Analysis of the Metabolites of Isorhamnetin 3-O-Glucoside Produced

Mar 6, 2014 - Isorhamnetin 3-O-glucoside, which is widely contained in many vegetables and rice, is expected to be metabolized by intestinal microbiot...
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Analysis of the Metabolites of Isorhamnetin 3-O-Glucoside Produced by Human Intestinal Flora in Vitro by Applying Ultraperformance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry Le-yue Du, Min Zhao, Jun Xu, Da-wei Qian, Shu Jiang,* Er-xin Shang, Jian-ming Guo, and Jin-ao Duan* Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210023, People’s Republic of China ABSTRACT: Isorhamnetin 3-O-glucoside, which is widely contained in many vegetables and rice, is expected to be metabolized by intestinal microbiota after digestion, which brings about the profile of its pharmacological effect. However, little is known about the interactions between this active ingredient and the intestinal flora. In this study, the preculture bacteria and GAM (general anaerobic medium) broth with isorhamnetin 3-O-glucoside were mixed for 48 h of incubation. Ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry was used for analysis of the metabolites of isorhamnetin 3-Oglucoside in the corresponding supernatants of fermentation. The parent and five metabolites were found and preliminarily identified on the basis of the chromatograms and characteristics of their protonated ions. Four main metabolic pathways, including deglycosylation, demethoxylation, dehydroxylation, and acetylation, were summarized to explain how the metabolites were converted. Acetylated isorhamnetin 3-O-glucoside and kaempferol 3-O-glucoside were detected only in the sample of Escherichia sp. 12, and quercetin existed only in the sample of Escherichia sp. 4. However, the majority of bacteria could metabolize isorhamnetin 3-O-glucoside to its aglycon isorhamnetin, and then isorhamnetin was degraded to kaempferol. The metabolic pathway and the metabolites of isorhamnetin 3-O-glucoside yielded by different isolated human intestinal bacteria were investigated for the first time. The results probably provided useful information for further in vivo metabolism and active mechanism research on isorhamnetin 3-O-glucoside. KEYWORDS: isorhamnetin 3-O-glucoside, human intestinal microbiota, metabolites, UPLC/Q-TOF-MS, metabolic pathway, polyphenols

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by the different isolated human intestinal flora are not well investigated. The intestinal microbiota is composed of 1013−1014 microorganisms, with at least 100 times as many genes as our genome, the microbiome. Its composition is individual-specific and ranges among individuals and also within the same individual during life.11 For many years, it was believed that the main function of the large intestine was the resorption of water and salt and the facilitated disposal of waste materials. However, this task definition was far from complete, as it did not consider the activity of the microbial content of the large intestine. Nowadays, the complex microbial ecosystem in our intestines should be considered as a separate organ within the body, with a metabolic capacity which exceeds that of the liver.12 These bacteria have enormous catalytic and hydrolytic potential due to their excellent enzymatic system. Furthermore, most traditional medicines are administrated orally. Their ingredients will inevitably come into contact with intestinal bacteria and subsequently be metabolized in the intestinal tract before absorption into the blood. Most polyphenolic compounds are present in traditional medicines and plant-derived foods as glycosides that, after ingestion,

INTRODUCTION Epidemiological studies have revealed that a diet rich in plantderived foods has a protective effect on human health. Flavonoids are universally found in plant metabolites and are an integral part of diets for humans and animals.1 So far, many studies have demonstrated that flavonoids possess multibeneficial biological activities such as anticancer, antioxidative, anti-inflammatory, and antimicrobial effects.2 Isorhamnetin 3-O-glucoside, one member of the flavonol compounds, is widely contained in many vegetables and rice such as atsumi-kabu (red turnip, Brassica campestris L.) leaves, onions, Brassica juncea, and Thai black rice.3−6 Additionally, isorhamnetin 3-O-glucoside is an important active ingredient of Traditional Chinese medicine and its compounds such as Ginkgo biloba L. and Yinxingye Capsule, which has been used clinically to treat cough, crown heart disease, hyperlipidemia, and angina for centuries.7 Modern research shows that isorhamnetin 3-O-glucoside has the pharmacological activities of alleviating oxidative stress and preventing liver injury induced by carbon tetrachloride in mice.3,8 Moreover, it may be a leading compound for further study as a new drug for the prevention and treatment of diabetes and its complications.9 Its aglycon isorhamnetin can protect endothelial cells from injury caused by oxidized low-density lipoprotein, decrease blood pressure, and alleviate the damage of ischemia− reperfusion to ventricular myocytes.10 However, the metabolic pathway and metabolites of isorhamnetin 3-O-glucoside yielded © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2489

