Flavonol 3-O-triglycosides from Actinidia species

Phyrochemlstry,Vol 29, No Prmted m Great Brltam

1, pp

289-292,1990

FLAVONOL

003l-9422/90 $300 + 0.00 0 1989Pergamon Press plc

3-O-TRIGLYCOSIDES

FROM ACTINIDIA

SPECIES

ROSEMARY F WEBBY and KENNETH R. MARKHAM Chemistry Dlv~slon, DSIR, Private Bag, Petone, New Zealand (Recerved 17 May 1989)

Key Wbrd Index--Actrnh trlglycoslde acetate

arguta, A ermntha, A polygama,

Actnudlaceae, Iclwlfrult; flavonols, trlglycosldes,

Abstract-In the course of a chemotaxonomtc study of the genus Acttmdw, several new flavonol triglycostdes have been characterlsed by ‘H and 13C NMR spectroscopy. These are kaempferol and quercetin, 3-O-[cl-rhamnopyranosyl(1-4)-rhamnopyranosyl-( 1-6)-p-galactopyranoslde, kaempferol 3-O-[a-rhamnopyranosyl-(l-4)-rhamnopyranosyl(1-6)+glucopyranoslde], and kaempferol 3-O-[cc-rhamnopyranosyl-(1-4)-3”’-O-acetyl-~-rhamnopyranosyl-(l-6)P-galactopyranonde] Quercetm and lsorhamnetm analogues of the dirhamnosyl glucoside were also detected.

INTRODUCTION The genus Actmidq which mcludes 52 recognised species and many more varieties, is a large and taxonomically confused genus One of the species, A. delwosa (klwifrult) is a horticultural crop of major economic importance to New Zealand A study of the flavonolds of Actimdla was initiated m an attempt to clarify aspects of the taxonomy. Flavonolds have proven to be valuable taxonomlc characters in previous investigations of New Zealand plants [l, 23, and to our knowledge have not been studied in Actmldm species previously, although work on condensed tannins has been reported [3,4]. In the course of the chemotaxonomic survey of Actvmfra, several new flavonol triglycosldes have been encountered and the details of their structural elucidation are presented here.

RESULTS AND

DISCUSSION

A chemotaxonomlc survey of Acttmdw species has resulted m the discovery of new flavonol gtycosldes in three species. The glycosldes were Isolated from an ethanol-water extract by column (polyamide) and paper chromatography Fmal clean-up was by column chromatography on C-8 reversed phase. Actinuim arguta accumulated four high RI (PC, 15% HOAc) glycosides. On acid hydrolysis, two gave quercetin and two kaempferol All four also yielded rhamnose and galactose m a ratio of 2 1. On deacylatlon, the two higher moblhty (WA) gIycosldes, one quercetm and one kaempferol, were converted to the two lower moblhty glycosides 1 and 2 respectively Absorption spectroscopy of the two unacylated compounds showed that only the 3hydroxyls were substituted, thus defining these compounds as 3-0-glycosldes Llquld secondary ion mass spectroscopy (SIMS) on the quercetm glycoslde (I) defined the M, as 756 (M + 1 = 757) consistent with a dlrhamnogalactoslde formulation The ‘H NMR spectrum also supported the presence of two rhamnoses and one galactose with rhamnose related signals at 4 42 and 4.73 (H-l, d, J = < 2 5 Hz) and 0 98 289

