Gut Metabolism of Furanocoumarins: Proposed Function of Co O-Methyltransferase.

Gut metabolism of natural products is of great interest due to the altered biological activity of the metabolites. To study the gut metabolism of the dietary furanocoumarins, the biotransformation of Angelica dahurica was studied with human gut microbiota. The major components of Avenula dahurica, including xanthotoxin (1), bergapten (2), imperatorin (3), isoimperatorin (4), oxypeucedanin (5), and byakangelicol (6), were all metabolized by the human fecal sample, and each furanocoumarin was also biotransformed by Blautia sp. MRG-PMF1 responsible for intestinal O-demethylation. Oxypeucedanin (5) and byakangelicol (6) were converted to oxypeucedanin hydrate (9) and desmethylbyakangelicin (12), respectively. The gut microbial conversion of xanthotoxin (1) and bergapten (2) with the MRG-PMF1 strain resulted in the production of xanthotoxol (7) and bergaptol (8), respectively, due to the methyl aryl ether cleavage by O-methyltransferase. Unexpectedly, the biotransformation of prenylated furanocoumarins, imperatorin (3), and isoimperatorin (4) resulted in the corresponding deprenylated furanocoumarins of xanthotoxol (7) and bergaptol (8), respectively. The cleavage of the prenyl aryl ether group by gut microbiota was unprecedented metabolism. Our data presented the first deprenylation of prenylated natural products, presumably by the anaerobic prenyl aryl ether cleavage reaction catalyzed by Co-corrinoid enzyme.

Introduction

Furanocoumarins are bioactive polyphenolic compounds common in various vegetables and fruits, such as citrus, parsnips, parsley, celery, figs, carrots, and grapefruit. The bioactivity of dietary furanocoumarins, including anti-inflammatory, anticancer, and neuroprotective effects, are well documented.[1] However, furanocoumarins can be also phototoxic and photomutagenic after oral intake, differentiating them from other polyphenolics.[2] For example, furanocoumarins in bergamot, lime, and lemon oils can cause photodermatitis upon subsequent exposure to UV light after ingestion.[3] It is widely believed that furanocoumarins play an important role in plant defense mechanisms against herbivores. In turn, some insects also developed an adaptation mechanism through the enzymatic metabolism of the toxic furanocoumarins.[4] Regardless of the known phototoxicity, vegetables and fruits rich in furanocoumarins are still widely consumed, whereas human toxicity from their ingestion is not widespread.

Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. is a perennial plant belonging to the family of APIACEAE. The rhizome has traditionally been used for the treatment of headache, stuffy nose, toothache, and acne in Asia.[5] Various biological activities of the crude extract and furanocoumarin isolated from Avenula dahurica have been reported. For example, isoimperatorin (4) is suggested as the active compound of A. dahurica, which is effective against animal influenza and coronavirus infection.[6] It also inhibits airway inflammation and mucus hypersecretion and exhibits synergic anti-mycobacterial effects in combination with drugs.[7,8] Imperatorin (3) has also been reported to exhibit anticancer,[9] antiviral,[10] antilipogenic,[11] and antihypertensive effects (Figure ).[12]

Figure 1

Molecular structures of dietary furanocoumarins isolated from A. dahurica. The number for each compound is equal to the HPLC peak number.

While little is known about the absorption and distribution of A. dahurica extract, the major furanocoumarins were mainly found in the liver, kidney, and stomach tissues of rat after oral intake.[13] Interestingly, imperatorin (3) was reported to be metabolized to xanthotoxol (7) before urine excretion.[14,15] Xanthotoxol (7) can be more readily removed from the body due to the increased solubility, and it was suggested as a detoxification mechanism. However, the detailed metabolic pathway from imperatorin (3) to xanthotoxol (7) still remains unknown. From the extensive HPLC-MS2 analysis of rat urine and plasma, deprenylation of isoimperatorin (4), resulting in bergaptol (8), was also reported without identification of the metabolic pathway.[16]

Since the study of in vitro gut metabolism can have significant affects on the human gut metabolism,[17−20] we hypothesized that an investigation of the gut metabolism of dietary furanocoumarins could play a key role in the rightful evaluation of their biological activity. Here, we have investigated the gut metabolism of A. dahurica with human fecal samples and Blautia sp. MRG-PMF1.

