Activity-guided isolation of an antiandrogenic compound of Pygeum africanum

Article abstract of DOI:10.1055/s-2006-941472

Inactivation of the androgen receptor (AR) through androgen ablation and treatment with antiandrogens is a major goal in the therapy for prostate hyperplasia and prostate cancer. Bioactivity-directed fractionation of a selective dichloromethane extract from the stem bark of Pygeum africanum led to the isolation of the antiandrogenic compound atraric acid. Its activity was examined by an androgen receptor responsive reporter gene assay. For lead structure optimization we transformed the natural occurring compound atraric acid into its ethyl, n-propyl and n-butyl esters and their antiandrogenic activities were examined as well. In addition, benzoic acid was isolated. The structures of all compounds were determined and characterized by means of ¹H- and ¹³C-NMR, HR-EI-mass, IR and UV spectroscopy. Georg Thieme Verlag KG Stuttgart.

Full text of DOI:10.1055/s-2006-941472

Sonja Schleich1
Maria Papaioannou2
Aria Baniahmad2
Activity-Guided Isolation of an Antiandrogenic
Rudolf Matusch1
Compound of Pygeum africanum
Abstract
well. In addition, benzoic acid was isolated. The structures of all
compounds were determined and characterized by means of
Inactivation of the androgen receptor (AR) through androgen ab- 1H- and 13C-NMR, HR-EI-mass, IR and UV spectroscopy.
lation and treatment with antiandrogens is a major goal in the
therapy for prostate hyperplasia and prostate cancer. Bioactiv- Key words
ity-directed fractionation of a selective dichloromethane extract Pygeum africanum ´ Prunus africana ´ Rosaceae ´ atraric acid ´
from the stem bark of Pygeum africanum led to the isolation of benzoic acid ´ antiandrogenic activity ´ androgen ´ hormone ther-
the antiandrogenic compound atraric acid. Its activity was exam- apy
ined by an androgen receptor responsive reporter gene assay. For
lead structure optimization we transformed the natural occur- Supporting information available online at
ring compound atraric acid into its ethyl, n-propyl and n-butyl http://www.thieme-connect.de/ejournals/toc/plantamedica
esters and their antiandrogenic activities were examined as
Introduction
BPH afflicts over 40% of men aged 60 years or older [2] and is clo-
sely related with the development of prostate cancer (PCa), which
547
Pygeum africanum (Hook. f.), an evergreen tree also known as is the most commonly diagnosed malignancy and second leading
Prunus africana (Hook. f.) Kalkm., is distributed across the entire cause of cancer death in men older than age 40 years in Western
continent of Africa in mountain regions. The tree is a member of countries [3]. Both prostatic diseases are associated with an enlar-
the Rosaceae family and its bark is traditionally used to treat gement of the prostate. The growth of this organ is regulated by
chest pain, malaria and fevers [1]. Nowadays chloroform extracts the male sexual hormones, the androgens. These bind to the AR,
from the stem barks of Pygeum africanum are applied for the which transactivates target genes. Therefore, the main target for
treatment of patients suffering from benign prostatic hyperpla- the treatment of prostate enlargement, especially the malignant
sia (BPH) [2]. The extract from the pulverized bark is incorporat- type PCa, is the down-regulation of androgens realized by the use
ed into capsules and sold under various trade names, among of antiandrogenic agents like the well-known non-steroidal anti-
others the French drug Tadenanꢀ of Debat Laboratoires, which is androgen Bicalutamid (Casodexꢀ), for example.
a widespread phytotherapeutic agent in the therapy for BPH, par-
ticularly in the United States.
