16

2)an

dwas

selected

forfrag

men

tatio

nin

theMS2


Phytochem. Anal. 2011, 22, 516–525 Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pca

519

OO

O

OO

OHCN

C

OOH

OH

OH

CO

CO

OH

OH

OH

OH

OHHO

C O

OHHO

HO

133

769

152

750

617

732170

A)

7

O

O

CO

HOOH

OH

O

OCO

HO

HO

HO

O

OC

OOH

OH

OHCO

CO

HO

HOOH

OH

OHHO

152

787

170

769

617

152

170599

B)

14

O

O

O

O

O

OH3CO

O

OH

HOHO

OHO

327

162

15

312

28

284

44

18

C)

O

O

O

O

OCH3

OCH3H3CO

O

OH

HOHO

OHO

162

343

15

328

15

313

19

Figure 2. Schematic fragmentation of prunasin (A) 2′,3′,4′,6′‐tetra‐O‐gallate (7), (B) 1,2,3,4,6‐penta‐O‐galloyl‐β‐D‐glucose (14), (C) 3′‐O‐methyl‐3,4‐methylenedioxyellagic acid 4′‐O‐β‐D‐glucopyranoside (18) and 3,3′,4‐tri‐O‐methylellagic acid 4′‐O‐β‐D‐glucopyranoside (19) in negative mode ESI‐MSn.

T. Hooi Poay et al.

520

Liquid chromatography and mass spectrometry

The chromatographic analysis was performed using the Accela™ U‐HPLCsystem (Thermo Scientific, San Jose, CA, USA) equipped with aquaternary pump, a built‐in degasser, a PDA detector and an auto‐sampler. A Hypersil Gold RP C8 column (3 μm, 2.1 mm i.d. × 150 mm)was used. The mobile phase consisted of acetonitrile (A) and waterwith 0.1% formic acid (B) at a flow‐rate of 200 μL/min, with acombination of step‐gradient and isocratic elution, A:B as follows:0 min, 0:100; 12 min, 1:99; 13 min, 15.4:84.6; 15 min, 15.4:84.6; 100 min,16:84; 150 min, 18:82; 205 min, 20:80; 255 min, 28:72; 265 min, 30:70,maintained for 5 min. The effluent from the LC column was directedinto the ESI probe.

The MSn analysis was performed on a LTQ Orbitrap massspectrometer (Thermo Fisher Scientific, Bremen, Germany) equippedwith an electrospray ionisation probe. A negative ion mode wasemployed in the analysis because this mode afforded the best limits ofdetection for the compounds. The MSn data were acquired by OrbitrapFTMS analyser operating at full scan with a target mass resolution of30000 and data‐dependent MSn method with dynamic exclusionenabled and a repeat count of 2. In this method, the most intenseparent ion was isolated and fragmented twice to generate the MS2 data.Then the most intense daughter ion was also isolated and fragmentedtwice to produce the MS3 data. The spectra were recorded in the rangeof m/z 100–1000 and the collision energy for MSn was adjusted to35–40%. The optimised parameters in the negative ion mode were asfollows: ion spray voltage, 3.5 kV; sheath gas (N2), 40 arbitrary units;auxiliary gas (N2), 20 arbitrary units; capillary temperature, 285 °C. Masscalibration was performed according to the manufacturer’s guidelinesusing a manufacturer defined mixture of caffeine, sodium dodecyl

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sulphate, sodium taurocholate, the tetrapeptide MRFA and Ultramark1621. All data were processed using Qual browser (Thermo FisherScientific, San Jose, CA, USA).

Nuclear magnetic resonance spectroscopy

All the compounds except compounds 18 and 19 were dissolvedin Me2CO‐d6 with droplets of D2O to enhance the solubility ofthe samples. Compounds 18 and 19 were dissolved in DMSO‐d6. The1H‐ and 13C‐NMR spectral data were recorded using a Bruker DRX 300NMR spectrometer (300 MHz for 1H‐NMR and 75 MHz for 13C‐NMR).

