A new stilbene glycoside from Elephantorrhiza goetzei

Article abstract of DOI:10.1016/S0367-326X(01)00295-7

A new stilbene glycoside, 5-methoxy-(E)-resveratrol 3-O-rutinoside (1) was isolated from the root bark of Elephantorrhiza goetzei, along with known compounds, namely gallic acid, (E)-resveratrol, (±)-catechin, (±)-gallocatechin and oleanene triterpenoids. The combined ethyl acetate-methanol extract exhibited high lethality against brine shrimp larvae (LC₅₀ 10.8 ppm) compared to the isolates, some of which were not active. Copyright

Full text of DOI:10.1016/S0367-326X(01)00295-7

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Fitoterapia 72 2001 649᎐655
A new stilbene glycoside from
Elephantorrhiza goetzei
Cornelius C.W. Wanjala, Runner R.T. MajindaU
Department of Chemistry, Uni¨ersity of Botswana, Pri¨ate Bag 00704, Gaborone, Botswana
Received 24 January 2001; accepted in revised form 19 March 2001
Abstract
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.
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A new stilbene glycoside, 5-methoxy- E -resveratrol 3-O-rutinoside 1 was isolated from
the root bark of Elephantorrhiza goetzei, along with known compounds, namely gallic acid,
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E -resveratrol, " -catechin, " -gallocatechin and oleanene triterpenoids. The combined
ethyl acetate-methanol extract exhibited high lethality against brine shrimp larvae LC50
10.8 ppm compared to the isolates, some of which were not active. ᮊ 2001 Elsevier Science
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B.V. All rights reserved.
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Keywords: Elephantorrhiza goetzei; Stilbenes; Flavonoids; Phenolics; Triterpenoids; 5-Methoxy- E -
resveratrol 3-O-rutinoside
1. Introduction
The genus Elephantorrhiza consists of shrubs, small trees or low bushes springing
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from underground rhizomes 1 . In Southern Africa four species of Elephantorrhiza,
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namely E. burkei, E. elephantina, E. goetzei and E. suffruticosa are known 2 . E.
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goetzei Harms Fabaceae is a tree that grows to over 8 m, found in North Eastern
Botswana and is used by the local communities as a remedy for sores of the penis
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x
and vulva, irregular menstruation and for cleansing the womb after abortion 2,3 .
E. goetzei root previously investigated was found to contain flavan-3-ols and the
extractives showed activity against gram-positive and gram-negative bacteria and
U Corresponding author. Tel.: q267-355-2503; fax: q267-355-2836.
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E-mail address: majindar@mopipi.ub.bw R.R. Majinda .
0367-326Xr01r$ - see front matter ᮊ 2001 Elsevier Science B.V. All rights reserved.
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PII: S 0 3 6 7 - 3 2 6 X 0 1 0 0 2 9 5 - 7

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C.C.W. Wanjala, R.R.T. Majinda rFitoterapia 72 2001 649᎐655
650
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fungi 4 . In this paper, we report the isolation of stilbenes, flavan-3-ols, triter-
penoids and gallic acid.
2. Experimental
2.1. General methods
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Melting point: Stuart Scientific SMP1 melting point apparatus; Specific rota-
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D: Polatronic-D Schimdt q Haensch polarimeter; UV: Shimadzu UV-
tion
␣
2101PC spectrophotometer; IR: Perkin-Elmer 2000 FT-IR spectrometer. The one-
dimensional H 300 MHz , C 75.4 MHz , DEPT and two-dimensional COSY,
HMQC, HMBC spectra acquired on Bruker Avance DPX 300 spectrometer and
1
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referenced to residual solvent signal. MS: HRMS was done on autospec TOF
spectrometer. EI and ESIMS on Finnigan MAT SSQ 700 single quadrupole
instrument. Column chromatography-silica gel 60 particle size 0.040᎐0.063 mm for
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column chromatography Merck ; vacuum liquid chromatography VLC -silica gel
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HF254 5᎐15 ␮m mesh Merck ; Sephadex LH-20 Sigma ; preparative TLC-silica
gel 60 PF254 for preparative layer chromatography Merck ; analytical TLC-TLC
silica gel 60-F254 pre-coated alumina sheets Merck and visualized using UV 254
and 366 nm and vanillin-sulfuric acid spray.
