48 P/ante Medica 56(1990)
A Novel N6-Substituted Adenosine Isolated from Mi Huan Jun (Armillaria mellea) as a Cerebral-Protecting Compound Nuoharu Watanahe '. Tadashi Obuchi1 Masaharu Tarnai , H/ron/clAra/cl . Sadafwni Gm urn Yang Jun-S/tan2. Yu De-Quan2. LinngXiao-Tian'. and Huan Jun-Hue2 2
Research Center, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Omiya, Saitama 330, Japan Institute of Materia Medica, Chinese Academy of Medical Sciences, I Xian Nong Tan Street, Beijing, Peop]e's Republic of China Address for correspondence
Materials and Methods Fermentation ofA. me/lea
Abstract Successive purification of a crude extract of cultured Mi Huan Jun mycelia, followed by an assay of the effect on complete ischemia in mice, led to the isolation of a new compound with cerebral protecting activity, hereafter designated as AMG-1. The structure of AMG-1
was proposed as being 6-(5-hydroxy-2-pyridyl-
methylamino)-9--rihofuranosylpurine (1) en the basis of its 1JV, mass, 1H-NMR, and 'tC-NMR spectra.
Arm/liar/a me/lea Vahl. ex Fr. Quel. (Tricholomataceae) (Mi Huan Jun) was cultured as reported by Yang et al. (1). The rnycelia obtained by filtration of cultured broth were airdried at 65—75 °C.
Bioassay Ten male ICR strain mice, weighing 20—30g. were used for each group. Cerebral ischemia was produced by de-
capitation according to the method described by HolowachThurston et al. (4). The mice were decapitated caudal to the intersec-
Key words
Arm/liar/a me//ca, cytokinin, N6-substituted adenosine, cerebral-protecting compound.
tion of the medulla and the spinal cord. Crude or purilied materials, equivalent to 10—20 g of dried mycelia were dissolved in 10 ml of sahne and were administered subcutaneously in a volume of 10 mI/kg 30 minutes before decapitation. Gasping elicited by decapitation began several seconds after decapitation. Gasping was
considered to have terminated (the last gasp) when respiration ceased and the tongue protruded. The time (duration of gasping) between decapitation and the last gasp was determined. In this
Introduction
model, some antidepressants. neuroleptics and tranquilizers
Mi lluan Jun. a fungus IAr,nillaria mel/en
ing (6).
Vahi. cx Fr. Quel. (Tricholomataceae)J symbiotic with Gastrod/a data Blume, was first cnltured by us (1) to serve for a wide range of clinical uses in China. Cultured Mi Huan Jun mycelia are used in the form of tablets to treat geriatric pa-
tients with palsy, dizziness, headacbe, neurasthenia, insomnia, numbness in limbs, and infantile convulsion, as is also the well-known Tian Ma, a symbiont of Mi Huan Jun and G. claw Blume (2).
In tbe course of our pharmacological studies on the extracts of Mi iluan Jun, cerebral-protecting activities were found (3) in the decapitation-induced complete ischemia model (4). Little is known about the effective principles of the extract of Mi Huan Jun but it is known to contain antimicrobial sesquiterpenoid aromatic esters (5). In this paper, we describe the isolation of a cerebral-protecting principle, designated as AMG-1, from the extract of Mi Huan Jun. and its structure elucidation by spectral analyses.
significantly and dose-dependently prolonged the duration of gasp-
Isolation oj'/tMG-I 'l'he dried mycella (1.25 kg) of A. melee were extracted three times with 61 of 50% aqueous methanol at 60°C for 1 h. The combined extracts were concentrated under reduced pressure to a volume of 21. The aqueous layer obtained frooi partition with ethyl acetate was concentrated to 500 ml, diluted to 61 with water, and was submitted to chromatography on a H-I 03 (polystyrene absorbeot, Nankai University, China) column (colunui bed volume: Vt = 1.51. 8.0 a 29.9cm). After washing with 31 ofwator, the column was eluted with 31 of methanol. Evaporation of the solvent in eacuo gave 46.8 g of a residue (A3). which was dissolved in 21 of water and subjected to a liowex SOW (H' form, Vt 980 ml, 5.5 x 41,3cm) column after removal of the insoluble materials. The column was developed with 21 of water and 41 oft M NH4OH solu-
tion, succesively. Biologically active fractions doted with I M NII4OH solution were collected and concentrated to yield crude material (A3B3, 23.4 g). Further purification was achieved by preparative HPLC (Waters Prep LC/System 500 equipped with a Prep Pak-500/C18 cartridge) using a stepwise gradient of methanol in water at a flow rate of 200 mI/mm. The active fraction ehited with 50% methanol was concentrated in vecuo to dryness. The dried material (A3B3C6, 3.01 g) was dissolved in 10 ml of water, and purified by Sephadex (i-lU (Pharmacia) column IV! 480 ml, 4.Os 38.2 cm) chromatography using water as the eluant.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Received: January 9, 1989
Planta Medica 56(1990) 49
A NovelN6-SubstitutedAdenosine Isolated from Mi Huan Jun (Armillaria mellea)
The fractions containing the active compound,
(A3B3C6D6E5 + A3B3C6D7F6) was achieved by HPLC [Waters 6000A equipped with a column: Nucleosil 5C18 (10 x 250 mm) and a (IV detector at 215 nm; eluted with 15% CH3CNI. Homogeneity of AMG-1 was ascertained in analytical HPLC (Nucleosil5C18, 10% CH3CN in 0.1 % trifluoroacetic acid) and by TLC (F. Merck Silica gel
eluted in a range ofVe (elution volume)IVt 2.8—3.7 and 3.7—5.7, were collected and evaporated to dryness. These active materials
(A3B3C6D6, 45.8mg and A3B3C6D7, 23.2mg) were further purified by HPLC [Waters 6000A equipped with a column: Develosil ODS-5 (10 x 250mm) and a (IV detector at 215nm] using
60F254, Art. 5715: chloroform-methanol 10: 3). The yield was 1.2mg. The isolation procedure for AMG-1 is summarized in Fig. l.
