APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1977, p. 500-505 Copyright i 1977 American Society for Microbiology
Vol. 34, No. 5 Printed in U.S.A.
Degradation of the Plant Flavonoid Phellamurin by Aspergillus niger SAEKO SAKAIt Department of Biology, Faculty of Science, Tokyo Metropolitan University, Setagaya-ku, Tokyo, 158, Japan Received for publication 18 April 1977
We have previously described the structure of phellamurin, a plant flavonoid, 3,4',5,7-tetrahydroxy-8-isoprenylflavanone-7-0-glucoside (17). Degradation of phellamurin by Aspergillus niger, using modified Czapek-Dox medium as well as phellamurin or one of its degradation products as a sole carbon source, is reported here. Eleven compounds are identified from phellamurin degradation products. A. niger apparently decomposes phellamurin by first removing glucose with f,-glucosidase; neophellamuretin is the first degradation product. Fission of the heterocycic ring of (5"-hydroxyisopropyl-4",5"-dih'drofurano)[2",3"-h]3,4',5-trihydroxyflavanone, which is obtained from neophellamuretin through a few alterations of the side chain, is followed by cleavage of a C-C bond between C==O and carbon at a-position and conversion of (5"-hydroxyisopropyl4",5"-dihydrofurano)[2",3"-d]-2',4,6',a-tetrahydroxychalcone to p-hydroxymandelic acid (B-ring) and 2,4,6-trihydroxy-5-carboxyphenylacetic acid (A-ring). It is suggested that p-hydroxymandelic acid is oxidized to p-hydroxybenzoic acid. 2,4,6-Trihydroxy-5-carboxyphenylacetic acid is metabolized to phloroglucinol carboxylic acid, which subsequently is decarboxylated to phloroglucinol. These results provided new information on the isoprene unit metabolism of the side chain of phellamurin and firmly established the degradation pathway of phellamurin by A. niger. as
The ability of microorganisms to enzymatically transform naturally occurring organic compounds to other substances is well known and has been the subject of numerous studies (8, 10, 19, 22). The importance of these studies is obvious, since the turnover of chemical substances throughout the world is attributed to metabolism by microorganisms. It is similarly well known that plant and animal remains are decomposed by microbes both on and under the ground. This decomposition pattern is also the case for flavonoids, which are common constituents of higher plants. In recent years, considerable information has become available concerning microbial degradation of aromatic compounds (5, 18, 19). Aromatic compound degradation involves hydroxylation of the aromatic ring to form dihydroxy compounds, followed by ring cleavage to yield compounds that can be utilized via the tricarboxylic acid cycle (2, 6). Udupa et al. (21, 22) incubated (±)-flavanone with Gibberella fujikuroi and obtained several compounds: (-)-flavan-4a-ol; 2'-hydroxychalcone; 2'-4-dihydroxydihydrochalcone; 2',4-dihydroxychalcone; (±)4'-hydroxyflavanone; and (-)-4'-hydroxyflavant Present address: National Cancer Institute, National Institutes of Health, Bethesda, MD 20014.
500
4a-ol. Jeffrey et al. (11) have shown that dihydrogossypetin is a metabolite in bacterial (Pseudomonas species) degradation of (-)-taxifolin. Cell-free extracts from the same Pseudomonas species further oxidized dihydrogossypetin via cleavage of the A-ring to form oxaloacetic acid together with 5-(3,4-dihydrophenyl)-4-hydroxy3-oxo-valero-S-lactone (10). However, microbial transformation of flavanone has not been extensively studied. Phellodendron amurense leaves, a tree of Rutaceae, contain a large quantity of phellamurin (7). We have previously described that the aglycone of phellamurin formed by Aspergillus niger (neophellamuretin) is 3,4',5,7-tetrahydroxy-8isoprenylflavanone, and the structure of phellamurin should be the corresponding 7-0-glucoside (17). Up until now, we have found no investigation on the microbial degradation of the isoprenyl group associated with the benzene ring. The present paper reports results of studies on the metabolism of the flavonoid phellamurin by A. niger. MATERIALS AND METHODS Culture. Stock culture maintained on agar slants. the modified Czapek-Dox croelements (FeCl3 6H20,
of A. niger IAM-25 was The growth medium was medium with some mi20 mg; ZnSO4 7H20, 10
VOL. 34, 1977 mg; MnSO4 4H20, 3 mg; Na2MoO4 2H20, 1.5 mg; CuSO4 5H20, 1 mg) as well as 20 g of glucose and 0.1 g of phellamurin per liter; its pH was adjusted to 4.5 with HCI. The phellamurin solution and all remaining ingredients were sterilized separately and combined aseptically in flasks before inoculation. One liter of the liquid culture medium was inoculated with spores grown on five slants. Two liters of the liquid culture medium was incubated for 4 to 25 days at 25°C. After incubation, the medium was decanted, and mycelial mats were washed three times with sterilized water and replaced with a solution of either 0.1% phellamurin or 0.1% degradation product in water. The solution of 2 liters was incubated under the same conditions as described above. Extraction and fractionation. Two liters of the culture filtrate was acidified with dilute HCI to pH 2 and thoroughly extracted with diethyl ether. The etheral extract was back-extracted with 1% sodium bicarbonate to yield a neutral and an acidic fraction. The mother liquid, after the ether extraction, was reextracted with ethyl acetate (ethyl acetate portion). The neutral portion was evaporated, and the residue was dissolved in a small volume of ethanol. The ethanolic solution was then applied to a polyamide column (Woelm) (25 by 170 mm) and eluted successively with 100-ml volumes of each of the following aqueous ethanol mixtures: 0, 20, 40, 60, 80, and 100%. The fractions were concentrated and examined by thinlayer chromatography on silica gel plates GF254 with the solvent chloroform-ethyl acetate-formic acid (5:4:1). After being dried, chromatograms were exposed under ultraviolet light, and fluorescent spots were marked. Compounds isolated from silica gel plates with ethanol were recrystallized from ethanolwater. The acidic and ethyl acetate portions were evaporated, and the residues, dissolved in a small volume of ethanol, were applied to two polyamide columns and eluted with absolute ethanol. The degradation products were isolated by thin-layer chromatography, as described above. Time course of appearance of some degradation products by A. niger. Twelve-day-old mycelial mats were washed at least three times with sterilized water and replaced with 200 ml of distilled water
containing phellamurin or degradation product at a concentration of 1 mg/ml. After incubation for 2, 5, 7, and 10 days, each resting-cell culture medium was extracted with ether. The etheral extracts were evaporated, and the remaining residue was applied to a polyamide column and eluted with 100 ml of absolute ethanol. The ethanolic fraction was evaporated, dissolved in 2 ml of ethanol, and examined by thin-layer chromatography. Compounds were isolated quantitatively from silica gel plates with ethanol. The quantity of these compounds was estimated from the optical density at 300 nm. Chemicals. Phellamurin was isolated from P. amurense leaves by the method of Hasegawa and Shirato (7). Other chemicals were obtained commercially. Chromatographic examination. Samples, together with known compounds as required, were applied to either Whatman no. 1 filter paper or a silica gel GF254 (nach Stahl; E. Merck AG, Darmstadt, Germany) plate by means of a capillary pipette, and
DEGRADATION OF PHELLAMURIN
501
the chromatogram was developed. The solvents used for paper chromatography were: (i) n-butanol-glacial acetic acid-water (6:1:2), (ii) 6% acetic acid, and (iii) benzene-glacial acetic acid-water (6:7:3); the solvent systems for thin-layer chromatography were: (iv) chloroform-ethyl acetate-formic acid (5:4:1) and (v) petroleum ether-chloroform-ethyl acetate-formic acid (10:5:4:1). After drying at room temperature, chromatograms were exposed under ultraviolet light, and fluorescent spots were marked. Spectrometry. Mass and nuclear magnetic resonance spectra were measured by Hitachi RMS-4 and Hitachi-Perkin-Elmer 60 MHz, respectively.
