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Mangostanol, a prenyl xanthone from Garcinia mangostana
Pergamon PIh S0031-9422(96)00410-4
Phvtochemistry, Vol. 43, No. 5, pp. 1099-1102, 1996 Copyright (~ 1996 Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/96 $15.00 + 0.00
MANGOSTANOL, A PRENYL XANTHONE FROM GARCINIA MANGOSTANA NATTAYACHAIRUNGSRILERD,*KAZUYATAKEUCHI,YASUSHIOH1ZUMI* SHIGEO NOZOE and TOMIH1SAOHTAt Department of Pharmacognosy; *Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980, Japan
(Received in revisedform 7 May 1996) Key Word Index--Garcinia mangostana; guttiferae; xanthones; mangostanol; cAMP phosphodiesterase inhibition; {3,5,9-trihydroxy-2,2-dimethyl-8-methoxy-7-(3-methylbut-2-enyl)-2H,6H3,4-dihydropyrano[3,2-b]xanthen-6-one}.
Abstract--During studies for identification of biologically active components from natural sources, a new polyoxygenated xanthone mangostanol was isolated from the fruit hull of Garcinia mangostana, along with known xanthones, ce-mangostin, y-mangostin, gartanin, 8-deoxygartnin, 5,9-dihydroxy-2,2-dimethyl-8-methoxy-7-(3methylbut-2-enyl)-2H,6H-pyrano[3,2-b]xanthen-6-one, garcinone E and 2-(y,y-dimethylallyl)-l,7-dihydroxy-3methoxyxanthone and epicatechin. Spectroscopic analysis mainly by 1D and 2D NMR spectroscopy, established the structure of mangostanol {3,5,9-trihydroxy-2,2-dimethyl-8-methoxy-7-(3-methylbut-2-enyl)-2H,6H-3,4dihydropyrano[3,2-b]xanthen-6-one}. Mangostanol, and c~- and y-mangostin show moderate inhibitory effects on cAMP phosphodiesterase. Copyright © 1996 Published by Elsevier Science Ltd
INTRODUCTION
Garcinia mangostana (Guttiferae) is a tree, known for its medicinal properties. The fruit hull (pericarp) of this plant is used as an antiinflammatory agent, astringent and in the treatment of diarrhoea. The fruit hull of this plant has been reported to contain the major products, c~- (2) and y-mangostin (3), and minor xanthones [I-5]. Recently, we have found that a yellowish excretion of the fruit hull of G. mangostana, a crystalline mixture consisting mainly a- (2) and y-mangostin (3), showed an inhibitory effect on cAMP phosphodiesterase. To obtain the active component, we fractionated the methanol extract of the fruit hull. Repeated chromatography on silica gel gave the main components, c~-mangostin (2) and related congeners. Preparative HPLC of a polar fraction gave a new component, mangostanol 1 (Fig. 1). The present paper reports the isolation and structure elucidation of a new polyoxygenated xanthone, mangostanol (1), from the fruit hull, along with other known xanthones. Inhibitory activities of the isolated xanthones on cAMP phosphodiesterase are also described.
RESULTS
AND DISCUSSION
The n-BuOH soluble portion of the methanol extract of G. mangostana was fractionated by a combination of column chromatographic steps using silica-gel (dichloromethane-methanol elution with increasing polartAuthor to whom correspondence should be addressed.