20% methanol to give fractions (A3B3C6D6E5. A3B3C6D7F6) con-
taining AMG-1. Isolation of AMG-1 from combined fractions Dried Mycelia (1,250 g)
extracted with 50% methanol
Crude extract washed with ethylacetate Aqueous layer
H-103 column chromatography
Step 1
MeOH fraction
Al
A2
Fig. 1 Summary of the isolation procedures for the cerebral protecting compound.
—: active fraction.
A3
Dowex SOW column chromatography
Step 2
1M, NH4OH fraction
A3B1 -B2
-B3 HPLC Prep Pat ODS
-C2
A3B3C1
Step 3
-d6
-d3
Step 4
G-10 column
Step 5 Develosil ODS
IPLC
Step 6
Nucleosil 5C18
Fig. 2 Preparative HPLC of A3B3C6D6 (A) and A3B3C6DJ (B). -E5, -F6: active fractions.
(A)
(B) - F6
-E5
A)
k&JJ I
0
25
I
1
1
.
tR mm
50
0
I
25
tR ml n
50
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_______
50 Planta Medica 56(1990)
Naoharu Watanabe et al.
UV spectra were recorded by a Hitachi model 220A spectrophotometer. 1H-NMR and '3C-NMR spectra were recorded in CD3OD using a JEOL JNM-GX400 spectrometer and a Va-
nan XL 300 spectrometer, respectively. Chemical shifts (1) are given in ppm from TMS as the internal standard and coupling con-
stants (I) in Hz. Secondary ion mass spectra (SIMS) were determined with a Hitachi M-80A mass spectrometer with a Hitachi M003 data acquisition and processing system.
GC/MS A JEOL JMS-DX303 gas chromatograph-mass spectrometer was used. Conditions for analysis were as follows: methylsiicone (0.53mm x 10 m, Hewlett Packard) column, at 300 °C; He flow 20 mI/mm; separator temperature 300 °C; injection
temperature 320 °C; electron energy 70 eV, accelerating voltage 2.5 kV. Mass measurements and determination of the elemental composition of ions were carried out using a JEOL JMA-DA5 000 mass data system.
Table 1 1H-NMR and 13C-NMR assignments of AMG-1 in CD3OD at 20°C (°at 60°C).
'H
Carbon No.
shift
multiplicity
s
2
8.21
4
—
5
—
6 8 1'
8.27
J
154.3 145.7 121.5
159.7 142.5
—
2'
5.96 4.76
s d dd
3'
4.32
dd
4'
4.17
dt
5'
HA 3.74
dd dd
6.2 6.2,5.2 5.2,2.8 2.8,2.5 2.5,12.8 2.5,12.8
HB3.88
13C
shift
multiplicity d
s s
s
76.3
d d d
73.5
d
89.0
d
92.1
64.3
t
2"
—
7.28
dd
8.0,0.9
155.5 124.8
s
3" 4"
7.18
dd
8.0,2.8
125.9
d
5" 6" 7"
—
150.4
s
138.4
d
8.06
dd
4.78°
broads
2.8,0.9
45.8
d
t
AMG-1 (1)
Results Isolation of the cerebral-protecting compound
Colourless powder; UV: ?max (90% CH3OH) = 268nm; ?max (90% CH3OH, acidic) =265nm; ?max (90% CH3OH, basic) = 264nm; SIMS: ,n/z 375 (MH), 397 (M + NaY; 'H-NMR (CD3OD) and 13C-NMR (CD3OD): see Table 1.
Table 2 shows the enrichment and localization of cerebral protecting activity in each step of purification and isolation.
Tetrakis-trimethylsilyl-AMG-1: TMS4-AMG- I
Trimethylsilylation of AMG-1 with bis(trimethylsilyl)-acetamide in acetonitrile (1: 2) at 70°C for 15 mm in a sealed vial gave TMS4-AMG-1 with a trace ofTMS5-AMG-1 (m/z 734, M). GC/MS of TMS4-AMG-1 (retention time on GC trace = 4.04 mm): in/z 662 (Mt, 20%). 647 (M — CH3, 9%), 572 (M — TMSOH, 2%), 557 (M — CH3 TMSOH, 3%), 444 EM - C3H4(OTMS)2,
-
2%], 429 EM — C3H30(OTMS)2, 5%], 343 EM — C4H4(OTMS)3, 40%], 314 EM — C5H50(OTMS)3, 45%], 348 [C5H50(OTMS)3, 4%], 259(348
The crude fraction A3, with the cerebral protecting activity at a dose of 377mg (equivalent to lOg of dried mycelia)/kg s.c., was purified to yield two active fractions: A3B3C6D6 and A3B3C6D7 in four steps by H-103 column, Dowex 50W column, preparative HPLC and Sephadex G-10 column.
