Minor biflavonoids from Lophira alata leaves

Article abstract of DOI:10.1021/np050169w

The leaves of the Cameroonian medicinal plant Lophira alata afforded two new biflavonoids, lophirone L (1) and lophirone M (2), and the known luteolin and lithospermoside. Both biflavonoids were obtained in small quantities, and their structures show some

Full text of DOI:10.1021/np050169w

1206
J. Nat. Prod. 2006, 69, 1206-1208
Minor Biflavonoids from Lophira alata Leaves
Anastasie Ewola Tih,† Raphael Tih Ghogomu,*,† Beibam Lucas Sondengam,† Christelle Caux,‡ and Bernard Bodo‡
UniVersity of Yaounde I, P.O. Box 812, Yaounde, Cameroon, and ESA 5154, USM 502, 63 Rue Buffon, 75005 Paris, France
ReceiVed May 17, 2005
The leaves of the Cameroonian medicinal plant Lophira alata afforded two new biflavonoids, lophirone L (1) and
lophirone M (2), and the known luteolin and lithospermoside. Both biflavonoids were obtained in small quantities, and
their structures show some new and unusual biflavonoid diversity.
Lophira alata Banks ex Gaertn (Ochnaceae) grows as tall trees
in the tropical rain forest of Africa. Its stem bark is exploited for
medicinal uses, and its heartwood is sold as first-grade timber. While
searching for plant extracts in the Cameroon flora with antitumor
activity, the MeOH extract of the stem bark of L. alata showed
significant suppressive activity on Epstein Barr virus.1 Some of
the polyflavonoids identified in earlier phytochemical investigations
show significant antimicrobial activity.2-9 Our investigation has
been extended to the leaf extract (MeOH), from which luteolin and
lithospermoside10 were identified as major constituents. We have
now carried out further examination on the same extract obtained
using plant material from a different habitat and characterized four
constituents. Two are new biflavonoids, for which structures 1 and
2 have been assigned from spectroscopic and chemical data.
The solvent-free MeOH extract of air-dried leaves of L. alata
was washed with warm EtOAc to give a soluble EtOAc fraction.
After concentration to dryness, the resultant powder was fractionated
into four fractions (F1, F2, F3 and F4) by column chromatography
on Sephadex LH-20 (MeOH). The purification of fraction F4 by
silica gel column chromatography first gave luteolin and litho-
spermoside, both identified by comparison with authentic samples.
The resultant unseparated subfractions were purified by repeated
preparative TLC on silica gel plates. The technique of multiple
migration led to the isolation of compounds 1 and 2. Both gave a
brick red color with Mg/HCl and a deep blue color with aqueous
FeCl3 solution, suggesting that they are flavonoids.
Compound 1, obtained as an amorphous yellow solid, is a
biflavonoid with molecular mass 538. Its molecular formula,
C30H18O10, accounting for 22 unsaturation sites, is deduced from
its [M + H]+ ion, which appeared at 539.0974 in the HRCIMS. It
showed intense IR absorptions indicative of the presence of
hydroxyls (3390 and 3250 cm-1), conjugated and hydrogen-bonded
carbonyls (1640 and 1634 cm-1), a conjugated double bond (1620
cm-1), and aromatic rings (1605 and1504 cm-1).
for five acetyl methyls at 2.22 (3H, s), 2.33 (3H, s), 2.34 (6H, s),
and 2.35 (3H, s) in its H NMR spectrum further confirms the
1
presence of five acetate groups on 1a.