RESULTS AND DISCUSSION Degradation of phellam , a plant flavonoid, was investigated. The following compounds (Fig. 1, A through K) were obtained from phellamurin as the degradation products. The metabolic pathway of phellamurin is also proposed (Fig. 1). Identification of degradation products. Compound A was isolated from the neutral portion. The properties of this compound are completely identical to those of neophellamuretin (17) by comparison of behavior on thin-layer chromatography, melting point, and infrared, mass, and nuclear magnetic resonance spectra. The structure of compound A was 3,4',5,7-tetrahydroxy-8-isoprenylflavanone, as described previously (17). Fraction B gave a colorless crystalline solid (melting point, 140 to 142°C) from the neutral portion, and its elementary analysis was consistent with C20H2208H20. The mass spectrum of compound B shows a parent ion peak at m/e 390 (14%). Ions at m/e 372 and 354 arise from the loss of 18 (M - H20) and 36 (M - 2H20) mass units, respectively, from the molecular ion. It should be pointed out that compound B has two hydroxyl groups different from those of neophellamuretin. An ethanolic solution of this compound gave a purplish-brown coloration with ferric chloride. It produced a reddish-purple color by a reduction test with either magnesium or zinc powder and concentrated hydrochloric acid. This color is considered characteristic of flavanonols (16). This compound also showed ultraviolet absorption peaks (in ethanol) at 300 and 340 nm, and the former peak undergoes the bathochromic shift of 24 and 36 nm after addition of aluminum chloride (12) and sodium acetate (14), respectively. It is suggested that the hydroxyl groups are present at the C5 and C7 positions, while a 4'-substituted flavanone was supported by an infrared absorption at 830 cm-'. These results showed that the possible binding sites for two hydroxyl groups are the positions of the side chains. A similar hydroxylation re-
502
SAKAI
M3C
APPL. ENVIRON. MICROBIOL.
CM3 C
H3C
3C3
CH
Phellamurin
CH3 COH
IOCH
Comp. A
CH
CH2
04< O O O/ON
G10
Comp. B
CH2 0
HO
0-
OH
GIO OH
ON N
11
0
OH
3C\ /C3 COOH
Comp.
HO
Comp. H OH
CM2
HO
0
Comp. C
NC-HN2
ON
ON O
O
ONH
ONH
OH
OH
Comp E HO
OH
i
Comp. D
-aI--COON
COON
/CH3
C-OH
HC NC- 2
ON
N..
OH
H3 C\
C-OH
0
0
Camp F
Camp. G
H
ON
nOOC-C ;;
ON
-
-
OHC
/
OH
-
HOOC
/
OH
OH
Comp. J
FIG. 1. Proposed pathway for phellamurin degradation in A. niger.
action was observed by Suzuki et al. (20). The infrared spectrum indicated that the binding sites are not at a dimethyl group, strongly suggesting that the binding sites should be at C-,8 and C--y. From the above results, the structure of compound B is thought to be 3,4',5,7-tetra-
hydroxy-8-(,B,y-dihydroxyisovaleryl)-flavanone. Compound C gave a pale-yellow solid (melting point, 126 to 1270C) from the neutral portion, and its elementary analysis was consistent with C20H2007. The mass spectrum of compound C shows a parent ion peak at m/e 372 (9%).
In the ultraviolet spectrum, pronounced absorption maxima were observed at 300 and 340 nm. The presence of a chelated OH group was indicated by a positive ferric chloride reaction (23) and a bathochromic shift in the ultraviolet absorption maximum after addition of aluminum chloride (12). When reduced with either magnesium or zinc powder and concentrated hydrochloric acid, a reddish-purple color developed, characteristic of flavanonols (16). The infrared spectrum of compound C shows the presence of C=O and OH groups. The high-field signals (0.98 and 1.118) in the nuclear magnetic resonance spectrum of compound C acetate are attributed to protons of the gem-dimethyl groups that have a long-range coupling with the methine proton at 4.528 (1). A signal at 1.528 is due to a tertiary alcohol. A signal at 2.838 shows methylene protons of benzyl structure that split
into a doublet by coupling to the next methine proton, a triplet (J = 15 Hz) at 4.52. Two pairs of doublets (6.89 and 7.24; J = 8 Hz) represent an A2B2 system of H-2',6' and H-3',5' protons. The protons at 5.118 (d) and 5.45 (d, J2,3 = 10 Hz) exhibit the AB system of H-2 and H-3 (1, 4, 15). The proton at 6.06 (singlet) is a signal of H-6 (A-ring). One acetyl group derived from the C3 hydroxyl group is 1.77 ppm. The two methyl groups at 2.06 and 2.12 are due to aromatic acetyl groups, which are C4' and C5 positions, as indicated previously (17) in neophellamuretin. As compared with that of compound A acetate, only two aromatic acetyl groups were observed. From the results of the nuclear magnetic resonance studies, the presence of hydroxyl groups at the C3 and C5 positions was confirmed. Protons of the B-ring were the same as the ones on neophellamuretin, so the loss of the hydroxyl group is obviously due to the hydroxyl group at the C7 position. The above data indicate that the structure of compound C is (5"-hydroxyisopropyl-4",5"-dihydro-
furano)[2",3"-h]-3,4',5-trihydroxyflavanone. Compound D was also obtained from the neutral portion as a yellow product (melting point, 185°C). Compound D gave a brown color with
alcoholic ferric chloride (23) and a negative color with the HCl-Mg reduction test (16). It produced an orange solution on addition of concentrated sulfuric acid, which turned colorless when dis-
VOL. 34, 1977
tilled water was added. These color reactions indicate that compound D is chalcone (22). The ultraviolet spectrum (absorption maxima at 273, 300, and 378 nm) also suggested a chalcone structure (14). These absorptions each undergo bathochromic shifts of 8, 15, and 62 nm, respectively, with addition of aluminum chloride (14), indicating the presence of a hydroxy group at the 6'-position. There was no shift after addition of sodium acetate, probably indicating the ring fusion between the 4'-hydroxyl position and the 3'-side chain. The infrared spectrum of compound D showed the presence of a hydroxyl, a carbonyl, and a substituted aromatic ring. The mass spectra of compound C {(5"-hydroxyisopropyl - 4", 5"-dihydrofurano) [2", 3"- h] - 3,4',5trihydroxyflavanone} and compound D have been determined. Both compounds C and D gave the molecular ion m/e 372. In compound C, peaks at m/e 236 (100%), m/e 176 (33%), m/e 164 (55%), and m/e 133 (59%) were observed. In compound D, peaks at mWe 221 (38%), m/e 177 (28%), mWe 165 (100%), and m/e 134 (57%) were observed. Compound C showed a peak at m/e 236 as the base peak; compound D gave a base peak at m/e 165. The difference between the two compounds is mainly due to fission of the side chain [(5"-hydroxyisopropyl-4",5"-dihydrofurano) group], but the process of cleavage is very similar (9). However, other peaks at m/e 264 (12%), m/e 249 (30%), and 219 (23%) in compound C were never observed in spectra of compound D. The above results confirm that compound D is not a flavanone but a chalcone. Compound C was converted to the corresponding chalcone. Compound D was estimated as (5"-hydroxyisopropyl-4",5"-dihydrofurano)[2", 3" -d]-2',4,6',a-tetrahydroxychalcone. From the acidic portion, compound E was isolated. It gave a blue color with ferric chlorideferricyanide reagent (23) and a yellow color with bromocresol green (23), suggestive of a hydroxyphenylcarboxylic acid. It failed to give a color with diazotized benzidine. It showed ultraviolet absorption maxima at 278 and 284 (shoulder) nm. Compound E had Rf values of 0.81 (solvent i), 0.86 (solvent ii), 0.04 (solvent iii), and 0.50 (solvent iv). Compound E coincides in all its properties with p-hydroxymandelic acid. By ultraviolet absorption spectrum and paper and thin-layer chromatographies, identity was confirned. Compound F was detected from the neutral fraction. It exhibited an ultraviolet maximum at 290 nm. The compound turned to a brown color with alcoholic ferric chloride (23) and a blue color with ferric chloride-ferricyanide reagent (23), suggestive of a hydroxyphenyl compound. Compound F gave an orange color with 2,4-