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2.2. Plant material
E. goetzei roots were collected from Mapoka, North East District, Botswana, in
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August 1997 and identified by Dr L.M. Turton. A voucher specimen No. 3393 was
deposited at the University of Botswana Herbarium.
2.3. Extraction and isolation
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Air-dried and powdered root bark 2.0 kg was extracted sequentially to exhaus-
tion with CH2Cl2 , EtOAc and MeOH. The solvents were removed to give the
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crude extracts 5, 10 and 25 g , respectively. The EtOAc and MeOH extracts, giving
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relatively similar TLC profiles CHCl3rMeOHrHOAcrH2O 70:26:2:2 , were
combined 35 g and part of this 15 g was subjected to VLC using CH2Cl2 with
increasing amounts of MeOH stepwise to give fractions A CH2Cl2rMeOH 1:1, 3
g and B MeOH, 4 g . Fraction A was further separated by gel filtration on
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Sephadex LH-20 with CHCl3rMeOH 1:1 and the concentrated eluate was resolved
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by PTLC with CHCl3rMeOHrHOAcrH2O 70:26:2:2 , to give E -resveratrol 80
. w x
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mg 5 and gallic acid 110 mg . Fraction B was further separated by gel filtration
on Sephadex LH-20 with MeOH to give fractions B1 and B2. Fraction B1 was
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resolved by PTLC CHCl3rMeOHrHOAcrH2O 70:26:2:2 to give " -catechin
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. w x Ž w x
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. w
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70 mg 6,7 , " -gallocatechin 85 mg 6,8 and the triterpenoids sericoside 9
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w
x Ž w x Ž
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100 mg , bellericoside 10 20 mg and arjungenin 9 40 mg . Fraction B2 was
further purified on gradient Si-gel CC and PTLC by multiple development

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C.C.W. Wanjala, R.R.T. Majinda rFitoterapia 72 2001 649᎐655
651
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CHCl3rMeOHrH2OrHOAc 70:26:2:2 to give 5-methoxy- E -resveratrol 3-O-
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rutinoside 1 90 mg and E -resveratrol 3-O-rutinoside 11 400 mg .
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2.4. Methylation of E -res¨eratrol 3-O-rutinoside
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Diazomethane in ether 12,13 100 ml was added dropwise with swirling to an
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ice-cooled solution of E -resveratrol 3-O-rutinoside 200 mg . After 6 h, at
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complete reaction of the starting product TLC monitored , the mixture was
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concentrated and resolved by PTLC to afford 4Ј,5-dimethoxy- E -resveratrol 3-O-
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rutinoside 150 mg 14 , 5-methoxy- E -resveratrol 3-O-rutinoside 1 43 mg and
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4Ј-methoxy- E -resveratrol 3-O-rutinoside 2 9 mg .
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E -Res¨eratrol 3-O-rutinoside. White crystals MeOH , mp 188᎐190ЊC uncor-
.
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␣
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y30.2 c 0.1,
rected ; Rf 0.30, CHCl3rMeOHrHOAcrH2O 70:26:2:2;
D
.
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MeOH ; UV max MeOH : 317 log ␧ 4.41 , 305 4.42 ; qNaOMe 336, 340 sh ;
y1
1
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qNaOAc 317, 306 nm; IR bands KBr : 3353, 2920, 1599 cm ; H-NMR 300
.
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.
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MHz, DMSO-d6 : H-2 6.65, br s , H-4 6.38, br s , H-6 6.65, br s , H-␣ 6.87, d, J
.
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16.3 Hz , H-␤ 7.02, d, J 16.3 Hz , H-2Ј,6Ј 7.42, d, J 8.3 Hz , H-3Ј,5Ј 6.78, d, J 8.3
.
