Degradation of the Plant Flavonoid Phellamurin by Aspergillus Niger

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1977, p. 500-505 Vol. 34, No. 5 Copyright i 1977 American Society for Microbiology Printed in U.S.A. Degradation of the Plant Flavonoid Phellamurin by Aspergillus niger SAEKO SAKAIt Department ofBiology, Faculty ofScience, 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, as 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"-hydroxyisopropyl- 4",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. The ability of microorganisms to enzymati- 4a-ol. Jeffrey et al. (11) have shown that dihy- cally transform naturally occurring organic com- drogossypetin is a metabolite in bacterial (Pseu- pounds to other substances is well known and domonas species) degradation of (-)-taxifolin. has been the subject of numerous studies (8, 10, Cell-free extracts from the same Pseudomonas 19, 22). The importance of these studies is ob- species further oxidized dihydrogossypetin via vious, since the turnover of chemical substances cleavage of the A-ring to form oxaloacetic acid throughout the world is attributed to metabo- together with 5-(3,4-dihydrophenyl)-4-hydroxy- lism by microorganisms. It is similarly well 3-oxo-valero-S-lactone (10). However, microbial known that plant and animal remains are de- transformation of flavanone has not been exten- composed by microbes both on and under the sively studied. ground. This decomposition pattern is also the Phellodendron amurense leaves, a tree ofRu- case for flavonoids, which are common constit- taceae, contain a large quantity of phellamurin uents of higher plants. (7). We have previously described that the agly- In recent years, considerable information has cone ofphellamurin formed byAspergillus niger become available concerning microbial degra- (neophellamuretin) is 3,4',5,7-tetrahydroxy-8- dation of aromatic compounds (5, 18, 19). Aro- isoprenylflavanone, and the structure of phella- matic compound degradation involves hydrox- murin should be the corresponding 7-0-glucoside ylation of the aromatic ring to form dihydroxy (17). Up until now, we have found no investiga- compounds, followed by ring cleavage to yield tion on the microbial degradation of the iso- compounds that can be utilized via the tricar- prenyl group associated with the benzene ring. boxylic acid cycle (2, 6). Udupa et al. (21, 22) The present paper reports results of studies on incubated (±)-flavanone with Gibberella fuji- the metabolism of the flavonoid phellamurin by kuroi and obtained several compounds: (-)-fla- A. niger. van-4a-ol; 2'-hydroxychalcone; 2'-4-dihydroxy- MATERIALS AND dihydrochalcone; 2',4-dihydroxychalcone; (±)- METHODS 4'-hydroxyflavanone; and (-)-4'-hydroxyflavan- Culture. Stock culture of A. niger IAM-25 was maintained on agar slants. The growth medium was t Present address: National Cancer Institute, National In- the modified Czapek-Dox medium with some mi- stitutes of Health, Bethesda, MD 20014. croelements (FeCl3 6H20, 20 mg; ZnSO4 7H20, 10 500 VOL. 34, 1977 DEGRADATION OF PHELLAMURIN 501 mg; MnSO4 4H20, 3 mg; Na2MoO4 2H20, 1.5 mg; the chromatogram was developed. The solvents used CuSO4 5H20, 1 mg) as well as 20 g of glucose and 0.1 for paper chromatography were: (i) n-butanol-glacial g of phellamurin per liter; its pH was adjusted to 4.5 acetic acid-water (6:1:2), (ii) 6% acetic acid, and (iii) with HCI. The phellamurin solution and all remaining benzene-glacial acetic acid-water (6:7:3); the solvent ingredients were sterilized separately and combined systems for thin-layer chromatography were: (iv) chlo- aseptically in flasks before inoculation. One liter of roform-ethyl acetate-formic acid (5:4:1) and (v) petro- the liquid culture medium was inoculated with spores leum ether-chloroform-ethyl acetate-formic acid grown on five slants. Two liters of the liquid culture (10:5:4:1). After drying at room temperature, chromat- medium was incubated for 4 to 25 days at 25°C. ograms were exposed under ultraviolet light, and flu- After incubation, the medium was decanted, and orescent spots were marked. mycelial mats were washed three times with sterilized Spectrometry. Mass and nuclear magnetic reso- water and replaced with a solution of either 0.1% nance spectra were measured by Hitachi RMS-4 and phellamurin or 0.1% degradation product in water. Hitachi-Perkin-Elmer 60 MHz, respectively. The solution of 2 liters was incubated under the same conditions as described above. RESULTS AND DISCUSSION