The well-proven use of Pygeum extracts in the treatment of BPH
evokes the question whether the described plant contains com-
Affiliation
1 Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marburg, Germany
2 Genetisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany
Correspondence
Aria Baniahmad ´ Genetisches Institut ´ Justus-Liebig-Universität Giessen ´ Heinrich-Buff-Ring 58±62 ´
35392 Giessen ´ Germany ´ Phone: +49-641-99-35468 ´ Fax: +49-641-99-35469 ´
E-mail: aban@mti.uni-jena.de
& Dept. of Biochemistry University of Kuopio POB. 1627, 70211 Kuopio, Finland
Received September 27, 2005 ´ Accepted December 20, 2005
Bibliography
Planta Med 2006; 72: 547±551 ꢁ Georg Thieme Verlag KG Stuttgart ´ New York
DOI 10.1055/s-2006-941472 ´ Published online April 28, 2006
ISSN 0032-0943

pounds with antiandrogenic properties and, moreover, which is Materials and Methods
the active ingredient of Pygeum? Pharmacologically, Tadenanꢀ
has been shown to be a potent inhibitor of rat prostatic fibroblast General experimental procedures
proliferation in response to direct activators of protein kinase C, Melting points were determined on an HM-LUX microscope
in a variety of growth factors including basic fibroblast growth melting apparatus from Leitz (Bensheim, Germany). NMR spec-
factor (bFGF), epidermal growth factor (EGF), and insulin growth tra were recorded in CDCl3 using a JEOL Eclipse+ 500 MHz spec-
factor (IGF) [4]. Furthermore, in the evolution of prostatic growth trometer with TMS as internal standard. UV spectra were ac-
and BPH symptomatology, also inflammatory processes are in- quired with a Shimadzu 2101PC UV-VIS photometer. EI-MS were
volved [5]. Certain inflammatory cell types, such as macropha- obtained with a Vacuum Generators VG Micromass 7070 H mass
ges, are known to produce chemotactic mediators including leu- spectrometer employing an ionizing energy of 70 eV. HR-EI-MS
kotrienes. It has been demonstrated that P. africanum extracts were recorded on a Micromass AutoSpec spectrometer with an
cause a dose-dependent inhibition of leukotriene synthesis in ionizing energy of 70 eV. Preparative HPLC was carried out on a
human polymorphonuclear cell cultures stimulated by the cal- Waters prep LC 4000 system with a 990 diode array detector. IR
cium ionophore A 23187 [6].
spectra were recorded on a Nicolet FT-IR spectrometer type 510P.
Solvents for extraction and alcohols for esterification with the
In addition, P. africanum has been described to have a phytoes- quality ªextra pureº were purchased from Merck KG (Darmstadt,
trogenic activity (by either activating or inhibiting the estrogen Germany). Solvents for chromatography had the quality ªLiChro-
receptor(s)) on the prostate resulting in a reduction of volume solvꢀº and were purchased from Merck KG. KOH for esterifica-
of prostatic hypertrophy [7], [8].
tion with ªp.a.º quality was purchased from Roth (Karlsruhe,
Germany). Reference substance 6 was purchased from Aldrich
Previously realized analytical studies focusing on the bark mate- Chem. Co. (Hamburg, Germany).
rial of Pygeum africanum have revealed the presence of steroids,
saturated and unsaturated fatty acids [9] and triterpenic acids Plant material
esterified with trans-ferulic acid [10]. But none of these ingredi- Bark material of Pygeum africanum was a gift of Euromed S.A.,
ents could be attributed definitely to the bioactivity of the de- Barcelona, Spain. A voucher specimen (No. 8438102) is depos-
scribed plant in the treatment of BPH and, therefore, the exact ited at the Department of Pharmaceutical Chemistry, Philipps-
mechanism of action is still unknown [11].
University of Marburg, Germany.