DEGRADATION OF PHELLAMURIN
503
dinitrophenylhydrazine, characteristic of either ketones or aldehydes (3). Compound F had Rf values of 0.90 (solvent i), 0.75 (solvent ii), 0.59 (solvent iii), and 0.90 (solvent iv). The above data suggested the product to be p-hydroxybenzaldehyde, which was confirmed by ultraviolet absorption spectrum and paper and thin-layer chromatographies with an authentic sample. Compound G was detected in the acidic portion. An ethanolic solution of this compound gave a brown color with FeCl3 (23), a blue color with ferric chloride-ferricyanide reagent, and a yellow color with bromocresol green (23), indicating the presence of phenolic hydroxyl and carboxyl groups. It exhibited an ultraviolet absorption maximum at 255 nm. Compound G had Rf values of 0.92 (solvent i), 0.63 (solvent ii), 0.35 (solvent iii), and 0.40 (solvent v). This compound was identified asp-hydroxybenzoic acid by paper and thin-layer chromatographies with an authentic sample. Compound H (melting point, 230°C), obtained from the ethyl acetate fraction, gave a colorless crystalline solid. Compound H failed to react with either magnesium or zinc and hydrochloric acid (16). This compound gave a purple color in the pine-shaving reaction, a reddish-orange color with diazotized benzidine (23), and a yellow color with bromocresol green. The ultraviolet spectrum showed prominent absorption at 295 nm and two inflections at 255 and 263 nm. The former peak undergoes bathochromic shifts of 20 and 5 nm with addition of aluminum chloride (12) and sodium acetate (14), respectively. The most likely structure for this compound seemed to be a phloroglucinol carboxylic derivative. It had Rf values of 0.75 (solvent ii), 0.00 (solvent iii), and 0.20 (solvent iv). The mass spectrum of compound H showed a parent ion peak at m/e 228, and M - 1 ion at m/e 227. The base peak was M - 19, which was due to further loss of an hydrogen atom from the M - H20 ion. The second most prominent peak in the spectrum was m/e 226, which was due to further dehydrogenation from the M - 1 ion. These pathways were ascertained by the presence of metastable ions. Other ions of importance in the spectrum of this compound were the M - 46 (m/e 182) ion, which probably arose by the decarboxylation from the M - 1 ion, and the M - 63 (m/e 165) ion, which was formed by the loss of OH from the M - 46 ion. Moreover, the M - 75 (m/e 153) ion was due to the loss of CH2 from the M - 63 ion and hydrogenation to the same ion at m/e 126, that is, phloroglucinol. The phenylcation (m/e 77) was due to the loss of 3(OH)
for phloroglucinol. From the above results, compound H is thought to be 2,4,6-trihydroxy-5carboxyphenylacetic acid.
504
SAKAI
APPL. ENVIRON. MICROBIOL.
Compound I was identified from the acidic (jmmoles) portion. It gave a deep-pink color in the pineshaving raction and a red color with diazotized 100 benzidine. The principle of coloration by diazonium reagents is that a diazo-coupling occurs when the para-position to a phenolic hydroxyl group is free (10). If there were at least one vacant position in either the phloroglucinol or phloroglucinol derivatives, a pine-shaving reac50 (pmoles) tion would give a reddish- or bluish-purple color. to be phloroglucinol Compound I was expected carboxylic acid from the above color reactions. Compound I showed ultraviolet absorption -i1 0.5 peaks at 270 and 320 nm. It had Rf values of 0.75 (solvent i), 0.65 (solvent ii), and 0.00 (solvent 0.0 0 iii). It was identified as phloroglucinol carboxylic acid by paper chromatography with an authentic 0 2 6 10 4 8 sample. DAYS Compound J was isolated from the acidic porFIG. 2. Change with time of phellamurin metabotion. It gave a purple color in the pine-shaving lites, using replacement culture containing 0.2 g (ca. reaction and strong reddish-brown color with 400 wnol) ofphellamurin. (A) Compound A; (B) comdiazotized benzidine (10). It also gave a strong pound B; (C) compound C; (D) compound D. blue color with ferric chloride-ferricyanide reagent (23). These observations indicate that the tions of compounds A, B, and C were observed compound is phloroglucinol. In the ultraviolet after 7 days of incubation and that of compound spectrum, pronounced absorption maxima were D after 10 days. However, in the presence of observed at 255 (shoulder), 269, and 272 (shoul- glucose, the peak of maximum accumulation of der) nm. Compound J had Rf values of 0.75 (sol- degradation products was generally delayed. To vent i), 0.60 (solvent ii), 0.00 (solvent iii), and study the pathway for the formation of the com0.55 (solvent iv). From the paper and thin-layer pounds from A, the replacement culture medium chromatographic comparison with authentic containing 0.1% compound A was used. Comsamples, compound I is consistent with phloro- pounds B, C, and D were obtained from compound A as degradation products after incubaglucinol. A small amount of compound K was isolated tion for 7 days. Compound D was enzymatically from the ethyl acetate fraction. Compound K formed from compound C, and this was further coincided in all its properties with phellamurin supported by an experiment with the replacement culture medium containing 0.1% comsupplied as the substrate. Fungal degradation of phellamurin. The pound C; that is, compound D was obtained fungal degradation usually involves an initial from compound C as the only degradation prodrelease of sugars by endogenous glycosidases, uct after incubation for 4 days. Since it is followed by hydrolytic cleavage of the hetero- thought from the structures of compounds A cyclic ring of the aglycone (13). A. niger appar- and C that conversion of compound A to C is ently degrades phellamurin by first removing not a direct reaction, compound A is probably glucose with fi-glycosidase. Therefore, the first converted to compound B by adding two moledegradation product of phellamurin is com- cules of water to the side chain of compound A pound A, that is, neophellamuretin (3,4',5,7-te- through two unidentified intermediates. Dehytrahydroxy-8-isoprenylflavanone). After incu- droxylation and the ring fusion in the side chain bation for 2, 5, 7, and 10 days, using a resting- of compound B occur at the same time to procell culture medium containing 0.1% phellamu- duce compound C. After fission of the heterorin, the quantity of degradation products (com- cyclic ring of compound C, followed by cleavage pounds A, B, C, and D) was estimated from of the C-C bond between carbonyl and carbon the optical density at 300 nm (Fig. 2). Compound at the a-position, the conversion of compound A, B [3,4',5,7-tetrahydroxy-8-(,8,y-dihydroxyiso- D to E (p-hydroxymandelic acid) and compound valeryl)-flavanone], C [(5"-hydroxyisopropyl- H (2,4,6-trihydroxy-5-carboxyphenylacetic acid) 4",5"-dihydrofurano)[2",3"-h]-3,4',5-trihydroxy- occurred. Compound E was derived from the Bflavanone], and D [(5"-hydroxyisopropyl-4",5"- ring, and compound H was derived from the Adihydrofurano)[2",3"-d]-2',4,6',a-tetrahydroxy- ring. The fornation of benzoic acid from manchalcone] were obtained from phellamurin as delic acid in a microorganism system (8) is docdegradation products. The highest accumula- umented. It is thought that compound E (p-hy-
VOL. 34, 1977
droxymandelic acid) is oxidized to compound G (p-hydroxybenzoic acid) by the same enzymes of the mandelate pathway (8), compound F (p-hydroxybenzaldehyde) and compound G being intermediates in this pathway. On the other hand, after cleavage of the side chain, compound H is metabolized to compound I (phloroglucinol carboxylic acid), which subsequently is decarboxylated to compound J (phloroglucinol). These metabolic pathways are illustrated in Fig. 1. The results of this study provide evidence for the metabolism of the isoprene unit of the side chain of pheliamurin and establish the degradation pathways of phellamurin by A. niger.