ity) and reversed phase HPLC on ODS-silica gel (aq. methanol elution). The major components a - (2) and y-mangostin (3) and related xanthone congeners were isolated from early fractions. HPLC separation of a polar fraction using a reversed phase column gave mangostanol (1). Mangostanol (1) was isolated as a fine yellow powder, [ce]o + 16.3 °. Its UV absorption maxima (242, 306 and 333 nm) were characteristic of an oxygenated xanthone. The ~H NMR spectrum showed signals due 3 to two tertiary methyls (6 1.34 and 1.46) on sp quaternary carbons, two olefinic methyls (6 1.67 and 1.82), two methylenes (6 2.55; 2.91 and 4.05), a methoxyl (6 3.75), a methine (6 3.78), an olefinic methine (6 5.29) and two aromatic methines (6 6.32 and 6.66). The ~3C NMR spectrum of mangostanol showed 24 signals due to four methyl, two methylene, one methoxyl, two carbinols, fourteen olefinic and a carbonyl carbon. The 13C-tH correlations for all protonated carbons of mangostanol (1) were observed in the HMQC spectrum. The observed NMR signals are characteristic of 1,3,6,7-tetraoxygenated xanthone such as cr-mangostin (2) [1] and 5,9-dihydroxy-2,2dimethyl - 8 - methoxy - 7 - (3 - methylbut - 2 - eny) - 2H,6Hpyrano[3,2-b]xanthen-6-one [5]. The major difference to those compounds is that mangostanol (1) has a carbinol group at C-3. The carbon networks were assigned on the basis of 13C-~H long range correlation data in the HMBC spectrum as shown in Fig. 2. The correlation network from geminal methyls to a phenyl via methine and methylene, was observed as follows. The geminal methyl signals at 6H 1.34 and
1099
1100
N. CHAIRUNGSRILERDet
4' 5' H3C~/CH3
2'
I'
H3CO
0
7
OH 6
10
11
~
~,,~
12
1
4) 5) H3C~./CH 3 i~f3, 2' "~,l I)
R
O
4" CH3 O
l
l
OH
2"[' ' ~ /
"
6 HO
3 5
3~OH
O 10
4
OH
2 : R = CH 3 3:R=H
"?3
al.
1.46, two of the four methyl signals at higher field than 2.0, correlated each other and with two carbinol carbon signals (C-3 and C-2) at 6c 69.6 and 79.5. Those two carbons are connected with methylene hydrogens (6H 2.56; 2.92) that showed correlation peaks with three quaternary aromatic carbons (5, 4a, 12a) at ~c 156.2, 105.4 and 162.2. The latter two signals showed cross peaks with an aromatic hydrogen signal (C-12) at 6H 6.32. This and the other (6H 6.66) isolated aromatic hydrogens (C-10 and C-12) showed four crosspeaks with aromatic carbon signals, respectively. The fact that the two aromatic hydrogens interacted with different aromatic carbons, respectively, indicated similar but differently penta-substituted phenyl groups. The correlation network (B) from the aromatic hydrogen (H-10) to the isoprenyl groups and the carbon (C-8) bearing a methoxyl substituent was assigned as follows. The hydrogen signal (H-10) at 6 6.66 showed a cross peak with the carbon signal (C-8) at ~5 144.9 that was correlated with the O-methyl signal at 6 3.75 and with the methylene signals (H2-1') at 6 4.03 and 4.07. NOE enhancement between methyls in methoxyl and dimethylallyl moieties were also observed. Two partial structures thus were derived by HMBC analysis. Judging from the unsaturation unit in the molecular formula and ~3C chemical shifts, the two partial structures were assembled on either side of the ketone (~c 178.9), thereby constructing a xanthone skeleton. Consequently, the gross structure 1 {3,5,9 trihydroxy - 2,2 - dimethyl - 8 - methoxy - 7 - (3 -
~
3
O/~_~O O~ I B
A
~'~ •
denotesquarternarycarbon Fig. 1. Partial structures of mangostanol (1).
HMBC NOE
Mangostanol, a prenyl xanthone from Garcinia mangostana 120
8o 6o
! 40
,
I 20
i
I 40
,
I 60
,
I 80
i
I 100
Sampleconcentration(p_g/ml) Fig. 2. Inhibitory activities of (73) mangostanol (1), (A) a-mangostin (2) and (0) y-mangostin (3) on cAMP phosphodiesterase, using glycyrhetic acid as control. methylbut - 2 - enyl) - 2H,6H - 3,4 - dihydropyrano[3,2 - b]xanthen - 6 - one} was established for mangostanol as illustrated in Fig. 1. Because of limited amounts of mangostanol (1), the configuration at C-3 has not been determined. Isolated xanthones (1, 2 and 3) showed inhibitory activities against cAMP phosphodiesterase. This enzyme plays an important role in the hydrolysis of the intracellular cyclic AMP. It is considered that the actions of papaverine, dipyridamole, caffeine and theophylline are due to the inhibition of this enzyme [6, 7]. Fig. 2 showed inhibitory activities of xanthones on cAMP phosphodiesterase. Mangostanol (1), a-mangostin (2) and y-mangostin (3) showed IC5o less than 47, 24 and 50 /.tM, respectively.