—
TMSO, 10%), 245 (348 — CH2OTMS, 17%), 230 [C4H5(OTMS)2, 38%], 217 [C3H3(OTMS)2, 13%], 195 (NH-CH2-C5H3N-OTMS, 13%), 180 (CH2-C5-H3N-OTMS, 15%) (see Fig. 3).
180
379CH2OTMS- 482 OTMS393
'46 166
1662:Mf--3...647
— 195; HNLcH2--,_)--OTMS
-TM50H -TMS0H
572
557
377TMS0H-467f N
L
.,LN -CH2Ofl-IS 245
TMSO
103,..559
-H20
541
34 3(•H)
QTMS\OTMS - 320
234 429
,-TMSOH
230 217
(- H)
444
Fig.3 Proposed ElMS fragmentations for TMS4-AMG-1.
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Instrumental methods
Planta Med/ca 56(1990) 51
A Novel N6-Substitu ted Adenosine Isolated from Mi Huan Jun (Armillaria mellea)
Step
1
activefr. A3
2
4
3
A3B3
6
5
A3B3C6
-C6D6
-C6D7
-D6E5
-D7F6
AMG-1
fr.wt.
1240g
1200g
1160g
fr.wt.b
46.8g
23.4g
3.Olg
ilOOg 45.8mg
ilOOg 23.2mg
1050g 2.5mg
1050g 1.7mg
l000g 1.2mg
10
10
10
20
20
20
20
20
377
195
25.9
0.833
0.422
0.048
0.032
0.024
gekg mg/kg
Table 2 The enrichment and localization of cere bral-protecting activity of the purified sample by each purification step.
Fraction weight represented as the reduced weight of dried mycelia. Fraction weight represented as the weight of active fraction. Minimum effective dose represented as the reduced weight of dried mycelia. Minimum effective dose represented as the weight of active fraction.
When administered s.c., AMG-1 showed marked dose-related efficacy between 12 and 48 .tg/kg (Table 3). Table 3 Effect of AMG-1 (1) on persistent time of gasping of isolated heads of mice by decapitation. dose .sg/kg s.c.
gasping duration mean (sec)
24.22 0.59"
48
Statistical significance was assessed using a one.way analysis of variance followed
by the Dunnet test (20). ' p < 0.05, " p < 0.001
Structure elucidation AMG-1 (1) exhibited the following UV spectral characteristics: Xmx (90% CH3OH, neutral) = 268 nm; max (90% CH3OH, acidic) = 265nm; max (90% CH3OH, basic) = 264 nm, indicative of a 6,9-disubstituted adenine (7, 8). The 1H-NMR spectrum showed two signals at ô = 8.21
(2-H, s) and 8.27 ppm (8-H, s), three methine protons at ô = 4.76(2'-H, dd,J= 6.2, 5.2), 4.32 (3'-H, dd, J= 5.2, 2.8), and 4.17 ppm (4'-H, dt, I = 2.8, 2.5), ABX-type coupled hydroxymethyl protons ô 3.74 (5'-HA, dd, J= 2.5, 12.8) and
3.88ppm(5'-HB, dd, J= 2.5,12.8), andan anomericproton at ö = 5.96 ppm (1'-H, d, 1= 6.2). These results, as well as the '3C-NMR spectral data (Table 1) indicated the structure I as being N6-substituted 93-ribofuranosyladenine.
H
N'' H
ion at m/z = 662.2941 corresponding to C16H14N605TMS4 (calcd. 662.2919). Thus, the formula of AMG-1 was deter-
mined to be C16H18N605 FM. = 374; SIMS: m/z = 375 (MH)I.
By subtracting the composition (C10F112N504) of the identified adenosine moiety from the empirical formula ofAMG1, the N6-substituent was deduced to have the composition C6H6NO. The fragment ion at ,n/z = 180, corresponding to C6H5NOTMS, was observed in the ElMS spectrum of TMS4-
AMG-1. This was regarded as being strong evidence that the unidentified N6-substitutent has a mass of 108 a.m.u. The proposed MS fragmentation (9, 10) scheme for TMS4AMg-1 is shown in Fig. 3.
S.E.M.
20.33 0.38 19.57 0.42 21.93±0.71'
water control 12 24
The high resolution mass spectrum ofAMG1 as a trimethylsilyl (TMS) derivative yielded a molecular
The signals attributed to the unidentified unit in the 1H-NMR spectrum of AMG-1 were mainly ob-
served in the aromatic region: ô 7.18 (4"-H, dd, J= 8.0, 2.8), 7.28 (3"-H, dd, J= 8.0, 0.9), and 8.06 ppm(6"-H, dd, I = 2.8, 0.9). These signals were indicative of the presence of a 2,5-disubstituted pyridine in the N6-substituent. Besides these protons, a signal at b = 4.78 ppm (7"-H2, br. s) as well as a signal at ô = 45.8 ppm in the 13C-NMR were detected. These were characteristic of the -CH2- group adjacent to the
N6. The introduction of a TMS group into the N6-substituent, as was observed in the ElMS of TMS4-AMG-1, indi-
cates that one hydrexy group is present on an N6-substituent. Thus, 5-hydroxy-2-pyridylmethyl or 2-hydroxy-5pyridylmethyl moieties are likely for the N6-substituent.