Extraction and fractionation. Two liters of the culture filtrate was acidified with dilute HCI to pH 2 Degradation ofphellam , a plant flavonoid, and thoroughly extracted with diethyl ether. The eth- was investigated. The following compounds (Fig. eral extract was back-extracted with 1% sodium bicar- bonate to yield a neutral and an acidic fraction. The 1, A through K) were obtained from phellamurin mother liquid, after the ether extraction, was reex- as the degradation products. The metabolic tracted with ethyl acetate (ethyl acetate portion). The pathway of phellamurin is also proposed neutral portion was evaporated, and the residue was (Fig. 1). dissolved in a small volume of ethanol. The ethanolic Identification of degradation products. solution was then applied to a polyamide column Compound A was isolated from the neutral por- (Woelm) (25 by 170 mm) and eluted successively with tion. The properties of this compound are com- 100-ml volumes of each of the following aqueous pletely identical to those of neophellamuretin ethanol mixtures: 0, 20, 40, 60, 80, and 100%. The (17) by comparison of behavior on thin-layer fractions were concentrated and examined by thin- melting point, and infrared, layer chromatography on silica gel plates GF254 with chromatography, the solvent chloroform-ethyl acetate-formic acid mass, and nuclear magnetic resonance spectra. (5:4:1). After being dried, chromatograms were ex- The structure of compound A was 3,4',5,7-tet- posed under ultraviolet light, and fluorescent spots rahydroxy-8-isoprenylflavanone, as described were marked. Compounds isolated from silica gel previously (17). plates with ethanol were recrystallized from ethanol- Fraction B gave a colorless crystalline solid water. The acidic and ethyl acetate portions were (melting point, 140 to 142°C) from the neutral evaporated, and the residues, dissolved in a small portion, and its elementary analysis was consist- volume of ethanol, were applied to two polyamide ent with C20H2208H20. The mass spectrum of columns and eluted with absolute ethanol. The deg- compound B shows a parent ion peak at m/e radation products were isolated by thin-layer chro- Ions at 372 354 arise from matography, as described above. 390 (14%). m/e and Time course of appearance of some degrada- the loss of 18 (M - H20) and 36 (M - 2H20) tion products by A. niger. Twelve-day-old mycelial mass units, respectively, from the molecular ion. mats were washed at least three times with sterilized It should be pointed out that compound B has water and replaced with 200 ml of distilled water two hydroxyl groups different from those of neo- containing phellamurin or degradation product at a phellamuretin. An ethanolic solution of this concentration of 1 mg/ml. After incubation for 2, 5, compound gave a purplish-brown coloration 7, and 10 days, each resting-cell culture medium was with ferric chloride. It produced a reddish-purple extracted with ether. The etheral extracts were evap- a reduction test with either magnesium orated, and the remaining residue was applied to a color by polyamide column and eluted with 100 ml of absolute or zinc powder and concentrated hydrochloric ethanol. The ethanolic fraction was evaporated, dis- acid. This color is considered characteristic of solved in 2 ml of ethanol, and examined by thin-layer flavanonols (16). This compound also showed chromatography. Compounds were isolated quantita- ultraviolet absorption peaks (in ethanol) at 300 tively from silica gel plates with ethanol. The quantity and 340 nm, and the former peak undergoes the of these compounds was estimated from the optical bathochromic shift of 24 and 36 nm after addi- density at 300 nm. tion of aluminum chloride (12) and sodium ace- Chemicals. Phellamurin was isolated from P. amu- tate (14), respectively. It is suggested that the rense leaves by the method of Hasegawa and Shirato hydroxyl groups are present at the C5 and C7 (7). Other chemicals were obtained commercially. flavanone was Chromatographic examination. Samples, to- positions, while a 4'-substituted gether with known compounds as required, were ap- supported by an infrared absorption at 830 cm-'. plied to either Whatman no. 1 filter paper or a silica These results showed that the possible binding gel GF254 (nach Stahl; E. Merck AG, Darmstadt, sites for two hydroxyl groups are the positions Germany) plate by means of a capillary pipette, and of the side chains. A similar hydroxylation re- 502 SAKAI APPL. ENVIRON. MICROBIOL.

