MICROBIAL TRANSFORMATION OF 2,5 DIHYDROXYCINNAMIC ACID BY ASPERGILLUS NIGER AND RHIZOPUS ORYZAE

2,5-dihydroxy cinnamic acid (1), when fermented with fungal culture, Rhizopus oryzae RCMB 014002 gave mainly two metabolites; 2,5-dihydroxy cinnamoyl alcohol (CinRM-1) and 2-Hydroxy-4,5-dimethoxy cinnamoyl alcohol (Cin-RM-2). Aspergillus niger RCMB002007, however, transformed 2,5-dihydroxy cinnamic acid (1), into a major metabolite;2-hydroxy-5-methoxy cinnamic acid (Cin-AM-3). The structures of the metabolic products were elucidated by means of spectroscopic data. The significance of the metabolites as antioxidants using DPPH radical scavenging assay and lipid peroxidation assay by thiobarbituric acid-reactive substances (TBARS) method using rat tissue homogenates in relation to their structure was discussed.


INTRODUCTION
Cinnamic acid, the deaminated product of phenyl-alanine in plant tissue, a frequent first step is the elimination of ammonia from the side-chain to generate the appropriate trans (E) cinnamic acid.Other cinnamic acids are obtained by further hydroxylation and methylation reactions, sequentially building up substitution patterns typical of shikimate pathway metabolites, i.e. an ortho oxygenation pattern.Cinnamic acid has a long history of human use as a component of plant-derived scents and flavorings (Hoskins, 1984).Cinnamic acid is also an auxin, a class of plant hormones regulating cell growth and differentiation (Thiman, 1969).Several biological activities including antioxidant, anti-inflammatory, cytotoxic, hepatoprotective, immunosuppressive, anti-cholesterolemic, antimicrobial and antiviral activities have been attributed to this class of compounds (Natella et al., 1999;Kim et al., 2005;Miles et al., 2005;Fernández-Martínez et al., 2007;Gravina et al., 2011;Prateek , 2011;Luana Dalbem et al., 2012) .
Biotransformations are useful techniques for producing medicinal and agricultural chemicals from both active and inactive materials.These reactions are an important route for introducing chemical functions into inaccessible sites of molecules and thereby to produce rare structures.The biotransformation reactions can involve high degree of regio-and stereospecificity and require mild reaction conditions.Many researchers have reported the production of drug metabolites using microbial biotransformations (Grogan, 2009).The use of microorganisms may be utilized as models of drug metabolism to predict the fate of xenobiotics in mammalian systems (Smith and Rosazza, 1982), since this method often gives sufficient quantities of metabolites, complete chemical structure and pharmacological activities could be determined.In our ongoing research on microbial transformation of phenolic compounds (Hosny et al., 2001), 2,5-dihydroxycinnamic acid (1), was screened using 11 different microorganisms.Of the organisms which effected transformation, scale up studies was carried out with selected cultures to isolate the maximum number of metabolites in reasonable yields.Structure elucidation of the isolated metabolites and their possible impact on the antioxidant activity in relation to structure are reported herein.

General Experimental Procedures:
Infra-red spectra were recorded using a Bruker Tensor 27 FT-IR (Bruker OpticsGmbH, Ettlingen, Germany) spectrometer with KBr pellets and UV spectra were determined JASCO V-520 UV/VIS spectrophotometer.JEOL NMR spectrometer operating at 500MHz for 1 H-NMR spectra were obtained in CDOD or CDCl 3 using TMS as an internal standard with the chemical shifts expressed in δ and coupling constants (J) in Hertz.El-MS (VG-ZAB-H F), X-mass (158.64,800.00) (VGA analytical, Inc.).Silica gel column chromatography (CC) was performed on silica gel 60 (E.Merck, Darmstadt, Germany).TLC was carried out on pre-coated silica gel 60 F 254 (Merck) plates.Developed chromatograms were visualized by spray with 1% vanillin/H 2 SO 4 , followed by heating at 100 o C for 3 min.TLC plates were developed with solvent systems: A (EtOAc:Hexane, 1:1, v:v) or B (CHCl 3 :MeOH, 8.5:1.5, v:v).2,5-dihydroxy cinnamic acid (1), used in this study was kindly given as a gift from Prof. Mohammed Hosny, Al-Azhar University, Faculty of Pharmacy, Pharmacognosy Department, Cairo, Egypt.The purity of the substrates was confirmed by TLC and 1 H-NMR.