Comparison studies on the 1H-NMR and 13C-NMR of the aromatic region of AMG-1 and 2-methyl-5hydroxypyridine were carried out. The 1H-NMR signals [ô = 7.04 (3-H, dd, J = 8.5, 0.9), 7.13 (4-H, dd, I = 8.5, 2.6), 7.95 ppm (6-H, dd, J= 2.6, 0.9)] and 13C-NMR signals [ö = 126.05 (C-3, d), 126.11 (C-4, d), 137.78 (C-6, d), 150.28 (C-
5, s), 154.40 ppm (C-2, s)J of 2-methyl-5-hydroxypyridine showed close similarity to those of AMG-1 (see Table 1). From this, a 5-hydroxy-2-pyridylmethylamino moiety was ascertained to he the partial structure of AMG-1, rather than a 2-hydroxy-5-pyridylmethylamino moiety. All these data suggest AMG-1 as being 6-(5hydroxy-2-pyridy1rnethylamino)-9-ribofuranosylpurine (1).
HO HO OH
1
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Preparative HPLC on Develosil ODS columns of A3B3C6D6 and A3B3C6D7 gave active fractions (-E5, -F6) with the same retention time as shown in Fig. 2. This demonstrated that one compound was responsible for the cerebral-protecting activity of these fractions. Preparative HPLC on a Nucleosil 5C15 column of combined -E5 and -F6 yielded an active principle, AMG-1.
Naoharu Watanabe et al.
52 Planta Medico 56 (1990)
Discussion
The authors are grateful to Dr. K. Kushida (Varian Instruments Ltd.) for measuring the 13C-NMR spectra, and Dr. D. C. Yue (Institute of Material Medica, Chinese Academy of Medical Sciences) for the cultivation of Mi Huan Jun.
References
In the complete ischemic test in mice, prolongation of gasping is one measure of the anti-ischemic effect. Gasping is regulated by nerve cells in the respiratory
center. A sustained neuronal function in the respiratory center would lead to prolonged gasping. Thus, the cerebralprotecting effect of the crude extract of Mi Huan Jun as well as AMG-1 will derive from the influence on nerve cells of the respiratory center in this experimental model.
Table 2 shows that the cerebral-protecting activity was concentrated to AMG-1. However, it is presum-
able that inhibitors or other active compounds are present in the biologically inactive fractions which were discarded during isolation of AMG-1. In fact, the presence of various
Yang, Y. P., Yue, D.C., Huo, Z. M. (1976) Acta Micro. Sinica 16,206— 2
Araki, H., Tsuchida, K., Hosoda, K., Aihara, H., Tamai, M., 'I
6
8
kinds ofpurine analogs in Mi Huan Jun mycelia is suggested
by our preliminary experiments (data are not shown), and xanthine analogs, which increased the cerebral metabolic rate and shortened the duration of gasping (6).
10
Further studies on these possible biologically active compounds in the extract will clarify the degree of the contribution of AMG-1 to the cerebral-protecting activity of Mi Huan Jun mycelia.
AMG-1 was suggested to be an N6-substituted adenosine, based on its spectral data. Some N6-substituted adenosines are well known as cytokinins (11, 12) and as one of the growth regulators of plants. 6-(O-Hydroxybenzylamino)-9[3-D-ribofuranosylpurine (13, 14, 15), a compound quite similar to AMG-1, has been isolated from the mature leaves of poplar (Populus x robusta Schneid) as a cytokinin. This has medium activity in the soybean callus
test but shows high activity in the radish leaf senescence test, suggesting that AMG-1 shows cytokinin activities. This will be discussed in detail elsewhere.
N6-Substituted adenosine also affects the cellular receptors of adenosine to cause a potentiation of histamine release, inhibition of platelet aggregation, inhibition ofpurinergic neurons, and/or sedation of the brain (16, 17). In order to secure further information on the role of the N6-substituent of adenosine, many anologs have been prepared and examined for their biological properties (18). Among them, AMG- 1 might be regarded as one ofthe potent cerebral-protecting compounds in the ischemia or hypoxia model.
The total synthesis of AMG-1 is under way to afford a substantial amount of AMG-1 for use in pharmacological evaluation for a comparison with the other N6substituted adenosines. This will also result in a chemical confirmation of the structure of AMG-1, which was considered to be most likely from its spectral data. This will be reported in the following paper (19).
213. Huang, Z. L. (1985) Chinese Journal of Integrated Traditional and Western Medicine 5, 251—257.
12 13 14
16
17 18
19
20
Watanabe, N., Omura, S., in preparation. Holowach-Thurston, J., Hanhart, R. E., Jones, E. M. (1987) Pediat. Res. 8, 238—243. Yang, J. S., Chen, Y. W., Feng, X. Z., Yu, D. Q., Liang, X. T. (1984) Planta Med. 50, 288—290. Araki, H., Karasawa, Y., Nojiri, M., Aihara. H. (1988) Meth. Find. Exptl. Clin. Pharmacol. 10, 349—356. Townsend, L. B., Robins, R. K., Loeppky, H. N.. Leonard, N. J. (1964) J. Am. Chem. Soc. 86, 5320—5325. Leonard, N. J., Carraway, K. L., Helgeson, J. P. (1965) J. Heterocycl. Chem. 2,291—297. McCloskey, J. A., Lawson, K. T., Krueyer, P.M., StilIwell, R. N. (1968) J. Am. Chem. Soc. 90. 4182—4184. Upper, C. D., Helgeson, J. P., Schmidt, C. J. (1970) Plant Growth Substances 798—807. Fleysher, M. H., Bloch, A., Flakala, T., Nichol, C. A. (1969) J. Med. Chem. 12, 1056—1061. Fleysher, M. H. (1972)J. Med. Chem. 15, 187—191. Hewett, E. W., Wareing, P. F. (1973) Planta (Berl.) 112, 225—233. Horgan, H., Hewett, E. W., Purse, J. G., Wareing, P. F. (1973) Tetrahedron Lett. 2827—2828. Horgan, R., Hewett, E. W., Horgan, J. M., Purse, J. G., Wareing, P. F. (1975) Phytochemistry 14, 1005—1008. Daly, J. W. (1982) J. Med. Chem. 25. 197—207. Snyder,S.H.,Katims,J.J.,Annau,Z..Bruns,R.F.,Daly,J.W.(1981) Proc. Natl. Acad. Sci. USA 78. 3260—3264. Kusachi, S., Thompson, R. D., Yamada, N., Daly, D. T., Olsson, R. A. (1986) J. Med. Chem. 29, 989—996. Hayashi, M., Araki, H., Watanabe, N., Tamai, M., Yokomori, S., Hatayama, K., Sota, K., Yang, J. S., Yu, D. Q., Liang, X. T., Huang, J. H. Planta Med., to be submitted. Dunnett, C. W. (1955) Am. Statist. Assoc. J. 50, 1096—1121.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
The complete ischemia-induced decapitation model used for the examination of purified materials from Mi-Huan Jun resulted in the isolation of AMG-1 with cerebral-protecting activity.
