Chinese Journal of Natural Medicines 2013, 11(6): 0684−0689
Chinese Journal of Natural Medicines
Selective hydrolysis of flavonoid glycosides by Curvularia lunata LIU Jing-Yuan 1, 2, YU He-Shui 1, 2, FENG Bing 1, KANG Li-Ping 1, 2, PANG Xu 1, XIONG Cheng-Qi 1, ZHAO Yang 1, LI Chun-Mei 2, ZHANG Yi 2, MA Bai-Ping 1* 1 2
Beijing Institute of Radiation Medicine, Beijing 100850, China; Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China Available online 20 Nov. 2013
[ABSTRACT] Twelve flavonoid glycosides were involved in the biotransformation of the glycosyl moieties by Curvularia lunata 3.4381, and the products were analyzed by UPLC/PDA-Q-TOF-MSE. Curvularia lunata displayed hydrolyzing activities on the terminal Rha or Glc units of some flavonoid glycosides. Terminal Rha with a 1→2 linkage of isorhamnetin-3-O-neohesperidoside and typhaneoside could be hydrolyzed by Curvularia lunata, but terminal Rha with a 1→6 linkage of rutin, typhaneoside, and quercetin-3-O-apiosyl-(1→2)-[rhamnosyl-(1→6)]-glucoside could not be hydrolyzed. Curvularia lunata could also hydrolyze the Glc of icariin, floramanoside B, and naringin. This is the first report of the hydrolysis of glycosyl units of flavonoid glycosides by Curvularia lunata. A new way to convert naringin to naringenin was found in this research. [KEY WORDS] Biotransformation; Flavonoid glycosides; Curvularia lunata; Rhamnosyl; Glucosyl; Selective Hydrolysis
[CLC Number] R284
1
[Document code] A
[Article ID] 1672-3651(2013)06-0684-06
Introduction
As is well-known, flavonoids exist in plants and fruits all around the world. Many flavonoids have important biological activities, such as antioxidant [1-2], antibacterial [1], and anticancer [3]. Most flavonoids are found as glycosides in nature, and the glucose-rhamnose residue is quite common in this kind of compound. However, many flavonoids present a better bioavailability after the sugar chain or one of the sugar residues was hydrolyzed [2, 4-5]. Hence, it is important to find a way to hydrolyze the glycosyl unit in flavonoid glycosides. Synthesis depending on biological catalysts tends to be more selective and less toxic than that depending on chemical catalysts and reagents. A lot of research has been reported on hydrolyzing glycosyl units by various enzymes and microorganisms [6-7]. In previous studies, a special glucoamylase with steroidal saponin-rhamnosidase activity was purified from Curvularia lunata 3.4381 [8]. Curvularia lunata is known for possessing structural modification activity, for instance, the extensive structural modification on taxane hemiacetals [9] and the hydroxylation of steroids [10]. In this work, it was found that Curvularia [Received on] 28-Feb.-2013 [*Corresponding author] MA Bai-Ping: Prof., E-mail: [email protected], Tel: 86-10- 68210077x930265, Fax: 86-10-68214653 These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
lunata had the activity to selectively hydrolyze the Rha or Glc residue of some flavonoid glycosides. LC-MS is an effective analytical technique widely used in rapidly screening and identifying compounds of interest [11]. The UPLC/ PDA-Q-TOF-MSE technique was applied in this study to identify the products of the biotransformations.
2 2.1
Materials and Methods
Chemicals Acetonitrile, HPLC grade, was purchased from Fisher Scientific Co. (Loughborough, UK). Formic acid was purchased from Acros Co. Ltd. (NJ, USA), HPLC grade. Deionized water was bought from Hangzhou Wahaha Group Co., Ltd. Privacy Policy. Other reagents of analytical grade were commercially available. 2.2 Substrate, microorganism and enzyme Dioscin, isorhamnetin-3-O-neohesperidoside, typhaneoside, vitexin, and isovitexin were prepared by this laboratory. Icariin was purchased from Nanjing TCM Institute of Chinese Materia Medica. Naringin was purchased from Shanghai Winherb Medical Science Co., Ltd. Quercetin-3-O-apiosyl-(1→2)[rhamnosyl-(1→6)]-glucoside and rutin were obtained from Beijing Institute of Pharmacology and Toxicology. Quercetin3-O-β-D-xylopyranosyl-(1→2)-β-D-galactopyranoside, quercetin3-O-β-D-glucopyranosyl, myricetin-3-O-β-D-glucopyranosyl, and floramanoside B [12] were obtained from Tianjin University of Traditional Chinese Medicine. Curvularia lunata 3.4381 was obtained from the Institute of Microbiology, Chinese
LIU Jing-Yuan, et al. /Chinese Journal of Natural Medicines 2013, 11(6): 684−689
