SUPPLEMENTARY MATERIAL
Bioactive glycosides from the African medicinal plant Boerhavia erecta L.
Ari S. Nugrahaa, Adama Hiloub*, Nicholas Vandegraaffc, David I. Rhodesc, Rachada
Haritakund and Paul A. Kellera*
a
School of Chemistry, University of Wollongong, Wollongong, New South Wales,
Australia 2522.
b
Département de biochimie-microbiologie, UFR/VT, Université de Ouagadougou,
Burkina Faso, Africa 03
c
Avexa Limited, 576 Swan St, Richmond, Victoria, Australia 3121
d
National Centre for Genetic Engineering and Biotechnology (BIOTEC), National
Science and Technology Development Agency (NSTDA), 113 Phaholyothin Road,
Klong1, Klong Luang, Pathumanthani 12120 Thailand
Abstract
Phytochemical studies of the previously unexplored stem of Boerhavia erecta from
Burkina Faso-Africa, resulted in the isolation of an unreported glycoside 4, 2,3dihydroxypropylbenzoate-3-O-β-[4''-methoxy] glucoronide as well as seven known
glycosides (1-3, 5-8). The molecular structure of 4 was elucidated by using
comprehensive spectroscopic and spectrometric including TOCSY and MS/MS spectra.
The major isolate 5 and 8 indicated a significant inhibition against HIV-integrase (IC50
10 and 22 μg/mL, respectively). The extracts and isolates were also tested for antimalarial activity but insignificant activity was observed.
Keywords: African medicinal plant, Boerhavia erecta, Glycosides, Anti-HIV integrase
activity, Anti-malarial activity, Burkino Faso
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Table of contents
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Experimental details
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1H-NMR
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spectrum of compound 4
13APT-NMR
spectrum of compound 4 (in absolute mode)
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gCOSY spectrum of compound 4
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gTOCSY spectrum of compound 4
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gHSQC spectrum of compound 4
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gHMBC spectrum of compound 4
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gHMBC spectrum (zoom) for specific H-C correlation in sugar ring
of compound 4 (CD3OD)
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HRESIMS compound 4
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MS/MS compound 4
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Semi Prep-HPLC profile of isolation of polar constituents from Boerhavia erecta
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Experimental
General experimental procedures
Silica gel (Merck Silica Gel 60, 0.063-0.200 mm). Analytical HPLC was performed in a
Waters 1525 binary HPLC pump coupled with Waters 2487 dual λ absorbance detector.
A symmetry ® C18 column was used (4.9 x 150 mm, 5 μm). Semi-preparative HPLC
was performed in a Waters LC system coupled with Waters 2489 UV/Visible detector.
An OBD SunfireTM C18 semi-preparative HPLC column (19 x 150 mm, 5 μm) was used.
ESI-MS spectra were collected from Waters platform LCZ mass spectrometer (low
resolution) and Waters QTof Ultima mass spectrometer (high resolution). MS/MS
experiment were carried out on Waters (micromass) Quattro microTM. NMR spectra
were obtained from Varian Unity Inova-500 MHz NMR spectrometer. Biological assays
for anti-HIV activity were peformed at Avexa Pty. Ltd in Melbourne, Victoria. Testing
for anti-malarial activities were performed at National Centre for Genetic Engineering
and Biotechnology, Thailand.
Plant material
B. erecta L. plant samples were collected in the experimental garden of the institute for
rural development (IDR), University of Ouagadougou, Burkina Faso (West Africa),
2008, and identified by Pr. Millogo-Rasolodimby Jeanne. A voucher specimen was
deposited in the herbarium of the Université de Ouagadougou, Burkina Faso, Africa
under accession numbers Hilou A02. The bark was removed from the stems with a
knife, and the stem dried in the laboratory at 25-30 ºC for 36 h, and pulverized in a
laboratory mortar. The ground materials were packaged out of light and dampness.
Extraction and isolation
A slurry of the powdered stembark of B. erecta L. (350 g), in methanol (1.0 L) in a
round bottom flask (2.0 L) was stirred for 48 h and then vacuum filtered and evaporated
to dryness (repeated twice) resulting in 45.0 g of dried extract. These extracts were
further solid-liquid fractionated with EtOAc:CH2Cl2:glacial acetic acid (4.75:4.75:0.5,
15 x 20 mL). The supernatants were vacuum-dried to produce a less polar fraction A
(2.0 g) and the remaining solid was dried to give the polar fraction B (41.4 g). The polar
fraction B (each 2.0 g to a total of 12.0 g), was further chromatographed over silica gel
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(20 x 300 mm, 0.063-0.200 mm; Merck) eluted with ACN (400 mL); ACN: MeOH:H2O
(9:0.5:0.5, 200 mL); ACN: MeOH:H2O (8.5:0.75:0.75, 400 mL) to produce fraction B1
(0.38 g), B2 (0.76 g), B3 (2.93 g), B4 (0.82 g) which were separated based on colour;
brown, green, brown-yellow and yellow, respectively. The most abundant fraction, B3,
was further chromatographed by semi-preparative HPLC with solvent A (0.1% TFA in
H2O) and solvent B (0.1% TFA in 90% ACN in H2O) as eluent developed in gradient
90% A to 75% A at a flow rate 15.5 mL/min to obtain compound 1 at tR 6.8 minutes
(0.29 mg/g dried plant sample), 2 at tR = 14.7 minutes (0.17 mg/g dried plant sample), 3
at tR = 19.4 minutes (0.27 mg/g dried plant sample), 4 at tR = 27.5 minutes (0.17 mg/g
dried plant sample), 5 at tR = 31.2 minutes (2.70 mg/g dried plant sample), 6 at tR = 33.8
minutes (0.28 mg/g dried plant sample), 7 at tR = 37.2 minutes (0.40 mg/g dried plant
sample) and compound 8 at tR = 37.8 minutes (1.23 mg/g dried plant sample).