1
The H NMR spectra (1D and 2D COSY) clearly showed that
Its 13C NMR spectrum revealed the presence of 30 sp2-hybridized
carbons, including two carbonyls (181.0 and 183.0 ppm); 10
quaternary carbons each substituted by an oxygen atom at 144.8,
149.1, 156.0, 158.9, 159.2, 162.9, 163.1, 164.4, 165.0, and 165.1
ppm; five quaternary carbons at 105.2, 106.0, 122.2, 127.2, and
129.0 ppm; and 13 tertiary carbons at 94.8, 94.9, 99.8, 100.0, 105.1,
116.0, 117.0 (×2), 122.0 (×2), 129.1, 131.0, and 156.1.
the following proton systems are implicated in the structure: one
AA′BB′ spin system of four aromatic protons (ring B2) [δH 6.88
(2H, d, 8.9 Hz) and 7.02 (2H, d, 8.9 Hz)]; an ABX spin system of
three aromatic protons (ring B1) [δH 6.90 (1H, d, 8.8 Hz), 8.03
(1H, dd, 2.4, 8.8 Hz), and 8.14 (1H, d, 2.4 Hz)]; and two
tetrasubstituted aromatic rings each with a pair of meta-coupled
protons: δH 6.25 and 6.53 (both 1H, d, J ) 2.1 Hz) (ring A1) and
δH 6.29 and 6.45 (each 1H, d, J ) 2.1 Hz) (ring A2). The two
singlets at δH 6.75 (1H) and 8.35 (1H) are indicative of the
characteristic chemical shifts of H-3 in flavones and H-2 in
isoflavones, respectively.
The complete assignments of the protonated carbon resonances
were made using the HSQC spectrum of 1, while a detailed analysis
of the HMBC data (Table 1, Supporting Information) led to the
definition of two substructures, I and II. Observed cross-peaks
indicated connections of the quaternary oxygen-bearing carbon at
165.0 ppm (C-2) and H-3 (δH 6.75, δC 105.1) and H-2′ (δH 8.14,
δC 131.0, ring B1), while the CdO at 183.0 ppm was correlated to
Evidence that compound 1 has five phenolic groups came from
its complete acetylation (Ac2O/pyridine), which gave the derivative
1a (C40H28O15), the IR spectrum of which showed no residual OH
absorption. Its HRCIMS spectrum displayed the [M + H]+ ion at
m/z 749.1502 (calculated for C40H29O15, 749.1506), in agreement
with a penta-acetate of lophirone L. The presence of sharp singlets
* To whom correspondence should be addressed. E-mail:
ghogomut@uycdc.uninet.cm; ghogomut@yahoo.fr.
† University of Yaounde I.
‡ ESA, Paris, France.
10.1021/np050169w CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/15/2006

Notes
Journal of Natural Products, 2006, Vol. 69, No. 8 1207
H-3 (δH 6.75), clearly suggesting the existence of a flavone moiety
as partial structure I. Similarly, connections observed for the
quaternary carbon at 122.2 (C-3′′) and H-2′′′ (δH 7.02, δC 122.0,
ring B2) and H-2′′ (δH 8.35, δC-2′′ 156.1) and also for the CdO at
181.0 and H-2′′ (δH 8.35) defined an isoflavonoid moiety as the
second partial structure, II. The peri OH groups at C-5 (ring A1)
and C-5′ (ring A2) gave separate singlets at δ 12.92 and 12.82,
respectively.
Partial structures I and II can only be linked by an ether bond
engaging one of the oxygen substituents. This was confirmed from
the NOESY spectrum of 1 in which a strong correlation was
observed between H-2′ (δH 8.14 ppm, ring B1) and H-6′′ (δH 6.29
ppm, ring A2), leading to a biflavonyl ether with structure 1 for
lophirone L.
Figure 1. Proton systems and substructures in 1 and 2.
The EIMS of 1 had intense retro Diels-Alder peaks, giving rise
to the base peak at m/z 152 and the ion at m/z 386.
Scheme 1
It is important to emphasize that 1 is the first report of a
biflavonyl ether with mixed flavone-isoflavone substructures from
natural sources and also the first report of the existence of a natural
biflavonyl ether in the genus Lophira. Our results also rule out
earlier impressions that biflavonoid ethers are only typical of Asian
Ochnaceae.