Results and Discussion

ESI‐MSn analysis of isolated compounds

A total of 20 compounds (Fig. 1) were isolated from the leaves ofP. rotundifolia and characterised using negative mode ofionisation by ESI‐MSn. The experimental m/z values of themolecular ions together with calculated mass accuracy and theirMS2 and MS3 fragment ions are tabulated in Table 1. Fragmentions of galloylated cyanogenic glucosides were found to besimilar for the isomeric compounds such as prunasin–digallate(2–4) and prunasin–trigallate (5–6). The trigalloyl‐glucoseanalogues (compounds 10–12) exhibited similar daughterions in the MS2 spectra except that compound 12 showed adominant fragment ion at m/z 483, which enables it to bedifferentiated from the other two analogues. The ellagitannins


C35H26NO17

m/z 732

C28H22NO13

m/z 580

C21H18NO9

m/z 428

-152 (galloyl)

-152 (galloyl)

-152 (galloyl)

C13H11NO9

m/z 276

-170 (gallate)

-170 (gallate)

-170 (gallate)

-152 (galloyl)

-152 (galloyl)

-152 (galloyl)

Prunasin-tetragallate C42H32NO22

m/z 902

Prunasin-trigallate C35H28NO18

m/z 750

Prunasin-digallate C28H24NO14

m/z 598

Prunasin-monogallate C21H20NO10

m/z 446

-133 (Cyanohydrin)

-133 (Cyanohydrin)

-133 (Cyanohydrin)

-133 (Cyanohydrin)

C34H25O21

m/z 769

C27H21O17

m/z 617

C20H17O13

m/z 465

C13H13O9

m/z 313

C7H5O5

m/z 169

C6H5O3

m/z 125

-152 (galloyl)

-152 (galloyl)

-152 (galloyl)

-170 (gallate)

-170 (gallate)

-170 (gallate)

C27H19O16

m/z 599

C20H15O12

m/z 447

C13H11O8

m/z 295

-152 (galloyl)

-152 (galloyl)

-144 (dehydrated hexosyl)

-18 (H2O)

- -

- -

--

-

-

-

-

--

-

-

-

-

-

Figure 3. Fragmentation pathways of galloylated cyanogenic glucosides (1–7) in negative mode ESI‐MSn.


(compounds 15–17) yielded a product ion at m/z 301,representing the fragment ion of ellagic acid and it wasdominant in compound 15 and 16 (Table 1). The loss of m/z 44[M – H – COO]– in gallic acid (20) results in a product ion at m/z125 (He and Xia, 2007; Mämmelä et al., 2000; Meyers et al., 2006;Nuengchamnong and Ingkaninan, 2009; Soong and Barlow, 2005;Zywicki et al., 2002). The MSn fragmentations of each galloylatedcyanogenic glucosides, gallotannins, ellagitannins and ellagicacid derivatives are discussed in the following sections.

52

Galloylated cyanogenic glucosides

A previously reported MSn analysis in positive mode forcyanogenic glucosides gave [M + Na]+ and [M + NH4]

+ ions,while the collision‐induced dissociation (CID) showedfragmentation of HCN and aglycone from the sodium adductparent ion and subsequently derived [M + Na – HCN]+ and[dehydrated glucose +Na]+ (Franks et al., 2005). In this study,the galloylated cyanogenic glucosides [prunasin 6′‐O‐gallate(1), prunasin 2′,6′‐di‐O‐gallate (2), prunasin 3′,6′‐di‐O‐gallate (3),prunasin 4′,6′‐di‐O‐gallate (4), prunasin 2′,3′,6′‐tri‐O‐gallate (5),prunasin 3′,4′,6′‐tri‐O‐gallate (6) and prunasin 2′,3′,4′,6′‐tetra‐O‐gallate (7)] were fragmented using negative ionisationmode which generated deprotonated parent ion [M – H]–.Compounds 1, 2–4, 5–6 and 7 are referred to as prunasin–monogallate, prunasin–digallate, prunasin–trigallate andprunasin–tetragallate, respectively, in Fig. 3. The ESI‐MSspectrum of prunasin–tetragallate showed [M – H]– at 902,while the fragment ions at [M – H – 133]–, [M – H – 152]– and[M – H – 170]– indicated the mass loss of cyanohydrin, galloyl


and gallate moieties respectively (Figs 2A and 3). Similarfragmentation patterns were also observed in prunasin–trigallate(m/z 750) and prunasin–digallate (m/z 598) but not in prunasin–monogallate (m/z 446). The major fragment ion of prunasin–monogallate at m/z 313 [M – H – 133]– underwent a neutral massloss of 144 (dehydrated hexosyl) following MS3, indicating scissionof a hexosyl unit to yield deprotonated gallic acid (Fig. 3 andTable 1). The deprotonated gallic acid at m/z 169 furtherunderwent decarboxylation with mass loss of 44 to form atrihydroxyphenol moiety atm/z 125. Product ions atm/z 769, 617,465 and 313 were also found in the fragmentation scheme ofgallotannins (Figs 3 and 4). These product ions of galloylatedcyanogenic glucosides are believed to demonstrate a similarfragmentation pathway, as presented in Fig. 4.