Academy of Sciences, AS, Beijing, China. The glucoamylase from Curvularia lunata was purified by this laboratory and stored at 4 °C. 2.3 Preparation of crude enzyme, extracellular enzyme, and intracellular enzyme Curvularia lunata was rejuvenated on potato dextrose agar slants, and then was grown in 500 mL corn steep liquor media (6 g corn steep liquor, 5 g Glc, 1 g yeast extract, 2.5 g (NH4)2SO4, 500 mL deionized water) in a 1 L Erlenmeyer flask at 30 ± 1 °C, 150 r·min−1 for 5 days in a constant temperature shaker. The fermentation broth and mycelium was disrupted by sonication. The cell debris was removed by centrifugation (4 160 × g, at 4 °C for 20 min). (NH4)2SO4 was added to the supernatant to 80% slowly with stirring. The mixture was stored at 4°C overnight. Protein precipitate was obtained from the mixture by centrifugation (9 000 × g, at 4 °C for 20 min). The crude protein was dissolved in the buffer (0.1 mol·L−1 citrate-sodium citrate buffer, pH 6) and was dialyzed against deionized water for 24 h. The solution was lyophilized as crude enzyme. It was found that the crude enzyme could hydrolyze isorhamnetin-3-O-neohesperidoside to isorhamnetin-3-Oglucoside, the crude enzyme activity was determined in terms of isorhamnetin-3-O-glucoside-liberating activity using isorhamnetin-3-O-neohesperidoside as a substrate. After optimizing, the best conditions for the conversion was in the citrate phosphate buffer of pH 4.0 at (50 ± 1) °C. For the assay, the crude enzyme (0.3 mg) and the substrate (0.2 mg isorhamnetin-3-O-neohesperidoside) were incubated in the optimized conditions mentioned above in a total volume of 500 μL for 24 h. The mixture was detected by HPLC with a PDA detector at 254 nm. One unit of crude enzyme activity (1 U) was defined as the amount of the crude enzyme necessary to convert 1 μmol isorhamnetin-3-O-neohesperidoside to isorhamnetin-3-O-glucoside per hour. The lyophilized crude enzyme activity was analyzed as 10 U·mg–1. In the preparation for the extracellular enzyme and intracellular enzyme, the fermentation broth and mycelium were separated by filtering with gauze. The fermentation broth was added with (NH4)2SO4 to 80% and dialyzed and lyophilized as mentioned above as the extracellular enzyme. Mycelium was suspended in the buffer (0.1 mol·L−1 citrate-sodium citrate buffer, pH 6), disrupted, centrifuged, added with (NH4)2SO4 to 80% and dialyzed and lyophilized as mentioned above as the intracellular enzyme. 2.4 Biotransformation procedures 2.4.1 Biotransformation of flavonoid glycosides by crude enzyme, intracellular enzyme, and extracellular enzyme The enzyme and substrate (0.1 mg) in the experiments were given as crude enzyme/substrate (3 : 2, m/m), intracellular enzyme/substrate (3 : 2, m/m), and extracellular enzyme/substrate (3 : 1, m/m). The biotransformation experiments were conducted in 1.5 mL UP tubes with the whole volume of 0.5 mL in the buffer (citrate phosphate buffer, pH 4.0) at (50 ± 1) °C for 24 h. The parallel controls with no substrate or no enzyme were maintained in the same conditions. All of the experiments were performed three times.
Samples were extracted by water-saturated n-butanol after conversion. The n-butanol fraction was dried under nitrogen. The methanol extract of the residue was analyzed by UPLC/PDA-Q-TOF-MSE. 2.4.2 Biotransformation of flavonoid glycosides by glucoamylase Isorhamnetin-3-O-neohesperidoside, typhaneoside, and naringin (0.1 mg each) were converted separately by 10 mg stored glucoamylase in the buffer (citrate phosphate buffer, pH 4.0) at (50 ± 1) °C for 24 h. Dioscin was converted under the same conditions to ensure the activity of the stored glucoamylase. The results were detected by TLC. 2.5 TLC and UPLC/PDA-Q-TOF-MSE analysis 2.5.1 TLC analysis TLC analysis of the samples was carried out on silica gel GF254 plates (Yantai Jiangyou Silica Gel Development Co., Ltd.). 2.5.2 UPLC/PDA-Q-TOF-MSE analysis Samples were analyzed by UPLC with a Waters Acquity UPLCTM system. The chromatography was performed on a Waters Acquity UPLC® HSS T3 column (2.1 mm × 100 mm, 1.8 μm). The mobile phase consisted of (A) 0.1% formic acid in water and (B) ACN containing 0.1% formic acid. Samples were detected at 254 nm and 280 nm in PDA, and the TOF MS detection was performed on a Synapt MS system with the data acquisition mode on MSE. The experiment was performed in the ESI (–) ionization mode with the data acquisition range from 100 to 1 500 Da. The source was 100 °C, and the desolvation was 450 °C with desolvation gas flow of 800 L·h–1. Leucine encephalin (200 pg·μL-1) was used as the lock mass. The capillary voltage was 3.0 KV, and the cone voltage was 25 V. The collision energy was 6 eV (trap) and 4 eV (transfer) for low-energy scans, and 45–60 eV ramp (trap) and 12 eV (transfer) for high-energy scans. The instrument was controlled by Masslynx 4.1 software (Waters Corporation).