November 27, 2013 March 6, 2014 March 6, 2014 March 6, 2014 dx.doi.org/10.1021/jf405261a | J. Agric. Food Chem. 2014, 62, 2489−2495

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mobile phase. HPLC was run in gradient mode (solvent system A from 10% to 40% in 0−7.5 min, from 40% to 90% in 7.5−9 min, held at 90% for 9−10 min, and back to 10% in 10−11 min for equilibration of the column) at a flow rate of 0.4 mL/min. The column temperature was set at 35 °C, while the autosampler temperature was maintained at 4 °C. MS Analysis. Selected incubation supernatants from degradation experiments were applied for the parent and metabolit identification by electrospray ionization mass spectrometry (ESI-MS) in negative mode. Leucine-enkephalin was used as the lock mass to generate an [M − H]− ion (m/z 554.2615). The source temperature was 120 °C, while the desolvation temperature was maintained at 350 °C. The cone and capillary voltages were 40 V and 3.0 kV. Meanwhile, the gas (N2) flows of the cone and desolvation were 50 and 600 L/h, respectively. Furthermore, the MSE experiment was carried out to obtain fragment mass information of the parent and metabolites, which was performed in two scan functions (the mass-scan range, scan time, interscan delay, and collision energy of function 1 and function 2 were m/z 100−1000, 0.5 s, 0.02 s, and 6 V and m/z 50−1000, 0.3 s, 0.02 s, and 15−45 V, respectively). All data collected were analyzed by a Waters Metabolynx (version 4.1) program.

undergo hydrolysis before absorption. Depending upon the glycoside moiety, hydrolysis and subsequent absorption can occur in either the small or the large intestine.13 The importance of intestinal bacteria for polyphenol metabolism was highlighted by the fact that germ-free or antibiotic-treated animals no longer form the phenolic acid metabolites (ring-fission products) of (+)-catechin, apigenin, myricetin, hesperidin, naringin, rutin, and 3′,4′,5,7-tetra-O-β-hydroxyethylrutoside.14 Moreover, the pharmacological effects of the polyphenols might be stronger after being degraded by human intestinal bacteria. For instance, the effect on platelet function of anthocyanins and their metabolites was stronger in combination.15 In this study, we attempted to isolate different intestinal bacteria from human feces and carry out research on their abilities and characteristics in the metabolism of isorhamnetin 3O-glucoside. To further clarify the metabolic profile of isorhamnetin 3-O-glucoside, rapid and sensitive ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) with automated data analysis (MetaboLynx) was used to analyze its metabolites.16,17 As far as we know, this was the first research on the metabolites of isorhamnetin 3-O-glucoside by human intestinal flora which could provide a powerful analytical methodology for the identification of multiple metabolites of natural products.

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RESULTS Metabolic Profile of Isorhamnetin 3-O-Glucoside by the Isolated Human Intestinal Bacteria. After optimization of the UPLC and MS conditions, negative ion electrospray tandem mass spectrometry and Metabolynx software were used to obtain a general survey of the isorhamnetin 3-O-glucoside metabolic profile. As a result of our study, the parent and five metabolites of isorhamnetin 3-O-glucoside (Table 1) were