EXPERIMENTAL
The spectroscopic data were measured by using the following instruments: IR spectra, JASCO A-100s IR spectrometer; UV spectra, Hitachi U-3200 spectrophotometer; optical rotation values, JASCO DIP-340 polarimeter; mass spectra, JEOL DX-303 spectrometer; ~H and 13C NMR spectra, JEOL GX-500 (500 MHz) or Hitachi R-3000 (300 MHz) using tetramethylsilane (TMS) as an internal standard. Thin layer chromatography (TLC) analyses were performed on Kieselgel 60 F254 (MERCK) with detection by UV irradiation and by heating on a hot plate after spraying with anisaldehyde reagent. Plant material. The fruits of G. mangostana were purchased in Bangkok, Thailand. A voucher specimen is deposited in the Herbarium, Botanical Garden, National Cancer Institute (NCI) in Bangkok, Thailand under No. NCI 011094. Isolation procedure. The fresh fruit hull ( 1 kg) of G. mangostana was extracted with MeOH (1 l × 2) at room temp. for l week. The combined extract was concd under red. pres. and then the residual aq. suspension (ca 200 ml) was extracted with EtOAc (150 ml × 2) and n-BuOH (150 ml × 2) successively. The
1101
EtOAc and n-BuOH frs were concd under red. pres. to give a gummy syrup (9.83 g and 22.16 g). The n-BuOH extract (5.87 g) was applied to a silica-gel (120 g, 33 cm i.d. × 3.8 cm) column 1 and eluted with CHzC12, CHEC1E-MeOH (9:1), CH2CI2-MeOH (4:1) and CH2C12-MeOH ( I : 1 ) respectively. Relatively less polar frs gave known compounds, a-mangostin (2, l g), y-mangostin (3, 40 mg), gartanin (10 mg), 8deoxygartanin (10 mg), 5,9 - dihydroxy - 2,2 - dimethyl - 8 - methoxy - 7 - (3 - methylbut - 2 - enyl) - 2H,6H pyrano[3,2 - b]xanthen - 6 - one (10 mg), garcinone E (3.3 mg), 2 - (y,y - dimethylallyl) - 1,7 - dihydroxy - 3 - methoxyxanthone (1.1 mg), epicatechin (40 mg). Known compounds were identified from their MS, IR, 1H and ~3C NMR spectra. Successive purification of a polar fr. by reversed phase HPLC using aq. MeOH as an eluent gave mangostanol (1, 9.4 mg). Mangostanol (1) was recrystallized from CHECI ~MeOH as a fine yellow powder (9.4 mg); [a]~ ~ + 16.3 ° (c = 1.04, MeOH); UV/~maxMeOHnm (log e), 333 (3.94), KBr - I 306 (4.25), 242 (4.44), 208 (4.39). IR Vmax cm " 3418, 1609, 1460, 1275. HREI-MS m/z: 426.1674 [M] +, (Calcd for C 2 4 H 2 6 0 7 : 426.1679). ~H NMR and 13C NMR are shown in Table 1. Bioassay. Phosphodiesterase activity was determined from the amount of phosphate liberated, measured by the malachite green method [8, 9] which is highly sensitive to inorganic phosphate. Phosphodiesterase assay solutions were as follows. (a) The enzyme solution contains phosphodiesterase (0.037 unit ml-~), 5'-nucleotidase (1.67 unit ml-~), MgCI 2 (5 mM) and Tris-HC1 (0.2 M). (b) The reaction mixture A contains malachite green (0.33 mM), polyvinyl alcohol (3.87 g 1 ') and ammonium molybdate in 6N HC1 (8.33 mM). Sample was dissolved in 1.5% dimethyl sulphoxide. The reaction was started by the addition of cyclic AMP (10 mM, 100 tzl) to the enzyme solution (400 #1) at 30 °. After that, sample solution (500 #1), the reagent mixture A (1.0 ml) and 25% sodium citrate (200 /.tl) were added to the above solution successively every 5 min. The absorbance of the colour complex was measured at 630 nm using a UV spectrophotometer (HITACHI, U-2000) referenced against a mixed reagent blank. A calibration curve, obtained by this procedure using potassium dihydrogen phosphate solutions of known concentrations, was used to determine the amount of phosphorus present in the assay. In the control experiment, dimethyl sulphoxide was added instead of the solution of sample to minimize the effect of the vehicle solvent. Here we compare the effects of samples with glycyrrhetinic acid (reference compound for phosphodiesterase assay). All reagents were prepared freshly and distilled water was used in making these reagents. Acknowledgements--We thank Dr Kiti Chindavijak and Ms Pongpun Siripong (National Cancer Institute, Bangkok, Thailand) for providing us with an extraction facility. This work was supported in part by a Grant-inAid for Scientific Research (06672084 and 06303014)