The Co O-methyltransferase expressed by Blautia sp. MRG-PMF1 catalyzes the unique methyl aryl ether cleavage by utilizing the Co-corrinoid cofactor in the absence of oxygen.[18,21] The nucleophilicity of the reactive Co(I) species formed during the catalysis is strong enough to displace the methyl ether and known as supernucleophile.[22] Chemically, cleavage of prenyl aryl ether functional group, also found in imperatorin (3) and isoimperatorin (4), could follow the same nucleophilic substitution mechanism. Therefore, the metabolism of these two O-prenylated furanocoumarins by Blautia sp. MRG-PMF1 was also tested. The gut metabolism of furanocoumarins has never been studied at the molecular level, to the best of our knowledge.

Results and Discussion

Biotransformation of A. dahurica by the Mixed Cell Culture Prepared from Human Fecal Sample

The supercritical CO2 extract of A. dahurica was found to contain xanthotoxin (1), bergapten (2), imperatorin (3), isoimperatorin (4), oxypeucedanin (5), and byakangelicol (6) (Figure ). The concentration of the major furanocoumarins in the extract followed the order of oxypeucedanin (5) > imperatorin (3) > byakangelicol (6) > isoimperatorin (4) (see the Supporting Information). While imperatorin (3) and isoimperatorin (4) were generally recognized as major bioactive compounds of A. dahurica, it was reported that different compositions of furanocoumarins were found depending on the varieties, cultivation area, and cultivars of A. dahurica. For example, the rhizome of A. dahurica grown in Korea was rich in oxypeucedanin (5) and byakangelicol (6),[23] which was also confirmed by our study.

From the HPLC analysis of the biotransformation product obtained from the reaction between crude A. dahurica extract and fecal microbiota under anaerobic condition (Figure ), it was observed that byakangelicol (6, retention time; 16.8 min), oxypeucedanin (5, 17.0 min), and imperatorin (3, 24.0 min) were completely metabolized by the mixed cell culture within 3 days. Biotransformation of isoimperatorin (4, 28.5 min) was relatively slower in the mixed cell culture compared to the other furanocoumarins, and the HPLC peaks were observed from the culture medium, even after 7 days. Because the major furanocoumarins were metabolized by gut bacteria, biotransformation of each furanocoumarin was further investigated.

Figure 2

HPLC analysis (250 nm) of the A. dahurica extracts biotransformed by the human fecal sample. Three furanocoumarins of byakangelicol (6), oxypeucedanin (5), and imperatorin (3) at the retention times of 16.8, 17.0, and 24.0 min, respectively, in the chromatograms were completely metabolized, whereas isoimperatorin (4) at the retention time of 28.5 min was partially metabolized even in 7 days.

Biotransformation of A. dahurica Furanocoumarins by Blautia sp. MRG-PMF1

From the previous study,[18]Blautia sp. MRG-PMF1 strain was reported to metabolize polymethoxyflavones to the corresponding flavones by the cleavage of methyl aryl ether groups. The unique methyl ether cleavage is achieved by the action of supernucleophilic Co(I) reactive species generated in the Co-corrinoid O-methyltransferase enzyme.[24] Since three A. dahurica furanocoumarins, compounds 1, 2, and 6 in Figure , contain methyl aryl ether functional group, each furanocoumarin was reacted to Blautia sp. MRG-PMF1 strain to examine whether the strain is responsible for the conversion.

First, xanthotoxin (1) and bergapten (2) were reacted with MRG-PMF1 for 24 h, and the bioconversion products in the media were analyzed by HPLC. Xanthotoxin (1) at the retention time of 12.1 min was completely metabolized and the new peak at the retention time of 8.7 min was found. This peak was identified as xanthotoxol (7), from the comparison of its retention time and UV spectrum with those of the reference compound (Figure A). Likewise, bergaptol (8) at the retention time of 9.9 min was the only metabolite produced from the biotransformation of bergapten (2) (Figure B). Both metabolites, xanthotoxol (7) and bergaptol (8), were the demethylated metabolites from the corresponding furanocoumarins. The methyl aryl ether cleavage reaction by Blautia sp. MRG-PMF1 strain is now the well-established gut metabolism of polyphenolics.[19] It is also evident that human intestinal bacterium MRG-PMF1 can metabolize the methoxyfuranocoumarins in A. dahurica.