Gallotannins

Gallotannins or galloylated esters of glucose are knownto show elimination of multiple galloyl [M – H – 152]– and gallate[M – H – 170]– moieties in MSn analysis (Meyers et al., 2006;Salminen et al., 1999; Soong and Barlow, 2005). In this study,schematic fragmentation of 1,2,3,4,6‐penta‐O‐galloyl‐β‐D‐glucose(14, m/z 939) as presented in Fig. 2(B) together with itsMSn spectra in Table 1 also showed mass loss of several galloyl(m/z 152) and gallate (m/z 170) groups. Fragmentationpathways of the gallotannins are shown in Fig. 4, where themonogalloyl‐glucose, digalloyl‐glucose, tetragalloyl‐glucose andpentagalloyl‐glucose represent 6‐O‐galloyl‐D‐glucose (8), 3,6‐di‐O‐galloyl‐D‐glucose (9), 1,2,3,6‐tetra‐O‐galloyl‐β‐D‐glucose(13) and 1,2,3,4,6‐penta‐O‐galloyl‐β‐D‐glucose (14), respectively,


1

Pentagalloyl-glucose C41H31O26

(m/z 939)

-071- 152

(m/z 769)

(m/z 725)

tetragalloyl-glucose C34H27O22

(m/z 787)

-44 -170

(m/z 599)

-152

(m/z 617)

-170 -152

trigalloyl-glucose C27H23O18

(m/z 635) -152 -170 -152

(m/z 465)

-170 -152

digalloyl-glucose C20H19O14

(m/z 483)

-44

(m/z 439)

-152

monogalloyl-glucose C20H19O14

(m/z 331)

-60

(m/z 423)

-152

-170

(m/z 313)

-152-170

(m/z 295)

-60

(m/z 271)

-44

(m/z 125)

-60

(m/z 211)

-18

(m/z 405) -102

(m/z 169)

-18 (m/z 447)

-152

-18

-44

(m/z 573)

-126-152

(m/z 287)

-144

(m/z 169)

-162

Figure 4. Fragmentation pathways of gallotannins (8–14) in negative mode ESI‐MSn.

T. Hooi Poay et al.

522

while 1,2,3‐tri‐O‐galloyl‐β‐D‐glucose (10), 1,4,6‐tri‐O‐galloyl‐β‐D‐glucose (11) and 3,4,6‐tri‐O‐galloyl‐D‐glucose (12) werecategorised as the isomeric trigalloyl‐glucose. The pentagalloyl‐glucose (m/z 939) shows consistent mass loss of galloyl moietiesto tetragalloyl‐glucose (m/z 787), trigalloyl‐glucose (m/z 635),digalloyl‐glucose (m/z 483) and monogalloyl‐glucose (m/z 331).Additionally, the neutral mass loss of gallic acid [M – H – 170]–

moieties in pentagalloyl‐glucose (m/z 939), tetragalloyl‐glucose(m/z 787), trigalloyl‐glucose (m/z 635), digalloyl‐glucose (m/z 483)are also illustrated. Fragmentation of digalloyl‐glucose (9) at m/z483 contributed to two major product ions at m/z 423 and m/z439. The product ion at m/z 423 was suggested to be derivedfrom the removal of two formaldehyde moieties [M – H – 60]–

(Taylor et al., 2005) from glucose moiety, whereas product ion atm/z 439 was formed as a result of decarboxylation [M – H – 44]–.The fragment ion at m/z 423 then underwent independentlosses of [M – H – 152]– (m/z 271) and [M – H – 18]– (m/z 405)(Fig.4 and Table 1). Elimination of glucosyl moiety frommonogalloyl‐glucose was observed and subsequently formed thedeprotonatedgallic acid atm/z 169 (Fig. 4). Fragment ions atm/z 271[M – H – 60]– and at m/z 211 [M – H – 60 – 60]– were the majorproduct ions in MS2 and MS3 of monogalloyl‐glucose, respectively


(Fig. 4 and Table 1). The observation of intermediate ions atm/z 271andm/z 211 formonogalloyl‐glucose and digalloyl‐glucose had alsobeendescribed previously (Mämmelä et al., 2000; Soong andBarlow,2005; Zywicki et al., 2002). The fragment ion at m/z 211 wassuggested to be due to the removal of two formaldehyde (CH2O)groups in the glucosemoiety. Them/z 271 ionwas further subjectedto fragmentation to produce signal atm/z 169, formed by the loss ofa glucosyl unit (He and Xia, 2007; Kool et al., 2010, Mullen et al., 2003;Simirgiotis and Schmeda‐Hirschmann, 2010). The m/z 169 ion thenunderwent removal of CO2 to generate an ion at m/z 125.