Analytical-Scale Biotransformation of 2,5dihydroxycinnamic acid (1):
A two-stage fermentation protocol (Hosny, and Rosazza 1999), was used for analytical and preparative scale formation of (1) metabolites.For screening experiments, solid cultures kept on either potato dextrose agar or sabaraud maltose agar of the following organisms was used: Cunninghamella elegans (RCMB 012001), Cunninghamella echinulata (RCMB 012002), Mucorrouxii (RCMB 015004), Absidia corymbifera (RCMB 051002), Penicillium notatum (RCMB 001023), Penicillium aurantiogriseum (RCMB), Candida albicans (RCMB 005004), Rhodotorula glutins (RCMB 028001), Rhizopus oryzae (RCMB 014002), Aspergillus niger (RCMB002007(5)001002(2) and Aspergillus flavus RCMB002002(3).Each culture was used separately to inoculate 100 ml flasks containing one fifth of their volume of the following medium: 5% (w/v) soybean meal, 0.5% yeast extract, 0.5% NaCl, 0.5% K 2 HPO 4 , and 2% dextrose per 1 L of distilled water, adjusted to pH 7.0 with 6 N HCl, was autoclaved at 121 o C for 15 min.Analytical incubations were conducted in 25 mL of sterile medium held in 125 mL stainless steel-capped Delong culture flasks that were incubated for 72 h at 28°C on a rotary shaker operating at 250 rpm.A 10% inoculum derived from 72 h old stage I cultures was used to initiate stage II cultures, which were incubated for 24 h more before receiving 5 mg of 1 in Tween 80-H 2 O (0.5 mL; 1∶3 V/V), and incubations was continued.Substrate controls consisted of sterile medium and substrate incubated under the same conditions but without microorganism.Samples of 3 mL were withdrawn for analysis at 24, 48, 72, and 144 h after substrate addition, extracted with 1 mL of EtOAc: n-BuOH (9:1).The organic layer was separated from aqueous medium by centrifugation at 1,200 x g in a desktop centrifuge and 60 L samples were spotted onto TLC plate developed with solvent system using CH 2 Cl 2 : MeOH : CH 3 COOH (15: 0.5: 0.3 ml) as developing solvents.The developed chromatograms were visualized by spraying with vanillin/H 2 SO 4 , followed by heating with a heating gun until maximum development of the spots color.On the basis of screening experiments, two metabolites were reproducibly formed by Rhizopus oryzae (RCMB 014002), and one metabolite formed by Aspergillus niger (RCMB002007(5)001002(2) after 72 h.
The EtOAc extract from scaling up the reaction of (1) with Aspergillus niger (340 mg) was chromatographed on a silica gel column (1.5 x 100 cm, 100 g) eluted with mixtures of CH 2 Cl 2 and MeOH.Fractions eluted with 3% MeOH in CH 2 Cl 2 (52 mg) contained one major spot (R f 0.55, CH 2 Cl 2 : MeOH: Acetic acid (15: 0.5: 0.3 ml) and several minors which are more polar.Re-chromatography on silica gel column using the same solvent system afforded metabolite Cin-AM-3 (16 mg).
Animals: Male Westar rats (250-300 g) were handled according to international regulations.They were allowed to take standard laboratory diet and water ad libitum, and the animals were maintained at 24 °C with 12 h light period.