Acknowledgements
A Novel N6-Substituted Adenosine Isolated from Mi Huan Jun (Armillaria mellea) as a Cerebral-Protecting Compound Nuoharu Watanahe '. Tadashi Obuchi1 Masaharu Tarnai , H/ron/clAra/cl . Sadafwni Gm urn Yang Jun-S/tan2. Yu De-Quan2. LinngXiao-Tian'. and Huan Jun-Hue2 2
Research Center, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Omiya, Saitama 330, Japan Institute of Materia Medica, Chinese Academy of Medical Sciences, I Xian Nong Tan Street, Beijing, Peop]e's Republic of China Address for correspondence
Materials and Methods Fermentation ofA. me/lea
Abstract Successive purification of a crude extract of cultured Mi Huan Jun mycelia, followed by an assay of the effect on complete ischemia in mice, led to the isolation of a new compound with cerebral protecting activity, hereafter designated as AMG-1. The structure of AMG-1
was proposed as being 6-(5-hydroxy-2-pyridyl-
methylamino)-9--rihofuranosylpurine (1) en the basis of its 1JV, mass, 1H-NMR, and 'tC-NMR spectra.
Arm/liar/a me/lea Vahl. ex Fr. Quel. (Tricholomataceae) (Mi Huan Jun) was cultured as reported by Yang et al. (1). The rnycelia obtained by filtration of cultured broth were airdried at 65—75 °C.
Bioassay Ten male ICR strain mice, weighing 20—30g. were used for each group. Cerebral ischemia was produced by de-
capitation according to the method described by HolowachThurston et al. (4). The mice were decapitated caudal to the intersec-
Key words
Arm/liar/a me//ca, cytokinin, N6-substituted adenosine, cerebral-protecting compound.
tion of the medulla and the spinal cord. Crude or purilied materials, equivalent to 10—20 g of dried mycelia were dissolved in 10 ml of sahne and were administered subcutaneously in a volume of 10 mI/kg 30 minutes before decapitation. Gasping elicited by decapitation began several seconds after decapitation. Gasping was
considered to have terminated (the last gasp) when respiration ceased and the tongue protruded. The time (duration of gasping) between decapitation and the last gasp was determined. In this
Introduction
model, some antidepressants. neuroleptics and tranquilizers
Mi lluan Jun. a fungus IAr,nillaria mel/en
ing (6).
Vahi. cx Fr. Quel. (Tricholomataceae)J symbiotic with Gastrod/a data Blume, was first cnltured by us (1) to serve for a wide range of clinical uses in China. Cultured Mi Huan Jun mycelia are used in the form of tablets to treat geriatric pa-
tients with palsy, dizziness, headacbe, neurasthenia, insomnia, numbness in limbs, and infantile convulsion, as is also the well-known Tian Ma, a symbiont of Mi Huan Jun and G. claw Blume (2).
In tbe course of our pharmacological studies on the extracts of Mi iluan Jun, cerebral-protecting activities were found (3) in the decapitation-induced complete ischemia model (4). Little is known about the effective principles of the extract of Mi Huan Jun but it is known to contain antimicrobial sesquiterpenoid aromatic esters (5). In this paper, we describe the isolation of a cerebral-protecting principle, designated as AMG-1, from the extract of Mi Huan Jun. and its structure elucidation by spectral analyses.