3
Results
The substrates and products involved in the study are listed in Table 1. The peaks presenting in the reaction samples were evaluated by comparing retention times and peak areas with those of the control samples. The fragmentation patterns of substrates served as templates in the structure elucidation of the products. 3.1 Structural identification of products hydrolyzed by crude enzyme Twelve substrates participated in the biotransformation by the crude enzyme. Five of them had hydrolyzed products (Fig. 1). 3.1.1 Product from the biotransformation of isorhamnetin-3-O-neohesperidoside Isorhamnetin-3-O-neohesperidoside showed a deprotonated ion at m/z 632.161 4 [M – H]–, and the product showed a deprotonated ion at m/z 477.105 6 [M – H]– (a Rha less than isorhamnetin-3-O-neohesperidoside). The product produced an aglycone fragment ion at m/z 315.050 0 [M – H – Glc]– and radical aglycone fragment ion at m/z 314.042 4 [M – H – · Glc]– . Other fragment ions of the product were displayed at m/z 271.024 8 [M – H – Glc – CO2]–, 243.028 7, and 151.0045 (cleavage of RDA). The fragmentation pattern of
LIU Jing-Yuan, et al. /Chinese Journal of Natural Medicines 2013, 11(6): 684−689
Fig. 1
UPLC-PDA chromatograms of the products transformed by crude enzyme
A. Chromatogram of transformed mixture of isorhamnetin-3-O-neohesperidoside and typhaneoside (a. Isorhamnetin-3-O-neohesperidoside. b. transformed mixture of isorhamnetin-3-O-neohesperidoside. c. typhaneoside. d. transformed mixture of typhaneoside). B. Chromatogram of transformed mixture of naringin (e. naringin. f. transformed mixture of naringin). C. Chromatogram of transformed mixture of floramanoside B (g. floramanoside B. h. transformed mixture of floramanoside B). D. Chromatogram of transformed mixture of icariin (i. icariin. j. transformed mixture of icariin).
the product was the same as isorhamnetin-3-O- neohesperidoside (Fig. 2). It was deduced that the terminal rhamnose Rha of isorhamnetin-3-O-neohesperidoside was hydrolyzed. The product was identified as isorhamnetin-3-O-glucoside (P1). 3.1.2 Product from the biotransformation of typhaneoside The product of typhaneoside produced the same deprotonated ion as isorhamnetin-3-O-neohesperidoside at m/z 623.163 4 [M – H]– and showed different retention times in the chromatogram (Fig. 1). It was identified as a structural isomer of isorhamnetin-3-O-neohesperidoside. Typhaneoside has one more 1, 6-linkage Rha than isorhamnetin-3O-neohesperidoside. It was deduced that the crude enzyme hydrolyzed the terminal Rha with a 1,2-linkage to the sugar residue of typhaneoside. The product of typhaneoside was identified as isorhamnetin-3-O-rutinoside (P2). 3.1.3 Products from the biotransformation of naringin, floramanoside B, and icariin Following the method mentioned above, the product from the biotransformation of naringin was identified as naringenin (P3). The product from the biotransformation of floramanoside B was identified as 3, 3', 4', 5', 5, 7, 8-hepta-hy droxyl flavone (P4), and the product from the biotransformation of icariin was identified as icariside II (P5). 3.2 Hydrolysis by intracellular enzyme, extracellular enzyme, and glucoamylase Isorhamnetin-3-O-neohesperidoside, typhaneoside, naringin, and icariin were converted by intracellular and extracellular enzyme to locate the active enzyme. All of the compounds could be hydrolyzed by both the intracellular and extracellular enzyme (Fig. 3), so the enzyme existed both in the intracellular and extracellular matrix. No hydrolysis
products were found in the biotransformation of isorhamnetin-3-O-neohesperidoside, typhaneoside, and naringin by glucoamylase.
4
Discussion
Based on this study, the enzymes extracted from Curvularia lunata 3.438 1 have displayed three kinds of activities: hydrolyzing Rha of some steroid saponins [8], hydrolyzing Rha of some flavonoid glycosides, and hydrolyzing Glc of some flavonoid glycosides. The crude enzyme could hydrolyze the terminal Rha which had a 1→2 linkage to the sugar residues of isorhamnetin-3-O-neohesperidoside and typhaneoside. No hydrolysis occurred on the terminal Rha with a 1→6 linkage to the sugar residues of rutin, typhaneoside, and quercetin-3-O-apiosyl-(1→2)-[rhamnosyl-(1→6)]- glucoside. The terminal xyl of quercetin-3-O-β-D- xylopyranosyl(1→2)-β-D-galactopyranoside with a 1→2 linkage to the sugar residues could not be hydrolyzed. Glc of icariin, floramanoside B, and naringin could be hydrolyzed by the crude enzyme, while no hydrolysis occurred on the Glc of myricetin 3-O-Glc, quercetin 3-O-Glc, vitexin, and isovitexin. Biotransformation rules for hydrolyzing flavonoid glycosides were not as specific as those for hydrolyzing steroid saponins. But it still displayed some characteristics. The enzymes could hydrolyze the terminal Rha that had a 1→2 linkage to the sugar residues of flavonoid glycosides, but could not hydrolyze the terminal Rha that had a 1→6 linkage to the residue. It suggested that the biotransformation patterns of hydrolyzing terminal Rha of flavonoid glycosides might be related to the connective modality of the sugar chain. The active enzyme reacting on the Rha in the flavonoid glycosides was not the glucoamylase purified previously [8]. The
LIU Jing-Yuan, et al. /Chinese Journal of Natural Medicines 2013, 11(6): 684−689
Fig. 2
ESI-MS spectra of isorhamnetin-3-O-neohesperidoside and P1
A. MS spectrum of isorhamnetin-3-O-neohesperidoside detected under low energy scan; B. MS spectrum of P1 detected under low energy scan. C. MS spectrum of isorhamnetin-3-O-neohesperidoside detected under high energy scan; D. MS spectrum of P1 detected under high energy scan.