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Hz , glucosyl H-1 4.84, d, J 7.2 Hz , H-2 3.30᎐3.33 , H-3 3.30᎐3.33 , H-4
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3.40᎐3.45 , H-5 3.40᎐3.45 , H-6 3.86, 3.40᎐3.45 , rhamnosyl H-1 4.60, br s , H-2
3.40᎐3.45 , H-3 3.68 m , H-4 3.30᎐3.33 , H-5 3.40᎐3.45 , H-6 1.12, d, J 6.0 Hz ;
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C-NMR 75.4 MHz, DMSO-d6 : C-1 140.2 , C-2 107.6 , C-3 159.7 , C-4 103.7 ,
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C-5 159.2 , C-6 106.3 , C-␣ 126.0 , C-␤ 129.4 , C-1Ј 128.8 , C-2Ј,6Ј 128.9 ,
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C-3Ј,5Ј 116.5 , C-4Ј 158.2 , glucosyl C-1 101.5 , C-2 74.1 , C-3 77.3 , C-4 70.4 ,
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C-5 76.2 , C-6 67.1 , rhamnosyl C-1 101.5 , C-2 71.6 , C-3 71.2 , C-4 73.0 , C-5
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69.2 , C-6 18.7 ; HRTOFMS mrz: 536.1631 calcd. for C26 H32O12 , 536.1682 .
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5,4Ј-Dimethoxy- E -res¨eratrol 3-O-rutinoside. White crystals MeOH , mp
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w x
126᎐129ЊC uncorrected ; Rf 0.35, CHCl3rMeOHrHOAcrH2O 70:26:2:2; ␣
D

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C.C.W. Wanjala, R.R.T. Majinda rFitoterapia 72 2001 649᎐655
652
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y14.3 c 0.3, MeOH ; UV max MeOH : 316 log ␧ 4.58 305 4.58 ; qNaOMe
y1
1H-NMR 300 MHz,
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;
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316, 305 nm; IR bands KBr : 3422, 2922, 1594 cm
CD3OD : H-2 6.84, br s , H-4 6.60, br s , H-6 6.78, br s , H-␣ 6.95, d, J 16.4 Hz ,
H-␤ 7.07, d, J 16.4 Hz , H-2Ј,6Ј 7.47, d, J 8.6 Hz , H-3Ј,5Ј 6.93, d, J 8.6 HZ ,
4Ј-MeO 3.79, s , 5-MeO 3.82, s , glucosyl H-1 4.90, d, J 7.2 Hz , H-2 3.30᎐3.33 ,
H-3 3.30᎐3.33 , H-4 3.40᎐3.45 , H-5 3.40᎐3.45 , H-6 3.86, 3.40᎐3.45 , rhamnosyl
H-1 4.73, br s , H-2 3.40᎐3.45 , H-3 3.68 m , H-4 3.30᎐3.33 , H-5 3.40᎐3.45 ,
H-6 1.12, d, J 6.2 Hz ; C-NMR 75.4 MHz, CD3OD : C-1 141.1 , C-2 108.6 ,
C-3 162.2 , C-4 103.2 , C-5 160.8 , C-6 106.5 , C-␣ 127.2 , C-␤ 129.9 , C-1Ј
.
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13
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131.2 , C-2Ј,6Ј 128.9 , C-3Ј,5Ј 115.1 , C-4Ј 160.2 , 4Ј-MeO 55.7 , 5-MeO 55.9 ,
glucosyl C-1 102.3 , C-2 74.8 , C-3 77.8 , C-4 71.3 , C-5 76.7 , C-6 67.5 ,
rhamnosyl C-1 102.0 , C-2 72.3 , C-3 72.0 , C-4 74.0 , C-5 69.7 , C-6 17.9 ;
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HRTOFMS mrz: 564.2206 calcd. for C28 H36O12 , 564.2207 ; EIMS mrz: 564 70 ,
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.
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418 15 , 270 65 228 75 .
.
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5-Methoxy- E -res¨eratrol 3-O-rutinoside 1 . White crystals MeOH , mp
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.
w x
160᎐162ЊC uncorrected ; Rf 0.33, CHCl3rMeOHrHOAcrH2O 70:26:2:2; ␣
D
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.
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.
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.
20.3 c 0.2, MeOH ; UV max MeOH 317 log ␧ 4.15 , 304 4.29 ; qNaOMe 345,
337 sh nm; IR bands KBr : 3413, 2921, 1590 cmy1; HRTOFMS mrz: 550.2053
.
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.
calcd. for C27 H34O12 , 550.2050 ; H and 13C-NMR: see Table 1.