A. DPPH radical scavenging activity
The ability of the extracts to scavenge free radicals was determined according to the method of De La Torre Boronat and Lopez Tamames (1997).In a 96-well plate, 10 μL of each sample or standard dissolved in ethanol (100 μg/mL) was added to 190 μL of 316 μM/mL DPPH solution.A blank was prepared using ethanol.After incubation at 30 °C for 30 min, the absorbance of each solution was measured at 517 nm.DL-α-tocopherol and BHT were used as positive controls.The scavenging activity of the samples was calculated as a percentage of free radical inhibition according to the formula:

% inhibition = A blank -A sample × 100 A blank
Where A blank is the absorbance of the blank at zero time and A sample is the absorbance of the sample after 30 min.All experiments were carried out in triplicate.Male Westar rats (250-300 g) were sacrificed, and the rat tissues (brain, heart and liver: 0.3-0.5 g) were rapidly removed and homogenized in 10 volumes of 15 mM Krebs buffer.Homogenates were centrifuged at 3000 x for 10 minutes at 4 °C to give supernatants containing (1.2 mg of protein/ ml; brain), (1.7 mg of protein/ ml; heart) and (2.5 mg of protein/ ml; liver) using Coomassie plus protein assay reagent and albumin standard as determined by the Bradford method (1976).During aerobic incubation of the tissue homogenates, MDA released reacts with thiobarbituric acid (TBA) to give a pink color.The capability of the samples to inhibit MDA formation is used as a measure of their antioxidant activity.The pink color complex of thiobarbituric acid reacting substance (TBARS) is measured at 532 nm for the test samples and positive standards (DL-α-tocopherol and BHT) (200 μg/mL), as well as, 2,5dihydroxycinnamic acid (1) and their metabolites (Cin-M-1-M-3, 100 μg/mL).The results were expressed as nano-moles of MDA equivalents per milligram of protein of rat (brain, heart and liver) homogenates.All measurements were done in triplicate.The capability to inhibit MDA formation was calculated using the following equation:

Statistical Analysis
All data were expressed as mean ± SE.Student's t-test [33] was applied for detecting the significance of difference between each sample; P < 0.05 was taken as the level of significance.