DEGRADATION OF PHELLAMURIN
505
teriol. 91:1140-1154. 9. Itagaki, Y., T. Kurokawa, S. Sasaki, Chin-Te Chang, and Fa-Ching Chen. 1966. The mass spectra of chalcones, flavones and isoflavones. Bull. Chem. Soc. Jpn. 39:538-543. 10. Jeffrey, A. M., D. M. Jerina, R. Self, and W. C. Evans. 1972. The bacterial degradation of flavonoids. Biochem. J. 130:383-390. 11. Jeffrey, A. M., K. Knight, and W. C. Evans. 1972. The bacterial degradation of flavonoids. Biochem. J. 130:373-381. 12. Jurd, L 1961. Spectral properties of flavonoid compounds, p. 108-154. In T. A. Geissman (ed.), The chemistry of flavonoid compounds. Pergamon Press, Oxford. 13. Krishnamurty, H. G., K. J. Cheng, G. A. Jones, F. J. Simpson, and J. E. Watkin. 1970. Identification of products produced by the anaerobic degradation of rutin and related flavonoids by Butyrivibrio sp. C:i. Can. J. Microbiol. 16:759-767. ACKNOWLEDGME NTIS 14. Mabry, T. J., K. R. Markham, and M. B. Thomas. I would like to express my sincere thanks to M. Hasegawa 1970. The structure analysis of flavonoids by ultraviolet and S. Yoshida of the Department of Biology, Faculty of spectroscopy, p. 165-230. In The systematic identificaScience, Tokyo Metropolitan University, for their kind guidtion of flavonoids. Springer-Verlag, Berlin, Heidelberg, ance and encouragement throughout this investigation. I New York. would also like to express cordial thanks to T. Inoue of the 15. Mabry, T. J., K. R. Markham, and M. B. Thomas. Hoshi College of Pharmacy for measurement of the mass and 1970. The structure analysis of flavonoids by proton infrared spectra, and T. Sato of the Department of Chemistry, nuclear magnetic resonance spectroscopy, p. 260-273. Faculty of Science, Tokyo Metropolitan University, for reIn The systematic identification of flavonoids. Springercording the nuclear magnetic resonance spectrum. Finally, I Verlag, Berlin, Heidelberg, New York. would like to thank S. S. Thorgeirsson and P. J. Wirth of the 16. Pew, J. C. 1948. A flavanone from douglas-fir heartwood. National Cancer Institute, National Institutes of Health, for J. Am. Chem. Soc.70:3031-3034. critical reading of this manuscript. 17. Sakai, S., and M. Hasegawa. 1973. Structure of phellamurin. Phytochemistry 13:303-304. LITElRATURE CITED 18. Seidman, M. M. 1969. Influence of side-chain substituents 1. Barnes, C. S. 1963. The structure of munetone. Tetraon the position of cleavage of the benzene ring by hedron Lett. 5:281-288. Pseudomonas fluorencens. J. Bacteriol. 97:1192-1197. 2. Blakley, E. R. 1967. The metabolism of aromatic com- 19. Subba Rao, P. V., B. Fritig, J. R. Vose, and G. H. N. pounds with different side chains by a Pseudomonas. Towers. 1971. An aromatic 3,4-oxygenase from TilleCan. J. Microbiol. 13:761-769. tiopsis washingtonensis-oxidation of 3,4-dihydroxy3. Block, R. J., E. L Durrum, and G. Zweig. 1958. Kephenyl acetic acid to 16-carboxymethylmuconolactone. tones and aldehydes, p. 340-345. In A manual paper Phytochemistry 10:51-56. chromatography and paper electrophoresis. Academic 20. Suzuki, Y., K. Imai, and S. Marumo. 1974. Trans and Press Inc., New York. cis hydration of racemic 10,11-epoxyfarnesol into opti4. Braga de Oliveira, A., L G. Fonseca e Silva, and 0. cally active glycols by fungus. J. Am. Chem. Soc. R. Gottlieb. 1972. Flavonoids and coumarins from Pla96:3703-3705. tymiscium praecox. Phytochemistry 11:3515-3519. 21. Udupa, S. R., A. Banerji, and M. S. Chandha. 1968. 5. Clifford, D. R., J. K. Faulkner, J. R. L. Walker, and Microbiological transformation of flavanone. TetrahedD. Woodcock. 1969. Metabolism of cinnamic acid by ron Lett. 37:4003-4005. Aspergillus niger. Phytochemistry 8:549-552. 22. Udupa, S. R., A. Banerji, and M. S. Chadha. 1969. 6. Gibson, D. T. 1968. Microbial degradation of aromatic Microbiological transformations of flavonoids-II. compounds. Science 161:1093-1097. Transformation of (±) flavanone. Tetrahedron 7. Hasegawa, IL, and T. Shirato. 1953. Two new flavo25:5415-5419. noids from the leaves of Phelodendron amurense Ru- 23. Zweig, G., and J. Sherma (ed.). 1972. Detection reprecht. J. Am. Chem. Soc. 75:5507-5511. agents for paper and/or thin-layer chromatography, p. 8. Hegeman, G. D. 1966. Synthesis of the enzymes of the 111-170. In Handbook of chromatography. CRC Press, mandelate pathway by Pseudomonas putida. J. BacCleveland.