Fig. 3 UPLC-PDA chromatograms of the products transformed by crude enzyme, extracellular enzyme, and intracellular enzyme A. Chromatogram of transformed mixture of isorhamnetin-3-O-neohesperidoside; B. Chromatogram of transformed mixture of typhaneoside. C. Chromatogram of transformed mixture of naringin; D. Chromatogram of transformed mixture of icariin.
enzyme existed both in the intracellular and extracellular preparations. Since no hydrolysis occurred on the terminal Xyl of quercetin-3-O-β-D-xylopyranosyl-(1→2)-β-D-galactopyranoside, not all terminal glycosyls with a 1→2 linkage to the sugar
residues of flavonoid glycosides could be hydrolyzed by the crude enzyme. The enzyme displayed hydrolysis activity on Rha and Glc in some flavonoid glycosides which needs to be further investigated. Whether it was caused by one enzyme or
LIU Jing-Yuan, et al. /Chinese Journal of Natural Medicines 2013, 11(6): 684−689
several enzymes is still to be explored. Hydrolyzing the sugar chains of some flavonoid glycosides could improve their bioavailability [2, 5]. These results have provided a new way to accomplish this kind of structural modification. Another discovery in this research was that a new ap-
Table 1
proach in converting naringin to naringenin was found. This conversion is widely applied in food and pharmaceutical industries. It is usually finished by naringinase (EC 3.2.1.40) which is mainly secreted by fungi from Penicillium sp. and Aspergillus sp. [13].
Substrates and their products Substrates
Products
Substrates
Products
—
Isorhamnetin-3-O-neohesperidoside
Isorhamnetin-3-O-glucoside
Vitexin
—
—
Typhaneoside
Isorhamnetin-3-O-rutinoside
Rutin
—
—
Icariin
Icariside II
Quercetin-3-O-apiosyl-(1→2)-[rhamnosyl-(1→ 6)]-glucoside
—
—
Floramanoside B
3, 3', 4', 5', 5, 7, 8-heptahydroxyl flavone
Myricetin-3-O-β-D-glucopyranosyl
—
—
Naringin
Naringenin
Quercetin-3-O-β-D-glucopyranosyl
—
—
Isovitexin
5
—
Conclusions
Curvularia lunata displayed activities on hydrolyzing Rha and Glc units of some flavonoid glycosides. The crude enzyme extracted from Curvularia lunata could hydrolyze the terminal Rha that had a 1→2 linkage to the sugar residue, but could not hydrolyze a 1→6 linkage of flavonoid glycosides. These results supplemented the biotransformation
—
Quercetin-3-O-β-D-xylopyranosyl-(1→2)β-D-galactopyranoside
—
functions of Curvularia lunata and provided a new possibility to hydrolyze the sugar chains of flavonoid glycosides in order to enhance their bioavailability.
Acknowledgements The authors would like to thank ZHAO Yi-Min from the Pharmacology and Beijing Institute of Pharmacology and Toxicology for offering substrates generously.