M3C CM3 H3C CH3 C 3C3 COH

CH Phellamurin CH Comp. A IOCH Comp. B CH2 CH2

0 G10 04< O 0- HO OH GIOOH O O/ON ON N 0 11 OH 0

3C\ /C3 H3 C\ /CH3 i COOH C-OH C-OH Comp. Comp. H Comp. D Comp. C CM2 HC NC-2 NC-HN2

HO OH HO ON 0 ON ON N.. -aI--- O COON COON O ONH OH OH ONH OH 0

Comp E Camp F Camp. G HO OH H

nOOC-C ;; ON - OHC / OH - HOOC / OH - ON OH Comp. J FIG. 1. Proposed pathway forphellamurin degradation in A. niger. action was observed by Suzuki et al. (20). The into a doublet by coupling to the next methine infrared spectrum indicated that the binding proton, a triplet (J = 15 Hz) at 4.52. Two pairs sites are not at a dimethyl group, strongly sug- of doublets (6.89 and 7.24; J = 8 Hz) represent gesting that the binding sites should be at C-,8 an A2B2 system of H-2',6' and H-3',5' protons. and C--y. From the above results, the structure The protons at 5.118 (d) and 5.45 (d, J2,3 = 10 of compound B is thought to be 3,4',5,7-tetra- Hz) exhibit the AB system of H-2 and H-3 (1, hydroxy-8-(,B,y-dihydroxyisovaleryl)-flavanone. 4, 15). The proton at 6.06 (singlet) Compound C gave a pale-yellow solid (melting is a signal of H-6 (A-ring). One acetyl group point, 126 to 1270C) from the neutral portion, derived from the C3 hydroxyl group is 1.77 ppm. and its elementary analysis was consistent with The two methyl groups at 2.06 and 2.12 are due C20H2007. The mass spectrum of compound C to aromatic acetyl groups, which are C4' and shows a parent ion peak at m/e 372 (9%). C5 positions, as indicated previously (17) in neo- In the ultraviolet spectrum, pronounced ab- phellamuretin. As compared with that of com- sorption maxima were observed at 300 and 340 pound A acetate, only two aromatic acetyl nm. The presence of a chelated OH group was groups were observed. From the results of the indicated by a positive ferric chloride reaction nuclear magnetic resonance studies, the pres- (23) and a bathochromic shift in the ultraviolet ence of hydroxyl groups at the C3 and C5 posi- absorption maximum after addition of alumi- tions was confirmed. Protons of the B-ring were num chloride (12). When reduced with either the same as the ones on neophellamuretin, so magnesium or zinc powder and concentrated the loss of the hydroxyl group is obviously due hydrochloric acid, a reddish-purple color devel- to the hydroxyl group at the C7 position. The oped, characteristic of flavanonols (16). The in- above data indicate that the structure of com- frared spectrum of compound C shows the pres- pound C is (5"-hydroxyisopropyl-4",5"-dihydro- ence of C=O and OH groups. The high-field furano)[2",3"-h]-3,4',5-trihydroxyflavanone. signals (0.98 and 1.118) in the nuclear magnetic Compound D was also obtained from the neu- resonance spectrum of compound C acetate are tral portion as a yellow product (melting point, attributed to protons of the gem-dimethyl 185°C). Compound D gave a brown color with groups that have a long-range coupling with the alcoholic ferric chloride (23) and a negative color methine proton at 4.528 (1). A signal at 1.528 is with the HCl-Mg reduction test (16). It produced due to a tertiary alcohol. A signal at 2.838 shows an orange solution on addition of concentrated methylene protons of benzyl structure that split sulfuric acid, which turned colorless when dis- VOL. 34, 1977 DEGRADATION OF PHELLAMURIN 503 tilled water was added. These color reactions dinitrophenylhydrazine, characteristic of either indicate that compound D is chalcone (22). The ketones or aldehydes (3). Compound F had Rf ultraviolet spectrum (absorption maxima at 273, values of 0.90 (solvent i), 0.75 (solvent ii), 0.59 300, and 378 nm) also suggested a chalcone (solvent iii), and 0.90 (solvent iv). The above structure (14). These absorptions each undergo data suggested the product to bep-hydroxyben- bathochromic shifts of 8, 15, and 62 nm, respec- zaldehyde, which was confirmed by ultraviolet tively, with addition of aluminum chloride (14), absorption spectrum and paper and thin-layer indicating the presence of a hydroxy group at chromatographies with an authentic sample. the 6'-position. There was no shift after addition Compound G was detected in the acidic por- of sodium