Vol. 34, No. 5 Printed in U.S.A.
Degradation of the Plant Flavonoid Phellamurin by Aspergillus niger SAEKO SAKAIt Department of Biology, Faculty of Science, Tokyo Metropolitan University, Setagaya-ku, Tokyo, 158, Japan Received for publication 18 April 1977
We have previously described the structure of phellamurin, a plant flavonoid, 3,4',5,7-tetrahydroxy-8-isoprenylflavanone-7-0-glucoside (17). Degradation of phellamurin by Aspergillus niger, using modified Czapek-Dox medium as well as phellamurin or one of its degradation products as a sole carbon source, is reported here. Eleven compounds are identified from phellamurin degradation products. A. niger apparently decomposes phellamurin by first removing glucose with f,-glucosidase; neophellamuretin is the first degradation product. Fission of the heterocycic ring of (5"-hydroxyisopropyl-4",5"-dih'drofurano)[2",3"-h]3,4',5-trihydroxyflavanone, which is obtained from neophellamuretin through a few alterations of the side chain, is followed by cleavage of a C-C bond between C==O and carbon at a-position and conversion of (5"-hydroxyisopropyl4",5"-dihydrofurano)[2",3"-d]-2',4,6',a-tetrahydroxychalcone to p-hydroxymandelic acid (B-ring) and 2,4,6-trihydroxy-5-carboxyphenylacetic acid (A-ring). It is suggested that p-hydroxymandelic acid is oxidized to p-hydroxybenzoic acid. 2,4,6-Trihydroxy-5-carboxyphenylacetic acid is metabolized to phloroglucinol carboxylic acid, which subsequently is decarboxylated to phloroglucinol. These results provided new information on the isoprene unit metabolism of the side chain of phellamurin and firmly established the degradation pathway of phellamurin by A. niger. as
The ability of microorganisms to enzymatically transform naturally occurring organic compounds to other substances is well known and has been the subject of numerous studies (8, 10, 19, 22). The importance of these studies is obvious, since the turnover of chemical substances throughout the world is attributed to metabolism by microorganisms. It is similarly well known that plant and animal remains are decomposed by microbes both on and under the ground. This decomposition pattern is also the case for flavonoids, which are common constituents of higher plants. In recent years, considerable information has become available concerning microbial degradation of aromatic compounds (5, 18, 19). Aromatic compound degradation involves hydroxylation of the aromatic ring to form dihydroxy compounds, followed by ring cleavage to yield compounds that can be utilized via the tricarboxylic acid cycle (2, 6). Udupa et al. (21, 22) incubated (±)-flavanone with Gibberella fujikuroi and obtained several compounds: (-)-flavan-4a-ol; 2'-hydroxychalcone; 2'-4-dihydroxydihydrochalcone; 2',4-dihydroxychalcone; (±)4'-hydroxyflavanone; and (-)-4'-hydroxyflavant Present address: National Cancer Institute, National Institutes of Health, Bethesda, MD 20014.
500
4a-ol. Jeffrey et al. (11) have shown that dihydrogossypetin is a metabolite in bacterial (Pseudomonas species) degradation of (-)-taxifolin. Cell-free extracts from the same Pseudomonas species further oxidized dihydrogossypetin via cleavage of the A-ring to form oxaloacetic acid together with 5-(3,4-dihydrophenyl)-4-hydroxy3-oxo-valero-S-lactone (10). However, microbial transformation of flavanone has not been extensively studied. Phellodendron amurense leaves, a tree of Rutaceae, contain a large quantity of phellamurin (7). We have previously described that the aglycone of phellamurin formed by Aspergillus niger (neophellamuretin) is 3,4',5,7-tetrahydroxy-8isoprenylflavanone, and the structure of phellamurin should be the corresponding 7-0-glucoside (17). Up until now, we have found no investigation on the microbial degradation of the isoprenyl group associated with the benzene ring. The present paper reports results of studies on the metabolism of the flavonoid phellamurin by A. niger. MATERIALS AND METHODS Culture. Stock culture maintained on agar slants. the modified Czapek-Dox croelements (FeCl3 6H20,
of A. niger IAM-25 was The growth medium was medium with some mi20 mg; ZnSO4 7H20, 10
VOL. 34, 1977 mg; MnSO4 4H20, 3 mg; Na2MoO4 2H20, 1.5 mg; CuSO4 5H20, 1 mg) as well as 20 g of glucose and 0.1 g of phellamurin per liter; its pH was adjusted to 4.5 with HCI. The phellamurin solution and all remaining ingredients were sterilized separately and combined aseptically in flasks before inoculation. One liter of the liquid culture medium was inoculated with spores grown on five slants. Two liters of the liquid culture medium was incubated for 4 to 25 days at 25°C. After incubation, the medium was decanted, and mycelial mats were washed three times with sterilized water and replaced with a solution of either 0.1% phellamurin or 0.1% degradation product in water. The solution of 2 liters was incubated under the same conditions as described above. Extraction and fractionation. Two liters of the culture filtrate was acidified with dilute HCI to pH 2 and thoroughly extracted with diethyl ether. The etheral extract was back-extracted with 1% sodium bicarbonate to yield a neutral and an acidic fraction. The mother liquid, after the ether extraction, was reextracted with ethyl acetate (ethyl acetate portion). The neutral portion was evaporated, and the residue was dissolved in a small volume of ethanol. The ethanolic solution was then applied to a polyamide column (Woelm) (25 by 170 mm) and eluted successively with 100-ml volumes of each of the following aqueous ethanol mixtures: 0, 20, 40, 60, 80, and 100%. The fractions were concentrated and examined by thinlayer chromatography on silica gel plates GF254 with the solvent chloroform-ethyl acetate-formic acid (5:4:1). After being dried, chromatograms were exposed under ultraviolet light, and fluorescent spots were marked. Compounds isolated from silica gel plates with ethanol were recrystallized from ethanolwater. The acidic and ethyl acetate portions were evaporated, and the residues, dissolved in a small volume of ethanol, were applied to two polyamide columns and eluted with absolute ethanol. The degradation products were isolated by thin-layer chromatography, as described above. Time course of appearance of some degradation products by A. niger. Twelve-day-old mycelial mats were washed at least three times with sterilized water and replaced with 200 ml of distilled water
containing phellamurin or degradation product at a concentration of 1 mg/ml. After incubation for 2, 5, 7, and 10 days, each resting-cell culture medium was extracted with ether. The etheral extracts were evaporated, and the remaining residue was applied to a polyamide column and eluted with 100 ml of absolute ethanol. The ethanolic fraction was evaporated, dissolved in 2 ml of ethanol, and examined by thin-layer chromatography. Compounds were isolated quantitatively from silica gel plates with ethanol. The quantity of these compounds was estimated from the optical density at 300 nm. Chemicals. Phellamurin was isolated from P. amurense leaves by the method of Hasegawa and Shirato (7). Other chemicals were obtained commercially. Chromatographic examination. Samples, together with known compounds as required, were applied to either Whatman no. 1 filter paper or a silica gel GF254 (nach Stahl; E. Merck AG, Darmstadt, Germany) plate by means of a capillary pipette, and
DEGRADATION OF PHELLAMURIN
501
the chromatogram was developed. The solvents used for paper chromatography were: (i) n-butanol-glacial acetic acid-water (6:1:2), (ii) 6% acetic acid, and (iii) benzene-glacial acetic acid-water (6:7:3); the solvent systems for thin-layer chromatography were: (iv) chloroform-ethyl acetate-formic acid (5:4:1) and (v) petroleum ether-chloroform-ethyl acetate-formic acid (10:5:4:1). After drying at room temperature, chromatograms were exposed under ultraviolet light, and fluorescent spots were marked. Spectrometry. Mass and nuclear magnetic resonance spectra were measured by Hitachi RMS-4 and Hitachi-Perkin-Elmer 60 MHz, respectively.