A-Structure Elucidation of isolated metabolites
Of 11 microorganisms screened for their abilities to catalyze the bioconversion of 2,5dihydroxy cinnamic acid (1), Rhizopus oryzae (RCMB 014002) and Aspergillus niger (RCMB002007 (5)001002(2) reproducibly formed after 72 h of incubation two major metabolites [Cin-RM-1 and Cin-RM-2] from Rhizopus oryzae and [Cin-AM-3] from Aspergillus niger in good yield.None of the observed metabolites were formed in control cultures or in media containing no microorganisms but incubated under the same conditions.Following solvent extraction and column chromatographic purification, samples of metabolites were subjected to spectral analysis.Spectra (UV, IR, NMR, and mass spectrometry) for isolated metabolites were established by comparing their spectral data to those given in the literature.-RM-1): was obtained as a colorless oily product.It gave a bluish green color with vanillin/sulfuric acid.The UV spectrum recorded in MeOH showed absorption maxima attributable to a conjugated aromatic ring at  max 267 and 292 nm.The IR spectrum showed characteristic bands accounting for a phenolic group at 3350cm -1 (-OH), 3030cm -1 (=C-H), 1515, 1590cm -1 (aromatic C=C), 2870cm -1 (CH), 2980,1550cm -1 (C=C), 1430, 1390, 1100cm -1 (C-O).Addetionally, in IR spectrum, peak at 1680 cm -1 (C=O) in (1) was disappeared and a broad peak appeared at 3350 cm -1 (OH).The empirical formula was determined by accurate mass measurement as C 9 H 10 O 3 based on the molecular ion base peak atm/z 166 (M) + with 14 mass unit lower than of parent molecule (substrate, 180 mass unit), It also showed fragment ions at m/z 150 (M-OH) + , 137 (M-CH 2 OH + H) + , 122 (M-44 [C 2 H 4 O from side chain) + and 109 (M-58 [C 3 H 6 O, prop-2-en-1-ol] + H) + by EI-MS.
Metabolites (Cin-RM-2): was obtained as a colorless amorphous solid.It gave a blue color with vanillin/sulfuric acid.The UV spectrum recorded in MeOH showed absorption maxima attributable to a conjugated aromatic ring at  max 265 and 298 nm.The IRspectrum displayed characteristic absorption bands at 3330 cm -1 for hydroxyl group.The absorption band near 1595, 1504, 1465 cm -1 is for aromatic C=C stretching vibration and the band at 2875 cm -1 is for C-H bending vibration of aromatic compound.The absorption band at 2980, 1560 cm -1 for -C=C-stretching.The bands at 2820 and 1176-1323 cm -1 showed presence of methoxy group (-C-O stretching frequency) in the molecule.The empirical formula was determined by accurate mass measurement as C 11 H 14 O 4 based on the molecular ion base peak atm/z 210 (M) + with 30 mass unit over that of substrate (180 mass unit) and 44 mass unit over that of metabolite (Cin-RM-1) (166 mass unit).It also showed fragment ion peaks at m/z 195 (M- The 1 H-NMR spectrum of [Metabolite-RM-2], displayed two meta-coupled singlets each for 1H, at  H 6.74 (H-3) and  H 6.98 (H6)in the aromatic region indicating the presence of a tetra-substituted aromatic ring in the molecule.It also displayed a typical signals of resonances at H 6.32 (1H, d, J =16.5 Hz, H-7), 6.83 (1H, ddd, J = 16.5, 7.8, 5.8 Hz, H-8), 4.19 (1H, dd, J = 14.2, 4.7 Hz, H9a) and 3.70 (1H, dd, J = 14.2, 4.7 Hz, H9b).Coupling constant 16.5 Hz between olefinic protons H-7 and H-8 suggests clearly them being intrans geometry to each other's.Additionally, two sharp proton signals for two OCH 3 groups at  H 3.77 (3H, s, 4-OCH 3 ) and 3.72(3H, s, 5-OCH 3 ) were also observed.
The molecular formula was determined as C 10 H 10 O 4 on the basis of the molecular ion peaks observed at m/z 194 (M) + , 177 (M-H 2 O + H) + , 163 (M-OCH 3 + H) + , 148 (M-OCH 3 -OH) + , 134 (M-carboxylic acid -OH) + , 121 (M-OCH 3 -carboxylic acid + H) + , 107 (M-OCH 3 -OH -carboxylic acid) + by EI-MS.This formula was in good agreement with the 13 C-NMR spectrum, which showed 10 signals containing five methines, one methoxyl and four quaternary carbons.The molecular ion peak atm/z 194 (M) + with 14 mass unit over that of substrate and the 1 H-and 13 C-NMR spectra of [Cin-AM-3]revealed that this metabolite had the same carbon skeleton as [substrate], the only difference [Cin-AM-3] possessed one methoxyl group versus a hydroxyl group in [substrate].
The 1 H-and 13 C-NMR spectra of [Cin-AM-3], exhibited three aromatic proton and carbonsignals as an ABX spin-systemat  H 6.85 (1H, d, J = 9.0 Hz, H-3,  C 117.98), 6.81 (1H, d, J = 2.8 Hz, H-4,  C 113.72) and 6.97 (1H, dd, J = 2.8, 9.0 Hz, H-6,  C 112.60), indicating the presence of a trisubstituted aromatic ring.Two olefinic protons and carbons as an AB spin-system at  H 7.92 (1H, d, J = 16.2Hz, H-7,  C 140.07) and 6.41 (1H, d, J = 16.2Hz, H-8,  C 118.29), the large value of coupling constant (16.2 Hz) indicated the presence of transdisubstituted ethylene moiety in the molecule.The downfield signal for three hydrogens at  H 3.85, C 56.50) indicates that methyl group is attached to electron withdrawing oxygen atom of OCH 3 group at C-5.The 13 C chemical shifts of a carbon at  C 169.78 indicated the presence of carboxylic functional group in the molecule.The upfield chemical shifts of one of the ethyleniccarbon (C-8) and a quaternary carbon at  C 123.56 (C-1) indicated that the carboxylic group is located at C-8 position.The 13 C-chemical shifts of carbon atoms at  C 152.09 (C-5) versus 150.39 (C-5) in2,5-dihydroxy cinnamic acid, indicated that the methoxyl group was attached at C-5 position.
In conclusion, the results obtained with isolated metabolites have indicated that scavenging effects is dependent on their chemical structure and thought to be due to their hydrogen donating activity.In general phenolic OH is known as scavenger of free radicals and it consequently exhibits antioxidative activity (Hosny, and Rosazza, 2002).Especially, in regards to substitution on the phenyl ring several studies have reported that the existence of an electron donating group such as methoxyl substitution as with several metabolites obtained in this study enhances antioxidant effectiveness (Hosny, and Rosazza, 2002), claimed that the phenolic group is essential for the free-radical-scavenging activity and that the presence of the methoxy group further increased the activity.Lipid peroxidation is a free radical mediated process which has been implicated in a variety of disease states.It involves the formation and propagation of lipid radicals, uptake of oxygen, a re-arrangement of the double and unsaturated lipids that results in a variety of degraded products (e.g., alkenes, malondialdehyde (MDA), lipid hydroperoxides and conjugated dienes) that eventually causes destruction of membrane lipids.Thus lipid peroxidation and conjugated diene measurement plays important role along with MDA assay (Halliwell and Chirico, 1993).The increased peroxidation can result in changes in cellular metabolism of the hepatic and extra-hepatic tissues.Increase in accumulation of MDA, conjugated diene and hydro-peroxides in cells can result in cellular dehydration and whole cell deformity and death (Halliwell and Chirico, 1993).Itis well known that defense mechanism in liver, kidney, heart, brain and lungs are prone to oxidative damage.Alteration of fatty acid composition by increased lipid levels may contribute for lowering the resistance of tissues and higher rate of oxidative stress.