N. CaAmUNGSRILERDet al.
1102
Table 1. ~H and ~3C NMR spectral data for mangostanol (1) in CD3OD Position
~3C
2 3 4
79.5 69.6 27.1
4a 5 5a 6 6a 7 8 9 10 10a lla
105.4 156.2 107.6 178.9 114.9 138.3 144.9 155.7 102.3 156.8 158.4
12
94.4
12a 1'
162.2 27.1
2' 3' 4' 5' 2-CH 3
125.7 131.4 18.4 26.0 20.7 25.6 61.3
8-OCH3
~H*
HMBC correlationst
3.78(1H, dd, J = 5.5, 7.3) 2.56(1H, dd, J = 7.3, 17.3) 2.92(1H, dd, J = 5.5, 17.3)
C-2, 3, 4a, 5, 12a C-2, 3, 4a, 5, 12a
6.66(1H, s)
C-6a, 8, 9, 10a
6.32(1H, s)
C-4a, 5a, lla, 12a
4.03(1H, dd, J = 7.0, 14.0) 4.07(1H, dd, J = 7.0, 14.0) 5.30(1H, dd, J = 1.5, 7.0)
C-6a, 7, 8, 2', 3' C-4', 5'
1.82(3H, s) 1.67(3H, s) 1.34(3H, s) 1.46(3H, s) 3.75(3H, s)
C-2', 3', 5' C-2', 3', 4' C-2, 3, 2-CH 3 C-2, 3, 2-CH 3 C-8
*Coupling constants (J) are given in Hz. tHMBC spectrum was taken with maximum value of 9 Hz for nJcn. from the Ministry of Education, Science, Sports and Culture, Japan. REFERENCES
1. Sakai, S., Katsura, M., Takayama, H., Aimi, N., Chokethawom, N. and Suttajit, M. (1993) Chem. Pharm. Bull. 41, 958. 2. Parveen, M. and Ud-Din Khan, N. (1988) Phytochemistry 27, 3694. 3. Balasubramanian, K. and Rajagopalan, K. (1988) Phytochemistry 27, 1552. 4. Mahabusarakam, W., Wiriyachitra, P. and Taylor, W. C. (1987) J. Nat. Prod. 50, 474.
5. Sen, A. K., Sarkar, K. K., Mazumder, P. C., Banerji, N., Uusvuori, R. and Hase, T. A. (1982) Phytochemistry 21, 1747. 6. Weinryb, I., Chasin, M., Free, C. A., Harris, D. N., Goldenberg, H., Michel, I. M., Paik, V. S., Phillips, M., Samaniego, S. and Hess, S. M. (1972) J. Pharm. Science 61, 1556. 7. Nikaido, T., Ohmoto, T., Noguchi, H., Kinoshita, T., Saitoh, H. and Sankawa, U. (1981) Planta Med. 43, 18. 8. Chan, K. M., Delfert, D. and Junger, K. D. (1986) Analyt. Biochem. 157, 375. 9. Lanzetta, E A., Alvarez, L. J., Reinach, E S. and Candia, O. A. (1979) Analyt. Biochem. 100, 95.