Compound 2, a pale yellow, amorphous solid, had an [M + H]+
peak at m/z 541.1139 in the HRCIMS (541.1135 calculated for
C30H21O10). This implied a biflavonoid with molecular mass of 540
and molecular formula of C30H20O10, accounting for 21 unsaturation
sites. Its IR spectrum showed intense absorption bands for hydroxyl
groups (3412 and 3250 cm-1), a conjugated and hydrogen-bonded
carbonyl (1634 cm-1), a conjugated double bond (1620 cm-1), and
aromatic rings (1590 and 1503 cm-1).
Its complete acetylation (Ac2O/pyridine) gave 2a (C40H30O16),
which showed no residual IR hydroxyl absorption. It had an [M +
H]+ ion at m/z 767.1615 in its HRCIMS spectrum, clearly indicating
a hexa-acetate (calculated for C40H31O16: 767.1612). This was
confirmed by six acetyl methyl singlets at δ 2.16, 2.24, 2.26, 2.29,
(δH 7.30) was correlated to the carbon C-2′′ (δc 164.8), hence
leading to partial structure IV. This is a flavone residue, as can be
deduced from the characteristic chemical shift of the H-3′′ proton
(δH 6.53). The peri OH groups in substructures III and IV appeared
as singlets respectively at δ 12.96 and 12.23.
Further examination of the HMBC spectrum of 2 clearly showed
connections between C-3′ (δC 124.1) and both H-8′′ (δH 7.48) and
H-6′′ (δH 7.24) (Table 1). This unambiguously established the
interflavanyl link between C-3′ and C-7′′.
The preferential susceptibility of the 3-hydroxyflavanone ring
in its EIMS is explained by a retro Diels-Alder fragmentation
giving two intense peaks at m/z 152 (100%) and m/z 389 (92%).
The ionization of the molecule probably involves both flavonoid
substructures since ions from a second retro Diels-Alder frag-
mentation appeared at m/z 118 and 270. The presence of ions at
m/z 388, 270, and 118 (Scheme 1) further confirms the absence of
an oxygenated function at C-7.
The circular dichroism (CD) spectrum of 2 showed a positive
Cotton effect at λ366 nm (∆ꢀ ) +2.684) and a negative Cotton effect
at λ288 nm (∆ꢀ ) -6.968). Both values implied the absolute
configurations 2R, 3R for the two asymmetric carbons in lophirone
M in accordance with literature.11
1
2.32, and 2.34 in the H NMR spectrum of 2a.
The 13C NMR spectrum of 2 had signals for 30 carbons, among
which 28 were sp2 hybridized. This includes two conjugated and
hydrogen-bonded carbonyl carbons at 198.0 and 183.0; eight
quaternary carbons each substituted by an oxygen atom at δ 158.6,
159.2, 162.0,164.2, 164.8, 165.1, 165.3, and 167.5; six quaternary
carbons at δ 103.0, 106.8, 124.0, 124.1, 128.0, and 130.4; and 12
tertiary carbons at δ 96.0, 97.0, 104.4, 108.3, 110.1, 115.9(×2),
116.7, 128.2, 130.0, and 131.1(×2). Two sp3 tertiary oxygen-bearing
carbon atoms gave signals at δ 83.3 and 85.5. All the chemical
shifts of protonated carbons were confirmed using the HSQC
spectrum of 2.
1
The analysis of the 1D and 2D COSY H NMR spectra of 2
revealed that the following proton systems were incorporated in
its structure: an AA′BB′ spin system [δH 6.74 (2H, d, 8.4 Hz) and
7.30 (2H, d, 8.4 Hz) (ring B2)]; an ABX aromatic ring system [ δH
6.96 (1H, d, 9.1 Hz), 7.75 (1H, dd, 2.1, 9.1 Hz), and 7.74 (1H, d,
2.4 Hz) (ring B1)]; and two sets of isolated meta-coupled aromatic
protons [δH 5.99 and 6.01 (both 1H, d, 1.9 Hz), ring A1; and δH
7.24 and 7.48 (both 1H, d, 1.9 Hz), ring A2]. An AB system
comprising two deshielded aliphatic protons belonging to two
oxymethine groups gave doublets at δH 4.77 and 5.93 (J ) 12 Hz),
suggesting the presence of a 3-hydroxyflavanone unit in this
structure.