LIU Jing-Yuan, et al. /Chinese Journal of Natural Medicines 2013, 11(6): 684−689
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新月弯孢霉对黄酮苷的选择性水解 刘静媛 1, 2，余河水 1, 2，冯 张 祎 2，马百平 1*
冰 1，康利平 1, 2, 庞
1
北京放射医学研究所，北京 100850；
2
天津中医药大学，天津 300193
旭 1，熊呈琦 1，赵
阳 1，李春梅 2，
【 摘 要】 利用新月弯孢霉（Curvularia lunata 3.4381）对 12 个黄酮苷类化合物进行了生物转化研究，并利用 UPLC/PDA-Q-TOF-MSE 对转化产物进行分析鉴定。结果显示，新月弯孢霉对一些黄酮苷类化合物的末端鼠李糖基或葡萄糖基表 现出了水解活性。新月弯孢霉可以水解异鼠李素-3-O-新橙皮苷和香蒲新苷中 1→2 连接的末端鼠李糖基，但不能水解芦丁、香 蒲新苷和槲皮素-3-O-芹菜糖基芦丁糖苷中 1→6 连接的末端鼠李糖基。新月弯孢霉还可以水解淫羊藿苷、Floramanoside B 和柚 皮苷中的葡萄糖基。首次报道了新月弯孢霉水解黄酮苷类化合物糖基的研究，并发现了一种将柚皮苷转化为柚皮素的新途径。 【关键词】 生物转化；黄酮苷；新月弯孢霉；鼠李糖基；葡萄糖基；水解
Chinese Journal of Natural Medicines
Selective hydrolysis of flavonoid glycosides by Curvularia lunata LIU Jing-Yuan 1, 2, YU He-Shui 1, 2, FENG Bing 1, KANG Li-Ping 1, 2, PANG Xu 1, XIONG Cheng-Qi 1, ZHAO Yang 1, LI Chun-Mei 2, ZHANG Yi 2, MA Bai-Ping 1* 1 2
Beijing Institute of Radiation Medicine, Beijing 100850, China; Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China Available online 20 Nov. 2013
[ABSTRACT] Twelve flavonoid glycosides were involved in the biotransformation of the glycosyl moieties by Curvularia lunata 3.4381, and the products were analyzed by UPLC/PDA-Q-TOF-MSE. Curvularia lunata displayed hydrolyzing activities on the terminal Rha or Glc units of some flavonoid glycosides. Terminal Rha with a 1→2 linkage of isorhamnetin-3-O-neohesperidoside and typhaneoside could be hydrolyzed by Curvularia lunata, but terminal Rha with a 1→6 linkage of rutin, typhaneoside, and quercetin-3-O-apiosyl-(1→2)-[rhamnosyl-(1→6)]-glucoside could not be hydrolyzed. Curvularia lunata could also hydrolyze the Glc of icariin, floramanoside B, and naringin. This is the first report of the hydrolysis of glycosyl units of flavonoid glycosides by Curvularia lunata. A new way to convert naringin to naringenin was found in this research. [KEY WORDS] Biotransformation; Flavonoid glycosides; Curvularia lunata; Rhamnosyl; Glucosyl; Selective Hydrolysis
[CLC Number] R284
1
[Document code] A
[Article ID] 1672-3651(2013)06-0684-06
Introduction
As is well-known, flavonoids exist in plants and fruits all around the world. Many flavonoids have important biological activities, such as antioxidant [1-2], antibacterial [1], and anticancer [3]. Most flavonoids are found as glycosides in nature, and the glucose-rhamnose residue is quite common in this kind of compound. However, many flavonoids present a better bioavailability after the sugar chain or one of the sugar residues was hydrolyzed [2, 4-5]. Hence, it is important to find a way to hydrolyze the glycosyl unit in flavonoid glycosides. Synthesis depending on biological catalysts tends to be more selective and less toxic than that depending on chemical catalysts and reagents. A lot of research has been reported on hydrolyzing glycosyl units by various enzymes and microorganisms [6-7]. In previous studies, a special glucoamylase with steroidal saponin-rhamnosidase activity was purified from Curvularia lunata 3.4381 [8]. Curvularia lunata is known for possessing structural modification activity, for instance, the extensive structural modification on taxane hemiacetals [9] and the hydroxylation of steroids [10]. In this work, it was found that Curvularia [Received on] 28-Feb.-2013 [*Corresponding author] MA Bai-Ping: Prof., E-mail: [email protected], Tel: 86-10- 68210077x930265, Fax: 86-10-68214653 These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
lunata had the activity to selectively hydrolyze the Rha or Glc residue of some flavonoid glycosides. LC-MS is an effective analytical technique widely used in rapidly screening and identifying compounds of interest [11]. The UPLC/ PDA-Q-TOF-MSE technique was applied in this study to identify the products of the biotransformations.
2 2.1
Materials and Methods
Chemicals Acetonitrile, HPLC grade, was purchased from Fisher Scientific Co. (Loughborough, UK). Formic acid was purchased from Acros Co. Ltd. (NJ, USA), HPLC grade. Deionized water was bought from Hangzhou Wahaha Group Co., Ltd. Privacy Policy. Other reagents of analytical grade were commercially available. 2.2 Substrate, microorganism and enzyme Dioscin, isorhamnetin-3-O-neohesperidoside, typhaneoside, vitexin, and isovitexin were prepared by this laboratory. Icariin was purchased from Nanjing TCM Institute of Chinese Materia Medica. Naringin was purchased from Shanghai Winherb Medical Science Co., Ltd. Quercetin-3-O-apiosyl-(1→2)[rhamnosyl-(1→6)]-glucoside and rutin were obtained from Beijing Institute of Pharmacology and Toxicology. Quercetin3-O-β-D-xylopyranosyl-(1→2)-β-D-galactopyranoside, quercetin3-O-β-D-glucopyranosyl, myricetin-3-O-β-D-glucopyranosyl, and floramanoside B [12] were obtained from Tianjin University of Traditional Chinese Medicine. Curvularia lunata 3.4381 was obtained from the Institute of Microbiology, Chinese
LIU Jing-Yuan, et al. /Chinese Journal of Natural Medicines 2013, 11(6): 684−689