Phvtochemistry, Vol. 43, No. 5, pp. 1099-1102, 1996 Copyright (~ 1996 Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/96 $15.00 + 0.00
MANGOSTANOL, A PRENYL XANTHONE FROM GARCINIA MANGOSTANA NATTAYACHAIRUNGSRILERD,*KAZUYATAKEUCHI,YASUSHIOH1ZUMI* SHIGEO NOZOE and TOMIH1SAOHTAt Department of Pharmacognosy; *Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980, Japan
(Received in revisedform 7 May 1996) Key Word Index--Garcinia mangostana; guttiferae; xanthones; mangostanol; cAMP phosphodiesterase inhibition; {3,5,9-trihydroxy-2,2-dimethyl-8-methoxy-7-(3-methylbut-2-enyl)-2H,6H3,4-dihydropyrano[3,2-b]xanthen-6-one}.
Abstract--During studies for identification of biologically active components from natural sources, a new polyoxygenated xanthone mangostanol was isolated from the fruit hull of Garcinia mangostana, along with known xanthones, ce-mangostin, y-mangostin, gartanin, 8-deoxygartnin, 5,9-dihydroxy-2,2-dimethyl-8-methoxy-7-(3methylbut-2-enyl)-2H,6H-pyrano[3,2-b]xanthen-6-one, garcinone E and 2-(y,y-dimethylallyl)-l,7-dihydroxy-3methoxyxanthone and epicatechin. Spectroscopic analysis mainly by 1D and 2D NMR spectroscopy, established the structure of mangostanol {3,5,9-trihydroxy-2,2-dimethyl-8-methoxy-7-(3-methylbut-2-enyl)-2H,6H-3,4dihydropyrano[3,2-b]xanthen-6-one}. Mangostanol, and c~- and y-mangostin show moderate inhibitory effects on cAMP phosphodiesterase. Copyright © 1996 Published by Elsevier Science Ltd
INTRODUCTION
Garcinia mangostana (Guttiferae) is a tree, known for its medicinal properties. The fruit hull (pericarp) of this plant is used as an antiinflammatory agent, astringent and in the treatment of diarrhoea. The fruit hull of this plant has been reported to contain the major products, c~- (2) and y-mangostin (3), and minor xanthones [I-5]. Recently, we have found that a yellowish excretion of the fruit hull of G. mangostana, a crystalline mixture consisting mainly a- (2) and y-mangostin (3), showed an inhibitory effect on cAMP phosphodiesterase. To obtain the active component, we fractionated the methanol extract of the fruit hull. Repeated chromatography on silica gel gave the main components, c~-mangostin (2) and related congeners. Preparative HPLC of a polar fraction gave a new component, mangostanol 1 (Fig. 1). The present paper reports the isolation and structure elucidation of a new polyoxygenated xanthone, mangostanol (1), from the fruit hull, along with other known xanthones. Inhibitory activities of the isolated xanthones on cAMP phosphodiesterase are also described.
RESULTS
AND DISCUSSION
The n-BuOH soluble portion of the methanol extract of G. mangostana was fractionated by a combination of column chromatographic steps using silica-gel (dichloromethane-methanol elution with increasing polartAuthor to whom correspondence should be addressed.
