Notes
Journal of Natural Products, 2004, Vol. 67, No. 11 1937
70% with H2O and partitioned with EtOAc. Both fractions
were subjected to chromatographic separations to isolate the
individual components. The hexane fraction (25 g) was chro-
matographed on a silica gel column using hexane/EtOAc
mixtures of increasing polarity (0 to 100% EtOAc) to yield five
secondary fractions (H1-H5). Fraction H4 was chromato-
graphed on a silica gel column eluted with hexane/EtOAc (8:
2) to yield pure 3 (201 mg, 0.01%). Fraction H2 (1.950 g) was
chromatographed under the same conditions given above,
which yielded three fractions, H21-H23. Fraction H22 (1.370
g) was crystallized from EtOAc/hexane mixtures and yielded
lupeol (1.250 g, 0.062%). Fraction H3 (140 mg) was purified
on a silica gel preparaive-TLC plate using toluene (100%),
which yielded pure 2 (36 mg, 0.0018%). The EtOAc partition
(16 g) was chromatographed on a silica gel column eluted with
hexane/EtOAc mixtures of increasing polarity, which yielded
six fractions (E1-E6). Fraction E6 was chromatographed on
a silica gel column using CHCl3/MeOH mixtures as eluents,
which resulted in three fractions, E61-E63. Fraction E62 (56
mg) crystallized from MeOH and yielded 1 (30 mg, 0.0015%).
Fraction E2 (789 mg) was chromatographed on a silica gel
column using hexane/EtOAc (9:1) and yielded betulin (564 mg,
0.028%).
Octahydroeuclein (1,8,1′,5′-tetrahydroxy-6,3′-dimeth-
yl-2,3,6′,7′-tetrahydro-1H,5′H-[2,2′]binaphthalenyl-4,8′-di-
one) (1): amber yellow crystals; mp 238-242 °C; [R]D25 +33.0°
(c 0.03; CHCl3); IR (KBr) νmax 3500, 3310, 2930, 2860, 1680,
1595, 1265, 1180 cm-1; UV (MeOH) λmax 267 nm (log ꢀ 3.43),
336 nm (log ꢀ 1.88); 1H NMR (500 MHz, CDCl3) δ 12.2 (1H, s,
OH-1′), 6.93 (1H, s, H-4′), 6.69 (1H, s, H-7), 6.65 (1H, s, H-5),
4.73 (1H, dd, J ) 5.0, 6.0 Hz, H-5′), 4.70 (1H, d, J ) 3.4 Hz,
H-1), 4.10 (1H, dd, J ) 17.5, 13.5 Hz, H-3â), 3.70 (1H, ddd, J
) 13.5, 3.6, 3.4 Hz, H-2R), 2.90 (1H, ddd, 17.8, 7.3, 4.9 Hz,
H-7′a), 2.60 (1H, ddd, 17.8, 8.0, 4.9 Hz, H-7′b), 2.50 (1H, dd, J
) 17.5, 3.6 Hz, H-3R), 2.30 (3H, s, H-11′), 2.28 (3H, s, H-11),
2.25 (1H, m, H-6′a), 2.06 (1H, m, H-6′b); 13C NMR (75 MHz,
CDCl3) δ 204.7 (s, C-8′), 204.3 (s, C-4), 160.7 (s, C-1′, C-8), 148.5
(s, C-6), 147.4 (s, C-3′), 144.7 (s, C-10′), 144.1 (s, C-9), 125.3
(s, C-2′), 121.1 (d, C-4′), 120.5 (d, C-5), 117.8 (d, C-7), 113.8 (s,
C-9′), 112.9 (s, C-10), 71.5 (d, C-1), 66.5 (d, C-5′), 40.3 (d, C-2),
36.2 (t, C-3), 34.7 (t, C-7′), 31.1 (t, C-6′), 21.7 (q, C-11, C-11′);
EIMS m/z 382 [M]+ (9), 364 (100), 346 (31), 329 (4), 219 (5),
201 (12), 188 (9), 177 (11), 164 (9), 135 (21), 107 (5); HREIMS
m/z 382.14217 [M]+ (calcd for C22H22O6, 382.14164).
Figure 1. HMBC correlations of 1.
(δΗ 1.00, 0.95, 0.93, 0.90, 0.88, and 0.77), including an
isopropenyl group [δΗ 1.66 (Me-30), 4.52 and 4.65 (H-29)],
an (E)-isoferulate moiety [δ 3.90 (OMe), 6.26 and 7.56 (H-
8′, H-7′), three aromatic protons 6.88 (H-5′), 7.01 (H-2′),
and 7.04 (H-6′)], a methine proton bearing an ester δ 4.45
(obscured by olefinic protons), and a typical lupenol Hâ-19
proton signal (δΗ 2.33).15 By comparison of the NMR data
with those of lupeol, which has been isolated from the same
source, compound 2 was assigned as a lupeol derivative
with an extra (E)-isoferuloyl moiety at C-3. The position
of the methoxy group of the isoferuloyl moiety at C-4′ was
confirmed by a HMBC experiment, which showed correla-
tions of MeO/C-4′, H-2′/C-4′, and H-6′/C-4′. The relative
configurations of C-3 could not determined from the
recorded spectra of 2; however, hydrolysis of 2 gave lupeol
with a OH-3â configuration, which was identified on the
basis of comparison of its mp, [R]D, and 1H NMR spectrum
with literature values.15
All compounds were tested for their antibacterial and
antimycobacterial (Mycobacterium tuberculosis) activity,
but only shinanolone (3) exhibited inhibitory activity
against Gram-positive bacteria (Bacillus pumilus ATCC
27142, Bacillus subtilis ATCC 6633, Staphylococcus aureus
ATCC 25923, and Streptococcus faecalis) and a drug-
sensitive strain of M. tuberculosis (MRC strain no. H37Rv
ATCC27294), at a concentration of 0.1 mg/mL. The new
binaphthoquinone, octahydroeuclein (1), was not active,
while the new triterpenoid 20(29)-lupene-3â-isoferulate (2)
showed activity only against B. pumilus (0.1 mg/mL). The
reference antibiotic, streptomycin sulfate, inhibited the
growth of all bacterial species tested in this study at 0.01
mg/mL, except Pseudomonas aeruginosa and Serratia
marcescens, which were inhibited at 0.05 and 0.1 mg/mL,
respectively. M. tuberculosis was found to be susceptible
to the drugs streptomycin and ethambutol at concentra-
tions of 0.004 and 0.006 mg/mL, respectively.