In the present study we initially assayed several selective ex- Extraction and isolation
tracts from the stem bark of Pygeum africanum and we found a Dried plant material (1.73 kg) was powdered and homogenized
strong antiandrogenic activity of the selective dichloromethane in n-hexane (ice-cooling) using an Ultra Turrax and percolated
extract using the androgen receptor (AR) responsive MMTV-Luc successively and exhaustively with n-hexane (25.0 L), CH2Cl2
reporter gene assay. For this reason we performed an activity- (26.0 L), MeOH (25.0 L), MeOH-H2O 1:1 (26.0 L) and H2O (12.5
guided fractionation of this extract resulting in the isolation of L) at room temperature. After evaporation of solvents under va-
the antiandrogenic natural compound atraric acid (1), which cuum at 30 8C, the bioactive CH2Cl2 residue (11.0 g of green dry
was discovered next to benzoic acid (5) in this plant for the first mass) was fractionated over silica gel (Macherey-Nagel; Düren,
time (Fig.1). For the purpose of lead structure optimization we Germany; Si60, 15±25 mm). For this purpose the extract was dis-
investigated a method for the transesterification of 1 into its solved in 2000 mL CH2Cl2 and filtered through 0.7 mm filter paper
ethyl (2), n-propyl (3) and n-butyl (4) ester derivatives and we (Schleicher & Schüll; Dassel, Germany). 25 g of silica gel (Merck
analyzed their antiandrogenic activities with regard to the Si60, 0.063±0.200 mm) were added to the extract and then the
length of ester chain and found an even higher bioactivity of 2 solvent was evaporated under vacuum at 30 8C. The coated silica
548
and 3.
gel was placed on the top of a dry-packed silica gel column
(Merck Prepbarꢀ 400”100 mm) and eluted with a linear gradient
at a flow rate of 120 mL ´ min±1; gradient: 0 min hexane (100:0),
50 min hexane (100:0), 350 min CH2Cl2 (100:0), 500 min CH2Cl2
(100:0), 700 min CH2Cl2-MeOH (80:20), 750 min MeOH (100:0),
800 min MeOH (100:0), 850 min H2O (100:0), 885 min H2O
(100:0). The obtained fractions from 239 min to 293 min were
bioactive and contained 1 and 5. These compounds were then
isolated by preparative HPLC [250”21 mm, 100±5 C18 HD Ma-
cherey-Nagel, 22 mL´min-1; gradient: 0 min ACN-H2O (with addi-
tion of TFA 0.1%) (20:80), 40 min ACN-H2O (80:20), 45 min ACN
(100:0)]. Compound 5 was collected from min 10±13, bioactive
1 was collected from min 23±25. 11.0 g of CH2Cl2 extract contain
17.6 mg of 1 ( = 0.16%).
Fig. 1 Chemical
structures of the
isolated and deriva-
tized compounds.
Compound 1, atraric
acid, was isolated in
an activity-guided
manner from Py-
geum africanum, 2±
4 represent deriva-
tized compounds,
benzoic acid was
also identified in ex-
tracts from Pygeum
africanum and com-
Preparation of the esters 2, 3 and 4
pound 6 , identical in molecular weight and side-chains compared to
1 but with a different structure, has also been tested.
300 mg of 1 were stirred with 90 mg KOH overnight in 10 mL of a
solution of the corresponding alcohol (EtOH, PrOH, n-BuOH). The
esters were purified by preparative RP-HPLC [250”21 mm, 100±
Schleich S et al. Activity-Guided Isolation¼ Planta Med 2006; 72: 547±551

5 C18 HD Macherey-Nagel, 22 mL ´ min±1, isocratic: MeOH-H2O Benzoic acid (5): See Supporting Information.
(70:30) for the purification of 2, MeOH-H2O (72:28) to afford 3,
MeOH-H2O (75:25) to afford 4].
Cell culture and AR responsive MMTV-Luc reporter gene assay
The plasmid pMMTV-luc, which contains a luciferase reporter
gene driven by the mouse mammary tumor virus long terminal
Compound characterization
Methyl 2,4-dihydroxy-3,6-dimethylbenzoate = atraric acid (1): repeats, is described in [12]. The expression vector for the human
See Supporting Information.