It is important to note the absence of an oxygenated substituent
at C-7 in 2. Generally, flavonoid biosynthesis has always implicated
the presence of an oxygen atom at C-7 (ring A), but only very few
exceptions such as 3 and 4 have so far been reported in Artemisia
campestris.12 These are flavanones with unusual ring A hydroxyl-
ation, as they lack the C-7 hydroxyl group. 2 is, thus, the first report
of a biflavonoid without an oxygenated function at C-7.
Cross-peaks in the HMBC spectrum of lophirone M (2) defined
two substructures, designated III and IV (Figure 1). HMBC
connections were observed between the carbonyl at δ 198.0 and
H-2 (δH 5.93) and H-3 (δH 4.77). H-2 (δH 5.93) was connected to
C-2′ (δC 131.0), confirming the presence of a 3-hydroxyflavanone
moiety as partial structure III. The relative trans configuration of
H-2 and H-3 was confirmed from the large coupling constant (12
Hz). C-1′′′ (δC 130.0) was connected to H-3′′ (δH 6.53), while H-2′′′
Experimental Section
General Experimental Procedures. IR spectra were recorded using
KBr disks on a JASCO FTIR-300E spectrometer. UV spectra were
obtained on a Kontron-Uvikon 930 spectrophotometer, and optical
1
rotations were measured on a Perkin-Elmer 341 polarimeter. H (400
MHz) and 13C (100 MHz) NMR spectra were obtained on a Bruker
WM 400 spectrometer with Me2CO-d6 as solvent and TMS as internal

1208 Journal of Natural Products, 2006, Vol. 69, No. 8
Notes
standard. For HMBC spectra the delay was 70 µs with JCH ≈ 7 Hz.
Sephadex LH-20 (Pharmacia Fine Chemicals) and silica gel 60 (Merck)
were used for CC. Solvent used for TLC was a CH2Cl2/MeOH mixture
(10:1, v/v). Preparative TLC plates on a glass support were coated with
fluorescent Si gel (F254). HRESI mass spectra were registered using
NH3 as ionizing gas on an applied biosystems APIQ-STAR PULSAR
spectrometer. EIMS were recorded on a Nermag R10-10 apparatus.
Plant Material. Lophira alata Banks ex Gaertn (Ochnaceae) leaves
were harvested in Mbankomo district near Yaounde, Cameroon, in
December 2002. Its identification was made by Mr. Paul Mezili, botanist
in the National Herbarium in Yaounde (Cameroon), where a voucher
specimen (no. PM 3481) was deposited.
Extraction and Isolation. The air-dried plant material was reduced
to a fine powder (1.5 kg) and extracted at room temperature in a
percolator, first with CH2Cl2 and then with MeOH, to give two extracts.
Removal of MeOH from the second extract gave a brown gum (143
g), which was further washed with warm EtOAc. The solvent was
removed from the former to give the crude brown EtOAc extract (38
g). Fractionating by CC (Sephadex LH-20 with MeOH as eluent) gave
168 fractions of 50 mL each. They were combined into four fractions:
F1 (23.1 g), F2 (12.2 g), F3 (1.4 g), and F4 (0.3 g).
