K.L.,Ravenscroft, N.,Farrant, J.M.,Lindsey, G.G. and Brandt, W.F. (2004) The ... John P Moore1, Neil Ravenscroft2, George G Lindsey1, Jill M Farrant1 and Wolf F ...
The polyphenols of Myrothamnus flabellifolius Galloylquinate ester : Anthocyanin complexes in the leaves of the desiccated resurrection plant John P Moore1, Neil Ravenscroft2, George G Lindsey1, Jill M Farrant1 and Wolf F Brandt1 Department of Molecular and Cellular Biology1, Department of Chemistry2, University of Cape Town, Private Bag, Rondebosch 7701, South Africa
INTRODUCTION The resurrection plant Myrothamnus flabellifolius (Figs 1 & 2) belongs to a unique group of Southern African angiosperms able to survive regular periods of dehydration to an air dry state1. The leaves have been noted to contain high levels of polyphenols. We have structurally charactarised the main phenolic compounds* and determined their ultrastructural location within the leaves. Upon dehydration the leaves become reddish in colour (Figs 3 & 4) due primarily to the abaxial accumulation of anthocyanin compounds (Figs 5 & 6). We have characterised the main anthocyanin compounds present and observed a possible interaction with the previously characterised galloylquinic acids* which could function in vivo2 during dehydration.
Fig.1. M. flabellifolius in the dehydrated state
Fig.2. M. flabellifolius in the hydrated state
METHODS AND RESULTS
Fig.3. The abaxial leaf surface of M.flabellifolius in the dehydrated state
Fig.4. The adaxial leaf surface of M.flabellifolius in the dehydrated state
Fig.5. A cross section through a leaf cluster of M.flabellifolius in the dehydrated state
Fig.6. A cross section through a leaf cluster at higher magnification demonstrating abaxial location of anthocyanins
The main polyphenol was purified via RP C18 HPLC and characterised to be 3,4,5 tri-O-galloylquinic acid (Figs 7 & 8) via 1D and 2D NMR with MALDI and ESI MS. Higher molecular weight compounds were shown to be galloylated (Fig 9) and ellagic acid (Fig 10) based derivatives of 3,4,5 tri-O-galloylquinic acid using MALDI TOF MS. Over 50 % of the dry leaf weight of M.flabellifolius are galloylquinic acids. HO
5
7
MALDI TOF MS analysis reveals polygalloyl ester chains
1
800
O OH R1
6
OH
4
HO
Depside Ester Bonds
500
B OH
6
5
1
OH HO
400
m/z 17
Core molecule
O
m/z 125
OH
m/z 153 m/z 169
OH
OH
O OH
O
O
O
OH
OH
O
O O
OH
600 O
O
HO
700
O O
O O
3
OOC
OH
900
OH
7
2
OH
1000
4
A 1
O
HO
OH 3
2
OH
O
300
O O
200
3
20
OH HO
HO OH
0
HO
OH O
OH
2
100
C
O
30
40
-100 Time (min)
Namibian : m/z at 647(3), 786(4), 936(5), 1570(9) & 1722(10) South African : additional late m/z at 1088(6)*, 675(3)*,936(5)*, 1322(10)* & 882(7)*
HO
O
R2 OH O OH
Fig.7. The main phenolic compound 3,4, 5 tri-O-gall oyl quini c a ci d
Fig.8. HPLC profile of galloylquinic acids present in the leaves of M.flabellifolius.
Fig.9. Polygalloyl ester chains revelead by MALDI MS composed of 3,4,5 tri-O-galloylquinic acid core
Fig.10. Putative ellagitannin substructure of certain galloylquinic acids present in the leaves of M.flabellifolius
Ultrastructural location was demonstrated via transmission electron microscopy (Figs 11 & 12). Anthocyanins were extracted and analysed via RP C18 HPLC and MALDI MS (Fig 13). The main compound is provisionally assigned to be cyanidin glucose (Fig 14B) and was shown to co-purify and co-pigment with galloylquinic acids (Fig 14). These compounds can possibly form ion pair interactions in the epidermal and endodermal leaf cells. OH
A
HO
OH
OH HO OH OD 520 RED HPLC H2O to MeOH TFA
300
O
O
O
HO 250
O
OH
O OH
200
O
OD 520
150
HO
OH O
100
OH
50
0 2000
B 2100
2200
2300
2400
2500
2600
2700
2800
2900
OH
3000
HO
-50
O
+
HO
-100 time (min)
O HO
Fig. 11. Dehydrated leaf mesophyll cell of M. flabellifolius
Fig. 12. Hydrated leaf mesophyll cell of M. flabellifolius
Fig.13. HPLC profile of anthocyanins present in the leaves of M.flabellifolius
( Micrograph Labels : P, polyphenols; V, vacuole; CW, cell wall; C, chloroplast; S, starch granule; n, nucleus, Bar = 3µm)
O
H
OH OH
H OH OH
H H
Fig.14. Ion pair complexes between (A) galloylquinic acids and (B) anthocyanins
CONCLUSIONS Galloylquinic acids stabilise liposome membranes against desiccation and are powerful anti-oxidants (shown to protect linoleic acid against free radical oxidation)3. In addition galloylquinic acids are UV absorbing compounds and so possibly function in the alleviation of light stress. Over 50 %of the dry leaf weight constituents are galloylquinic acids and with their vacuolar location they can function as powerful cellular anti-oxidants3. Anthocyanins (putatively assigned as cyanidin glucose) have been shown to increase upon desiccation in a light dependant manner and are responsible for the abaxial colouration of the leaves. Galloylquinic acids co-purify, co-pigment and possibly ion pair with anthocyanins so as to possibly enhance their stability and anti-oxidant/light shielding function.
REFERENCES
ACKNOWLEDGEMENTS
1. Sherwin, H.W., and Farrant, J.M. (1996). Differences in rehydration of three desiccation tolerant angiosperm species. Ann. Bot. 78, 703–710. 2. Kranner. I., Beckett, R.P., Wornik, S., Zorn, M., and Pfeifhofer, H.W. (2002). Revival of a resurrection plant correlates with its antioxidant status. Plant. J. 31, 13–24. 3. Moore. J.,Westall. K.L.,Ravenscroft, N.,Farrant, J.M.,Lindsey, G.G. and Brandt, W.F. (2004) The predominant polyphenol in the leaves of the resurrection plant Myrothamnus flabellifolius, 3, 4, 5 tri-O-galloylquinic acid, protects membranes against desiccation and free radical induced oxidation. (in review)
The authors would like to express their gratitude to the National Research Foundation and the University of Cape Town for financial support. We would also like to thank K.Cooper and R.Karreman for technical assistance. As well as M.Jaffer and M.Waldron of the UCT Electron Microscope Unit.