and 1.08 (H-6, J = 5.8 Hz), and the fi-galactose H-l at 5.33 (J = 7.5 Hz) Interglycosldlc linkage points were determined from the 13CNMR spectrum (see Table 1). The galactose moiety was represented by the same signals as m quercetm 3-0-galactoslde except that C-6 appeared 5 ppm downfield and C-5,2.2 ppm upfield Glycosylatlon of galactose by rhamnose at the 6-hydroxyl is thus evident. This was confirmed by pectmase hydrolysis which produced quercetin 3-0-galactoslde and a quercetin 3-0-rhamnosylgalactoslde which had R, values close to those of quercetm 3-0-rhamnosyl-( 1-6)-glucoslde The trlsaccharlde is therefore defined as a rhamnosylated robmoblose. The second rhamnose must be attached to the rhamnosyl portion of the robmoblose, since not only are the galactose signals equivalent to those seen m other 3-0-robmobiosldes (see Table l), but the rhamnose slgnals are not doubled (or double sized) as would be expected [S] for a branched cham tnglycoslde. For example, only a single rhamnose C-4 signal is seen at 72 ppm, the other appearing at 78 ppm. This 6 ppm downfield shift 1s consistent with the addltlonal rhamnose substltuent bemg attached to the hydroxyl at C-4 of the robmoblose rhamnose The structure proposed for 1 1s therefore the linear quercetm 3-O-[a-rhamnopyranosyl(1-4)-a-rhamnopyranosyl-(1-6)-B-galactopyranoslde]. By analogy, and by Its relatlonshlp to 3 (see below), the kaempferol dlrhamnogalactoslde, 2, 1s considered to be kaempferol 3-O-[a-rhamnopyranosyl-(14)+rhamnopyranosyl-( I-6)-/?-galactopyranoslde] The acylated derivatives of 1 and 2 were not avalIabIe m sufficient quantity from this plant to enable structural eluadation. Actmzdta polygama appeared (2D-PC) to contam the same unacylated trlglycosldes as A. arguta. Absorption spectroscopy, analysis of acid hydrolysis products and ‘HNMR spectroscopy, all indicated that the glycosyl moleties m both the quercetm and kaempferol glycosides are the same as m the A arguta equivalents, 1 and 2 13CNMR data confirmed that the mterglycosldlc linkages m the kaempferol glycoslde are the same as m 2 and It IS assumed that the same is true for the minor quercetm analogue Accordmgly these glycosldes are assigned

290

R F

WEBBY and

K R

MARKHAM

HO CH?-0

OH

1

R’=

0H.R2=

2

R’=

H.R2=

II

3

R’ =

H, R’ =

AC

OR2

OH OH

H

HO

4

structures 1 and 2 In addttion to 1 and 2, A. polygama accumulates two acylated kaempferol trtglycosides, one major (3) and another m trace quantities The major acylated glycoside, 3, was isolated in quantity by ID-PC, using BEW (n-butanol-HOAc-H,O 4: 1.2.2) which separated tt from the unacylated glycostdes. Deacylation converted 3 mto the unacylated glycoside 2 and actd hydrolyses produced kaempferol together with the sugars rhamnose and galactose m the ratto of 2 1 The “C NMR spectrum of 3 contained signals at 620.7 and 169 9 whtch were not present m the spectrum of 2. These Indicate the presence of an acetyl functton. The other visible difference between the ’ 3C NMR spectra of 2 and 3 was the upfield shaft of one signal from 678 to 76 on acylatton (Table 1) As mentioned above, the 678 signal represents the robmobrose rhamnose C-4, and a change m this stgnal therefore mdtcates that the acyl functton must be attached adjacent to this, t e at C-3 or C-5. As C-5 carries no hydroxyl the acetyl functton must be sited at C-3. The major acylated glycoside 3 IS therefore kaempferol 3-0-[a-rhamnopyranosyl-( l-4)-3”‘-O-acetylg-rhamnopyranosyl-(l-6)$galactopyranoside] The quercetm and kaempferol triglycosides from A. ermntha co-chromatographed (TLC) with 1 and 2 from A arquta and A polygama but sugar analysis showed the sugars to be rhamnose and glucose (2 1) rather than rhamnose and galactose A trace of tsorhamnetm aglycone was produced along with kaempferol and quercetm and it IS therefore concluded that an tsorhamnetin 3-0triglycostde may also be present The ’ 3C NMR spectrum of the kaempferol triglycostde 4 (Table 1) proved to be analagous to that of the kaempferol triglycoside ex A arqutu except that the galactose carbon stgnals are