androgen receptor pSG-hAR is described in [13]. Monkey kidney
CV1 cells were maintained in Dulbecco's modified Eagle's medi-
Ethyl 2,4-dihydroxy-3,6-dimethylbenzoate (2): White powder; um (DMEM, Invitrogen; Karlsruhe, Germany), supplemented
m.p. 127 8C; UV (MeOH): lmax (log e): 308 (2.89), 245 (3.40), 217 with 10% (v/v) fetal calf serum (Invitrogen; Karlsruhe, Germany),
nm (3.60); IR (KBr): lmax = 3450, 3100,1620,1310,1280, 800 cm±1; penicillin (100 IU/mL, Invitrogen; Karlsruhe, Germany) and
1H-NMR (500 MHz, CDCl3): d = 12.05 (1H, s, C-2 OH), 6.14 (1H, s, streptomycin (100 IU/mL, Invitrogen; Karlsruhe, Germany) at
H-5), 5.02 (1H, s, C-4 OH), 4.32 (2H, q, J = 7.0 Hz, H-1¢), 2.41 (3H, s, 37 8C and 5% CO2. For transfection experiments, cells were seed-
C-6 Me), 2.03 (3H, s, C-3 Me),1.34 (3H, t, J = 7.1 Hz, H-2¢); 13C-NMR ed onto 6-well tissue culture plates (Nunc; Roskilde, Denmark)
(125 MHz, CDCl3): see Table 1; EI-MS (70 eV): m/z (rel. int.) = 210 at 1.2”105 cells per well and grown in DMEM medium supple-
[M]+ (50), 164 (100), 136 (60); HR-EI-MS: m/z = 210.088; C11H14O4 mented with 10% (v/v) dextran-coated charcoal stripped serum
[M]+ requires 210.0892.
(Invitrogen; Karlsruhe, Germany) [14]. Six hours later cells were
transfected by using the Ca3(PO4)2 method [14]. The hAR expres-
n-Propyl 2,4-dihydroxy-3,6-dimethylbenzoate (3): White powder; sion vector (0.2 mg) was cotransfected with 1 mg of the reporter
m.p. 134 8C; UV (MeOH): lmax (log e): 307 (2.89), 242 (3.42), 217 plasmid MMTV-Luc and 0.2 mg of the cytomegalovirus (CMV)-
nm (3.63); IR (KBr): lmax = 3450, 3000, 1650, 1310, 1200, 800 driven b-galactosidase expressions virus, as internal control for
cm±1; 1H-NMR (500 MHz, CDCl3): d = 12.09 (1H, s, C-2 OH), 6.14 transfection efficiency. After 14 hours media were replaced ei-
(1H, s, H-5), 5.02 (1H, s, C-4 OH), 4.23 (2H, t, J = 6.7 Hz, H-1¢), ther with or without the addition of 3”1010 M R1881 together
2.41 (3H, s, C-6 Me), 2.04 (3H, s, C-3 Me), 1.73 (2H, m, J = 7.0 Hz, with the indicated compounds. After additional 48 hours cells
H-2¢), 0.97 (3H, t, J = 7.2 Hz, H-3¢); 13C-NMR (125 MHz, CDCl3): were harvested and assayed for luciferase and b-galactosidase
see Table 1; EI-MS (70 eV): m/z (rel. int.) = 224 [M]+ (31), 164 activity. Each of the values obtained with the luciferase assay
(100), 136 (44); HR-EI-MS: m/z = 224.1051; C12H16O4 [M]+ re- was normalized to that obtained with the b-galactosidase activ-
quires 224.1049.
ity from the same cellular extract. Luciferase activity was deter-
mined by injecting luciferin and measuring light emission at 562
n-Butyl 2,4-dihydroxy-3,6-dimethylbenzoate (4): White powder; nm and expressed as relative light units (RLU). All transfection
m.p. 117 8C; UV (MeOH): lmax (log e): 308 (2.89), 245 (3.40), 217 assays shown were performed in duplicate and were repeated
nm (3.60). IR (KBr): lmax = 3440, 3000,1700,1310,1200, 800 cm±1; at least twice.
1H-NMR (500 MHz, CDCl3): d = 12.09 (1H, s, C-2 OH), 6.14 (1H, s,
549
H-5), 4.97 (1H, s, C-4 OH), 4.27 (2H, t, J = 7.0 Hz, H-1¢), 2.41 (3H, s,
C-6 Me), 2.04 (3H, s, C-3 Me), 1.69 (2H, m, J = 7.3 Hz, H-2¢), 1.42 Results and Discussion
(2H, m, J = 8.3 Hz, H-3¢), 0.90 (3H, t, J = 7.3 Hz, H-4¢); 13C-NMR
(125 MHz, CDCl3): see Table 1; EI-MS (70 eV): m/z (rel. int.) = 238 Dried bark of Pygeum africanum was extracted exhaustively with
[M]+ (31), 164 (100), 136 (30); HR-EI-MS: m/z = 238.1226; n-hexane, dichloromethane, methanol, methanol-water (1:1)
C13H18O4 [M]+ requires 238.1226.
and water. Among these the selective dichloromethane extract
revealed antiandrogenic activity. Therefore this extract was eva-
porated and fractionated on silica gel with a gradient system
containing n-hexane, dichloromethane, methanol and water.