Figure 3

HPLC analysis of xanthotoxin (1, A) and bergapten (2, B) biotransformation by the MRG-PMF1 strain. A; Xanthotoxin (1) at the retention time of 12.1 min was metabolized to xanthotoxol (7) for which the peak is found at the retention time of 8.7 min. B; Bergapten (2) at the retention time of 13.8 min was completely metabolized to bergaptol (8) at the retention time of 9.9 min in 24 h.

Xanthotoxin (1) and bergapten (2) were proposed as the major compounds resulting in phototoxic dermatitis among the celery pickers.[25] It is suggested from our study that the rapid metabolism of these phototoxic furanocoumarins by gut bacteria, such as MRG-PMF1 strain, could prevent the symptom.

When biotransformation of oxypeucedanin (5) by MRG-PMF1 was performed, oxypeucedanin hydrate (9) was produced as a product (Figure C). However, the production of oxypeucedanin hydrate (9), though in traces, was also observed from the control experiment, which was performed in the absence of MRG-PMF1 (see the Supporting Information). It is well known that epoxide ring is unstable in the acidic aqueous environment. Therefore, it was concluded that the conversion of oxypeucedanin (5) to oxypeucedanin hydrate (9) by MRG-PMF1 strain was not specifically by the action of the Co O-methyltransferase but by other unknown enzymes in MRG-PMF1. In fact, the same product, oxypeucedanin hydrate (9), was also produced by Lactococcus sp. MRG-IFC1, which does not express Co O-methyltransferase.[26]

Figure 4

Biotransformation of furanocoumarins by Blautia sp. MRG-PMF1. Xanthotoxin (1) and imperatorin (3) were metabolized to xanthotoxol (7) (A), and bergapten (2) and isoimperatorin (4) were metabolized to bergaptol (8) (B), respectively. Oxypeucedanin (5) was metabolized to oxypeucedanin hydrate (9) (C), but Co O-methyltransferase was not responsible for the biotransformation. Byakangelicol (6) was biotransformed to desmethylbyakangelicin (12) via either desmethylbyakangelicol (10) or byakangelicin (11) by demethylation or hydration reaction, respectively (D). Prenyl aryl ether cleavage reaction by Co O-methyltransferase is achieved by the ally aryl ether cleavage as evident from the conversion of 7-allyloxycoumarin (13) to 7-hydroxycoumarin (14) (E).

Biotransformation of byakangelicol (6) underwent O-demethylation and hydration, resulting in the three metabolites, desmethylbyakangelicol (10), byakangelicin (11), and desmethylbyakangelicin (12) (Figure D). Byakangelicin (11) isolated from A. dahurica was converted to desmethylbyakangelicin (12) in the rat as identified from the urine.[27] The new metabolite desmethylbyakangelicol (10) was proposed by HPLC–DAD-MS analysis.

Finally, biotransformation of imperatorin (3) and isoimperatorin (4) by MRG-PMF1 was performed. Substrates 3 and 4 have a prenyl aryl ether functional group instead of a methyl aryl ether functional group as shown in Figures and 4. Until now, it is known that the prenyl group in the plant-derived polyphenolic compounds can undergo only hepatic phase I metabolism, which involves Cyt P-450 type enzymes.[15] Anaerobic gut metabolism of the prenyl functional group has never been reported. To our surprise, imperatorin (3, 23.8 min) and isoimperatorin (4, 28.6 min) were biotransformed by MRG-PMF1, and xanthotoxol (7, 8.6 min) and bergaptol (8, 9.9 min) were identified as metabolites, respectively (Figure ). Although the formation of the products was slower than that of xanthotoxin (1) and bergapten (2), it was clear that both products accumulated over the reaction time. The production of deprenylated metabolites xanthotoxol (7) and bergaptol (8) can be achieved only by the cleavage of the prenyl aryl ether functional group. To the best of our knowledge, this transformation is unprecedented among the known gut metabolism. Such a reaction was only reported from the chemical transformations that utilize strong Lewis acids.[28,29] Recently, Giedyk et al. reported that the photoactivated vitamin B12, corresponding to the reduced Co(I) reactive species, can cleave the allyl aryl ether functional groups.[30]

Figure 5

HPLC analysis of imperatorin (3, A) and isoimperatorin (4, B) biotransformation by the MRG-PMF1 strain.