Compound 4
Pale yellow solid, 0.17 mg/g dried plant sample; m.p.: burnt at 295ºC; 1H-NMR
(CD3OD, 500 MHz), 8.06 (d, 3J = 7.5, Hz, 2H, H2' and H6'), 7.61 (dd, 3J = 7.5, 7.5 Hz,
1H, H4'), 7.48 (dd, 3J = 7.5, 7.5 Hz, 2H, H3' and H5'), 4.85 (d, 3J = 3.0, Hz, 1H, H1''),
4.41 (dd, 2J = 11.5, 3J = 4.5 Hz, 1H, H1a), 4.36 (dd, 2J = 11.0, 3J = 5.5 Hz, 1H, H1 a),
4.16 (m, 1H, H2), 4.05 (d, 3J = 10.0 Hz, 1H, H5''), 3.88 (dd, 2J = 9.5, 3J = 4.5 Hz, 1H,
H3 a), 3.72 (d, 3J = 3.5 Hz, 1H, H3''), 3.52 (dd, 2J = 9.5, 3J = 3.5 Hz, 1H, H3b), 3.46 (s,
OCH3), 3.45 (m, 1H, H2''), 3.27 (d, 3J= 10.0 Hz, 1H, H4''). 13C-NMR (CD3OD, 125
MHz), 173.0 (C6''a, C=O), 168.0 (C1'a, C=O), 134.3 (C4'), 131.2 (C1'), 130.7 (C2' and
C6'), 129.6 (C3' and C5'), 101.0 (C1''), 83.0 (C4''), 74.5 (C5''), 73.4 (C2''), 71.4 (C3''),
70.7 (C3), 69.6 (C2), 66.7 (C1), 60.8 (OCH3). ESIMS: m/z 409 (M+Na)+. HRESI MS:
calculated for C17H22O10Na (M+Na)+: 409.1106, found 409.1107.
Selected proton-carbon correlations in molecular structure of 4
Bioactivity testing
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Extract fractions were tested for anti-HIV integrase activity using purified HIV-1
integrase in a combined 3'-processing strand transfer assay as described by Ovenden et
al. without modification (Ovenden et al., 2004). Anti-malarial activity was determined
against Plasmodium falciparum K1 based on microculture radioisotope technique
described by Desjardins et al (Desjardins et al., 1979).
References
Desjardins RS, Canfeld CJ, Haynes JD, Chulay JD. 1979. Quantitative assessment of antimalarial activity
in vitro by a semi automated microdilution technique. Antimicrob. Agents Chemother. 16: 710-718.
Ovenden SPB, Yu J, San Wan S, Sberna G, Murray Tait R, Rhodes D, Cox S, Coates J, Walsh NG,
Meurer-Grimes BM. 2004. Globoidnan A: a lignan from Eucalyptus globoidea inhibits HIV
integrase. Phytochem. 65: 3255-3259.
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1H-NMR
spectrum of compound 4 (CD3OD)
-OCH3
δ 3.46 ppm
13APT-NMR
spectrum of compound 4 (in absolute mode, CD3OD)
-OCH3
δ 60.79 ppm
In the previously reported ester of glycerol α-D-glucoronide, the OCH3 was presented as singlet peak at δ
3.66 and 52.8 ppm in 1H and 13C-NMR, respectively (Maggi et.al, 2009).
Maggi A, Taskova R, Gotfredsen CH, Bianco A, Jensen SR. 2009. Chemical markers in Veronica sect.
Hebe. III. Biochem. System. Ecol. 37: 731–736
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gCOSY spectrum of compound 4 (CD3OD)
TOCSY spectrum of compound 4 (CD3OD)
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gHSQC spectrum of compound 4 (CD3OD)
gHMBC spectrum of compound 4 (CD3OD)
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gHMBC spectrum (zoom) for specific H-C correlation in sugar ring of compound 4
(CD3OD)
C4′′
OCH3
+
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HRESIMS spectrum of compound 4
ESIMS/MS spectrum of compound 4
Semi preparative HPLC profile of isolation of polar constituent from Boerhavia
erecta
5
8
1
2
3
4
Note: Chromatogram was collected at λ 254 nm
S 10
6
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