Ellagitannins

The ellagitannins (15–17) demonstrate the esterification ofthe HHDP group at the C‐2 and C‐3 of the glucose moiety(Fig. 1). 6‐O‐galloyl‐2,3‐O‐(S)‐hexahydroxy‐diphenoyl‐D‐glucose(15), praecoxin B (16) and pterocarinin C (17) were groupedas monogalloyl‐HHDP‐glucose, digalloyl‐HHDP‐glucose andtrigalloyl‐HHDP‐glucose respectively. The ESI‐MS depicted thesubsequent mass loss of galloyl (m/z 152), from trigalloyl‐HHDP‐glucose (m/z 937) to yield digalloyl‐HHDP‐glucose (m/z 785),then monogalloyl‐HHDP‐glucose (m/z 633) and finally HHDP‐


-galloyl

Spontaneous lactonization

-Hexosyl

[M-H-Hexosyl] ¯ : 319

-galloyl

[M-H-H2O] ¯ : 301

lactonization

[M-H-CO2] ¯ : 275

-HHDP -HHDP

decarboxylation

Trigalloyl-HHDP-glucose

[M-H] ¯ : 937

Digalloyl-HHDP- glucose

[M-H] ¯ : 785

Monogalloyl-HHDP- glucose

[M-H] ¯ : 633

HHDP-glucose[M-H] ¯ : 481

Trigalloyl-glucose[M-H-HHDP] ¯ : 635

Digalloyl-glucose[M-H-HHDP] ¯ : 483

O

OHOH

OHHO

O

HO

HO O

HO

O

OHOH

OHO

O

HO

HO OOOH

OHO

O

HO

HO

O

OO

OHO

O

HO

HO

-galloyl

Figure 5. Fragmentation pathways of ellagitannins (15–17). HHDP, hexahydroxydiphenolic acid.

0 50 100 150 200 250Time (min)

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

b)

18

19

3

42

65 7

10 20 30 40 50 60 70Time (min)

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

20 8 8

15

9

9

12

11

10 12

16 16

13

1

17 14

b)

a)

15

Figure 6. LC‐MS chromatographic profile of (a) Phyllagathis rotundifolia crude extract and (b) expanded chromatogram (3–72 min).


Phytochem. Anal. 2011, 22, 516–525 Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pca

523

T. Hooi Poay et al.

524

glucose (m/z 481) as shown in Fig. 5 and Table 1. Theellagitannins were also fragmented to become gallotannins bylosing the mass of 302 (HHDP), consequently resulting infragment ions at m/z 635 (trigalloyl‐glucose) and m/z 483(digalloyl‐glucose). These daughter ions are suggested toundergo similar fragmentation to the gallotannins (Fig. 4).The monogalloyl‐HHDP‐glucose did not show the massof monogalloyl glucose; instead two dominant fragment ionsat m/z 301 and m/z 275 were observed. The ion at m/z 301 (lossof glucose and galloyl groups) was the result of spontaneousdilactonisation of HHDP. The partial lactonisation of HHDP yieldsa product ion at m/z 319 and subsequent decarboxylation yieldsan ion at m/z 275 [M – H – 44]–. In Table 1, the MS3 of m/z 301 incompounds 15 and 16 gave prominent ions at m/z 273, 257 and229, like deprotonated ellagic acid (He and Xia, 2007; Mämmeläet al., 2000; Mullen et al., 2003; Simirgiotis and Schmeda‐Hirschmann, 2010; Soong and Barlow, 2005). These ions weresuggested to correspond to [M – H – CO]–, [M – H – CO2]

– and[M – H– CO2– CO]– respectively.