To confirm the prenyl aryl ether group cleavage by MRG-PMF1 Co O-methyltransferase, 7-allyloxycoumarin (13) was biotransformed by MRG-PMF1 (Figure E). The formation of 7-hydroxycoumarin (14) was confirmed and the reaction was much faster than that of imperatorin (3) and isoimperatorin (4) (see the Supporting Information). Therefore, it was concluded that the deprenylation of imperatorin (3) and isoimperatorin (4) resulted from the cleavage of allyl aryl ether functional group by Blautia sp. MRG-PMF1, which follows the same reaction mechanism as methyl aryl ether cleavage. Although we cannot completely exclude the deprenylation by an unknown enzyme, the biotransformation of the prenylated furanocoumarins (3 and 4) provides evidence that Co-corrinoid O-methyltransferase could catalyze the same reactions of methyl/allyl aryl ether cleavage as explored by the model complex.[30]

Gut Metabolism of Furanocoumarins

Gut metabolism of bioactive compounds has become more important than ever, since it affects human health in many ways.[31] We have been investigating the biotransformation of plant-derived polyphenolic compounds in the human gut microbiota to achieve molecular-level information on gut metabolism. The identification of the specific metabolites can be also applied to the exploration of new bioactive functional compounds. For example, isolation and biotransformation study of the S-equol-producing bacteria helped us to elucidate the gut metabolism of soybean isoflavones.[32]

When gut metabolism of polymethoxyflavones, common in citrus fruits and herbs, was investigated in the human intestine, Blautia sp. MRG-PMF1 has been found to catalyze the cleavage of the methyl aryl ether functional group.[21] The biotransformation by MRG-PMF1 is unique in that it shows activity toward a broad spectrum of substrates. It catalyzes the methyl ether cleavage of pentamethoxyflavone, curcumin, and icariin to quercetin, bisdemethoxycurcumin, and desmethylicariin, respectively.[17,18,20] The cleavage of the methyl aryl ether linkage is due to the Co O-methyltransferase expressed by the gut bacterium. The Co-corrinoid cofactor generates supernucleophilic Co(I) species, which attacks the methyl carbon to produce demethylated aryl ether (Figure A).[22]

Figure 6

Proposed mechanism of methyl aryl ether cleavage (A) and prenyl aryl ether cleavage (B) by Co-corrinoid O-methyltransferase. The Co(I)-corrinoid reactive species attacks allyl carbon to result in the ether bond cleavage as it catalyzes the methyl ether cleavage.

The recent growing interest in dietary furanocoumarins has attracted our attention. Especially, A. dahurica is an important economic crop in the region of Asia, including Korea. The major bioactive furanocoumarin in A. dahurica, such as imperatorin (3) and isoimperatorin (4), exhibits health-promoting effects, as well as toxicity. While the toxic effects of these dietary furanocoumarins are not general and often considered as allergic, gut metabolism after oral administration has never been investigated. When the extract of A. dahurica was reacted with the mixed cell culture prepared from the human fecal sample, metabolism of A. dahurica furanocoumarins was observed by HPLC analysis (Figure ). It was also found that the MRG-PMF1 strain was responsible for the conversion, and each furanocoumarin was biotransformed with the MRG-PMF1 strain (Figure ). Xanthotoxin (1) and bergapten (2) were converted to xanthotoxol (7) and bergaptol (8), respectively, due to the methyl aryl ether cleavage by MRG-PMF1 Co O-methyltransferase (Figure ). The biotransformation of imperatorin (3) and isoimperatorin (4) unexpectedly resulted in the production of xanthotoxol (7) and bergaptol (8), which suggested the prenyl aryl ether cleavage by MRG-PMF1.

The Co-corrinoid enzyme that catalyzed the prenyl group cleavage appears to follow the same reaction mechanism of methyl aryl cleavage reaction as shown in Figure . In the case of the methyl aryl ether cleavage reaction, the Co(I) reactive species attack methyl carbon to break the methyl C–O σ-bond. The electrons residing on the phenolic oxygen is later protonated to produce the aryl alcohol product (Figure A). Similarly, the nucleophilic attack by Co(I) could push the prenyl π-electron to break the allylic C–O σ-bond (Figure B). After the demethylation of methyl aryl ether, the Co(I) species form Co(III)–CH3, which is utilized for the biochemical C1-metabolism involving SAM and THF.