B.2-Ferrous sulphate-H2O2-stimulated lipid peroxidation in rat tissue homogenate.
There is good evidence that superoxidedismutase (SOD) and catalase are enzymes that scavenge free radicals during lipid peroxidation.The free radical chain reaction is widely accepted as a common mechanism of lipid peroxidation.Radical scavengers may directly react with and quench peroxide radicals to terminate the peroxidation chain reaction and improve the quality and stability of food products (Halliwell and Chirico, 1993).
A control experiment indicated that substrates and isolated fungal metabolites did not effect the measurement of TBARS because the absorbance at 532 nm was not effected by adding different substrates and isolated fungal metabolites to the rat tissue homogenate that already have been oxidatively modified because omission of rat homogenate from the reaction mixture abolished chromogen formation.D, L--tochopherol and BHT also inhibited this Fe 2+ -induced lipid peroxidation with IC 50 values in the range of (28.23 -35.10%), (22.05 -38.70%) and (33.15 -46.18%) in heart, brain and liver rat tissue homogenates, respectively.However, as shown in (Table 2), adding 200-500 g/mL of substrateson rat tissue homogenates (brain, heart and liver), reduce MDA formation in the presence of Fe 2+ -H 2 O 2 with IC 50 values (22.10%,10.20% and 32.25%)in heart, brain and liver rat tissue homogenatesrespectively, indicating lower anti-lipid peroxidation activities of substrates.The results obtained with the fungal metabolites; Cin-RM-1, Cin-RM-2 and Cin-AM-3 showedlow radical quenching for all tissue homogenate than those of such typical antioxidants D, L, -tocopherolbut its more than BHT (Table 2).
Our results clearly showed that lipid peroxidation in rat tissue homogenates (brain, heart and liver) induced by ferrous ion/H 2 O 2 as measured by MDA formation, was slightly inhibited by substrate and their metabolites.Since D,L,-tocopherol is thought to be associated with lipid-rich membranes, it is anti-oxidative is highly effective in protecting membranes against lipid peroxidation, as peroxyl and alkoxyl radicals.The data obtained from the present study indicates that the isolated metabolites hasanti-lipid peroxidative character with similar reaction mechanisms to those of D,L,-tocopherol and BHT for rat liver tissue homogenates.

Table ( 1
) Effects of substrates, metabolites and positive controls on the in vitro Free Radical Generation.

Table ( 2
) Inhibition Effect of substrates, metabolites and positive controls on FeSO 4 -H 2 O 2 Induced Lipid Peroxidation (MDA production)in Rat Tissue Homogenate.Values are presented as mean + SE of 3-test sample observation.P< 0.05 for all values. *