Lophirone M (2): C30H18O10, light yellow, amorphous powder;
25
[R]D +13 (MeOH, c 0.2); UV (MeOH) λmax (log ꢀ) 366(3.56),
288(4.35), 208(4.56); IR (KBr disk) ν cm-1 3412, 3250, 3005, 2935,
2852, 1634, 1590, 1503, 1450, 1361, 1250, 1162, 1096, 806; 1H NMR
(400 MHz) and 13C NMR (100 MHz), see Table 1; MS (70 eV, 110
°C) m/z (%) 540(10), 435(17), 415(21), 414(62), 388(92), 386(71),
384(6), 270(18), 254(10), 234(20), 206(23), 170(20), 152(100), 125(5),
118(12), 106(20), 66(14), 42(3); HRMS (NH3) [M + H]+ at 541.1434
(calcd for C30H21O10, 541.1439).
Acetylation of 2 (5 mg) following the same procedure as above gave
1
after workup 3.1 mg of 2a. C40H30O16: H NMR (400 MHz, acetone)
δ 7.73 (1H, dd, J ) 8.6, 2.4 Hz, H-6′), 7.64 (2H, d, J ) 8.5 Hz, H-2′′′,
H-6′′′), 7.23 (1H, d, J ) 8.6 Hz, H-5′), 7.21 (1H, d, J ) 2.4 Hz, H-2′),
7.20 (2H, d, J ) 8.5 Hz, H-3′′′ and H-5′′′), 7.19 (1H, d, 2.1 J ) Hz,
H-6); 7.16 (1H, d, J ) 2.1 Hz, H-8); 6.59 (1H, s, H-3′′); 6.28 (1H, d,
J ) 1.8 Hz, H-8′′); 6.23 (1H, d, J ) 1.8 Hz, H-6′′); 5.96 (1H, d, J )
12.5 Hz, H-3); 5.90 (1H, d, J ) 12.5 Hz, H-2); 2.36 (3H, s, CH3CO);
2.32 (3H, s, CH3CO); 2.31 (3H, s, CH3CO); 2.26 (3H, s, CH3CO);
2.24 (3H, s, CH3CO); 2.21 (3H, s, CH3CO); HRCIMS [M + H]+ at
m/z 767.1615 (calcd for C40H31O16, 767.1612).
Acknowledgment. We are indebted to M. Leonel Dubust for MS,
the IFS (grant F1389-2), and the French Ministry of National Education
for financial assistance.
Further purification of fraction F4 by CC on a silica gel support
with a gradient mixture of CH2Cl2/MeOH (from 0% to 20% MeOH)
gave 62 fractions of 25 mL each, combined into three subfractions,
Supporting Information Available: NMR data for compounds 1
and 2. This material is available free of charge via the Internet at http://
pubs.acs.org.
F
4a (124 mg), F4b (30 mg), and F4c (40 mg), from TLC analysis. Fraction
F4b was purified in a similar procedure to give luteolin (10 mg) and
lithospermoside (14 mg), identified by physical data and by co-
chromatography with reference samples.10
The purification of the more polar fraction F4c was carried out on
preparative TLC silica gel plates developed with a CH2Cl2/MeOH (10:
1) mixture and applying the multiple migration technique to give
compounds 1 (12 mg) and 2 (8 mg).
Lophirone L (1): (C30H18O10) yellow, amorphous powder; UV
(MeOH) c 0.2, λmax (log ꢀ) 333(4.45), 288(4.56), 211(4.85); IR (KBr
disk) ν cm-1 3390, 3240, 1634, 1605, 1600, 1204, 1441, 1291, 1225,
1080, 981; NMR (1H, 400 MHz and 13C, 100 MHz, see Table 1); MS
(70 eV, 110 °C) m/z (%) 538(30), 444(24), 430(41), 428(71), 402(26),
401(97), 388(30), 386(38), 385(10), 344(44), 277(30), 265(14), 250(16),
236(17), 221(15), 171(8), 152(100); [M + H]+ ion at m/z 539.0974
(calcd for C30H19O10, 539.0978).
