J Wood Sci (2003) 49:339–343 © The Japan Wood Research Society 2003 DOI 10.1007/s10086-002-0481-y ORIGINAL ARTICLE Yoshio Hirano · Ryuichiro Kondo · Kokki Sakai 5α-Reductase inhibitory tannin-related compounds isolated from Shorea laeviforia Received: March 4, 2002 / Accepted: August 26, 2002 Abstract Five tannin-related compounds – gallic acid, duction catalyzed by steroid 5α-reductase; excessive pro- flavogallonic acid dilactone, valoneic acid dilactone, duction of DHT is responsible for androgen-dependent dis- gallagyldilactone, ellagic acid – were isolated from the eases. A potent 5α-reductase inhibitor may be able to heartwood of Shorea laeviforia, and the inhibitory activity remedy or prevent diseases of this type, including male- of each against rat liver 5α-reductase was evaluated. pattern baldness, benign prostatic hyperplasia, acne, and Valoneic acid dilactone and gallagyldilactone exhibited female hirsutism. Two 5α-reductase isozymes are known, positive inhibitory activity, but gallic acid and ellagic acid and many steroidal 5α-reductase inhibitors have been in- did not. Flavogallonic acid dilactone stimulated 5α- vestigated. One of them, finasteride, a selective and com- reductase activity, even though this compound is structur- petitive inhibitor of type 2 isozyme, has shown strong ally similar to valoneic acid dilactone. The kinetic study of inhibitory activity1 and has been commercialized for the valoneic acid dilactone and gallagyldilactone indicated that treatment of benign prostatic hyperplasia and male-pattern the inhibitory behavior of 5α-reductase were not competi- baldness. This compound decreases the serum DHT con- tive against the substrate (testosterone) and were partially centration and increases the serum testosterone concentra- competitive against the cofactor (NADPH). Additionally, tion,2 but DHT levels do not reach zero because the type 1 double inhibition analysis of valoneic acid dilactone and isozyme produces DHT. On the other hand, some adverse NADPϩ showed synergetic inhibition. These results effects of finasteride (e.g., loss of libido, loss of erection, suggested that neither valoneic acid dilactone nor ejaculatory dysfunction, hypersensitivity reactions, gyneco- gallagyldilactone can affect the binding of testosterone but mastia, and severe myopathy) have been reported.3 There- that either compound could interact with an enzyme– fore, it is preferable to develop a therapeutic agent that can NADPϩ complex to inhibit 5α-reductase. inhibit the type 1 isozyme without such serious adverse effects. Key words Shorea laeviforia · Tannin-related compounds · Since the development of the nonsteroidal inhibitor Valoneic acid dilactone · Gallagyldilactone · 5α-Reductase ONO-3805,4 many nonsteroidal inhibitors have been inves- inhibitor tigated, and some have exhibited a manner of uncompeti- tive kinetics with the enzyme.5 On the other hand, the kinetics of the steroidal inhibitor epristeride have also been 6 Introduction reported as uncompetitive, whereas many steroidal inhibi- tors have been reported as competitive inhibitors versus a substrate.7 Uncompetitive inhibitors are thought to have α Testosterone is converted to a potent androgen 5 - advantages in vivo, as they cannot be affected directly by dihydrotestosterone (DHT) by reduced nicotinamide increasing the substrate concentration.8,9 adenine dinucleotide phosphate (NADPH)-mediated re- We previously reported on rat liver 5α-reductase (rat type 1 isozyme) inhibitory compounds from Shorea species; two dimeric, four trimeric, and two tetrameric stilbenes Y. Hirano (*) were isolated as inhibitory compounds from the heartwood Interior Design Research Institute, Fukuoka Industrial Technology of two Shorea species.10,11 Furthermore, dimeric stilbenes, Center, 405-3 Agemaki, Okawa, Fukuoka 831-0031, Japan Ϯ ϩ ϩ ( )-resveratrol trans-dehydrodimer, synthesized by a Tel. 81-944-86-3259; Fax 81-944-86-4744 α e-mail: hirano@fitc.pref.fukuoka.jp horseradish peroxidase/H2O2 system, showed strong 5 - reductase inhibitory activity.12 R. Kondo · K. Sakai Department of Forest and Forest Products Sciences, Faculty of We report here tannin-related compounds isolated from Agriculture, Kyushu University, Fukuoka 812-8581, Japan the heartwood of Shorea laeviforia (Dipterocarpaceae). We 340 evaluate each compound’s 5α-reductase inhibitory activity Valoneic acid dilactone (3) and kinetic profile. Brown solid: FAB-MS (negative ion mode) m/z: 469(M- Ϫ δ δ Љ H) , H 6.93 (s), 7.01 (s), 7.51 (s); C 106.9 (C-6 ), 108.0, 108.2, 108.4 (C-6, C-6Ј, C-1Љ), 110.5, 111.9 (C-1, C-1Ј), 113.8, Materials and methods 114.6 (C-5, C-5Ј), 135.1, 136.1, 136.5, 139.1, 139.4, 139.5, 140.4, 142.9, 148.4, 149.3 (C-2, C-3, C-4, C-2Ј, C-3Ј, C-4Ј, C- Sample wood 2Љ, C-3Љ, C-4Љ, C-5Љ), 159.0, 159.0 (C-7, C-7Ј), 165.6 (C-7Љ). A block of Shorea laevifolia was collected in Indonesia by Dr. Wasrin Syafii (Bogor Agricultural University, Gallagyldilactone (4) Indonesia). It was preserved at the herbarium of the De- partment of Forest and Forest Products Sciences, Kyushu Brown solid: FAB-MS (negative ion mode) m/z: 601(M- Ϫ δ δ Ј Ј University, Japan. The block was ground in a Wiley mill, H) , H 7.50; C 106.5, 107.4 (C-6, C-1 ), 110.1 (C-6 ), 112.0, and the meal was extracted with methanol for 24h at room 112.4, 122.7 (C-1, C-5, C-2Ј), 135.7, 136.1, 138.7, 139.1, 145.6, temperature. 