Academy of Sciences, AS, Beijing, China. The glucoamylase from Curvularia lunata was purified by this laboratory and stored at 4 °C. 2.3 Preparation of crude enzyme, extracellular enzyme, and intracellular enzyme Curvularia lunata was rejuvenated on potato dextrose agar slants, and then was grown in 500 mL corn steep liquor media (6 g corn steep liquor, 5 g Glc, 1 g yeast extract, 2.5 g (NH4)2SO4, 500 mL deionized water) in a 1 L Erlenmeyer flask at 30 ± 1 °C, 150 r·min−1 for 5 days in a constant temperature shaker. The fermentation broth and mycelium was disrupted by sonication. The cell debris was removed by centrifugation (4 160 × g, at 4 °C for 20 min). (NH4)2SO4 was added to the supernatant to 80% slowly with stirring. The mixture was stored at 4°C overnight. Protein precipitate was obtained from the mixture by centrifugation (9 000 × g, at 4 °C for 20 min). The crude protein was dissolved in the buffer (0.1 mol·L−1 citrate-sodium citrate buffer, pH 6) and was dialyzed against deionized water for 24 h. The solution was lyophilized as crude enzyme. It was found that the crude enzyme could hydrolyze isorhamnetin-3-O-neohesperidoside to isorhamnetin-3-Oglucoside, the crude enzyme activity was determined in terms of isorhamnetin-3-O-glucoside-liberating activity using isorhamnetin-3-O-neohesperidoside as a substrate. After optimizing, the best conditions for the conversion was in the citrate phosphate buffer of pH 4.0 at (50 ± 1) °C. For the assay, the crude enzyme (0.3 mg) and the substrate (0.2 mg isorhamnetin-3-O-neohesperidoside) were incubated in the optimized conditions mentioned above in a total volume of 500 μL for 24 h. The mixture was detected by HPLC with a PDA detector at 254 nm. One unit of crude enzyme activity (1 U) was defined as the amount of the crude enzyme necessary to convert 1 μmol isorhamnetin-3-O-neohesperidoside to isorhamnetin-3-O-glucoside per hour. The lyophilized crude enzyme activity was analyzed as 10 U·mg–1. In the preparation for the extracellular enzyme and intracellular enzyme, the fermentation broth and mycelium were separated by filtering with gauze. The fermentation broth was added with (NH4)2SO4 to 80% and dialyzed and lyophilized as mentioned above as the extracellular enzyme. Mycelium was suspended in the buffer (0.1 mol·L−1 citrate-sodium citrate buffer, pH 6), disrupted, centrifuged, added with (NH4)2SO4 to 80% and dialyzed and lyophilized as mentioned above as the intracellular enzyme. 2.4 Biotransformation procedures 2.4.1 Biotransformation of flavonoid glycosides by crude enzyme, intracellular enzyme, and extracellular enzyme The enzyme and substrate (0.1 mg) in the experiments were given as crude enzyme/substrate (3 : 2, m/m), intracellular enzyme/substrate (3 : 2, m/m), and extracellular enzyme/substrate (3 : 1, m/m). The biotransformation experiments were conducted in 1.5 mL UP tubes with the whole volume of 0.5 mL in the buffer (citrate phosphate buffer, pH 4.0) at (50 ± 1) °C for 24 h. The parallel controls with no substrate or no enzyme were maintained in the same conditions. All of the experiments were performed three times.
Samples were extracted by water-saturated n-butanol after conversion. The n-butanol fraction was dried under nitrogen. The methanol extract of the residue was analyzed by UPLC/PDA-Q-TOF-MSE. 2.4.2 Biotransformation of flavonoid glycosides by glucoamylase Isorhamnetin-3-O-neohesperidoside, typhaneoside, and naringin (0.1 mg each) were converted separately by 10 mg stored glucoamylase in the buffer (citrate phosphate buffer, pH 4.0) at (50 ± 1) °C for 24 h. Dioscin was converted under the same conditions to ensure the activity of the stored glucoamylase. The results were detected by TLC. 2.5 TLC and UPLC/PDA-Q-TOF-MSE analysis 2.5.1 TLC analysis TLC analysis of the samples was carried out on silica gel GF254 plates (Yantai Jiangyou Silica Gel Development Co., Ltd.). 2.5.2 UPLC/PDA-Q-TOF-MSE analysis Samples were analyzed by UPLC with a Waters Acquity UPLCTM system. The chromatography was performed on a Waters Acquity UPLC® HSS T3 column (2.1 mm × 100 mm, 1.8 μm). The mobile phase consisted of (A) 0.1% formic acid in water and (B) ACN containing 0.1% formic acid. Samples were detected at 254 nm and 280 nm in PDA, and the TOF MS detection was performed on a Synapt MS system with the data acquisition mode on MSE. The experiment was performed in the ESI (–) ionization mode with the data acquisition range from 100 to 1 500 Da. The source was 100 °C, and the desolvation was 450 °C with desolvation gas flow of 800 L·h–1. Leucine encephalin (200 pg·μL-1) was used as the lock mass. The capillary voltage was 3.0 KV, and the cone voltage was 25 V. The collision energy was 6 eV (trap) and 4 eV (transfer) for low-energy scans, and 45–60 eV ramp (trap) and 12 eV (transfer) for high-energy scans. The instrument was controlled by Masslynx 4.1 software (Waters Corporation).