significantly and dose-dependently prolonged the duration of gasp-
Isolation oj'/tMG-I 'l'he dried mycella (1.25 kg) of A. melee were extracted three times with 61 of 50% aqueous methanol at 60°C for 1 h. The combined extracts were concentrated under reduced pressure to a volume of 21. The aqueous layer obtained frooi partition with ethyl acetate was concentrated to 500 ml, diluted to 61 with water, and was submitted to chromatography on a H-I 03 (polystyrene absorbeot, Nankai University, China) column (colunui bed volume: Vt = 1.51. 8.0 a 29.9cm). After washing with 31 ofwator, the column was eluted with 31 of methanol. Evaporation of the solvent in eacuo gave 46.8 g of a residue (A3). which was dissolved in 21 of water and subjected to a liowex SOW (H' form, Vt 980 ml, 5.5 x 41,3cm) column after removal of the insoluble materials. The column was developed with 21 of water and 41 oft M NH4OH solu-
tion, succesively. Biologically active fractions doted with I M NII4OH solution were collected and concentrated to yield crude material (A3B3, 23.4 g). Further purification was achieved by preparative HPLC (Waters Prep LC/System 500 equipped with a Prep Pak-500/C18 cartridge) using a stepwise gradient of methanol in water at a flow rate of 200 mI/mm. The active fraction ehited with 50% methanol was concentrated in vecuo to dryness. The dried material (A3B3C6, 3.01 g) was dissolved in 10 ml of water, and purified by Sephadex (i-lU (Pharmacia) column IV! 480 ml, 4.Os 38.2 cm) chromatography using water as the eluant.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Received: January 9, 1989
Planta Medica 56(1990) 49
A NovelN6-SubstitutedAdenosine Isolated from Mi Huan Jun (Armillaria mellea)
The fractions containing the active compound,
(A3B3C6D6E5 + A3B3C6D7F6) was achieved by HPLC [Waters 6000A equipped with a column: Nucleosil 5C18 (10 x 250 mm) and a (IV detector at 215 nm; eluted with 15% CH3CNI. Homogeneity of AMG-1 was ascertained in analytical HPLC (Nucleosil5C18, 10% CH3CN in 0.1 % trifluoroacetic acid) and by TLC (F. Merck Silica gel
eluted in a range ofVe (elution volume)IVt 2.8—3.7 and 3.7—5.7, were collected and evaporated to dryness. These active materials
(A3B3C6D6, 45.8mg and A3B3C6D7, 23.2mg) were further purified by HPLC [Waters 6000A equipped with a column: Develosil ODS-5 (10 x 250mm) and a (IV detector at 215nm] using
60F254, Art. 5715: chloroform-methanol 10: 3). The yield was 1.2mg. The isolation procedure for AMG-1 is summarized in Fig. l.
20% methanol to give fractions (A3B3C6D6E5. A3B3C6D7F6) con-
taining AMG-1. Isolation of AMG-1 from combined fractions Dried Mycelia (1,250 g)
extracted with 50% methanol
Crude extract washed with ethylacetate Aqueous layer
H-103 column chromatography
Step 1
MeOH fraction
Al
A2
Fig. 1 Summary of the isolation procedures for the cerebral protecting compound.
—: active fraction.
A3
Dowex SOW column chromatography
Step 2
1M, NH4OH fraction
A3B1 -B2
-B3 HPLC Prep Pat ODS
-C2
A3B3C1
Step 3
-d6
-d3
Step 4
G-10 column
Step 5 Develosil ODS
IPLC
Step 6
Nucleosil 5C18
Fig. 2 Preparative HPLC of A3B3C6D6 (A) and A3B3C6DJ (B). -E5, -F6: active fractions.
(A)
(B) - F6
-E5
A)
k&JJ I
0
25
I
1
1
.
tR mm
50
0
I
25
tR ml n
50
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50 Planta Medica 56(1990)
Naoharu Watanabe et al.
UV spectra were recorded by a Hitachi model 220A spectrophotometer. 1H-NMR and '3C-NMR spectra were recorded in CD3OD using a JEOL JNM-GX400 spectrometer and a Va-
nan XL 300 spectrometer, respectively. Chemical shifts (1) are given in ppm from TMS as the internal standard and coupling con-
stants (I) in Hz. Secondary ion mass spectra (SIMS) were determined with a Hitachi M-80A mass spectrometer with a Hitachi M003 data acquisition and processing system.
GC/MS A JEOL JMS-DX303 gas chromatograph-mass spectrometer was used. Conditions for analysis were as follows: methylsiicone (0.53mm x 10 m, Hewlett Packard) column, at 300 °C; He flow 20 mI/mm; separator temperature 300 °C; injection
temperature 320 °C; electron energy 70 eV, accelerating voltage 2.5 kV. Mass measurements and determination of the elemental composition of ions were carried out using a JEOL JMA-DA5 000 mass data system.
Table 1 1H-NMR and 13C-NMR assignments of AMG-1 in CD3OD at 20°C (°at 60°C).
'H
Carbon No.
shift
multiplicity
s
2
8.21
4
—
5
—
6 8 1'
8.27
J
154.3 145.7 121.5
159.7 142.5
—
2'
5.96 4.76
s d dd
3'
4.32
dd
4'
4.17
dt
5'
HA 3.74
dd dd
6.2 6.2,5.2 5.2,2.8 2.8,2.5 2.5,12.8 2.5,12.8
HB3.88
13C
shift
multiplicity d
s s
s
76.3
d d d
73.5
d
89.0
d
92.1
64.3
t
2"
—
7.28
dd
8.0,0.9
155.5 124.8
s
3" 4"
7.18
dd
8.0,2.8
125.9
d
5" 6" 7"
—
150.4
s
138.4
d
8.06
dd
4.78°
broads
2.8,0.9
45.8
d
t
AMG-1 (1)
Results Isolation of the cerebral-protecting compound
Colourless powder; UV: ?max (90% CH3OH) = 268nm; ?max (90% CH3OH, acidic) =265nm; ?max (90% CH3OH, basic) = 264nm; SIMS: ,n/z 375 (MH), 397 (M + NaY; 'H-NMR (CD3OD) and 13C-NMR (CD3OD): see Table 1.
Table 2 shows the enrichment and localization of cerebral protecting activity in each step of purification and isolation.
