Structure–activity relationship for inhibition of 5α-reductase by triterpenoids isolated from Ganoderma lucidum
Graphical abstract
The structure–activity relationship of triterpenoids, which isolated from the ethanol extract if Ganoderma lucidum, is reported. Triterpenoids isolated from ethanol extracts of Ganoderma lucidum (Fr.) Krast (Ganodermataceae) inhibited 5α-reductase activity. The presence of the C-3 carbonyl group and of the C-26-α,β-unsaturated carbonyl group was characteristic of almost all inhibitors isolated from G. lucidum.
Introduction
The microsomal enzyme steroid 5α-reductase catalyzes the NADPH-dependent reduction of the Δ4,5 double bonds of a variety of 3-oxo-Δ4 steroids.1 It is well documented that androgen-responsive tissues such as prostate, seminal vesicle, epididymis, and skin metabolize the conversion of testosterone to 5α-dihydrotestosterone (DHT).2, 3 This process amplifies the androgenic response, perhaps because of the higher affinity of the androgen receptor for DHT than for testosterone.4 Both 5α-reductase and DHT perform critical roles physiologically and pathologically in humans. For example, DHT is necessary for adult prostate enlargement,5 for the development of the male genitalia, and for normal beard growth,6 while administration of DHT can enlarge the undetectable prostate7 in males born with a genetic 5α-reductase deficiency.8 Elevated DHT plasma levels have been reported in patients with either benign prostatic hyperplasia (BPH) or prostatic cancer.9 Therefore, inhibition of androgen action by 5α-reductase inhibitors is a logical treatment for 5α-reductase activity disorders. Furthermore, with the assistance of modern methods of molecular biology, two types of 5α-reductases, identified as types 110 and 2,11, 12 have been isolated from human and rat prostatic cDNA libraries, and the structures of both genes have been elucidated. The type 1 isozyme has a broad basic pH optimum and low affinity for testosterone (Km > 1 μM), while the type 2 isozyme has an acidic pH optimum and high affinity for testosterone (Km < 10 nM).13 The average sequence identity between isozymes within a given species is about 47%, while the sequence identity between the same isozyme across species is 60% for 5α-reductase type 1 and 77% for 5α-reductase type 2.14 Early reports found that the type 1 isozyme predominates in tissues such as liver, kidney, brain, lung, and skin, whereas the type 2 isozyme is more abundant in genital tissues such as the prostate. However, some recent evidence shows that, in the human prostate, type 1 is expressed mainly in the epithelial cells, whereas type 2 is localized mainly in the stromal compartment.13, 15 Consequently in advanced prostate cancer, which is characterized by the abnormal proliferation of epithelial cells, type 1 might become the predominant isozyme probably responsible for androgen metabolism. Moreover, it has been shown that 5α-reductase type 1 activity is three to four times greater in malignant hyperplasia than in BPH, but 5α-reductase type 2 activity is similar in both diseases. Therefore, we focused on 5α-reductase type 1 activity.
The inhibition of 5α-reductase with organic molecules has been studied for more than two decades. Numerous nonsteroidal and steroidal compounds have been designed and synthesized as competitive, noncompetitive or uncompetitive inhibitors of 5α-reductase. Among them, benzonolinones16, 17 and 4-azasteroids18, 19 have high inhibitory potencies to type 1 and/or type 2 enzyme(s) in vitro and/or in vivo. Finasteride, a synthetic 5α-reductase inhibitor, is currently used to treat BPH.20 However, it should be noted that these inhibitors have the potential to cause adverse effects such as those reported for finasteride21—that is, gynecomastia, impairment of muscle growth and severe myopathy—due to the structural similarity to steroidal hormones. Hence, the emergence of therapeutic materials having fewer side effects—preferably, edible natural products—would be highly desirable if their safety could be guaranteed.
For thousand of years, mushrooms have been known as a source of medicine. In East Asia, the fruiting body of the fungus Ganoderma lucidum has been used for centuries as a folk medicine to treat various human diseases such as cancer, hypertension, hepatitis, nephritis, and so on.22 Although the proliferation and migration of prostate cancer cells23 have been reported, the 5α-reductase inhibitory activity of these compounds from G. lucidum was reported only by our group. In our last paper, we revealed a characteristic fraction containing triterpenoids after separation by silica gel column chromatography that showed significant 5α-reductase inhibitory activity. We also reported the isolation of oxygenated lanostane-type triterpenoids with 5α-reductase inhibition, ganoderic acid TR, ganoderic acid DM, and 5α-lanosta-7,9(11),24-triene-15α,26-dihydroxy-3-one from G. lucidum, as well as their inhibitory effects on 5α-reductase.24, 25 In this paper, we investigated the ability of these compounds to inhibit 5α-reductase to determine what structural elements are important for the potent inhibition of 5α-reductase by this class of compounds in rat liver microsomes.
Section snippets
Chemistry
A variety of natural and synthetic compounds have been found to inhibit 5α-reductase. Here we isolated 13 species of triterpenoids from ethanol extracts of G. lucidem, and their structures are shown in Figure 1. The 13C NMR data for ganoderic acid TR (1), ganoderic acid DM (2),26 ganoderic acid A (3),27 ganoderic acid B (4),28 ganoderic acid C2 (5),29 ganoderic acid D (6),30 ganoderic acid I (7),29 5α-lanosta-7,9(11),24-triene-15α,26-dihydroxy-3-one (8),31 ganoderol B (9),32 ganodermatriol (10),
Results and discussion
Since the publication of papers describing the phenotypic characteristics of humans deficient in DHT,35 ample evidence has accumulated supporting the proposal that this product of testosterone metabolism is the principal androgen for trophic growth and for support of the prostate36 and the function of the sebaceous gland.37 In addition, unusually high levels of DHT have been correlated with diseases such as BPH,38 acne, male pattern baldness,39 and female hirsutism.40 Furthermore, recent
Conclusion
Factors independent from those related to enzyme-bound intermediates, such as the C-17 substituent, can greatly influence ligand affinity; within a series, however, those compounds that can best mimic the enolate intermediate (Fig. 4) in the testosterone-to-DHT transformation are the better 5α-reductase inhibitors. In this vein, the presence, placement, and degree of unsaturation have been shown to be critical to the potency of 5α-reductase inhibition by 26-carboxy triterpenoids. However, the
Chemistry
Ganoderma lucidum was obtained from Bisoken (Fukuoka, Japan). The mushroom was identified by Mr. Shuhei Kaneko, Fukuoka Prefecture Forest Research and Extension Center. The fruiting body was dried and ground to powder before use. Unless otherwise specified, chemicals were obtained from Sigma Aldrich Japan (Tokyo, Japan). Organic solvents were purchased from Wako Pure Chemical Industries (Osaka, Japan). [4-14C]Testosterone was obtained from PerkinElmer Japan (Kanagawa, Japan).
Ethanol extracts of G. lucidum
Dried and chipped
References and notes (50)
- et al.
Annu. Rev. Biochem.
(1994) - et al.
Nature
(1968) - et al.
J. Clin. Endocrinol. Metab.
(1972) - et al.
J. Biol. Chem.
(1973) - et al.
J. Clin. Endocrinol. Metab.
(1990) - et al.
J. Invest. Dermatol.
(1992) - et al.
Am. J. Med.
(1977) - et al.
Science
(1974) Endocr. Relat. Cancer
(1996)- et al.
Proc. Natl. Acad. Sci. U.S.A.
(1990)