3
Results
The substrates and products involved in the study are listed in Table 1. The peaks presenting in the reaction samples were evaluated by comparing retention times and peak areas with those of the control samples. The fragmentation patterns of substrates served as templates in the structure elucidation of the products. 3.1 Structural identification of products hydrolyzed by crude enzyme Twelve substrates participated in the biotransformation by the crude enzyme. Five of them had hydrolyzed products (Fig. 1). 3.1.1 Product from the biotransformation of isorhamnetin-3-O-neohesperidoside Isorhamnetin-3-O-neohesperidoside showed a deprotonated ion at m/z 632.161 4 [M – H]–, and the product showed a deprotonated ion at m/z 477.105 6 [M – H]– (a Rha less than isorhamnetin-3-O-neohesperidoside). The product produced an aglycone fragment ion at m/z 315.050 0 [M – H – Glc]– and radical aglycone fragment ion at m/z 314.042 4 [M – H – · Glc]– . Other fragment ions of the product were displayed at m/z 271.024 8 [M – H – Glc – CO2]–, 243.028 7, and 151.0045 (cleavage of RDA). The fragmentation pattern of
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Fig. 1
UPLC-PDA chromatograms of the products transformed by crude enzyme
A. Chromatogram of transformed mixture of isorhamnetin-3-O-neohesperidoside and typhaneoside (a. Isorhamnetin-3-O-neohesperidoside. b. transformed mixture of isorhamnetin-3-O-neohesperidoside. c. typhaneoside. d. transformed mixture of typhaneoside). B. Chromatogram of transformed mixture of naringin (e. naringin. f. transformed mixture of naringin). C. Chromatogram of transformed mixture of floramanoside B (g. floramanoside B. h. transformed mixture of floramanoside B). D. Chromatogram of transformed mixture of icariin (i. icariin. j. transformed mixture of icariin).
the product was the same as isorhamnetin-3-O- neohesperidoside (Fig. 2). It was deduced that the terminal rhamnose Rha of isorhamnetin-3-O-neohesperidoside was hydrolyzed. The product was identified as isorhamnetin-3-O-glucoside (P1). 3.1.2 Product from the biotransformation of typhaneoside The product of typhaneoside produced the same deprotonated ion as isorhamnetin-3-O-neohesperidoside at m/z 623.163 4 [M – H]– and showed different retention times in the chromatogram (Fig. 1). It was identified as a structural isomer of isorhamnetin-3-O-neohesperidoside. Typhaneoside has one more 1, 6-linkage Rha than isorhamnetin-3O-neohesperidoside. It was deduced that the crude enzyme hydrolyzed the terminal Rha with a 1,2-linkage to the sugar residue of typhaneoside. The product of typhaneoside was identified as isorhamnetin-3-O-rutinoside (P2). 3.1.3 Products from the biotransformation of naringin, floramanoside B, and icariin Following the method mentioned above, the product from the biotransformation of naringin was identified as naringenin (P3). The product from the biotransformation of floramanoside B was identified as 3, 3', 4', 5', 5, 7, 8-hepta-hy droxyl flavone (P4), and the product from the biotransformation of icariin was identified as icariside II (P5). 3.2 Hydrolysis by intracellular enzyme, extracellular enzyme, and glucoamylase Isorhamnetin-3-O-neohesperidoside, typhaneoside, naringin, and icariin were converted by intracellular and extracellular enzyme to locate the active enzyme. All of the compounds could be hydrolyzed by both the intracellular and extracellular enzyme (Fig. 3), so the enzyme existed both in the intracellular and extracellular matrix. No hydrolysis
products were found in the biotransformation of isorhamnetin-3-O-neohesperidoside, typhaneoside, and naringin by glucoamylase.
4
Discussion
Based on this study, the enzymes extracted from Curvularia lunata 3.438 1 have displayed three kinds of activities: hydrolyzing Rha of some steroid saponins [8], hydrolyzing Rha of some flavonoid glycosides, and hydrolyzing Glc of some flavonoid glycosides. The crude enzyme could hydrolyze the terminal Rha which had a 1→2 linkage to the sugar residues of isorhamnetin-3-O-neohesperidoside and typhaneoside. No hydrolysis occurred on the terminal Rha with a 1→6 linkage to the sugar residues of rutin, typhaneoside, and quercetin-3-O-apiosyl-(1→2)-[rhamnosyl-(1→6)]- glucoside. The terminal xyl of quercetin-3-O-β-D- xylopyranosyl(1→2)-β-D-galactopyranoside with a 1→2 linkage to the sugar residues could not be hydrolyzed. Glc of icariin, floramanoside B, and naringin could be hydrolyzed by the crude enzyme, while no hydrolysis occurred on the Glc of myricetin 3-O-Glc, quercetin 3-O-Glc, vitexin, and isovitexin. Biotransformation rules for hydrolyzing flavonoid glycosides were not as specific as those for hydrolyzing steroid saponins. But it still displayed some characteristics. The enzymes could hydrolyze the terminal Rha that had a 1→2 linkage to the sugar residues of flavonoid glycosides, but could not hydrolyze the terminal Rha that had a 1→6 linkage to the residue. It suggested that the biotransformation patterns of hydrolyzing terminal Rha of flavonoid glycosides might be related to the connective modality of the sugar chain. The active enzyme reacting on the Rha in the flavonoid glycosides was not the glucoamylase purified previously [8]. The
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Fig. 2
ESI-MS spectra of isorhamnetin-3-O-neohesperidoside and P1
A. MS spectrum of isorhamnetin-3-O-neohesperidoside detected under low energy scan; B. MS spectrum of P1 detected under low energy scan. C. MS spectrum of isorhamnetin-3-O-neohesperidoside detected under high energy scan; D. MS spectrum of P1 detected under high energy scan.