147.7 (C-2, C-3, C-4, C-3Ј, C-4Ј, C-5Ј), 157.6, 158.8 (C-7, C-7Ј). Extraction and separation of extractives of S. laevifolia Preparation of rat liver microsomes The methanol extract of milled heartwood (500g) of S. laeviforia was concentrated. After the same volume of wa- The livers of Sprague-Dawley female rats were removed ter was added, the mixture was partitioned with n-hexane, and homogenized in medium A (0.32M sucrose, 1mM diethylether, and n-BuOH. The n-BuOH-soluble fraction dithiothreitol, 20mM sodium phosphate, (pH 6.5) as de- 8,9 (64g) was subjected to column chromatography on a scribed in a previous report. The homogenate was centri- Sephadex LH-20 using EtOH as the eluent. Further purifi- fuged at 10000g for 10min at 0°C. The resulting pellets cation was performed by reverse-phase preparative high- were suspended in medium A and underwent centrifuga- performance liquid chromatography (HPLC). Gallic acid tion again. The microsomes were obtained as precipitates (1) 11mg, flavogallonic acid dilactone (2) 39mg, valoneic from further ultracentrifugation (105000g for 1h at 0°C) of acid dilactone (3) 14mg, gallagyldilactone (4) 18mg, and the preceding supernatants. The washed microsomes were ellagic acid (5) 27mg were isolated by preparative HPLC suspended in 10ml of medium A, and the microsomes were (GL Sciences) (C-18 column Inertsil PREP-ODS: 20mm dispersed using a syringe with 18-, 23-, and 26-gauge needles i.d. ϫ 250mm) using an acetonitrile–0.1% TFA gradient in succession. The microsome suspension was divided into (10%–50% acetonitrile) as eluent. small aliquots and stored at Ϫ80°C. The suspension was diluted with medium A just before use. Compounds isolated from S. laeviforia Determination of 5α-reductase inhibitory activity The 1H and 13C spectra were obtained with a JEOL JNM- AL400 spectrometer operating at 400MHz for 1H and at The standard reaction mixture, in a total volume of 0.3ml, 13 µ µ 100MHz for C, respectively. Dimethylsulfoxide (DMSO)- contained the microsomes, 150 M testosterone in 10 l µ 14 µ d6 was used as a solvent for nuclear magnetic resonance of ethanol containing 0.7 M [4- C]testosterone, 167 M (NMR) experiments. NADPH, and medium A, with or without the indicated 13 14 µ Flavogallonic acid dilactone, valoneic acid dilactone, amount of a sample in 10 l of DMSO. The amount of the and gallagyldilactone15 are known from the literature enzyme fraction was adjusted so around 70% of the test- and were identified by fast atom bombardment – mass osterone would be converted to DHT. Incubation was spectrometry (FAB-MS) data and by comparing their 1H- started by adding microsomes to the preheated reaction NMR data. Gallic acid and ellagic acid were identified by solution in a tube. After 10min the incubation was termi- µ comparing the spectroscopic data of commercial reagents nated by adding 10 l of 3M NaOH. (Sigma). The reaction mixture to determine the inhibitory behav- ior of 5α-reductase was the substrate that contained microsomes plus 150, 75, 50, or 37.5µM testosterone in 10µl Flavogallonic acid dilactone (2) of ethanol containing 0.70, 0.35, 0.23, or 0.18µM [4- 14C]testosterone, respectively, plus 167µM NADPH and Brown solid: FAB-MS (negative ion mode) m/z: 469(M- medium A, with or without the indicated amount of a Ϫ δ δ Ј µ H) , H 7.11 (s), 7.49 (s); C 107.3, 107.3 (C-1, C-1 ), 109.5, sample in 10 l of DMSO, in a total volume of 0.3ml. As 109.9 (C-6, C-6Ј), 111.3 (C-5), 112.5 (C-6Љ), 117.6, 120.0 (C- with the standard mixture described above, the amount of 5Ј, C-2Љ), 125.8 (C-1Љ), 135.2, 136.2, 136.7, 137.8, 138.9, 143.1, the enzyme fraction in each mixture was adjusted so around 143.7, 145.8, 147.5 (C-2, C-3, C-4, C-2Ј, C-3Ј, C-4Ј, C-3Љ, C- 70% of the testosterone would be converted to DHT. Incu- 4Љ, C-5Љ), 157.1, 158.9 (C-7, C-7Ј), 167.1 (C-7Љ). bation was started by adding microsomes to the preheated 341 reaction solution in a tube. After 2min the incubation was terminated by adding 10µl of 3M NaOH. The reaction mixture to determine the inhibitory behav- ior of 5α-reductase at the cofactor contained microsomes, 150µM testosterone in 10µl of ethanol containing 0.7µM [4- 14C]testosterone, plus 200, 150, 100, or 50µM NADPH, and medium A, with or without the indicated amount of a sample in 10µl of DMSO, in a final total volume of 0.3ml.