RESULTS AND DISCUSSION Degradation of phellam , a plant flavonoid, was investigated. The following compounds (Fig. 1, A through K) were obtained from phellamurin as the degradation products. The metabolic pathway of phellamurin is also proposed (Fig. 1). Identification of degradation products. Compound A was isolated from the neutral portion. The properties of this compound are completely identical to those of neophellamuretin (17) by comparison of behavior on thin-layer chromatography, melting point, and infrared, mass, and nuclear magnetic resonance spectra. The structure of compound A was 3,4',5,7-tetrahydroxy-8-isoprenylflavanone, as described previously (17). Fraction B gave a colorless crystalline solid (melting point, 140 to 142°C) from the neutral portion, and its elementary analysis was consistent with C20H2208H20. The mass spectrum of compound B shows a parent ion peak at m/e 390 (14%). Ions at m/e 372 and 354 arise from the loss of 18 (M - H20) and 36 (M - 2H20) mass units, respectively, from the molecular ion. It should be pointed out that compound B has two hydroxyl groups different from those of neophellamuretin. An ethanolic solution of this compound gave a purplish-brown coloration with ferric chloride. It produced a reddish-purple color by a reduction test with either magnesium or zinc powder and concentrated hydrochloric acid. This color is considered characteristic of flavanonols (16). This compound also showed ultraviolet absorption peaks (in ethanol) at 300 and 340 nm, and the former peak undergoes the bathochromic shift of 24 and 36 nm after addition of aluminum chloride (12) and sodium acetate (14), respectively. It is suggested that the hydroxyl groups are present at the C5 and C7 positions, while a 4'-substituted flavanone was supported by an infrared absorption at 830 cm-'. These results showed that the possible binding sites for two hydroxyl groups are the positions of the side chains. A similar hydroxylation re-
502
SAKAI
M3C
APPL. ENVIRON. MICROBIOL.
CM3 C
H3C
3C3
CH
Phellamurin
CH3 COH
IOCH
Comp. A
CH
CH2
04< O O O/ON
G10
Comp. B
CH2 0
HO
0-
OH
GIO OH
ON N
11
0
OH
3C\ /C3 COOH
Comp.
HO
Comp. H OH
CM2
HO
0
Comp. C
NC-HN2
ON
ON O
O
ONH
ONH
OH
OH
Comp E HO
OH
i
Comp. D
-aI--COON
COON
/CH3
C-OH
HC NC- 2
ON
N..
OH
H3 C\
C-OH
0
0
Camp F
Camp. G
H
ON
nOOC-C ;;
ON
-
-
OHC
/
OH
-
HOOC
/
OH
OH
Comp. J
FIG. 1. Proposed pathway for phellamurin degradation in A. niger.
action was observed by Suzuki et al. (20). The infrared spectrum indicated that the binding sites are not at a dimethyl group, strongly suggesting that the binding sites should be at C-,8 and C--y. From the above results, the structure of compound B is thought to be 3,4',5,7-tetra-
hydroxy-8-(,B,y-dihydroxyisovaleryl)-flavanone. Compound C gave a pale-yellow solid (melting point, 126 to 1270C) from the neutral portion, and its elementary analysis was consistent with C20H2007. The mass spectrum of compound C shows a parent ion peak at m/e 372 (9%).
In the ultraviolet spectrum, pronounced absorption maxima were observed at 300 and 340 nm. The presence of a chelated OH group was indicated by a positive ferric chloride reaction (23) and a bathochromic shift in the ultraviolet absorption maximum after addition of aluminum chloride (12). When reduced with either magnesium or zinc powder and concentrated hydrochloric acid, a reddish-purple color developed, characteristic of flavanonols (16). The infrared spectrum of compound C shows the presence of C=O and OH groups. The high-field signals (0.98 and 1.118) in the nuclear magnetic resonance spectrum of compound C acetate are attributed to protons of the gem-dimethyl groups that have a long-range coupling with the methine proton at 4.528 (1). A signal at 1.528 is due to a tertiary alcohol. A signal at 2.838 shows methylene protons of benzyl structure that split
into a doublet by coupling to the next methine proton, a triplet (J = 15 Hz) at 4.52. Two pairs of doublets (6.89 and 7.24; J = 8 Hz) represent an A2B2 system of H-2',6' and H-3',5' protons. The protons at 5.118 (d) and 5.45 (d, J2,3 = 10 Hz) exhibit the AB system of H-2 and H-3 (1, 4, 15). The proton at 6.06 (singlet) is a signal of H-6 (A-ring). One acetyl group derived from the C3 hydroxyl group is 1.77 ppm. The two methyl groups at 2.06 and 2.12 are due to aromatic acetyl groups, which are C4' and C5 positions, as indicated previously (17) in neophellamuretin. As compared with that of compound A acetate, only two aromatic acetyl groups were observed. From the results of the nuclear magnetic resonance studies, the presence of hydroxyl groups at the C3 and C5 positions was confirmed. Protons of the B-ring were the same as the ones on neophellamuretin, so the loss of the hydroxyl group is obviously due to the hydroxyl group at the C7 position. The above data indicate that the structure of compound C is (5"-hydroxyisopropyl-4",5"-dihydro-
furano)[2",3"-h]-3,4',5-trihydroxyflavanone. Compound D was also obtained from the neutral portion as a yellow product (melting point, 185°C). Compound D gave a brown color with
alcoholic ferric chloride (23) and a negative color with the HCl-Mg reduction test (16). It produced an orange solution on addition of concentrated sulfuric acid, which turned colorless when dis-
VOL. 34, 1977
tilled water was added. These color reactions indicate that compound D is chalcone (22). The ultraviolet spectrum (absorption maxima at 273, 300, and 378 nm) also suggested a chalcone structure (14). These absorptions each undergo bathochromic shifts of 8, 15, and 62 nm, respectively, with addition of aluminum chloride (14), indicating the presence of a hydroxy group at the 6'-position. There was no shift after addition of sodium acetate, probably indicating the ring fusion between the 4'-hydroxyl position and the 3'-side chain. The infrared spectrum of compound D showed the presence of a hydroxyl, a carbonyl, and a substituted aromatic ring. The mass spectra of compound C {(5"-hydroxyisopropyl - 4", 5"-dihydrofurano) [2", 3"- h] - 3,4',5trihydroxyflavanone} and compound D have been determined. Both compounds C and D gave the molecular ion m/e 372. In compound C, peaks at m/e 236 (100%), m/e 176 (33%), m/e 164 (55%), and m/e 133 (59%) were observed. In compound D, peaks at mWe 221 (38%), m/e 177 (28%), mWe 165 (100%), and m/e 134 (57%) were observed. Compound C showed a peak at m/e 236 as the base peak; compound D gave a base peak at m/e 165. The difference between the two compounds is mainly due to fission of the side chain [(5"-hydroxyisopropyl-4",5"-dihydrofurano) group], but the process of cleavage is very similar (9). However, other peaks at m/e 264 (12%), m/e 249 (30%), and 219 (23%) in compound C were never observed in spectra of compound D. The above results confirm that compound D is not a flavanone but a chalcone. Compound C was converted to the corresponding chalcone. Compound D was estimated as (5"-hydroxyisopropyl-4",5"-dihydrofurano)[2", 3" -d]-2',4,6',a-tetrahydroxychalcone. From the acidic portion, compound E was isolated. It gave a blue color with ferric chlorideferricyanide reagent (23) and a yellow color with bromocresol green (23), suggestive of a hydroxyphenylcarboxylic acid. It failed to give a color with diazotized benzidine. It showed ultraviolet absorption maxima at 278 and 284 (shoulder) nm. Compound E had Rf values of 0.81 (solvent i), 0.86 (solvent ii), 0.04 (solvent iii), and 0.50 (solvent iv). Compound E coincides in all its properties with p-hydroxymandelic acid. By ultraviolet absorption spectrum and paper and thin-layer chromatographies, identity was confirned. Compound F was detected from the neutral fraction. It exhibited an ultraviolet maximum at 290 nm. The compound turned to a brown color with alcoholic ferric chloride (23) and a blue color with ferric chloride-ferricyanide reagent (23), suggestive of a hydroxyphenyl compound. Compound F gave an orange color with 2,4-