References and Notes
(1) Murakami, A.; Ohigashi, H.; Nozaki, H.; Tada, T.; Kajim, X.;
Koshimizu, K. Agric. Biol. Chem. 1991, 55 (4), 1151-1154.
(2) Tih, A. E.; Ghogomu, T. R.; Sondengam, B. L.; Martin, M. T.; Bodo,
B. J. Nat. Prod. 1990, 53, 964-967.
(3) Tih, A. E.; Ghogomu, T. R.; Sondengam, B. L.; Martin, M. T.; Bodo,
B. Phytochemistry 1992, 31, 981-984.
(4) Tih, A. E.; Ghogomu, T. R.; Sondengam, B. L.; Martin, M. T.; Bodo,
B. Tetrahedron Lett. 1999, 40 (60), 4721-4924.
(5) Tih, A. E.; Martin, M. T.; Ghogomu, T. R.; Vindepot, I.; Sondengam,
B. L.; Bodo, B. Phytochemistry 1992, 31, 3595-3599.
(6) Tih, A. E.; Ghogomu, T. R.; Sondengam, B. L.; Martin, M. T.; Bodo,
B. Tetrahedron. Lett. 1988, 29 (45), 5797-5800.
(7) Murakami, A.; Tanaka, S.; Ohigashi, H.; Jisaka, M.; Hirota, M.; Irie,
R.; Takeda, N.; Tatematsu, A.; Koshimizu, K. Cancer Lett. 1991a,
58, 101-106.
Acetylation. Lophirone L (4 mg) was dissolved in a mixture of Ac2O
(1 mL) and dry pyridine (1 mL) in a 5 mL round-bottom flask The
well-corked flask was left in an oven at 60 °C for 4 h, after which it
was evaporated to dryness under vacuum. The resultant powder was
purified by CC on a Sephadex LH-20 support with MeOH as eluent to
give lophirone L penta-acetate (2.5 mg) as a white powder: C40H28O15,
(8) Murakami, A.; Tanaka, S.; Ohigashi, H.; Jisaka, M.; Hirota, M.; Irie,
R.; Takeda, N.; Tatematsu, A.; Koshimuzu, K. Phytochemistry 1991b,
31, 2689-2693.
(9) Murakami, A.; Tanaka, S.; Ohigashi, H.; Hirota, M.; Irie, R.;
Tatematsu, A.; Koshimizu, K. Biosci. Biotech. Biochem. 1992, 56,
769-772.
with [M + H]+ at m/z 749.1502 (calcd for C40H29O15); H NMR (400
1
MHz, Me2CO-d6) δ 8.39 (1H, s, H-2′′),8.25 (2H, J ) 8.5 Hz, H-3′′′,
H-5′′′), 8.15 (1H, dd, J ) 8.4, 2.4 Hz, H-6′), 7.50 (2H, d, J ) 8.5 Hz,
H-2′′′, H-6′′′), 7.19 (1H, d, J ) 8.8 Hz, H-5′), 7.10 (1H, d, J ) 2.4 Hz,
H-2′), 6.82 (1H, d, J ) 2.2 Hz, H-8), 6.79 (1H, s, H-2); 6.56 (1H, d,
J ) 2.2 Hz, H-6), 6.51 (1H, d, J ) 2.1 Hz, H-8′′), 6.48 (1H, d, J ) 2.1
Hz, H-6′′), 2.35 (3H, s, CH3CO); 2.34 (6H, s, 2 CH3CO); 2.33 (3H, s,
CH3CO); 2.22 (3H, s, CH3CO).
(10) Tih, A. E.; Ghogomu, T. R.; Sondengam, B. L.; Caux, C.; Bodo, B.
Biochem. Syst. Ecol. 2003, 31, 549-551.
(11) Gaffield, W. Tetrahedron 1970, 26, 4093-4108.
(12) Valent-Vetschera, K. M.; Fisher, R.; Wollenweber, E. Biochem. Syst.
Ecol. 2003, 31 (5), 487-498.
NP050169W