EXPERIMENTAL SECTION

Materials. HPLC-grade acetonitrile was obtained from TEDIA Co. Inc. (United States), along with the anaerobic glovebox. AnaeroPack rectangular jars and AnaeroPack-Anaero were purified by Mitsubishi Gas Chemical Co. Inc. (Japan). Formic acid was purchased from Merck KGaA (Germany). Isorhamnetin 3-O-glucoside was obtained from Shanghai Winherb Medical Science Co. (China). Ultrapure water was purified with an EPED superpurification system (China). Other reagents were of analytical grade. The general anaerobic medium (GAM) used for all fermentation experiments contains 0.3 g of L-cysteine hydrochloride, 0.3 g of sodium thioglycolate, 1.2 g of beef liver extract powder, 2.2 g of beef extract, 2.5 g of KH2PO4, 3.0 g of glucose, 3.0 g of soya peptone, 3.0 g of NaCl, 5.0 g of soluble starch, 5.0 g of yeast extract, 10.0 g of tryptone, 10.0 g of proteose peptone, 13.5 g of digestible serum powder, and 1000 mL distilled water. The pH was adjusted to 7.3. Bacterial Isolation and Culture Conditions. A bacterial suspension of fresh human feces was collected from a healthy female volunteer who had not taken any polyphenols before the experiment. The fecal sample was weighed and homogenized adequately with sterile physiological saline at a ratio of 1:4 (m/v), and then the mixture was centrifuged at 2000g for 10 min. The suspension was used as a human intestinal bacterial mixture. After being diluted in sterile water, the bacterial mixture was spread on GAM agar plates and then incubated in anaerobic gloveboxes under anaerobic conditions at 37 °C for 2 days. As a result of the incubation, about 150 different bacteria colonies were picked up and transferred to GAM agars on an inclined plane. Bacterial Incubation and Sample Preparation. Each bacterium was inoculated into 1.0 mL of GAM broth after being picked up from the GAM agar plate, followed by the anaerobic incubation of cultures at 37 °C for 24 h. A 0.1 mL sample of the preculture bacteria was inoculated into 0.9 mL of GAM broth with 0.1 mM isorhamnetin 3-O-glucoside. Then all samples which had been incubated for 48 h were extracted with ethyl acetate (1 volume of the culture) three times. Finally, the ethyl acetate layer was dried and redissolved in 0.3 mL of methanol.18 Each supernatant of the sample was used for UPLC/MS analysis. UPLC Analysis. The analysis of isorhamnetin 3-O-glucoside and its metabolites was carried out on a Waters ACQUITY UPLC system, equipped with a Syncronis C 18 column for separation. Acetonitrile (solvent system A) and ultrapure water/0.1% formic acid served as the

Table 1. Isorhamnetin 3-O-Glucoside and Its Metabolites (M1−M6) Produced by Human Intestinal Bacteria Using UPLC/Q-TOF-MS

a

no.

tRa/min

m/z, calcd

[M − H]−, found

M1

4.75

478.1111

477.0960

M2 M3 M4 M5

8.58 8.31 6.75 4.56

316.0583 286.0477 302.0427 448.1006

315.0415 285.0374 301.0359 447.0885

M6

6.03

520.1217

519.1136

identification isorhamnetin 3-O-glucoside isorhamnetin kaempferol quercetin kaempferol 3-O-glucoside acetylated isorhamnetin 3-O-glucoside

formula C22H22O12 C16H12O7 C15H10O6 C15H10O7 C21H20O11 C24H24O13

tR = retention time.

detected and identified in the different bacterial samples compared with the blank sample. These metabolites were probably isorhamnetin 3-O-glucoside (M1) catabolites: isorhamnetin (M2), kaempferol (M3), quercetin (M4), kaempferol 3-O-glucoside (M5), and acetylated isorhamnetin 3-O-glucoside (M6). The extracted ion chromatograms (XICs) from the total UPLC/MS chromatograms are shown in Figure 1. What is more, M2 and M3 were found in most of the samples of the isolated human intestinal bacteria, while M5 and M6 were obtained only in the sample of Escherichia sp. 12, and M4 existed only in the sample of Escherichia sp. 4. Identification of Metabolites. The characterization of metabolites in the fermentation supernatants was based on their ion fragmentation in ESI− mode. Parent. Isorhamnetin 3-O-glucoside (M1) was identified by comparing the UPLC retention time (4.75 min), accurate MS at m/z 477.0960 [M − H]−, and MS/MS spectra with those of the authentic standard (Figure 2a). 2490

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Figure 1. UPLC/MS chromatograms of isorhamnetin 3-O-glucoside and its metabolites: (a) strain 4 sample, (b) strain 12 sample, (c) strain 30 sample, (d) strain 46 sample.

Figure 2. Representative MS/MS spectrum: (a) M1 (m/z 477), (b) M2 (m/z 315), (c) M3 (m/z 285), (d) M4 (m/z 301), (e) M5 (m/z 447), (f) M6 (m/z 519).

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Figure 3. Proposed fragmentation pathways of isorhamnetin 3-O-glucoside and its metabolites.

Metabolite M2. According to Figure 2b, the [M − H]− ion of M2 was at m/z 315 with a retention time of 8.58 min, which was 162 Da lower than that of M1 isorhamnetin 3-O-glucoside. The MS/MS spectrum of M2 had product ions similar to those of M1. The high collision energy scan fragment ions were m/z 300,

which was produced by the loss of CH3 from m/z 315, and m/z 255, which was produced by the loss of O from m/z 271. The m/ z 151 ion was produced after retro-Diels−Alder (RDA) cleavage. The product ion at m/z 271 was formed by the elimination of CO2 from m/z 315, and then m/z 271 lost O to form m/z 255 2492

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Figure 4. Possible metabolic pathways of isorhamnetin 3-O-glucoside by the human intestinal bacteria: 1, deglycosylation; 2, demethoxylation; 3, acetylation; 4, dehydroxylation.