ity) and reversed phase HPLC on ODS-silica gel (aq. methanol elution). The major components a - (2) and y-mangostin (3) and related xanthone congeners were isolated from early fractions. HPLC separation of a polar fraction using a reversed phase column gave mangostanol (1). Mangostanol (1) was isolated as a fine yellow powder, [ce]o + 16.3 °. Its UV absorption maxima (242, 306 and 333 nm) were characteristic of an oxygenated xanthone. The ~H NMR spectrum showed signals due 3 to two tertiary methyls (6 1.34 and 1.46) on sp quaternary carbons, two olefinic methyls (6 1.67 and 1.82), two methylenes (6 2.55; 2.91 and 4.05), a methoxyl (6 3.75), a methine (6 3.78), an olefinic methine (6 5.29) and two aromatic methines (6 6.32 and 6.66). The ~3C NMR spectrum of mangostanol showed 24 signals due to four methyl, two methylene, one methoxyl, two carbinols, fourteen olefinic and a carbonyl carbon. The 13C-tH correlations for all protonated carbons of mangostanol (1) were observed in the HMQC spectrum. The observed NMR signals are characteristic of 1,3,6,7-tetraoxygenated xanthone such as cr-mangostin (2) [1] and 5,9-dihydroxy-2,2dimethyl - 8 - methoxy - 7 - (3 - methylbut - 2 - eny) - 2H,6Hpyrano[3,2-b]xanthen-6-one [5]. The major difference to those compounds is that mangostanol (1) has a carbinol group at C-3. The carbon networks were assigned on the basis of 13C-~H long range correlation data in the HMBC spectrum as shown in Fig. 2. The correlation network from geminal methyls to a phenyl via methine and methylene, was observed as follows. The geminal methyl signals at 6H 1.34 and
1099
1100
N. CHAIRUNGSRILERDet
4' 5' H3C~/CH3
2'
I'
H3CO
0
7
OH 6
10
11
~
~,,~
12
1
4) 5) H3C~./CH 3 i~f3, 2' "~,l I)
R
O
4" CH3 O
l
l
OH
2"[' ' ~ /
"
6 HO
3 5
3~OH
O 10
4
OH
2 : R = CH 3 3:R=H
"?3
al.
1.46, two of the four methyl signals at higher field than 2.0, correlated each other and with two carbinol carbon signals (C-3 and C-2) at 6c 69.6 and 79.5. Those two carbons are connected with methylene hydrogens (6H 2.56; 2.92) that showed correlation peaks with three quaternary aromatic carbons (5, 4a, 12a) at ~c 156.2, 105.4 and 162.2. The latter two signals showed cross peaks with an aromatic hydrogen signal (C-12) at 6H 6.32. This and the other (6H 6.66) isolated aromatic hydrogens (C-10 and C-12) showed four crosspeaks with aromatic carbon signals, respectively. The fact that the two aromatic hydrogens interacted with different aromatic carbons, respectively, indicated similar but differently penta-substituted phenyl groups. The correlation network (B) from the aromatic hydrogen (H-10) to the isoprenyl groups and the carbon (C-8) bearing a methoxyl substituent was assigned as follows. The hydrogen signal (H-10) at 6 6.66 showed a cross peak with the carbon signal (C-8) at ~5 144.9 that was correlated with the O-methyl signal at 6 3.75 and with the methylene signals (H2-1') at 6 4.03 and 4.07. NOE enhancement between methyls in methoxyl and dimethylallyl moieties were also observed. Two partial structures thus were derived by HMBC analysis. Judging from the unsaturation unit in the molecular formula and ~3C chemical shifts, the two partial structures were assembled on either side of the ketone (~c 178.9), thereby constructing a xanthone skeleton. Consequently, the gross structure 1 {3,5,9 trihydroxy - 2,2 - dimethyl - 8 - methoxy - 7 - (3 -
~
3
O/~_~O O~ I B
A
~'~ •
denotesquarternarycarbon Fig. 1. Partial structures of mangostanol (1).
HMBC NOE
Mangostanol, a prenyl xanthone from Garcinia mangostana 120
8o 6o
! 40
,
I 20
i
I 40
,
I 60
,
I 80
i
I 100
Sampleconcentration(p_g/ml) Fig. 2. Inhibitory activities of (73) mangostanol (1), (A) a-mangostin (2) and (0) y-mangostin (3) on cAMP phosphodiesterase, using glycyrhetic acid as control. methylbut - 2 - enyl) - 2H,6H - 3,4 - dihydropyrano[3,2 - b]xanthen - 6 - one} was established for mangostanol as illustrated in Fig. 1. Because of limited amounts of mangostanol (1), the configuration at C-3 has not been determined. Isolated xanthones (1, 2 and 3) showed inhibitory activities against cAMP phosphodiesterase. This enzyme plays an important role in the hydrolysis of the intracellular cyclic AMP. It is considered that the actions of papaverine, dipyridamole, caffeine and theophylline are due to the inhibition of this enzyme [6, 7]. Fig. 2 showed inhibitory activities of xanthones on cAMP phosphodiesterase. Mangostanol (1), a-mangostin (2) and y-mangostin (3) showed IC5o less than 47, 24 and 50 /.tM, respectively.