Meanwhile, xanthotoxol (7) in the rat urine was reported as a metabolite of imperatorin (3).[33] The authors proposed that the production of xanthotoxol (7) was due to the phase I metabolism, which involves the hydrolysis of the prenyl side chain of imperatorin (3). However, it is unlikely that Cyt P-450 type heme catalyzes the hydrolysis of prenyl aryl ether. Our data suggests that instead of phase I metabolism in the liver, gut metabolism is probably responsible for the formation of xanthotoxol (7). As shown from the liver microsomal metabolism of A. dahurica extracts, the hydroxylated and epoxidized metabolites were produced by Cyt P-450 type oxygenase.[34] Therefore, it is reasonable to suggest that some parts of furanocoumarin metabolism, such as demethylation and deprenylation, occur in the intestine before the absorption to the bloodstream.

In summary, the gut metabolism of furanocoumarins was elucidated in this report. We have reported that furanocoumarins in A. dahurica can be metabolized by human gut microbiota to produce demethylated and deprenylated metabolites. The results may be relevant to the detoxification pathway of the toxic dietary furanocoumarins. Besides, we have reported the new anaerobic gut metabolism of prenyl aryl ether cleavage by Co-corrinoid O-methyltransferase.

Experimental Section

Chemicals and Bacterium

Dried powder of Angelica dahurica (voucher deposit number; PBNV201812310001) was purchased from Phytobean AC Co. Ltd. (Yecheon-gun, Korea), and the extract was obtained from supercritical CO2 extraction (see the Supporting Information for the detailed experimental procedure). Reference furanocoumarins of xanthotoxin (1), bergapten (2), and imperatorin (3) were purchased from TCI (Tokyo, Japan). Isoimperatorin (4) and oxypeucedanin (5) were from the Korean Ministry of Food and Drug Safety (Ochang, Korea). Xanthotoxol (7) and bergaptol (8) were purchased from Alfa Aesar (Haverhill, MA). HPLC-grade MeOH, water, and MeCN were purchased from Burdick & Jackson Laboratories, Inc. (Muskegon, MI). EtOAc (99.5%) and DMF (99.5%) were purchased from Samchun Pure Chemicals (Pyeongtaek-si, Korea). Acetic acid for HPLC was purchased from Sigma-Aldrich (Buchs, Switzerland), while formic acid for mass spectrometry (∼98%) was purchased from Fluka (Darmstadt, Germany). GAM was from Nissui Pharmaceutical Co. (Tokyo, Japan). The GAM broth was prepared following the manufacturer’s instructions, and GAM plates were prepared by adding 1.5% (w/v) agar in GAM broth.

The human intestinal bacterium, Blautia sp. MRG-PMF1, isolated from our laboratory (GenBank accession number: KJ078647), was used in these experiments.[20] Bacterial growth and substrate conversion experiments were performed under anaerobic conditions, according to published methods.[17,18]

Byakangelicol (6) was isolated from the A. dahurica extract from the silica gel column chromatography using hexanes/ethyl acetate eluent. white powder; 1H NMR (600 MHz, CHCl3-d): δ 8.13 (1H, d, J = 9.6 Hz, H-4), 7.63 (1H, d, J = 2.4 Hz, H-2′), 7.01 (1H, d, J = 2.4 Hz, H-3′), 6.29 (1H, d, J = 9.6 Hz, H-3), 4.44 (2H, d, J = 5.4 Hz, H-1″), 4.19 (3H, s, 5-OCH3), 3.31 (1H, t, J = 5.4 Hz, H-2″) 1.34 (3H, s, H-4″), 1.26 (3H, s, H-5″).

HPLC Analysis

A Dionex UltiMate 3000 UHPLC (Thermo Fisher Scientific) with a PDA detector, equipped with a C18 Kinetex column (2.1 × 100 nm, 1.7 μm, Thermo Scientific, Waltham, MA) was used for HPLC analysis. Program setup, data collection, and analysis were conducted using Chromeleon Chromatography Data System (CDS) software version 6.80 (Thermo Fisher Scientific). The detector was set to record at 250 nm for furanocoumarin biotransformation products analysis, simultaneously with UV spectrum monitoring in the range of 190–380 nm. The injection volume was 1 μL, the flow rate was 0.2 mL/min, and the column temperature was 25 °C. HPLC eluents consist of 0.1% acetic acid (v/v) in water (A) and MeCN (B), and a multistep gradient program was employed.