20(29)-Lupene-3â-isoferulate (2): colorless crystals; mp
115-118 °C; [R]D25 +110.2° (c 0.05; CHCl3); IR (KBr) νmax 3500,
3310, 2930, 2860, 1680, 1595, 1265, 1180 cm-1; UV (MeOH)
λmax 294 nm (log ꢀ 1.8); 1H NMR (300 MHz, CDCl3) δ 7.56 (1H,
d, J ) 16.0 Hz, H-7′), 7.04 (1H, dd, J ) 8.1, 1.5 Hz, H-6′), 7.01
(1H, d, J ) 1.5 Hz, H-2′), 6.88 (1H, d, J ) 8.1 Hz, H-5′), 6.26
(1H, d, J ) 16 Hz, H-8′), 4.65 (1H, br s, H-29a), 4.52 (1H, br
s, H-29b), 4.45 (1H, m, H-3), 3.90 (1H, s, OMe), 2.33 (1H, td,
5.6, 5.6 Hz, H-19), 1.66 (3H, s, Me-30), 1.00 (3H, s, Me-24),
0.95 (3H, s, Me-23), 0.93 (3H, s, Me-26), 0.90 (3H, s, Me-27),
0.88 (3H, s, Me-25), 0.77 (3H, s, Me-28); 13C NMR (75 MHz,
CDCl3) δ 166.0 (s, C-9′), 150.8 (s, C-20), 146.8 (s, C-4′), 145.7
(s, C-3′, C-7′), 126.5 (s, C-1′), 126.1 (d, C-6′), 115.7 (d, C-8′),
113.6 (d, C-5′), 108.3 (d, C-2′), 108.2 (t, C-29), 79.1 (d, C-3),
54.9 (d, C-5), 54.8 (q, OMe), 49.4 (d, C-9), 47.3 (d, C-18, C-19),
42.0 (s, C-14), 41.8 (s, C-17), 40.7 (s, C-8), 39.9 (t, C-22), 38.4
(t, C-1), 37.4 (s, C-4, -13), 37.0 (s, C-10), 35.6 (t, C-16), 34.6
(t, C-7), 30.0 (t, C-21), 28.3 (q, C-23), 27.3 (t, C-15), 26.4 (t,
C-12), 23.9 (t, C-2), 22.5 (t, C-11), 19.9 (q, C-30), 18.3 (t, C-6),
17.2 (q, C-28), 15.8 (q, C-25), 15.6 (q, C-26), 15.0 (q, C-24), 14.5
(q, C-27); EIMS m/z 602 [M]+ (20), 379 (23), 203 (65), 177 (100),
149 (30), 135 (70); HREIMS m/z 602.44562 [M]+ (calcd for
C30H58O4, 602.43351).
Experimental Section
General Experimental Procedures. Melting points are
uncorrected. Optical rotations were measured with a Perkin-
Elmer 141 polarimeter. IR spectra were recorded on a Perkin-
Elmer 1310 spectrophotometer. UV spectra were recorded
using a Pharmacia LKB-ultraspec 111 UV spectrophotometer.
NMR spectra were recorded using a Bruker ARX 300 or a
Bruker Avance DRX 500 MHz. Mass spectra were obtained
with a JEOL JMS-AX505 W mass spectrometer. Silica gel
[Merck, Kieselgel 60 (0.063-0.200 mm) and (0.015-0.040
mm)] was used for column chromatography. Silica gel plates
(Merck, Kieselgel 60 F254) were used for TLC.
Plant Material. E. natalensis roots were collected from
Tembe Elephant Park in KwaZulu-Natal Province of South
Africa in May 2001. The plant material was identified kindly
by Prof. AE van Wyk, and a voucher specimen (N.L. 22) was
deposited in the Schweickerdt Herbarium (PRU), Pretoria,
South Africa.
25
1
Shinanolone (3):16 [R]D -8.6° (c 0.02; CHCl3); H NMR
(300 MHz, CDCl3) δ 12.37 (1H, s, OH-4), 6.82 (1H, s, H-5),
6.71 (1H, s, H-7), 4.85 (1H, dd, J ) 3.6, 7.4 Hz, H-4), 2.94 (1H,
ddd, 13.0, 8.2, 4.8 Hz, H-2â), 2.60 (1H, ddd, 13.0, 8.3, 3.8 Hz,
H-2R), 2.33 (3H, s, Me-11), 2.30 (1H, m, H-3a), 2.06 (1H, m,
H-3b); 13C NMR (75 MHz, CDCl3) δ 203.5 (s, C-1), 162.8 (s,
C-8), 148.7 (s, C-4a), 145.0 (s, C-6), 118.5 (d, C-5), 117.3 (d,
Extraction and Isolation. E. natalensis fresh root bark
was separated mechanically, homogenized with ethanol, and
extracted for 72 h (twice). The total ethanolic extracts were
combined and concentrated under reduced pressure, then
partitioned with n-hexane. The ethanolic layer was diluted to