198 Planta Medica 56(1990)
Armillaric Acid, A New Antibiotic Produced by
Armillaria mellea Tadashi Obuchi" ?, Hideaki Kondoh , Naoharu Watanabe , Masaharu Tamai , Sadafumi Omura',
Yang Jun-Shan2, andLiangXiao-Tian2 2
Research Center, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Omiya, Saitama 330, Japan Institute of Materia Medica, Chinese Academy of Medical Sciences, 1 Xian Nong Tan Street, Beijing, People's Republic of China Address for correspondence
Abstract A new antibiotic armillaric acid (2), has been isolated from the cultured mycelia of Armillaria mella (Vahl. ex Fr.) Quel (Trichlometaceae). Its structure
was elucidated on the basis of the spectral data. Compound 2 exhibits inhibitory activity against gram-positive bacteria and yeast. Key words
Armillaric acid, Armillaria mellea, Mi Huan Jun, antibiotic, NMR.
Materials and Methods Instrumentation UV spectra were recorded using a Hitachi UV22OA spectrophotometer. El-MS and Cl-MS were measured on a Hitachi-80A mass spectrometer and FD-MS on a Jeol JMS-DX303 mass spectrometer. 1H- and 13C-NMR were recorded with a Jeol JNM-GX400 NMR spectrometer at 400MHz and 100MHz using TMS as an internal standard.
Fungus material Cultured mycelia of Armillaria mellea were provided by the Jian Xi National Drug Factory.
Testing for antimicrobial activity Antimicrobial activities of 1, 2, 3, 4 and crude
Introduction The cultured mycelia of Mi Huan Jun (1), a (Vahi. ex Fr.) Quel (Trichlometaceae)I symbiotic with Gastrodia eleta Blume, are used in the form of tablets for the treatment of dizziness,
fungus [Armillaria rnellea
headache, neurasthenia, insomnia, numbness in limbs, and infantile convulsion, as well as Tian Ma, a symbiont of
Mi Huan Jun and G. elata (2). In the course of our pharmacological studies on the extracts of Mi Huan Jun, the cerebral-protecting activity and the effect on hyperlipemia
have been clarified (3). Recently, we have found that adenosine derivatives are the effective principles of the cerebral-protecting activity (4). We also reported the isolation and structural elucidation of several new protoilludene sesquiterpenoid aromatic esters, armillarin (3), melleolide (4) from the extract of Mi Huan Jun (5), Successive purification of the crude extract of cultured Mi Huan Jun to clarify the antibacterial principles followed by the assay for antibacterial activity, led to
the isolation of 2-hydroxy-4-methoxy-6-methylbenzoic acid (1) and armillaric acid (2). In this paper we describe the
isolation of compounds 1 and 2 with antifungal and antibacterial activity and their structural elucidation by spectral analyses.
samples were determined by the paper disk-agar diffusion method. Micrococcus luteus ATCC9341, Staphylococcus aureus 2O9PJC, Bacilus subtilis 6633, and Candida albicans NHL4O19 were used as test microorganisms.
Test plates (diameter: 9cm) for M. luteus and S.
aureus were prepared with lOml of Heart Infusion Agar (HIA, Eiken). inoculated with 0.1 ml of culture broth grown overnight in Heart Infusion Broth (HIB, Eiken). For C. albi cans, Yeast Morphology Agar (YMA, Difco) and Yeast Nitrogen Base (YNB, Difco) were
used intead of HIA and HIB, respectively. Test plate (diameter: 9cm) for B. subtilis was prepared with 10 ml of HIA inoculated with
io spores per ml. Paper disks (diameter: 8mm) with 50 l of the sample solution in a concentration range of 250—2000g of 1—4/ ml(in the case of crude samples: 1000—2000tg/ml) were applied to the test plates. The diameters of inhibition zones were measured after incubation for 18 hat 30°C.
Isolation of I and2 The dried mycelia (10 kg) of Armillaria mellea (Vahl ex Fr.) Quel were pulverized and extracted with 50% aqueous methanol (301 x 3) under reflux. After evaporation of methanol in vacuo, the residual solution (101) was extracted with ethyl acetate (101 x 2). The combined ethyl acetate layer was dried over anhy-
drous Na2SO4 and concentrated in uacuo to yield a brown paste (292.5 g). A portion of the dried material (49.73 g) was dissolved in 100 ml of chloroform and subjected to a silica gel (E. Merck) column (Vt = 700 ml. equilibrated with chloroform). The column was developed with 2100 ml of chloroform-methanol increasing the latter stepwise. Active fractions eluted by chloroform-methanol (95 : 5)
were combined and evaporated in vacuo to dryness, the residue
Downloaded by: University of Arizona Library. Copyrighted material.