Three of the resulting 35 fractions proved to be bioactive (data
not shown). The combined active fractions were subjected to
preparative RP-HPLC to yield 1 and 5. To avoid generation of arti-
facts, all procedures were carried out under mild conditions such
as low temperature, subdued light and nearly total exclusion of
oxygen. Structural elucidation of the isolated substances was ef-
fected by analyzing the 1H- and 13C-NMR spectroscopic data, HR-
EI-MS, IR and UV. The isolated compounds were individually
screened with the AR responsive MMTV-Luc reporter gene assay,
inhibiting the transactivation of human AR (Fig. 2) and 1 indicat-
ed potent antiandrogenic activity as well as the semisynthetic
esters.
Table 1 13C NMR spectral data of compounds 2±4 in CDCl3 at 125
MHz
Carbon
2
3
4
1
105.4
163.2
108.5
157.9
110.5
140.2
172.1
7.6
105.4
163.2
108.6
157.9
110.5
140.1
173.3
7.6
105.5
163.1
2
3108.5
4
157.8
110.5
140.1
167.9
7.5
5
6
7
3-Me
6-Me
1¢
24.2
24.2
24.1
66.3
30.5
19.3
13.5
Compound 1 was obtained with its known characteristics in-
cluding the solid state IR spectrum, 1H-NMR spectrum and 13C-
NMR spectrum [15], [16], [17].
61.2
67.0
2¢
14.2
22.0
3¢
10.8
4¢
Schleich S et al. Activity-Guided Isolation¼ Planta Med 2006; 72: 547±551

Fig. 2 Extension of the side chain of compound 1 enhances
the antiandrogenicity. Compounds 1±6 were analyzed for
their potency to inhibit human AR activated by the andro-
gen methyltrienolone (3”10±10 M) in a reporter-based cell
culture system. Indicated concentrations of 1±6 were test-
ed in the absence (white bars) or presence (black bars) of
methyltrienolone. As control solvent alone or untreated
tests are shown.
Fig. 3 Concentration-dependent inhibition of human AR
mediated transactivation. The indicated concentrations of
compound 1 was used for inhibition of human AR without
hormone (white bars) or activated by methyltrienolone
(black bars) in the experimental set-up shown in Fig. 2.
550
It is noteworthy that 1 was previously found as a degradation Benzoic acid (5) is a common natural compound occurring in dif-
product of various natural depsides and it occurs in its free state ferent plants such as Homalium cochinchinensis [23] and Uvaria
in lichens. But 1 has also been isolated from higher plants such as mocoli [24]. It appears in its free state or as a glycosyl conjugate
Newbouldia laevis [16], Alseodaphne andersonii [17], Acer and often serves as intermediate in the biosynthetic pathway of
nikoense [18] and Frullania brasiliensis [19]. The isolation of 1 salicylic acid [25]. Plants synthesize 5 starting from phenylala-
from the stem bark of Pygeum africanum begs the question nine via the intermediate cinnamic acid [26]. It is a matter of
whether 1 is a secondary metabolite of the plant itself or is the common knowledge that 5 has antimicrobial characteristics and
bark of the described tree colonized by lichens producing poly- serves as a food preservative in concentrations of 0.05±0.1%
ketides through the acetate-polymalonate pathway. For in- [27].
stance, the well-known lichen substance atranorin is a depside
of hematommic acid and 1 is biosynthesized through the polyke- The isolated compounds 1 and 5 and the semisynthesized esters
tide pathway by various lichen species [20]. Compound 1 and 2, 3 and 4 were subjected to the AR responsive reporter gene as-
other lichen products are described to have nematocidal activity say. Compound 5 did not indicate an antiandrogenic activity in
[21].