300 and m/z 151 (Figure 3). From this data analysis, M6 could be identified as acetylated isorhamnetin 3-O-glucoside. Analysis of the Metabolic Pathway of Isorhamnetin 3O-Glucoside by the Isolated Human Intestinal Microflora. According to our experimental results, the metabolic pathways of isorhamnetin 3-O-glucoside by the isolated human intestinal bacteria were proposed as displayed in Figure 4. Among all the isolated human intestinal bacteria, the majority of the strains, such as Escherichia sp. 12, Enterococcus sp. 30, and Bacillus sp. 46, could degrade isorhamnetin 3-O-glucoside (M1) to large amounts of isorhamnetin (M2) and kaempferol (M3), suggesting that this might be regarded as a common capability of intestinal bacteria. Additionally, Escherichia sp. 4 could also metabolize isorhamnetin 3-O-glucoside to isorhamnetin, which was further degraded to kaempferol and minor but detectable amounts of quercetin (M4). Isorhamnetin 3-O-glucoside was converted to kaempferol 3-O-glucoside (M5) and acetylated isorhamnetin 3-O-glucoside (M6) only by Escherichia sp. 12. Percentage of the Isolated Human Intestinal Bacteria That Yielded Each of the Metabolites. In terms of proportion, the parent, isorhamnetin 3-O-glucoside (M1), was detected in 92% of the isolated human intestinal flora. All of the isolated bacteria could yield the aglycon isorhamnetin (M2), suggesting that deglycosylation was the main metabolic pathway of isorhamnetin 3-O-glucoside. A total of 94% of the strains metabolized isorhamnetin-3-O-glucoside to kaempferol (M3). The metabolite quercetin (M4) was identified in 7.4% of the strains. However, only one sample of the isolated bacteria could degrade the parent to kaempferol 3-O-glucoside (M5) and acetylated isorhamnetin 3-O-glucoside (M6). Meanwhile, on the basis of the differences in metabolic ability, 28 samples of the isolated bacteria were identified for their great vitality to metabolic isorhamnetin 3-O-glucoside.

(Figure 3). In view of these results, we identified M2 as isorhamnetin, the deglycosylated product of the parent by loss of a glucose. Metabolite M3. Metabolite M3 was detected as deprotonated molecular ion [M − H]− at m/z 285 with a retention time of 8.31 min (Figure 2c). The product ion at m/z 255 was shaped by the elimination of CH2O from m/z 285 (Figure 3). The characteristic fragment ions were m/z 133 and 151 according to RDA cleavage. On the basis of the MS/MS spectrum, we determined M3 as kaempferol. Metabolite M4. Metabolite M4 showed a UPLC profile with a retention time of 6.75 min and an MS spectrum which gave an [M − H]− ion at m/z 301 (Figure 2d). The fragment ion m/z 151 corresponded to RDA cleavage from [M − H]− at m/z 301. The characteristic product ion at m/z 273 was formed by the elimination of CH2O from m/z 301, and then m/z 273 lost O to form m/z 257 (Figure 3). These results indicated that M4 was quercetin, which is the demethylated product of M2 isorhamnetin. Metabolite M5. Metabolite M5 with a retention time of 4.56 min produced a deprotonated molecular ion at m/z 447, a loss of CH2O (30 Da) from isorhamnetin 3-O-glucoside (M1). On the basis of the MS/MS spectrum in Figure 2e, M5 had product ions at m/z 285, 255, and 151, which also emerged in the product ion mass spectrum of M3 kaempferol. The product ion at m/z 151 was formed from the aglycon at m/z 285 by RDA cleavage (Figure 3). Thus, metabolite M5 was tentatively identified as kaempferol 3-O-glucoside. Metabolite M6. Metabolite M6 was found at 6.03 min in the UPLC system, and m/z 519 was its deprotonated molecular ion, which was 42 Da higher than that of isorhamnetin 3-O-glucoside. The MS/MS spectrum of M6 (Figure 2f) showed a product ion at m/z 477, which was generated from m/z 519 by loss of acetyl, and then m/z 477 lost a glucose to form the product ion at m/z 315. The other main product ions extracted from M6 were m/z 2493