replaced by glucose carbon signals The mterglycosidtc linkage points are therefore at the same sites and the structure of 4 1s thus defined as kaempferol 3-0-[arhamnopyranosyl-( I-4)-a-rhamnopyranosyl-( I-6)-p-glucopyranoside]. The quercetm and isorhamnetm trtglycosides were not available m sufficient quantities for i3C NMR studies, but their sugar components and chromatographic mobilmes relative to 1, 2 and 4 are consistent with them being the quercetm and tsorhamnetm analogues of 4 The kaempferol and quercetm triglycosides presented m thts paper have not been previously reported [9] and the trtsaccharide rha( I-4)rha( I-6)gal appears unknown m flavonol glycostdes In glycosidtc form thts trtsacchartde is claimed to have been found and has been referred to m some communications [e g 991 I] as the rhamnmostde However the structure of rhamnmostdes has recently been revised to the rha( I-3)rha( 1-6)gal[ 121 on the basis of 13C NMR data. Rtess-Maurer and Wagner [12] synthestzed rha( l-4)rha( I-6)gal glycostdes m order to fully identify the compounds isolated from Rhamnus, as previous mass spectroscoptc studies [ 10, 1l] did not fully identtfy the sugar linkages As a result of thts work, RtessMaurer and Wagner asstgned the rha( I-4)rha( l-6)gal structure to isorhamnmose whtch to date appears to be known only as the synthettc product These workers reported the kaempferol and quercetm 3-O-rhamnmostdes m species of Rhamnus together wtth an acetylated triostde of rhamnetin (rhamnetm-3-O-[a-L-rhamnopyranosyl-( l-3)-O-(4-O-acetyl-)-a-t rhamnopyranosyhl-6)b-D-galactopyranostde] There are no reports of lmear flavonol rha(l-4)rha( I-6)glucosides havmg been found previously [9]

1023 713 73 4 680 75 8 60 8

a-f Assignments

Gal 1” 2” 3” 4” 5” 6” Glc 1” 2” 3” 4” 5” 6” Rha 1”’ 2”’ 3”’ 4”’ 5” 6” Rha ,111 1 2”” ,111 3 4”’ 5”” 111, 6

3-O-gal

1014 74.2 77 2 70 1 76.5 610

3-0-glc

may be reversed

[6]

[6]

1006 70 3” 70.7” 72.0 68 1 17.4

101.5 74.2 76 5 70.1 75 8 66.9

3-0-rutm

[7]

1004 70.6” 70 7” 72.1 684 180

1020 713 73 2 68.3 73.8 654

[8]

1019 70 4a 70 6” 71.5 70 1” 17 3

3-0-rha

[6]

A arguta

100.2” 70’ 70’ 72 2 68” 176’

103 9” 71” 70* 78,l 68t’ 178’

102 4” 71 lb 73 1’ 68” 73 6 659

1

1 13C NMR data (ppm) for sugar moleties

3-0-robmoblose

Table

100 3” 70 4e 701’ 72 7 68j 17 5’

102 4” 71”lb 70* 78.1 68” 17.7’

102 4” 717b 73 1c 68 4d 73 6’ 662

2

-

100 2” 70 4’ 706 72 2 68 4d 17 5’

100.3’ 70* 71.4b 784 68” 17 7’

- I

-

A polygama

102 6” 71 8b 73.3 68 5d 73.6” 664

2 -.-

100.7” 70” 7oj 72 5’ 68 4c 174’

102 5” 70” 72 1’ 76 7 674 177’