contrast to the atratate esters 1±4 (Fig. 2). The isolated atraric
acid from Pygeum africanum revealed similar inhibition of AR
It was an aim of the present study to investigate a facile method function compared to that of the commercially available atraric
for the transesterification of 1 into other alkyl esters with possi- acid (Fig. 2). Concentration-dependent analyses revealed that 1
bly higher antiandrogenic activity. For this purpose 1 has to be inhibits the activity of human AR significantly at a concentration
stirred with potassium hydroxide overnight in a solution of the of 1.0 mM (Fig. 3). Further inhibition and complete inactivation of
corresponding alcohol with which the acid should be esterified. human AR was observed at 10 mM and 100 mM. Interestingly, the
The reaction product can be freed from not converted educt by modified compounds 2 and 3 showed even higher biological ac-
means of preparative RP-HPLC.
tivity compared to 1, both of them inhibited androgen-induced
luciferase expression potently at 1.0 mM, whereas 4 proved to
Compound 5 proved to be benzoic acid. Its IR, UV, EI-MS, 1H- and have a comparable activity to 1 (Fig. 2). Interestingly, 6 having
13C-NMR spectroscopically measured values are in good agree- an identical molecular weight and a similar structure as 1, did
ment with published data in the SDBS database [22].
not reveal a significant antiandrogenic capacity, strongly sup-
porting the selectivity of the test system and indicating the im-
Schleich S et al. Activity-Guided Isolation¼ Planta Med 2006; 72: 547±551

11
12
13
14
15
16
McClure MW. An overview of holistic medicine and complementary
and alternative medicine for the prevention and treatment of BPH,
prostatitis, and prostate cancer. World J Urol 2002; 20: 273±84
Gast A, Schneikert J, Cato AC. N-terminal sequences of the human an-
drogen receptor in DNA binding and transrepressing functions. J Ster-
oid Biochem Mol Biol 1998; 65: 117±23
Dotzlaw H, Moehren U, Mink S, Cato AC, Iniguez Lluhi JA, Baniahmad
A. The amino terminus of the human AR is target for corepressor ac-
tion and antihormone agonism. Mol Endocrinol 2002; 16: 661±73
Dotzlaw H, Papaioannou M, Moehren U, Claessens F, Baniahmad A.
Agonist-antagonist induced coactivator and corepressor interplay on
the human androgen receptor. Mol Cell Endocrinol 2003; 213: 79±85
Brehm L, Stoeckli-Evans H, Tabacchi R. The crystal structure of methyl b-
orcinol-carboxylate ( = methyl 2,4-dihydroxy-3,6-dimethylbenzoate).
Helv Chim Acta 1983; 66: 824±7
Gormann R, Kaloga M, Li XC, Ferreira D, Bergenthal D, Kolodziej H. Fur-
anonaphthoquinones, atraric acid and a benzofuran from the stem
barks of Newbouldia laevis. Phytochemistry 2003; 64: 583±7
Lee SS, Chang SM, Chen CH. Chemical constituents from Alseodaphne
andersonii. J Nat Prod 2001; 64: 1548±51
Nagumo S, Ishizawa S, Nagai M, Inoue T. Studies on the constituents of
Aceraceae plants. XIII. Diarylheptanoids and other phenolics from Acer
nikoense. Chem Pharm Bull 1996; 44: 1086±9
portance of the position of the methyl groups and the hydroxy
groups on the benzene ring.
Taken together, these results demonstrate that an ester chain
length of two or three carbon atoms effects the highest bioactiv-
ity for the atratate derivatives. These compounds may provide a
novel approach to the prevention and treatment of the prevalent
diseases benign prostatic hyperplasia (BPH) and human prostate
cancer (PCa).
Acknowledgements
The authors like to thank Alexander-Philipp Scheffler of Euromed
S.A., Barcelona, Spain, for providing the plant material.