For the analysis of the A. dahurica extract and biotransformation products of furanocoumarins, solution B was started at 10%, increased to 30% for 1 min, to 40% for 11 min, to 50% for 15 min, to 60% for 3 min, to 80% for 2 min, to 90% for 1 min, and held for 7 min.

HPLC-MS Analysis of Metabolites

The metabolites derived from the biotransformation of the furanocoumarins were analyzed by a Dionex Ultimate 3000 UHPLC (Thermo Fisher Scientific) equipped with a Hypersil GOLD column (3 μm particle size, 100 × 2.1 mm i.d., Thermo Fisher Scientific) and a DAD. A Thermo Fisher Scientific LCQ fleet instrument (Thermo Scientific, Waltham, MA) was coupled with the HPLC system for electrospray ionization mass spectrometry (ESI-MS) analysis. Program setup, data collection, and analysis were conducted using Xcalibur software (Thermo Fisher Scientific). The mobile phase consisted of 0.1% (v/v) formic acid in water (A) and 0.1% formic acid (v/v) in MeCN (B). Multistep gradient for HPLC-UV-ESI/MS analysis was programmed, starting at 20% B and held for 1 min, increased to 40% B for 4 min, to 50% B for 5 min, to 70% B for 5 min, and to 80% B for 5 min. The injection volume of the analyte was set at 2 μL. Helium was used as collision gas, while nitrogen was used as both sheath and auxiliary gas. The analysis was performed in positive-ion detection mode and the conditions were adjusted as follows: ion spray voltage, 5 kV; capillary temperature, 275 °C; capillary voltage, 19 V; tube lens offset, 90 V. Both full-scan and multistage mass spectrometry (MS) analysis mode were operated. The full-scan mode was programmed to perform scanning in the range of m/z 150–500. For MS analysis, a data-dependent program was used so that the most abundant ions in each scan were selected and subjected to MS analysis (up to n = 3), with a collision energy of 35 eV.

Biotransformation of the Angelica dahurica Extract

The experimental protocol was evaluated and approved by the Institutional Review Board of Chung-Ang University (Approval Number: 1041078–201502-BR-029–01).

All of the experimental procedures for the biotransformation were performed under anaerobic conditions (CO2 5%, H2 10%, N2 85%) at 35 °C, except for the metabolite analysis. A fresh fecal sample from a healthy volunteer was taken in a sterilized tube containing GAM broth topped with mineral oil and placed immediately in the anaerobic chamber. The mixture was kept inside the anaerobic chamber for 2 h. For the preparation of mixed cell cultures, 10 μL of the original mixture was added into 1 mL of sterile GAM broth and further stabilized inside the chamber for 2 h. The extract of A. dahurica was then added to the medium to initiate the biotransformation assay. The final concentration of the A. dahurica extract in the medium was 0.2 mg/mL. After 3 and 7 days, 300 μL of the mixtures was collected and the reaction was stopped by the addition of 1 mL of EtOAc. The mixture was vortexed for 1 min and centrifuged (10 770g) for 10 min. The organic layer (800 μL) was transferred into a new microcentrifuge tube and dried under a vacuum. The dried residue was dissolved in 300 μL of MeOH and filtered through a 0.2 μm PTFE filter (Advantec, Japan) for HPLC analysis.

Biotransformation of Furanocoumarins by Blautia sp. MRG-PMF1

For the biotransformation of each furanocoumarin, the anaerobic culture of Blautia sp. MRG-PMF1 was grown at 35 °C until it reaches OD600 of ∼0.6. To initiate the reaction, 18 μL of the substrate (10 mM in DMF) was added into the bacterial culture (1.8 mL). After the scheduled reaction times, aliquots (300 μL) of the reaction media were transferred to microcentrifuge tubes. The allocated culture was extracted twice with 1 mL of EtOAc, and the supernatant (800 μL) was collected after vortexing (1 min) and centrifugation (10 770g for 10 min). The supernatant was dried under a vacuum; then, the dried residue was dissolved in MeOH (300 μL). The MeOH solution was then filtered through a 0.2 μm PTFE filter (Advantec, Japan) for chromatography analysis.