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-hydroxyman- delic 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 car- boxylic 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 phella- murin 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.