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DISCUSSION The metabolites of isorhamnetin 3-O-glucoside yielded by human intestinal bacteria and its relevant metabolic process have not been reported in advance. In this study, we established a UPLC/Q-TOF-MS technique to detect and identify the metabolites of isorhamnetin 3-O-glucoside produced by the isolated human intestinal microflora. After incubation with different intestinal bacteria, parent isorhamnetin 3-O-glucoside and its five metabolites (M1−M6) were tentatively identified in the isolated bacterial samples according to their retention times, accurate molecular masses, and fragment ions. The metabolites were converted by four main metabolic pathways, including deglycosylation, demethoxylation, dehydroxylation, and acetylation. Though the intestinal microflora is composed of massive species, it is possible that a lot of flora conduct the same metabolic steps of isorhamnetin 3-O-glucoside. So far, it has been demonstrated that isorhamnetin could be rapidly absorbed and eliminated in rat plasma after oral administration of mulberry leaf extract. Additionally, double peaks were observed in the curves of mean plasma concentration for isorhamnetin. The first peak appeared at 0.19 h and the second peak at 7.25 h, which might be explained by enterohepatic recirculation.19 In addition, numerous preclinical studies have indicated that kaempferol possesses a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, anticancer, cardioprotective, neuroprotective, antiosteoporotic, anxiolytic, analgesic, estrogenic/antiestrogenic, and antiallergic activities.20 Moreover, the metabolism of quercetin was claimed to exert beneficial health effects, including protection against various diseases such as osteoporosis, certain forms of cancer, and pulmonary and cardiovascular diseases, and also protection against aging. Especially the ability of quercetin to scavenge highly reactive oxygen species such as peroxynitrite and the hydroxyl radical was suggested to be involved in these possible beneficial health effects.21 Some studies also reported that quercetin exhibits antiproliferative, antiatherosclerotic, and neuroprotective activities.22,23 More importantly, quercetin and isorhamnetin could enhance the oral absorption of each other in rats.24 We could arrive at a conclusion that the metabolites of isorhamnetin 3-O-glucoside such as isorhamnetin, kaempferol, and quercetin might have an impact on its biological effect, which would influence the clinical effects of traditional herbal medicines and medicinal plants. Additionally, these metabolites could improve the bioavailability for absorption of isorhamnetin 3-Oglucoside by colonic mucosa due to their lower polarity than that of the parent. It has been reported that the biological activities and bioavailability of flavonoid aglycons are usually higher than those of flavonoid glycosides.25 Daidzein exhibited more potent antioxidant, antitumor cytotoxic, and estrogenic effects than daidzin.26 The aglycon quercetin also had stronger antiinflammatory activity though inhibition of inducible nitric oxide synthase expression and nitrite production and antimicrobial and antioxidant activities than its glycosides.27−29 On the other hand, there are massive studies on the microbial metabolism of flavonoids. Moreover, the conversion profile obtained from in vitro incubation with fecal microbiota can elucidate the conversion route. Many flavonoids undergo a ring fission in which the C-ring is degraded; hydroxylated aromatic compounds are formed from the A-ring and phenolic acids from the B-ring. The phenolic acid metabolites of flavonols reflect the hydroxylation pattern of the B-ring structure.30 The primary metabolite of quercetin is 2-(3,4-dihydroxyphenyl)acetic acid.

Further dehydroxylation results in the formation of 2-(3hydroxyphenyl)acetic acid from both the dihydroxylated derivatives.31 Other metabolites derived from quercetin include phloroglucinol, 3,4-dihydroxybenzaldehyde, 3,4-dihydroxytoluene, butyrate, and acetate.32−36 What is more, Clostridium and Eubacterium genera, which are phylogenetically associated, are other common elements involved in the metabolism of many phenolics. The health benefits from phenolic consumption should be attributed to their bioactive metabolites and also to the modulation of the intestinal bacterial population.14 The metabolic pathway and metabolites of isorhamnetin 3-Oglucoside produced by isolated human intestinal bacteria shown here are very important for the evaluation of the absorption and metabolism of isorhamnetin 3-O-glucoside in vivo. Further research will be performed on the enzyme system which contributes to the bacterial metabolism of isorhamnetin 3-Oglucoside.

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AUTHOR INFORMATION

Corresponding Authors

*Phone/fax: +86 25 85811516. E-mail: [email protected]. *Phone/fax: +86 25 85811116 E-mail: [email protected]. Funding

This work was fiscally supported by the National Basic Research Program of China (973 Program) (Grants 2011CB505300 and 2011CB505303), the National Natural Science Foundation of China (Grants 81072996 and 81102743), Jiangsu Province Colleges and Universities Natural Science Major Basic Research Projects (Grant 10KJA360039), and the Construction Project for Jiangsu Key Laboratory for High Technology Research of TCM Formulae (Grant BM2010576). Notes

The authors declare no competing financial interest.

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