acetate, probably indicating the ring tion. An ethanolic solution of this compound fusion between the 4'-hydroxyl position and the gave a brown color with FeCl3 (23), a blue color 3'-side chain. The infrared spectrum of com- with ferric chloride-ferricyanide reagent, and a pound D showed the presence of a hydroxyl, a yellow color with bromocresol green (23), indi- carbonyl, and a substituted aromatic ring. The cating the presence of phenolic hydroxyl and mass spectra of compound C {(5"-hydroxyiso- carboxyl groups. It exhibited an ultraviolet ab- propyl-4", 5"-dihydrofurano)[2",3"-h] -3,4',5- sorption maximum at 255 nm. Compound G had trihydroxyflavanone} and compound D have Rf values of 0.92 (solvent i), 0.63 (solvent ii), 0.35 been determined. Both compounds C and D (solvent iii), and 0.40 (solvent v). This compound gave the molecular ion m/e 372. In compound was identified asp-hydroxybenzoic acid by paper C, peaks at m/e 236 (100%), m/e 176 (33%), m/e and thin-layer chromatographies with an au- 164 (55%), and m/e 133 (59%) were observed. thentic sample. In compound D, peaks at mWe 221 (38%), m/e Compound H (melting point, 230°C), obtained 177 (28%), mWe 165 (100%), and m/e 134 (57%) from the ethyl acetate fraction, gave a colorless were observed. Compound C showed a peak at crystalline solid. Compound H failed to react m/e 236 as the base peak; compound D gave a with either magnesium or zinc and hydrochloric base peak at m/e 165. The difference between acid (16). This compound gave a purple color the two compounds is mainly due to fission of in the pine-shaving reaction, a reddish-orange the side chain [(5"-hydroxyisopropyl-4",5"-di- color with diazotized benzidine (23), and a yellow hydrofurano) group], but the process of cleavage color with bromocresol green. The ultraviolet is very similar (9). However, other peaks at m/e spectrum showed prominent absorption at 295 264 (12%), m/e 249 (30%), and 219 (23%) in nm and two inflections at 255 and 263 nm. The compound C were never observed in spectra of former peak undergoes bathochromic shifts of compound D. The above results confirm that 20 and 5 nm with addition of aluminum chloride compound D is not a flavanone but a chalcone. (12) and sodium acetate (14), respectively. The Compound C was converted to the correspond- most likely structure for this compound seemed ing chalcone. Compound D was estimated as to be a phloroglucinol carboxylic derivative. It (5"-hydroxyisopropyl-4",5"-dihydrofurano)[2", had Rf values of 0.75 (solvent ii), 0.00 (solvent 3"-d]-2',4,6',a-tetrahydroxychalcone. iii), and 0.20 (solvent iv). The mass spectrum of From the acidic portion, compound E was compound H showed a parent ion peak at m/e isolated. It gave a blue color with ferric chloride- 228, and M - 1 ion at m/e 227. The base peak ferricyanide reagent (23) and a yellow color with was M - 19, which was due to further loss of bromocresol green (23), suggestive of a hydrox- an hydrogen atom from the M - H20 ion. The yphenylcarboxylic acid. It failed to give a color second most prominent peak in the spectrum with diazotized benzidine. It showed ultraviolet was m/e 226, which was due to further dehydro- absorption maxima at 278 and 284 (shoulder) genation from the M - 1 ion. These pathways nm. Compound E had Rf values of 0.81 (solvent were ascertained by the presence of metastable i), 0.86 (solvent ii), 0.04 (solvent iii), and 0.50 ions. Other ions of importance in the spectrum (solvent iv). Compound E coincides in all its of this compound were the M - 46 (m/e 182) properties with p-hydroxymandelic acid. By ul- ion, which probably arose by the decarboxyla- traviolet absorption spectrum and paper and tion from the M - 1 ion, and the M - 63 (m/e thin-layer chromatographies, identity was con- 165) ion, which was formed by the loss of OH firned. from the M - 46 ion. Moreover, the M - 75 Compound F was detected from the neutral (m/e 153) ion was due to the loss of CH2 from fraction. It exhibited an ultraviolet maximum at the M - 63 ion and hydrogenation to the same 290 nm. The compound turned to a brown color ion at m/e 126, that is, phloroglucinol. The phen- with alcoholic ferric chloride (23) and a blue ylcation (m/e 77) was due to the loss of 3(OH) color with ferric chloride-ferricyanide reagent for phloroglucinol. From the above results, com- (23), suggestive of a hydroxyphenyl compound. pound H is thought to be 2,4,6-trihydroxy-5- Compound F gave an orange color with 2,4- carboxyphenylacetic acid. 504 SAKAI APPL. ENVIRON. MICROBIOL. Compound I was identified from the acidic (jmmoles) portion. It gave a deep-pink color in the pine- shaving raction and a red color with diazotized benzidine. The principle of coloration by diazon- 100 ium 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 reaction would give a reddish- or bluish-purple color. 50 (pmoles) Compound I was expected to be phloroglucinol carboxylic acid from the above color reactions. Compound I showed ultraviolet absorption -i1 0.5 peaks at 270 and 320 nm. It had Rfvalues 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 4 6 8 10 sample. DAYS Compound J was isolated from the acidic por- FIG. 2. Change with time ofphellamurin metabo- tion. 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) com- diazotized 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 com- 0.55 (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. Com- samples, compound I is consistent with phloro- pounds B, C, and D were obtained from com- glucinol. pound A as degradation products after incuba- 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 replace- supplied as the substrate. ment culture medium containing 0.1% com- 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 prod- release 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 mole- degradation 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. Dehy- trahydroxy-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 pro- cell culture medium containing 0.1% phellamu- duce compound C. After fission of the hetero- rin, 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 B- flavanone], and D [(5"-hydroxyisopropyl-4",5"- ring, and compound H was derived from the A- dihydrofurano)[2",3"-d]-2',4,6',a-tetrahydroxy- ring. The fornation of benzoic acid from man- chalcone] were obtained from phellamurin as delic acid in a microorganism system (8) is doc- degradation products. The highest accumula- umented. It is thought that compound E (p-hy- VOL. 34, 1977 DEGRADATION OF PHELLAMURIN 505 droxymandelic acid) is oxidized to compound G teriol. 91:1140-1154. (p-hydroxybenzoic acid) by the same enzymes of 9. Itagaki, Y., T. Kurokawa, S. Sasaki, Chin-Te Chang, and Fa-Ching Chen. 1966. The mass spectra of chal- the mandelate pathway (8), compound F (p-hy- cones, flavones and isoflavones. Bull. Chem. Soc. Jpn. droxybenzaldehyde) and compound G being in- 39:538-543. termediates in this pathway. On the other hand, 10. Jeffrey, A. M., D. M. Jerina, R. Self, and W. C. Evans. after cleavage of the side chain, compound H is 1972. The bacterial degradation offlavonoids. Biochem. J. 130:383-390. metabolized to compound I (phloroglucinol car- 11. Jeffrey, A. M., K. Knight, and W. C. Evans. 1972. boxylic acid), which subsequently is decarbox- The bacterial degradation of flavonoids. Biochem. J. ylated to compound J (phloroglucinol). These 130:373-381. metabolic pathways are illustrated in Fig. 1. 12. Jurd, L 1961. Spectral properties of flavonoid com- The results of this pounds, p. 108-154. In T. A. Geissman (ed.), The chem- study provide evidence for istry of flavonoid compounds. Pergamon Press, Oxford. the metabolism of the isoprene unit of the side 13. Krishnamurty, H. G., K. J. Cheng, G. A. Jones, F. J. chain of pheliamurin and establish the degra- Simpson, and J. E. Watkin. 1970. Identification of dation pathways of phellamurin by A. niger. products produced by the anaerobic degradation of rutin and related flavonoids by Butyrivibrio sp. C:i. ACKNOWLEDGMENTIS Can. J. Microbiol. 16:759-767. 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 identifica- Science, Tokyo Metropolitan University, for their kind guid- tion 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 re- In The systematic identification offlavonoids. Springer- cording 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. 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