DEGRADATION OF PHELLAMURIN
503
dinitrophenylhydrazine, characteristic of either ketones or aldehydes (3). Compound F had Rf values of 0.90 (solvent i), 0.75 (solvent ii), 0.59 (solvent iii), and 0.90 (solvent iv). The above data suggested the product to be p-hydroxybenzaldehyde, which was confirmed by ultraviolet absorption spectrum and paper and thin-layer chromatographies with an authentic sample. Compound G was detected in the acidic portion. An ethanolic solution of this compound gave a brown color with FeCl3 (23), a blue color with ferric chloride-ferricyanide reagent, and a yellow color with bromocresol green (23), indicating the presence of phenolic hydroxyl and carboxyl groups. It exhibited an ultraviolet absorption maximum at 255 nm. Compound G had Rf values of 0.92 (solvent i), 0.63 (solvent ii), 0.35 (solvent iii), and 0.40 (solvent v). This compound was identified asp-hydroxybenzoic acid by paper and thin-layer chromatographies with an authentic sample. Compound H (melting point, 230°C), obtained from the ethyl acetate fraction, gave a colorless crystalline solid. Compound H failed to react with either magnesium or zinc and hydrochloric acid (16). This compound gave a purple color in the pine-shaving reaction, a reddish-orange color with diazotized benzidine (23), and a yellow color with bromocresol green. The ultraviolet spectrum showed prominent absorption at 295 nm and two inflections at 255 and 263 nm. The former peak undergoes bathochromic shifts of 20 and 5 nm with addition of aluminum chloride (12) and sodium acetate (14), respectively. The most likely structure for this compound seemed to be a phloroglucinol carboxylic derivative. It had Rf values of 0.75 (solvent ii), 0.00 (solvent iii), and 0.20 (solvent iv). The mass spectrum of compound H showed a parent ion peak at m/e 228, and M - 1 ion at m/e 227. The base peak was M - 19, which was due to further loss of an hydrogen atom from the M - H20 ion. The second most prominent peak in the spectrum was m/e 226, which was due to further dehydrogenation from the M - 1 ion. These pathways were ascertained by the presence of metastable ions. Other ions of importance in the spectrum of this compound were the M - 46 (m/e 182) ion, which probably arose by the decarboxylation from the M - 1 ion, and the M - 63 (m/e 165) ion, which was formed by the loss of OH from the M - 46 ion. Moreover, the M - 75 (m/e 153) ion was due to the loss of CH2 from the M - 63 ion and hydrogenation to the same ion at m/e 126, that is, phloroglucinol. The phenylcation (m/e 77) was due to the loss of 3(OH)
for phloroglucinol. From the above results, compound H is thought to be 2,4,6-trihydroxy-5carboxyphenylacetic acid.
504
SAKAI
APPL. ENVIRON. MICROBIOL.
Compound I was identified from the acidic (jmmoles) portion. It gave a deep-pink color in the pineshaving raction and a red color with diazotized 100 benzidine. The principle of coloration by diazonium reagents is that a diazo-coupling occurs when the para-position to a phenolic hydroxyl group is free (10). If there were at least one vacant position in either the phloroglucinol or phloroglucinol derivatives, a pine-shaving reac50 (pmoles) tion would give a reddish- or bluish-purple color. to be phloroglucinol Compound I was expected carboxylic acid from the above color reactions. Compound I showed ultraviolet absorption -i1 0.5 peaks at 270 and 320 nm. It had Rf values of 0.75 (solvent i), 0.65 (solvent ii), and 0.00 (solvent 0.0 0 iii). It was identified as phloroglucinol carboxylic acid by paper chromatography with an authentic 0 2 6 10 4 8 sample. DAYS Compound J was isolated from the acidic porFIG. 2. Change with time of phellamurin metabotion. It gave a purple color in the pine-shaving lites, using replacement culture containing 0.2 g (ca. reaction and strong reddish-brown color with 400 wnol) ofphellamurin. (A) Compound A; (B) comdiazotized benzidine (10). It also gave a strong pound B; (C) compound C; (D) compound D. blue color with ferric chloride-ferricyanide reagent (23). These observations indicate that the tions of compounds A, B, and C were observed compound is phloroglucinol. In the ultraviolet after 7 days of incubation and that of compound spectrum, pronounced absorption maxima were D after 10 days. However, in the presence of observed at 255 (shoulder), 269, and 272 (shoul- glucose, the peak of maximum accumulation of der) nm. Compound J had Rf values of 0.75 (sol- degradation products was generally delayed. To vent i), 0.60 (solvent ii), 0.00 (solvent iii), and study the pathway for the formation of the com0.55 (solvent iv). From the paper and thin-layer pounds from A, the replacement culture medium chromatographic comparison with authentic containing 0.1% compound A was used. Comsamples, compound I is consistent with phloro- pounds B, C, and D were obtained from compound A as degradation products after incubaglucinol. A small amount of compound K was isolated tion for 7 days. Compound D was enzymatically from the ethyl acetate fraction. Compound K formed from compound C, and this was further coincided in all its properties with phellamurin supported by an experiment with the replacement culture medium containing 0.1% comsupplied as the substrate. Fungal degradation of phellamurin. The pound C; that is, compound D was obtained fungal degradation usually involves an initial from compound C as the only degradation prodrelease of sugars by endogenous glycosidases, uct after incubation for 4 days. Since it is followed by hydrolytic cleavage of the hetero- thought from the structures of compounds A cyclic ring of the aglycone (13). A. niger appar- and C that conversion of compound A to C is ently degrades phellamurin by first removing not a direct reaction, compound A is probably glucose with fi-glycosidase. Therefore, the first converted to compound B by adding two moledegradation product of phellamurin is com- cules of water to the side chain of compound A pound A, that is, neophellamuretin (3,4',5,7-te- through two unidentified intermediates. Dehytrahydroxy-8-isoprenylflavanone). After incu- droxylation and the ring fusion in the side chain bation for 2, 5, 7, and 10 days, using a resting- of compound B occur at the same time to procell culture medium containing 0.1% phellamu- duce compound C. After fission of the heterorin, the quantity of degradation products (com- cyclic ring of compound C, followed by cleavage pounds A, B, C, and D) was estimated from of the C-C bond between carbonyl and carbon the optical density at 300 nm (Fig. 2). Compound at the a-position, the conversion of compound A, B [3,4',5,7-tetrahydroxy-8-(,8,y-dihydroxyiso- D to E (p-hydroxymandelic acid) and compound valeryl)-flavanone], C [(5"-hydroxyisopropyl- H (2,4,6-trihydroxy-5-carboxyphenylacetic acid) 4",5"-dihydrofurano)[2",3"-h]-3,4',5-trihydroxy- occurred. Compound E was derived from the Bflavanone], and D [(5"-hydroxyisopropyl-4",5"- ring, and compound H was derived from the Adihydrofurano)[2",3"-d]-2',4,6',a-tetrahydroxy- ring. The fornation of benzoic acid from manchalcone] were obtained from phellamurin as delic acid in a microorganism system (8) is docdegradation products. The highest accumula- umented. It is thought that compound E (p-hy-
VOL. 34, 1977
droxymandelic acid) is oxidized to compound G (p-hydroxybenzoic acid) by the same enzymes of the mandelate pathway (8), compound F (p-hydroxybenzaldehyde) and compound G being intermediates in this pathway. On the other hand, after cleavage of the side chain, compound H is metabolized to compound I (phloroglucinol carboxylic acid), which subsequently is decarboxylated to compound J (phloroglucinol). These metabolic pathways are illustrated in Fig. 1. The results of this study provide evidence for the metabolism of the isoprene unit of the side chain of pheliamurin and establish the degradation pathways of phellamurin by A. niger.