1
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4Ј-Methoxy- E -res¨eratrol 3-O-rutinoside 2 . White crystals MeOH , mp
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w x
166᎐169ЊC uncorrected ; Rf 0.33, CHCl3rMeOHrHOAcrH2O 70:26:2:2; ␣
D
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y27.1 c 0.3, MeOH ; UV max MeOH 317 log ␧ 4.21 , 305 4.30 ; qNaOMe
340, 332 sh nm; IR bands KBr : 3414, 2921, 1591 cmy1; HRTOFMS mrz:
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550.2053 calcd. for C27 H34O12 , 550.2051 ; H and 13C-NMR: see Table 1.
1
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2.5. Studied acti¨ity
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Cytotoxicity by brine shrimp Artemia salina lethality according to Meyer et al.
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15 on the combined ethyl acetate-methanol extract and isolated compounds.
3. Results and discussion
w
xq
HRTOFMS of 1 gave molecular ion peak mrz M 550.2053, consistent with
1
13
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the molecular formula C27 H34O12. H and C-NMR spectra Table 1 indicated 1
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had an E -resveratrol aglycone moiety by showing signals consistent with those
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published for E -resveratrol glucoside 5,11 . The assignments in Table 1 were
confirmed by HMQC, HMBC and COSY spectra. The H-NMR spectrum showed
1
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signals for two trans olefinic protons at ␦ 6.89 H-␣, d, J s 16.0 Hz and ␦ 7.05
H-␤, d, J s 16.0 Hz . The 13C-NMR of 1 gave 27 carbon signals and the DEPT
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spectrum together with HMQC data confirmed that 22 of these were protonated.
DEPT spectrum also showed two methyl, one methylene and 19 methine carbons.
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In the HMBC spectrum, the H-␣ proton showed a cross-peak to C-2 ␦ 108.5 ppm
and C-6 ␦ 106.4 ppm carbon signals of the A ring. The H-␤ proton signal in turn
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C.C.W. Wanjala, R.R.T. Majinda rFitoterapia 72 2001 649᎐655
653
Table 1
1
H 300 MHz and 13C-NMR data for compounds 1 and 2 in CD3ODa
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Position
1
2
␦
␦
␦
␦
C
H
C
H
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s
1
2
3
4
5
6
␣
141.3
s
141.2
Ž .
Ž .
108.4 d
6.82, br s
6.58, br s
6.76, br s
108.5 d
6.83, br s
6.59, br s
6.77, br s
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s
162.2
s
162.1
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103.3 d
103.0 d
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Ž .
s
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d
160.2
s
159.7
106.9
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106.4 d
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.
.
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.
.
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126.4 d
6.89, d 16.0
126.4 d
6.94, d 16.4
Ž
7.04, d 16.4
Ž
Ž .
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129.9 d
␤
7.05, d 16.0
130.2 d
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s
1Ј
130.1
s
130.3
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.
.
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.
.
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128.9 d
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116.7 d
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s
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55.7 q
2Ј,6Ј
3Ј,5Ј
4Ј
4Ј-OCH3
5-OCH3
7.39, d 8.4
128.9 d
7.45, d 8.6
Ž
Ž .
Ž
6.86, d 8.4
116.5 d
6.92, d 8.6
Ž .
s
158.4
160.5
3.79, s
Ž .
55.9 q
3.82, s
Glucose
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.
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.
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102.3 d
1
2
3
4
5
6
4.90 7.2
102.3 d
4.90 7.3
Ž .
74.8 d
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74.8 d
3.30᎐3.33
3.30᎐3.33
3.40᎐3.45
3.40᎐3.45
3.30᎐3.33
3.30᎐3.33
3.40᎐3.45
3.40᎐3.45
Ž .
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77.8 d
77.8 d
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71.3 d
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71.3 d
Ž .
Ž .
76.7 d
76.7 d
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67.5 t
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67.5 t
3.86, 3.40᎐3.45
3.86, 3.40᎐3.45
Rhamnose
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Ž .
102.0 d
1
2
3
4
5
6
4.73, br s
3.40᎐3.45
3.68, m
102.0 d
4.73, br s
3.40᎐3.45
3.68, m
3.30᎐3.33
3.40᎐3.45
Ž .
72.3 d
Ž .
72.3 d
Ž .
Ž .
72.0 d
72.0 d
Ž .
74.0 d
Ž .
74.0 d
3.30᎐3.33
3.40᎐3.45
Ž .
Ž .
69.7 d
69.7 d
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.