Tetrakis-trimethylsilyl-AMG-1: TMS4-AMG- I
Trimethylsilylation of AMG-1 with bis(trimethylsilyl)-acetamide in acetonitrile (1: 2) at 70°C for 15 mm in a sealed vial gave TMS4-AMG-1 with a trace ofTMS5-AMG-1 (m/z 734, M). GC/MS of TMS4-AMG-1 (retention time on GC trace = 4.04 mm): in/z 662 (Mt, 20%). 647 (M — CH3, 9%), 572 (M — TMSOH, 2%), 557 (M — CH3 TMSOH, 3%), 444 EM - C3H4(OTMS)2,
-
2%], 429 EM — C3H30(OTMS)2, 5%], 343 EM — C4H4(OTMS)3, 40%], 314 EM — C5H50(OTMS)3, 45%], 348 [C5H50(OTMS)3, 4%], 259(348
The crude fraction A3, with the cerebral protecting activity at a dose of 377mg (equivalent to lOg of dried mycelia)/kg s.c., was purified to yield two active fractions: A3B3C6D6 and A3B3C6D7 in four steps by H-103 column, Dowex 50W column, preparative HPLC and Sephadex G-10 column.
—
TMSO, 10%), 245 (348 — CH2OTMS, 17%), 230 [C4H5(OTMS)2, 38%], 217 [C3H3(OTMS)2, 13%], 195 (NH-CH2-C5H3N-OTMS, 13%), 180 (CH2-C5-H3N-OTMS, 15%) (see Fig. 3).
180
379CH2OTMS- 482 OTMS393
'46 166
1662:Mf--3...647
— 195; HNLcH2--,_)--OTMS
-TM50H -TMS0H
572
557
377TMS0H-467f N
L
.,LN -CH2Ofl-IS 245
TMSO
103,..559
-H20
541
34 3(•H)
QTMS\OTMS - 320
234 429
,-TMSOH
230 217
(- H)
444
Fig.3 Proposed ElMS fragmentations for TMS4-AMG-1.
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Instrumental methods
Planta Med/ca 56(1990) 51
A Novel N6-Substitu ted Adenosine Isolated from Mi Huan Jun (Armillaria mellea)
Step
1
activefr. A3
2
4
3
A3B3
6
5
A3B3C6
-C6D6
-C6D7
-D6E5
-D7F6
AMG-1
fr.wt.
1240g
1200g
1160g
fr.wt.b
46.8g
23.4g
3.Olg
ilOOg 45.8mg
ilOOg 23.2mg
1050g 2.5mg
1050g 1.7mg
l000g 1.2mg
10
10
10
20
20
20
20
20
377
195
25.9
0.833
0.422
0.048
0.032
0.024
gekg mg/kg
Table 2 The enrichment and localization of cere bral-protecting activity of the purified sample by each purification step.
Fraction weight represented as the reduced weight of dried mycelia. Fraction weight represented as the weight of active fraction. Minimum effective dose represented as the reduced weight of dried mycelia. Minimum effective dose represented as the weight of active fraction.
When administered s.c., AMG-1 showed marked dose-related efficacy between 12 and 48 .tg/kg (Table 3). Table 3 Effect of AMG-1 (1) on persistent time of gasping of isolated heads of mice by decapitation. dose .sg/kg s.c.
gasping duration mean (sec)
24.22 0.59"
48
Statistical significance was assessed using a one.way analysis of variance followed
by the Dunnet test (20). ' p < 0.05, " p < 0.001
Structure elucidation AMG-1 (1) exhibited the following UV spectral characteristics: Xmx (90% CH3OH, neutral) = 268 nm; max (90% CH3OH, acidic) = 265nm; max (90% CH3OH, basic) = 264 nm, indicative of a 6,9-disubstituted adenine (7, 8). The 1H-NMR spectrum showed two signals at ô = 8.21
(2-H, s) and 8.27 ppm (8-H, s), three methine protons at ô = 4.76(2'-H, dd,J= 6.2, 5.2), 4.32 (3'-H, dd, J= 5.2, 2.8), and 4.17 ppm (4'-H, dt, I = 2.8, 2.5), ABX-type coupled hydroxymethyl protons ô 3.74 (5'-HA, dd, J= 2.5, 12.8) and
3.88ppm(5'-HB, dd, J= 2.5,12.8), andan anomericproton at ö = 5.96 ppm (1'-H, d, 1= 6.2). These results, as well as the '3C-NMR spectral data (Table 1) indicated the structure I as being N6-substituted 93-ribofuranosyladenine.
H
N'' H
ion at m/z = 662.2941 corresponding to C16H14N605TMS4 (calcd. 662.2919). Thus, the formula of AMG-1 was deter-
mined to be C16H18N605 FM. = 374; SIMS: m/z = 375 (MH)I.
By subtracting the composition (C10F112N504) of the identified adenosine moiety from the empirical formula ofAMG1, the N6-substituent was deduced to have the composition C6H6NO. The fragment ion at ,n/z = 180, corresponding to C6H5NOTMS, was observed in the ElMS spectrum of TMS4-
AMG-1. This was regarded as being strong evidence that the unidentified N6-substitutent has a mass of 108 a.m.u. The proposed MS fragmentation (9, 10) scheme for TMS4AMg-1 is shown in Fig. 3.
S.E.M.