Received: March 16, 1989
Planta Medica 56(1990) 199
Armillaric Acid, A New Antibiotic Produced by Armillaria mellea
acid (2), armillarin (3), and melleolide 4.
o HO
The ethyl acetate fraction showed an-
HO Hlb34,.z3l21Hb:)
tibacterial and antifungal activity. From the ethyl acetate fraction, I and 2 were isolated as active principles by succesive column chromatography and preparative HPLC.
He1 isl"CH3 H1b CH3
R2(C-1
2 armillaric acid HO
Compound 1 was identified (C9H1004, Mr =
COOH
3
arrnitlarin
182) as 2-hydroxy-4-methoxy-6-methylbenzoic acid by di-
CHO
4
melLeotide
rect comparison of its spectral data with those of an authen-
CHO
tic specimen. The physicochemical properties of 2 are closely related to those of 3 and 4. The IJV absorption at 222nm and the JR band at 1630 cm1 in 2 indicated the
R1
H3CO HO
was dissolved in 10 ml of benzene-acetone (9 1) and subjected to a silica gel (E. Merck) column [Vt = 200 ml. equilibrated with benzene-acetone (9: 1)] chromatography. Combined active fractions
were concentrated to give a yellow powder (103.9 mg). Further
presence of an cz,)3-unsaturated carbonyl system in 2 as well
asin3 and 4. FD-MS and Cl-MS spectra gave the (M + HY
ous methanol at a flow rate of l5mlJmin; monitored at 254nm;
ion peak at m/z = 417 with the characteristic fragment ions at m/z = 249 and at m/z = 151 due to fission between the orsellinc acid and protoilluden part. As the relative molecular mass of 2 was found to be 16 amu higher than that of 4 in
Waters model 6000A] gave the active fractions which are eluted at
these MS, the molecular formula of 2 was estimated as
purification by Sephadex LH-20 (Pharmacia) column (Vt 200 ml) chromatography in methanol followed by preparative-HPLC [ODS: Nucleosil 5C18 (Macherey-Nagel) column 20 x 250 mm; 50% aque-
a retention time of 360—5.O4min (1) and 9.84—12.00mm (2). Evaporation of the solvent of these fractions afforded 33mg of 1 and 10mg of 2. 2-Hydroxy-4-methoxy-6-methylbenzoic acid (1):
light brown powder, UV: ?C) nm 254, 293; IR: vmcm' 3400 (broad s), 1590, 1370, 1200, 820, 700; MS: ElMS m/z 196 (M); CIMS m/z 197 (M + M) as methyl ester, 1H-NMR: 6 (CDC13). 2.57 (3H, br. s), 3.82 (3H, s), 6.33 (1H, dd, J= 2.0,
Armillaric Acid, A New Antibiotic Produced by
Armillaria mellea Tadashi Obuchi" ?, Hideaki Kondoh , Naoharu Watanabe , Masaharu Tamai , Sadafumi Omura',
Yang Jun-Shan2, andLiangXiao-Tian2 2
Research Center, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Omiya, Saitama 330, Japan Institute of Materia Medica, Chinese Academy of Medical Sciences, 1 Xian Nong Tan Street, Beijing, People's Republic of China Address for correspondence
Abstract A new antibiotic armillaric acid (2), has been isolated from the cultured mycelia of Armillaria mella (Vahl. ex Fr.) Quel (Trichlometaceae). Its structure
was elucidated on the basis of the spectral data. Compound 2 exhibits inhibitory activity against gram-positive bacteria and yeast. Key words
Armillaric acid, Armillaria mellea, Mi Huan Jun, antibiotic, NMR.
Materials and Methods Instrumentation UV spectra were recorded using a Hitachi UV22OA spectrophotometer. El-MS and Cl-MS were measured on a Hitachi-80A mass spectrometer and FD-MS on a Jeol JMS-DX303 mass spectrometer. 1H- and 13C-NMR were recorded with a Jeol JNM-GX400 NMR spectrometer at 400MHz and 100MHz using TMS as an internal standard.
Fungus material Cultured mycelia of Armillaria mellea were provided by the Jian Xi National Drug Factory.
Testing for antimicrobial activity Antimicrobial activities of 1, 2, 3, 4 and crude
Introduction The cultured mycelia of Mi Huan Jun (1), a (Vahi. ex Fr.) Quel (Trichlometaceae)I symbiotic with Gastrodia eleta Blume, are used in the form of tablets for the treatment of dizziness,
fungus [Armillaria rnellea
headache, neurasthenia, insomnia, numbness in limbs, and infantile convulsion, as well as Tian Ma, a symbiont of
Mi Huan Jun and G. elata (2). In the course of our pharmacological studies on the extracts of Mi Huan Jun, the cerebral-protecting activity and the effect on hyperlipemia
have been clarified (3). Recently, we have found that adenosine derivatives are the effective principles of the cerebral-protecting activity (4). We also reported the isolation and structural elucidation of several new protoilludene sesquiterpenoid aromatic esters, armillarin (3), melleolide (4) from the extract of Mi Huan Jun (5), Successive purification of the crude extract of cultured Mi Huan Jun to clarify the antibacterial principles followed by the assay for antibacterial activity, led to
the isolation of 2-hydroxy-4-methoxy-6-methylbenzoic acid (1) and armillaric acid (2). In this paper we describe the
isolation of compounds 1 and 2 with antifungal and antibacterial activity and their structural elucidation by spectral analyses.
samples were determined by the paper disk-agar diffusion method. Micrococcus luteus ATCC9341, Staphylococcus aureus 2O9PJC, Bacilus subtilis 6633, and Candida albicans NHL4O19 were used as test microorganisms.