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17.8 q
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1.21, d 6.3
Ž .
17.9 q
1.21, d 6.4
aAssignments were confirmed by HMQC, HMBC and DEPT experiments.
Ž
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showed HMBC correlations to carbon signals C-2Ј,6Ј ␦ 128.9 ppm , thus con-
firming the positions of the A and B aromatic rings. The glucopyranosyl anomeric
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proton signal ␦ 4.90 d, J s 7.2 Hz showed HMBC correlation with C-3 ␦ 162.2
.
ppm , confirming that the O-glucopyranose moiety which is attached to resveratrol
A ring through the anomeric carbon. The rhamnopyranosyl anomeric proton signal
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.
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␦ 4.73 br s showed HMBC correlation with glucose moiety C-6 ␦ 67.5 ppm ,
confirming that the O-rhamnopyranose moiety is 1 ª 6 attached to glucopyranose.
The carbon signal at ␦ 158.4 ppm was confirmed as the C-4Ј signal by its
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three-bond connectivity to the two proton doublet at ␦ 7.39 H-2Ј and H-6Ј which
are protons of the phenolic ring B and the methoxyl protons ␦ 3.82 showed HMBC
correlation with C-5 ␦ 160.2 . In contrast to the glycosylation of flavonoids which
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C.C.W. Wanjala, R.R.T. Majinda rFitoterapia 72 2001 649᎐655
654
causes an upfield shift of the ipso carbon, glycosylation in this stilbene has caused
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a downfield shift relative to the substituted C-5 MeO-bearing carbon 16,17 . The
␤-D-glucopyranosyl and ␣-L-rhamnopyranosyl moieties were evident from the 12
carbon signals from 101.5 to 18.7 ppm which were in agreement with literature
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x
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values 16,17 . The doublet ␦ 4.90 J s 7.2 Hz for the anomeric proton signal gave
confirmation of the ␤-anomeric configuration of glucopyranose and the broad
singlet ␦ 4.73 for the anomeric proton signal confirmed the ␣-anomeric configura-
tion of rhamnopyranose.
Ž .
Methylation of E -resveratrol 3-O-rutinoside was necessary in order to assign
the C-3, C-5 and C-4 carbons of the stilbenes isolated unambiguously with the help
of the HMBC spectral expansions.
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HRTOFMS of 2 gave molecular ion peak mrz M 550.2053, consistent with
13
the molecular formula C27 H34O12. The 1H and C-NMR spectra Table 1 of
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compound 2 were similar to those of 1 except for the MeO group showing HMBC
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correlation with C-4Ј ␦ 160.5 ppm . Thus, compound 2 was identified as 4Ј-
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methoxy- E -resveratrol 3-O-rutinoside.
Both compounds 1 and 2 showed no lethality on brine shrimp larvae at concen-
trations of up to 1000 ppm, while a LD50 of 10.8 ppm was determined for the
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combined ethyl acetate-methanol extract Table 2 .
Table 2
Lethality of extractives from Elephantorrhiza goetzei root bark to brine shrimp larvae on exposure for
24 h
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Extractive
LC50 24 ppm
Combined EtOAc-MeOH extract
10.8
25.3
20.3
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.
" -Catechin
.
" -Gallocatechin
.
E -Resveratrol
56.8
.
E -Resveratrol 3-O-rutinoside
398.7
4 107
4 107
4 107
126.3
213.2
245.5
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.
( )
( )
5-Methoxy- E -resveratrol 3-O-rutinoside 1
4Ј-Methoxy- E -resveratrol 3-O-rutinoside 2
4Ј,5-Dimethoxy- E -resveratrol 3-O-rutinoside
Ž
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.
Sericoside
Bellericoside
Arjungenin
Acknowledgements
C.C.W.W. would like to thank the UNESCO-ROSTA through Deutscher
Ž
.
Akademischer Austauschdienst DAAD , Germany for a Scholarship and Jomo
Kenyatta University of Agriculture and Technology, Kenya for a study leave and

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C.C.W. Wanjala, R.R.T. Majinda rFitoterapia 72 2001 649᎐655
655
Ž
R.R.T.M. would like to thank the IFS for a research grant. Dr L.M. Turton retired
.
botanistrchemist, University of Botswana is acknowledged for identifying the
plant.
References
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