102 9” 71 730 68,7’ 73” 66,2

3

1009” 70b 70.5b 72 3 68.6’ 176

1019 7op 7op 78.1 68 4’ 176

102 3” 743 76 5 70 9b 75.7 67,6

A erlantha 4

R F WEBB? and K R MARKHAM

292 EXPERIMENTAL

PIant materml Samples supphed by Dr A R Ferguson (DIVISION of Horticulture and Processmg, DSIR) were from horticultural specimens grown at Te Puke and Kumeu, New Zealand Sample extractmn and work-up 50 g dry wt oven-dried (100‘) fohage was ground to a powder and extracted overnight with EtOH-H,O (1 1) The extract was apphed to a polyamide column (Machery-Nagel) m H,O The solvent was changed to 100% EtOH once the eluate was clear This procedure removed tannm and polysaccharlde contammants The EtOH eluate was further purified by 1D or 2D paper chromatography (PC) using TBA (t-BuOH-HOAc-H,O (3 1 1)) and 159/o HOAc [6] Fmal clean up was by CC on a C-8 reversed phase column The fractron was applied m H,O and after elutlon to remove H,Osoluble lmpurltles the solvent was changed to MeOH to retrieve the flavonolds ‘H and 13C NMR spectra were measured m DMSO-d, solutlons at 80 and 20 MHZ respectively, ‘H spectra were also measured at 200 MHz 6 are expressed as ppm downfield from TMS Techmques such as absorption spectroscopy, acid hydrolyals and product analysis, deacylatlon, paper and thm layer chromatography were carried out as desLrlbed m [6] unless otherwise stated R, values were taken from 2D-PCs Sugar ratios were estimated by PC and confirmed by ‘H NMR spectroscopy Compound 1 RI 0 47 (TBA), 0 62 (15% HOAc) Acid hydrolySIS gave quercetm, rhamnose galactobe 2 I ~.i:p” nm 260, 268(sh), 3OO(sh), 362, (NaOMe) 274, 330, 412, (NaOAc) 26@sh), 274, 325(sh), 372, (NaOAc-H,BO,) 264, 294(sh), 380 Llquld SIMS M + 1757 ‘H NMR (DMSO-d,) 12 58 s (S-OH), 7 65d. J = 8 Hz (H-6’), 7 54 5(H-2’), 6 84 d, J = 8 1 Hz (H-5’). 6 40 d, J = ((1 2 5 Hz (H-8). 6 20 d, J = ca 2 5 Hz (H-6), 5 33 d. J = 7 5 Hz (Gall), 4 73 d, J = < 2 5 Hz (Rha-1), 4 42 d, J z < L 5 Hz (Rha-l), 108 d, J = 5 8 Hz (Rha-Me), 0 98, J = 5 8 Hz (Rha-Me) Pectmase treatment 3 hr, products cleaned up by passage through a C8 reversed phase column Compound 2 R, 0 60 (TBA), 0 70 (15% HOAc) Acid hydrolySISgave kaempferol, rhamnose galactose 2 1 i.z$“’ nm 268,300 (sh), 352, (NaOMe) 276, 328, 404. (NaOAc) 276, 306, 368, (NaOAc-H,BO,) 268, 300 (sh), 354 ‘H NMR (DMSO-d,) As for 3 below but lackmg the 6 1 97 slgnal and posaesamg a doublet at 64.72 m place of the 64 68-4 92 multlplet Compound 3 (acylated 2) gave the same acid hydrolysis products and UV absorption spectra as 2, R, 0 73 (TBA), 0 70 (15% HOAc) ‘HNMR (DMSO-d,) 13 85 s (5-OH), 8 05 d, J =87Hz(H-2’6’).686d.J=87Hz(H-3’5’),64ld,J=ccr25Hr (H-8),618d,J=ta25Hz(H_6),524d, J=ccr6Hz(Gal-1),

4 92-4 68 m (Rha-1 and Rha-3”‘), 4 49 d, J = unclear (Rha-1), 197 s (AC), 0 95 m (Rha-Me) Compound 4 R, 0 65 (TBA), 0 70 (15% HOAc) Acid hydrolySISgave kaempferol, rhamnose glucose 2 1 UV spectra ldentlcal to 2 ‘H NMR (DMSO-d,) 7 93 d, J = 8 3 Hz, (H-2,.6’), 6 85 d, J = 8 5 Hz (H-3,.5’). 6 23 d. .I = cu 2 5 Hz (H-X), 6 06 d, J = ca 2 5 Hz (H-6), 5 19 d, J : (u 5 1 Hz (Glu-1), 4 72. J < 2 5 Hz (Rha1). ca 4 4. pdrtlq obscured (Rhd-1) 098 hr (Rha-Me) R,s of other Isolated glycosldes .4 polyyamu mmor acylated kdempferol triglycoslde 0 X8 (TBA), 0 70 (15% HOAc), A uryuta acylated kaempfcrol trlglycoslde 0 74 (TBA), 0 70 (15% HOAc), acylated quercetm trlglycostde 0 54 (TBA). 0 63 (15% HOAc) A ermnthu quercetm 3-O-dlrhamnogluLoslde 0 52 (TBA). 0 62 (15% HOAc)

Acknowledgements The authors thank Dr A R Ferguson (Dlvlslon of Horticulture dnd Processmg, DSIR) for supplymg plant material, Drs H Wong and L J Porter for runnmg NMR spectra and hquld SIMS respectively

REFERENCES

I 2 3 4 5 6 7 8 9

10 11 12

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