20.33 0.38 19.57 0.42 21.93±0.71'
water control 12 24
The high resolution mass spectrum ofAMG1 as a trimethylsilyl (TMS) derivative yielded a molecular
The signals attributed to the unidentified unit in the 1H-NMR spectrum of AMG-1 were mainly ob-
served in the aromatic region: ô 7.18 (4"-H, dd, J= 8.0, 2.8), 7.28 (3"-H, dd, J= 8.0, 0.9), and 8.06 ppm(6"-H, dd, I = 2.8, 0.9). These signals were indicative of the presence of a 2,5-disubstituted pyridine in the N6-substituent. Besides these protons, a signal at b = 4.78 ppm (7"-H2, br. s) as well as a signal at ô = 45.8 ppm in the 13C-NMR were detected. These were characteristic of the -CH2- group adjacent to the
N6. The introduction of a TMS group into the N6-substituent, as was observed in the ElMS of TMS4-AMG-1, indi-
cates that one hydrexy group is present on an N6-substituent. Thus, 5-hydroxy-2-pyridylmethyl or 2-hydroxy-5pyridylmethyl moieties are likely for the N6-substituent.
Comparison studies on the 1H-NMR and 13C-NMR of the aromatic region of AMG-1 and 2-methyl-5hydroxypyridine were carried out. The 1H-NMR signals [ô = 7.04 (3-H, dd, J = 8.5, 0.9), 7.13 (4-H, dd, I = 8.5, 2.6), 7.95 ppm (6-H, dd, J= 2.6, 0.9)] and 13C-NMR signals [ö = 126.05 (C-3, d), 126.11 (C-4, d), 137.78 (C-6, d), 150.28 (C-
5, s), 154.40 ppm (C-2, s)J of 2-methyl-5-hydroxypyridine showed close similarity to those of AMG-1 (see Table 1). From this, a 5-hydroxy-2-pyridylmethylamino moiety was ascertained to he the partial structure of AMG-1, rather than a 2-hydroxy-5-pyridylmethylamino moiety. All these data suggest AMG-1 as being 6-(5hydroxy-2-pyridy1rnethylamino)-9-ribofuranosylpurine (1).
HO HO OH
1
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Preparative HPLC on Develosil ODS columns of A3B3C6D6 and A3B3C6D7 gave active fractions (-E5, -F6) with the same retention time as shown in Fig. 2. This demonstrated that one compound was responsible for the cerebral-protecting activity of these fractions. Preparative HPLC on a Nucleosil 5C15 column of combined -E5 and -F6 yielded an active principle, AMG-1.
Naoharu Watanabe et al.
52 Planta Medico 56 (1990)
Discussion
The authors are grateful to Dr. K. Kushida (Varian Instruments Ltd.) for measuring the 13C-NMR spectra, and Dr. D. C. Yue (Institute of Material Medica, Chinese Academy of Medical Sciences) for the cultivation of Mi Huan Jun.
References
In the complete ischemic test in mice, prolongation of gasping is one measure of the anti-ischemic effect. Gasping is regulated by nerve cells in the respiratory
center. A sustained neuronal function in the respiratory center would lead to prolonged gasping. Thus, the cerebralprotecting effect of the crude extract of Mi Huan Jun as well as AMG-1 will derive from the influence on nerve cells of the respiratory center in this experimental model.
Table 2 shows that the cerebral-protecting activity was concentrated to AMG-1. However, it is presum-
able that inhibitors or other active compounds are present in the biologically inactive fractions which were discarded during isolation of AMG-1. In fact, the presence of various
Yang, Y. P., Yue, D.C., Huo, Z. M. (1976) Acta Micro. Sinica 16,206— 2
Araki, H., Tsuchida, K., Hosoda, K., Aihara, H., Tamai, M., 'I
6
8
kinds ofpurine analogs in Mi Huan Jun mycelia is suggested
by our preliminary experiments (data are not shown), and xanthine analogs, which increased the cerebral metabolic rate and shortened the duration of gasping (6).
10
Further studies on these possible biologically active compounds in the extract will clarify the degree of the contribution of AMG-1 to the cerebral-protecting activity of Mi Huan Jun mycelia.
AMG-1 was suggested to be an N6-substituted adenosine, based on its spectral data. Some N6-substituted adenosines are well known as cytokinins (11, 12) and as one of the growth regulators of plants. 6-(O-Hydroxybenzylamino)-9[3-D-ribofuranosylpurine (13, 14, 15), a compound quite similar to AMG-1, has been isolated from the mature leaves of poplar (Populus x robusta Schneid) as a cytokinin. This has medium activity in the soybean callus
test but shows high activity in the radish leaf senescence test, suggesting that AMG-1 shows cytokinin activities. This will be discussed in detail elsewhere.
N6-Substituted adenosine also affects the cellular receptors of adenosine to cause a potentiation of histamine release, inhibition of platelet aggregation, inhibition ofpurinergic neurons, and/or sedation of the brain (16, 17). In order to secure further information on the role of the N6-substituent of adenosine, many anologs have been prepared and examined for their biological properties (18). Among them, AMG- 1 might be regarded as one ofthe potent cerebral-protecting compounds in the ischemia or hypoxia model.
The total synthesis of AMG-1 is under way to afford a substantial amount of AMG-1 for use in pharmacological evaluation for a comparison with the other N6substituted adenosines. This will also result in a chemical confirmation of the structure of AMG-1, which was considered to be most likely from its spectral data. This will be reported in the following paper (19).
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The complete ischemia-induced decapitation model used for the examination of purified materials from Mi-Huan Jun resulted in the isolation of AMG-1 with cerebral-protecting activity.
Acknowledgements