Test plates (diameter: 9cm) for M. luteus and S.
aureus were prepared with lOml of Heart Infusion Agar (HIA, Eiken). inoculated with 0.1 ml of culture broth grown overnight in Heart Infusion Broth (HIB, Eiken). For C. albi cans, Yeast Morphology Agar (YMA, Difco) and Yeast Nitrogen Base (YNB, Difco) were
used intead of HIA and HIB, respectively. Test plate (diameter: 9cm) for B. subtilis was prepared with 10 ml of HIA inoculated with
io spores per ml. Paper disks (diameter: 8mm) with 50 l of the sample solution in a concentration range of 250—2000g of 1—4/ ml(in the case of crude samples: 1000—2000tg/ml) were applied to the test plates. The diameters of inhibition zones were measured after incubation for 18 hat 30°C.
Isolation of I and2 The dried mycelia (10 kg) of Armillaria mellea (Vahl ex Fr.) Quel were pulverized and extracted with 50% aqueous methanol (301 x 3) under reflux. After evaporation of methanol in vacuo, the residual solution (101) was extracted with ethyl acetate (101 x 2). The combined ethyl acetate layer was dried over anhy-
drous Na2SO4 and concentrated in uacuo to yield a brown paste (292.5 g). A portion of the dried material (49.73 g) was dissolved in 100 ml of chloroform and subjected to a silica gel (E. Merck) column (Vt = 700 ml. equilibrated with chloroform). The column was developed with 2100 ml of chloroform-methanol increasing the latter stepwise. Active fractions eluted by chloroform-methanol (95 : 5)
were combined and evaporated in vacuo to dryness, the residue
Downloaded by: University of Arizona Library. Copyrighted material.
Received: March 16, 1989
Planta Medica 56(1990) 199
Armillaric Acid, A New Antibiotic Produced by Armillaria mellea
acid (2), armillarin (3), and melleolide 4.
o HO
The ethyl acetate fraction showed an-
HO Hlb34,.z3l21Hb:)
tibacterial and antifungal activity. From the ethyl acetate fraction, I and 2 were isolated as active principles by succesive column chromatography and preparative HPLC.
He1 isl"CH3 H1b CH3
R2(C-1
2 armillaric acid HO
Compound 1 was identified (C9H1004, Mr =
COOH
3
arrnitlarin
182) as 2-hydroxy-4-methoxy-6-methylbenzoic acid by di-
CHO
4
melLeotide
rect comparison of its spectral data with those of an authen-
CHO
tic specimen. The physicochemical properties of 2 are closely related to those of 3 and 4. The IJV absorption at 222nm and the JR band at 1630 cm1 in 2 indicated the
R1
H3CO HO
was dissolved in 10 ml of benzene-acetone (9 1) and subjected to a silica gel (E. Merck) column [Vt = 200 ml. equilibrated with benzene-acetone (9: 1)] chromatography. Combined active fractions
were concentrated to give a yellow powder (103.9 mg). Further
presence of an cz,)3-unsaturated carbonyl system in 2 as well
asin3 and 4. FD-MS and Cl-MS spectra gave the (M + HY
ous methanol at a flow rate of l5mlJmin; monitored at 254nm;
ion peak at m/z = 417 with the characteristic fragment ions at m/z = 249 and at m/z = 151 due to fission between the orsellinc acid and protoilluden part. As the relative molecular mass of 2 was found to be 16 amu higher than that of 4 in
Waters model 6000A] gave the active fractions which are eluted at
these MS, the molecular formula of 2 was estimated as
purification by Sephadex LH-20 (Pharmacia) column (Vt 200 ml) chromatography in methanol followed by preparative-HPLC [ODS: Nucleosil 5C18 (Macherey-Nagel) column 20 x 250 mm; 50% aque-
a retention time of 360—5.O4min (1) and 9.84—12.00mm (2). Evaporation of the solvent of these fractions afforded 33mg of 1 and 10mg of 2. 2-Hydroxy-4-methoxy-6-methylbenzoic acid (1):
light brown powder, UV: ?C) nm 254, 293; IR: vmcm' 3400 (broad s), 1590, 1370, 1200, 820, 700; MS: ElMS m/z 196 (M); CIMS m/z 197 (M + M) as methyl ester, 1H-NMR: 6 (CDC13). 2.57 (3H, br. s), 3.82 (3H, s), 6.33 (1H, dd, J= 2.0,