17
18
References
19
Bardón A, Bovi Mitre G, Kamiya N, Toyota M, Asakawa Y. Eremophila-
nolides and other constituents from the Argentine liverwort Frullania
brasiliensis. Phytochemistry 2002; 59: 205±13
1
Bombardelli E, Morazzoni P. Prunus africana (Hook. f.) Kalkm. Fito-
terapia 1997; 68: 205±18
2 Ishani A, MacDonald R, Nelson D, Rutks I, Wilt TJ. Pygeum africanum
for the treatment of patients with benign prostatic hyperplasia: a sys-
tematic review and quantitative meta-analysis. Am J Med 2000; 109:
654±64
20 Bourgeois G, Suire C, Vivas N. Atraric acid, a marker for epiphytic li-
chens in the wood used in cooperage: identification and quantifica-
tion by GC/MS/(MS). Analysis 1999; 27: 281±3
21
Ahad AM, Goto Y, Kiuchi F, Tsuda Y, Kondo K, Sato T. Nematocidal prin-
ciples in ªoakmoss absoluteº and nematocidal activity of 2,4-dihy-
droxybenzoates. Chem Pharm Bull 1991; 39: 1043±6
3 Gonzalgo ML, Isaacs WB. Molecular pathways to prostate cancer. J
Urol 2003; 170: 2444±52
4 Yablonsky F, Nicolas V, Riffaud JP, Bellamy F. Antiproliferative effect of
Pygeum africanum extract on rat prostatic fibroblasts. J Urol 1997; 157:
2381±7
22 National Institute of Advanced Industrial Science and Technology, Ja-
pan. SDBS database. Available at http://www.aist.go.jp/RIODB/SDBS/
menu-e.html. Accessed in 2004.
5 Anim JT, Udo C, John B. Characterisation of inflammatory cells in be-
nign prostatic hyperplasia. Acta Histochem 1998; 100: 439±49
6 Paubert-Braquet M, Cave A, Hocquemiller R, Delacroix D, Dupont C,
Hedef N et al. Effect of Pygeum africanum extract on A23187-stimulat-
ed production of lipoxygenase metabolites from human polymorpho-
nuclear cells. J Lipid Mediat Cell Signal 1994; 9: 285±90
7 Mathe G, Hallard M, Bourut CH, Chenu E. A Pygeum africanum extract
with so-called phyto-estrogenic action markedly reduces the volume
of true and large prostatic hypertrophy. Biomed Pharmacother 1995;
49: 341±3
23 Ishikawa T, Nishigaya K, Takami K, Uchikoshi H, Chen IS, Tsai IL. Isola-
tion of salicin derivates from Homalium cochinchinensis and their anti-
viral activities. J Nat Prod 2004; 67: 659±63
24 Fleischer TC, Waigh RD, Waterman PG. A novel retrodihydrochalcone
from the stem bark of Uvaria mocoli. Phytochemistry 1997; 47: 1387±
91
551
25 Coquoz JL, Buchala A, MØtraux JP. The biosynthesis of salicylic acid in
potato plants. Plant Physiol 1998; 117: 1095±101
26 Hertweck C, Jarvis AP, Xiang L, Moore BS, Oldham NJ. A mechanism of
benzoic acid biosynthesis in plants and bacteria that mirrors fatty acid
b-oxidation. Chembiochem 2001; 10: 784±6
8 Mathe G, Orbach-Arbouys S, Bizi E, Court B. The so-called phyto-estro-
genic action of Pygeum africanum extract. Biomed Pharmacother
1995; 49: 339±40
27 Tremblay GC, Qureshi IA. The biochemistry and toxicology of benzoic
acid metabolism and its relationshipto the elimination of waste nitro-
gen. Pharmacol Ther 1993; 60: 63±90
9 Longo R, Tira S. Constituents of Pygeum africanum bark. Planta Med
1981; 42: 195 ± 6
10
Fourneau C, Hocquemiller R, CavØ A. Triterpenes from Prunus africana
bark. Phytochemistry 1996; 42: 1387±9
Schleich S et al. Activity-Guided Isolation¼ Planta Med 2006; 72: 547±551