Fig. 3 UPLC-PDA chromatograms of the products transformed by crude enzyme, extracellular enzyme, and intracellular enzyme A. Chromatogram of transformed mixture of isorhamnetin-3-O-neohesperidoside; B. Chromatogram of transformed mixture of typhaneoside. C. Chromatogram of transformed mixture of naringin; D. Chromatogram of transformed mixture of icariin.
enzyme existed both in the intracellular and extracellular preparations. Since no hydrolysis occurred on the terminal Xyl of quercetin-3-O-β-D-xylopyranosyl-(1→2)-β-D-galactopyranoside, not all terminal glycosyls with a 1→2 linkage to the sugar
residues of flavonoid glycosides could be hydrolyzed by the crude enzyme. The enzyme displayed hydrolysis activity on Rha and Glc in some flavonoid glycosides which needs to be further investigated. Whether it was caused by one enzyme or
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several enzymes is still to be explored. Hydrolyzing the sugar chains of some flavonoid glycosides could improve their bioavailability [2, 5]. These results have provided a new way to accomplish this kind of structural modification. Another discovery in this research was that a new ap-
Table 1
proach in converting naringin to naringenin was found. This conversion is widely applied in food and pharmaceutical industries. It is usually finished by naringinase (EC 3.2.1.40) which is mainly secreted by fungi from Penicillium sp. and Aspergillus sp. [13].
Substrates and their products Substrates
Products
Substrates
Products
—
Isorhamnetin-3-O-neohesperidoside
Isorhamnetin-3-O-glucoside
Vitexin
—
—
Typhaneoside
Isorhamnetin-3-O-rutinoside
Rutin
—
—
Icariin
Icariside II
Quercetin-3-O-apiosyl-(1→2)-[rhamnosyl-(1→ 6)]-glucoside
—
—
Floramanoside B
3, 3', 4', 5', 5, 7, 8-heptahydroxyl flavone
Myricetin-3-O-β-D-glucopyranosyl
—
—
Naringin
Naringenin
Quercetin-3-O-β-D-glucopyranosyl
—
—
Isovitexin
5
—
Conclusions
Curvularia lunata displayed activities on hydrolyzing Rha and Glc units of some flavonoid glycosides. The crude enzyme extracted from Curvularia lunata could hydrolyze the terminal Rha that had a 1→2 linkage to the sugar residue, but could not hydrolyze a 1→6 linkage of flavonoid glycosides. These results supplemented the biotransformation
—
Quercetin-3-O-β-D-xylopyranosyl-(1→2)β-D-galactopyranoside
—
functions of Curvularia lunata and provided a new possibility to hydrolyze the sugar chains of flavonoid glycosides in order to enhance their bioavailability.
Acknowledgements The authors would like to thank ZHAO Yi-Min from the Pharmacology and Beijing Institute of Pharmacology and Toxicology for offering substrates generously.
LIU Jing-Yuan, et al. /Chinese Journal of Natural Medicines 2013, 11(6): 684−689
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新月弯孢霉对黄酮苷的选择性水解 刘静媛 1, 2，余河水 1, 2，冯 张 祎 2，马百平 1*
冰 1，康利平 1, 2, 庞
1
北京放射医学研究所，北京 100850；
2
天津中医药大学，天津 300193
旭 1，熊呈琦 1，赵
阳 1，李春梅 2，
【 摘 要】 利用新月弯孢霉（Curvularia lunata 3.4381）对 12 个黄酮苷类化合物进行了生物转化研究，并利用 UPLC/PDA-Q-TOF-MSE 对转化产物进行分析鉴定。结果显示，新月弯孢霉对一些黄酮苷类化合物的末端鼠李糖基或葡萄糖基表 现出了水解活性。新月弯孢霉可以水解异鼠李素-3-O-新橙皮苷和香蒲新苷中 1→2 连接的末端鼠李糖基，但不能水解芦丁、香 蒲新苷和槲皮素-3-O-芹菜糖基芦丁糖苷中 1→6 连接的末端鼠李糖基。新月弯孢霉还可以水解淫羊藿苷、Floramanoside B 和柚 皮苷中的葡萄糖基。首次报道了新月弯孢霉水解黄酮苷类化合物糖基的研究，并发现了一种将柚皮苷转化为柚皮素的新途径。 【关键词】 生物转化；黄酮苷；新月弯孢霉；鼠李糖基；葡萄糖基；水解