Ellagic acid derivatives

The ESI‐MSn of compounds 18 and 19 produced the deproton-ated parent ion [M – H]– at m/z 489 and 505, respectively(Table 1). The fragmentation pattern is presented in Fig. 2(C).Both compounds were dominated by intense ion peaks at m/z327 and m/z 343, respectively, due to the removal of glucosemoiety [M – H – 162]–. The major ion peak at m/z 327 in 18 wasfragmented to m/z 312 through the loss of a CH3 group [M – H –15]–. The subsequent fragmentation of this ion yielded productions at m/z 284 and 240, which indicates the mass loss of COand CO2 respectively (Fig. 2C). Similarly, fragmentation of themajor ion in 19 atm/z 343 showed the elimination of first CH3 atm/z 328 in MS2 followed by a second CH3 at m/z 313 in MS3 asdisplayed in Fig. 2(C).

HPLC‐ESI‐MSn analysis of the crude extract ofPhyllagathis rotundifolia

Fig. 6 shows the LC‐MS chromatographic profile of the methanolextract of P. rotundifolia. The peaks were identified bycomparing their mass spectra with those of isolated compoundsas described in the previous sections.

Structural identification of the compounds

The structures of the isolated compounds (1–20) weredetermined by proton and carbon spectroscopic analysis. Thecyanogenic glucosides were identified as prunasin 6′‐O‐gallate,prunasin 2′,6′‐di‐O‐gallate, prunasin 3′,6′‐di‐O‐gallate, prunasin4′,6′‐di‐O‐gallate, prunasin 2′,3′,6′‐tri‐O‐gallate, prunasin 3′,4′,6′‐tri‐O‐gallate and prunasin 2′,3′,4′,6′‐tetra‐O‐gallate (Isaza et al.,2001; Ling et al., 2002; Miller et al., 2006). The seven gallotanninswere identified as 6‐O‐galloyl‐D‐glucose (He et al., 2001; Nonakaand Nishioka, 1983), 3,6‐di‐O‐galloyl‐D‐glucose (He et al., 2001),1,2,3‐tri‐O‐galloyl‐β‐D‐glucose (Lee et al., 1990; Nawwar et al.,1994), 1,4,6‐tri‐O‐galloyl‐β‐D‐glucose (Lee et al., 1992; Nawwaret al., 1994; Nonaka et al., 1984), 3,4,6‐tri‐O‐galloyl‐D‐glucose(He et al., 2001; Lee et al., 1989, 1991; Wilkins, 1988), 1,2,3,6‐tetra‐O‐galloyl‐β‐D‐glucose (Duan et al., 2004; Lee et al., 1991;Nishizawa et al., 1983; Saijo et al., 1990; Yoshida et al., 1991a),and 1,2,3,4,6‐penta‐O‐galloyl‐β‐D‐glucose (Gao et al., 2007;


Nishizawa et al., 1983; Saijo et al., 1990; Tanaka et al., 1985) bycomparing their spectroscopic data with literature values. Thethree ellagitannins were identified as 6‐O‐galloyl‐2,3‐O‐(S)‐hexahydroxy‐diphenoyl‐D‐glucose (Yoshida et al., 1991a,1991b), praecoxin B (Hatano et al., 1991; Yoshida et al., 1991a)and pterocarinin C (Yoshida et al., 1991a, 1995). The twoellagic acid derivatives and one phenolic acid were identified as3 ′ ‐O‐methyl‐3,4‐methylenedioxyellagic acid 4 ′ ‐O‐β‐D‐glucopyranoside (Khallouki et al., 2007; Li et al., 1999), 3,3′,4‐tri‐O‐methylellagic acid 4′‐O‐β‐D‐glucopyranoside (Li et al., 1999)and gallic acid (Yoshida et al., 1991b; Chanwitheesuk et al.,2007). The 1H‐NMR and 13C‐NMR spectroscopic data forcompounds 1–20 are available in the Supplementary Material.

ConclusionThe fragmentation schemes for galloylated cyanogenic gluco-sides, gallotannins, ellagitannins and ellagic acid derivativesisolated from the leaves of P. rotundifolia provide detailed MSn

information which facilitates rapid monitoring of seasonalvariation, maturation process and quality evaluation of plantmaterial.

Supporting Information

Supporting information can be found in the online version ofthis article.

Acknowledgements

H. P. Tan would like to acknowledge Forest Research InstituteMalaysia (FRIM), Selangor, Malaysia for a scholarship and Mr.Cheah Hun Teong of Alpha Analytical (M) Sdn Bhd for assistancein LC‐MS instrumentation. This study was funded by a grant (02‐03‐10‐SF0066) from the Ministry of Science, Technology andInnovation and an IPPP research grant (PS332/2008C andPS351/2010A) from the University of Malaya.

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Characterisation of Galloylated Cyanogenic Glucosides and ...

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