DEGRADATION OF PHELLAMURIN
505
teriol. 91:1140-1154. 9. Itagaki, Y., T. Kurokawa, S. Sasaki, Chin-Te Chang, and Fa-Ching Chen. 1966. The mass spectra of chalcones, flavones and isoflavones. Bull. Chem. Soc. Jpn. 39:538-543. 10. Jeffrey, A. M., D. M. Jerina, R. Self, and W. C. Evans. 1972. The bacterial degradation of flavonoids. Biochem. J. 130:383-390. 11. Jeffrey, A. M., K. Knight, and W. C. Evans. 1972. The bacterial degradation of flavonoids. Biochem. J. 130:373-381. 12. Jurd, L 1961. Spectral properties of flavonoid compounds, p. 108-154. In T. A. Geissman (ed.), The chemistry of flavonoid compounds. Pergamon Press, Oxford. 13. Krishnamurty, H. G., K. J. Cheng, G. A. Jones, F. J. Simpson, and J. E. Watkin. 1970. Identification of products produced by the anaerobic degradation of rutin and related flavonoids by Butyrivibrio sp. C:i. Can. J. Microbiol. 16:759-767. ACKNOWLEDGME NTIS 14. Mabry, T. J., K. R. Markham, and M. B. Thomas. I would like to express my sincere thanks to M. Hasegawa 1970. The structure analysis of flavonoids by ultraviolet and S. Yoshida of the Department of Biology, Faculty of spectroscopy, p. 165-230. In The systematic identificaScience, Tokyo Metropolitan University, for their kind guidtion of flavonoids. Springer-Verlag, Berlin, Heidelberg, ance and encouragement throughout this investigation. I New York. would also like to express cordial thanks to T. Inoue of the 15. Mabry, T. J., K. R. Markham, and M. B. Thomas. Hoshi College of Pharmacy for measurement of the mass and 1970. The structure analysis of flavonoids by proton infrared spectra, and T. Sato of the Department of Chemistry, nuclear magnetic resonance spectroscopy, p. 260-273. Faculty of Science, Tokyo Metropolitan University, for reIn The systematic identification of flavonoids. Springercording the nuclear magnetic resonance spectrum. Finally, I Verlag, Berlin, Heidelberg, New York. would like to thank S. S. Thorgeirsson and P. J. Wirth of the 16. Pew, J. C. 1948. A flavanone from douglas-fir heartwood. National Cancer Institute, National Institutes of Health, for J. Am. Chem. Soc.70:3031-3034. critical reading of this manuscript. 17. Sakai, S., and M. Hasegawa. 1973. Structure of phellamurin. Phytochemistry 13:303-304. LITElRATURE CITED 18. Seidman, M. M. 1969. Influence of side-chain substituents 1. Barnes, C. S. 1963. The structure of munetone. Tetraon the position of cleavage of the benzene ring by hedron Lett. 5:281-288. Pseudomonas fluorencens. J. Bacteriol. 97:1192-1197. 2. Blakley, E. R. 1967. The metabolism of aromatic com- 19. Subba Rao, P. V., B. Fritig, J. R. Vose, and G. H. N. pounds with different side chains by a Pseudomonas. Towers. 1971. An aromatic 3,4-oxygenase from TilleCan. J. Microbiol. 13:761-769. tiopsis washingtonensis-oxidation of 3,4-dihydroxy3. Block, R. J., E. L Durrum, and G. Zweig. 1958. Kephenyl acetic acid to 16-carboxymethylmuconolactone. tones and aldehydes, p. 340-345. In A manual paper Phytochemistry 10:51-56. chromatography and paper electrophoresis. Academic 20. Suzuki, Y., K. Imai, and S. Marumo. 1974. Trans and Press Inc., New York. cis hydration of racemic 10,11-epoxyfarnesol into opti4. Braga de Oliveira, A., L G. Fonseca e Silva, and 0. cally active glycols by fungus. J. Am. Chem. Soc. R. Gottlieb. 1972. Flavonoids and coumarins from Pla96:3703-3705. tymiscium praecox. Phytochemistry 11:3515-3519. 21. Udupa, S. R., A. Banerji, and M. S. Chandha. 1968. 5. Clifford, D. R., J. K. Faulkner, J. R. L. Walker, and Microbiological transformation of flavanone. TetrahedD. Woodcock. 1969. Metabolism of cinnamic acid by ron Lett. 37:4003-4005. Aspergillus niger. Phytochemistry 8:549-552. 22. Udupa, S. R., A. Banerji, and M. S. Chadha. 1969. 6. Gibson, D. T. 1968. Microbial degradation of aromatic Microbiological transformations of flavonoids-II. compounds. Science 161:1093-1097. Transformation of (±) flavanone. Tetrahedron 7. Hasegawa, IL, and T. Shirato. 1953. Two new flavo25:5415-5419. noids from the leaves of Phelodendron amurense Ru- 23. Zweig, G., and J. Sherma (ed.). 1972. Detection reprecht. J. Am. Chem. Soc. 75:5507-5511. agents for paper and/or thin-layer chromatography, p. 8. Hegeman, G. D. 1966. Synthesis of the enzymes of the 111-170. In Handbook of chromatography. CRC Press, mandelate pathway by Pseudomonas putida. J. BacCleveland.