EXPERIMENTAL
The spectroscopic data were measured by using the following instruments: IR spectra, JASCO A-100s IR spectrometer; UV spectra, Hitachi U-3200 spectrophotometer; optical rotation values, JASCO DIP-340 polarimeter; mass spectra, JEOL DX-303 spectrometer; ~H and 13C NMR spectra, JEOL GX-500 (500 MHz) or Hitachi R-3000 (300 MHz) using tetramethylsilane (TMS) as an internal standard. Thin layer chromatography (TLC) analyses were performed on Kieselgel 60 F254 (MERCK) with detection by UV irradiation and by heating on a hot plate after spraying with anisaldehyde reagent. Plant material. The fruits of G. mangostana were purchased in Bangkok, Thailand. A voucher specimen is deposited in the Herbarium, Botanical Garden, National Cancer Institute (NCI) in Bangkok, Thailand under No. NCI 011094. Isolation procedure. The fresh fruit hull ( 1 kg) of G. mangostana was extracted with MeOH (1 l × 2) at room temp. for l week. The combined extract was concd under red. pres. and then the residual aq. suspension (ca 200 ml) was extracted with EtOAc (150 ml × 2) and n-BuOH (150 ml × 2) successively. The
1101
EtOAc and n-BuOH frs were concd under red. pres. to give a gummy syrup (9.83 g and 22.16 g). The n-BuOH extract (5.87 g) was applied to a silica-gel (120 g, 33 cm i.d. × 3.8 cm) column 1 and eluted with CHzC12, CHEC1E-MeOH (9:1), CH2CI2-MeOH (4:1) and CH2C12-MeOH ( I : 1 ) respectively. Relatively less polar frs gave known compounds, a-mangostin (2, l g), y-mangostin (3, 40 mg), gartanin (10 mg), 8deoxygartanin (10 mg), 5,9 - dihydroxy - 2,2 - dimethyl - 8 - methoxy - 7 - (3 - methylbut - 2 - enyl) - 2H,6H pyrano[3,2 - b]xanthen - 6 - one (10 mg), garcinone E (3.3 mg), 2 - (y,y - dimethylallyl) - 1,7 - dihydroxy - 3 - methoxyxanthone (1.1 mg), epicatechin (40 mg). Known compounds were identified from their MS, IR, 1H and ~3C NMR spectra. Successive purification of a polar fr. by reversed phase HPLC using aq. MeOH as an eluent gave mangostanol (1, 9.4 mg). Mangostanol (1) was recrystallized from CHECI ~MeOH as a fine yellow powder (9.4 mg); [a]~ ~ + 16.3 ° (c = 1.04, MeOH); UV/~maxMeOHnm (log e), 333 (3.94), KBr - I 306 (4.25), 242 (4.44), 208 (4.39). IR Vmax cm " 3418, 1609, 1460, 1275. HREI-MS m/z: 426.1674 [M] +, (Calcd for C 2 4 H 2 6 0 7 : 426.1679). ~H NMR and 13C NMR are shown in Table 1. Bioassay. Phosphodiesterase activity was determined from the amount of phosphate liberated, measured by the malachite green method [8, 9] which is highly sensitive to inorganic phosphate. Phosphodiesterase assay solutions were as follows. (a) The enzyme solution contains phosphodiesterase (0.037 unit ml-~), 5'-nucleotidase (1.67 unit ml-~), MgCI 2 (5 mM) and Tris-HC1 (0.2 M). (b) The reaction mixture A contains malachite green (0.33 mM), polyvinyl alcohol (3.87 g 1 ') and ammonium molybdate in 6N HC1 (8.33 mM). Sample was dissolved in 1.5% dimethyl sulphoxide. The reaction was started by the addition of cyclic AMP (10 mM, 100 tzl) to the enzyme solution (400 #1) at 30 °. After that, sample solution (500 #1), the reagent mixture A (1.0 ml) and 25% sodium citrate (200 /.tl) were added to the above solution successively every 5 min. The absorbance of the colour complex was measured at 630 nm using a UV spectrophotometer (HITACHI, U-2000) referenced against a mixed reagent blank. A calibration curve, obtained by this procedure using potassium dihydrogen phosphate solutions of known concentrations, was used to determine the amount of phosphorus present in the assay. In the control experiment, dimethyl sulphoxide was added instead of the solution of sample to minimize the effect of the vehicle solvent. Here we compare the effects of samples with glycyrrhetinic acid (reference compound for phosphodiesterase assay). All reagents were prepared freshly and distilled water was used in making these reagents. Acknowledgements--We thank Dr Kiti Chindavijak and Ms Pongpun Siripong (National Cancer Institute, Bangkok, Thailand) for providing us with an extraction facility. This work was supported in part by a Grant-inAid for Scientific Research (06672084 and 06303014)
N. CaAmUNGSRILERDet al.
1102
Table 1. ~H and ~3C NMR spectral data for mangostanol (1) in CD3OD Position
~3C
2 3 4
79.5 69.6 27.1
4a 5 5a 6 6a 7 8 9 10 10a lla
105.4 156.2 107.6 178.9 114.9 138.3 144.9 155.7 102.3 156.8 158.4
12
94.4
12a 1'
162.2 27.1
2' 3' 4' 5' 2-CH 3
125.7 131.4 18.4 26.0 20.7 25.6 61.3
8-OCH3
~H*
HMBC correlationst
3.78(1H, dd, J = 5.5, 7.3) 2.56(1H, dd, J = 7.3, 17.3) 2.92(1H, dd, J = 5.5, 17.3)
C-2, 3, 4a, 5, 12a C-2, 3, 4a, 5, 12a
6.66(1H, s)
C-6a, 8, 9, 10a
6.32(1H, s)
C-4a, 5a, lla, 12a
4.03(1H, dd, J = 7.0, 14.0) 4.07(1H, dd, J = 7.0, 14.0) 5.30(1H, dd, J = 1.5, 7.0)
C-6a, 7, 8, 2', 3' C-4', 5'
1.82(3H, s) 1.67(3H, s) 1.34(3H, s) 1.46(3H, s) 3.75(3H, s)
C-2', 3', 5' C-2', 3', 4' C-2, 3, 2-CH 3 C-2, 3, 2-CH 3 C-8
*Coupling constants (J) are given in Hz. tHMBC spectrum was taken with maximum value of 9 Hz for nJcn. from the Ministry of Education, Science, Sports and Culture, Japan. REFERENCES
1. Sakai, S., Katsura, M., Takayama, H., Aimi, N., Chokethawom, N. and Suttajit, M. (1993) Chem. Pharm. Bull. 41, 958. 2. Parveen, M. and Ud-Din Khan, N. (1988) Phytochemistry 27, 3694. 3. Balasubramanian, K. and Rajagopalan, K. (1988) Phytochemistry 27, 1552. 4. Mahabusarakam, W., Wiriyachitra, P. and Taylor, W. C. (1987) J. Nat. Prod. 50, 474.
5. Sen, A. K., Sarkar, K. K., Mazumder, P. C., Banerji, N., Uusvuori, R. and Hase, T. A. (1982) Phytochemistry 21, 1747. 6. Weinryb, I., Chasin, M., Free, C. A., Harris, D. N., Goldenberg, H., Michel, I. M., Paik, V. S., Phillips, M., Samaniego, S. and Hess, S. M. (1972) J. Pharm. Science 61, 1556. 7. Nikaido, T., Ohmoto, T., Noguchi, H., Kinoshita, T., Saitoh, H. and Sankawa, U. (1981) Planta Med. 43, 18. 8. Chan, K. M., Delfert, D. and Junger, K. D. (1986) Analyt. Biochem. 157, 375. 9. Lanzetta, E A., Alvarez, L. J., Reinach, E S. and Candia, O. A. (1979) Analyt. Biochem. 100, 95.