Constituents of Enzymatically Modified Isoquercitrin and Enzymatically Modified Rutin (Extract)

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1 54 J. Food Hyg. Soc. Japan Vol. 41, No. 1 Original Constituents of Enzymatically Modified Isoquercitrin and Enzymatically Modified Rutin (Extract) (Received October 6, 1999) Takuml AKIYAMA*1, Tsutomu WASHING*2, Takashl YAMADA*1, Takatoshi KoDA*2 and Tamio MAITANI*1 (*1National Institute of Health Sciences: , Kamiyoga, Setagaya-ku, Tokyo , Japan; *2San-Ei Gen F. F. I., Inc.: , Sanwa -cho, Toyonaka-shi, Osaka , Japan) Enzymatically modified isoquercitrin and enzymatically modified rutin (extract) were analyzed to determine the structures and contents of their constituents. NMR analysis revealed that the 4-hydroxyl group of glucose was glucosylated in the manufacture of enzymatically modified isoquercitrin. LC/MS analysis established that enzymatically modified isoquercitrin consists of isoquercitrin and its a-glucosylated derivatives with 1-7 additional glucose moieties. Similarly, one sample of enzymatically modified rutin (extract) was shown to consist of rutin and its a-glucosylated derivatives. Rutin derivatives with up to 32 additional glucose moieties were detected. A different sample of enzymatically modified rutin (extract) consisted of rutin, its derivative with one additional glucose and isoquercitrin. It was suggested that two additional enzymes, as well as cyclodextrin glucanotransferase, play roles in the manufacture of this product. HPLC was employed to evaluate the contents of quercetin glycosides, which should determine solubility and antioxidative activity, in the three samples. Key words: natural food additive; enzymatically modified isoquercitrin; enzymatically modified rutin (extract); cyclodextrin glucanotransferase; nuclear magnetic resonance; liquid chromatography/mass spectrometry Introduction The antioxidative activity of fiavonoids from natural resources has attracted much attention. Quercetin glycosides, isoquercitrin and rutin (Fig. 1), have strong antioxidative activity, but their use is limited because of their poor solubility in water. Enzymatically modified isoquercitrin and enzymatically modified rutin (extract) are water-soluble modifications of these antioxidants, which are manufactured by transglycosylation with cyclodextrin glucanotransferase (CGTase)'. These natural food additives are included in the List of Existing Food Additives2). In the list, however, enzymatically modified isoquercitrin and enzymatically modified rutin (extract) are merely described as being composed mainly of a-glucosylisoquercitrin and a-glucosylrutin, respectively. Their chemical compositions have not been investigated in detail. It has been suggested that the major constituents of enzymatically modified rutin (extract) are rutin and its maltooligosaccharides designated as RGns, where n shows the number of additional glucosyl moieties'. Similarly, enzymatically modified isoquercitrin should consist of isoquercitrin and IGns. In many cases, CGTase catalyzes a-d-glucosylation of the 4-hydroxyl group of the glucopyranoside moiety of the acceptor molecule. Thus, the additional glucose moieties of RGns and IGns could be bound to the 4-hydroxyl group of the glucose moiety of rutin and isoquercitrin, respectively (Fig. 1). Recently, it has been reported that CGTase from an alkalophilic Bacillus species glucosylates 3-OH of the glucose moiety of neohesperidin3). Therefore, it is necessary to clarify which hydroxyl groups of these quercetin glycosides are glucosylated. The chemical structure of RG1 was confirmed by spectrometric analyses1). Consequently, in the first part of this paper, the structure elucidation of IGns will be described. Enzymatically modified isoquercitrin and enzymatically modified rutin (extract) are mixtures of quercetin glycosides which have sugar moieties with different sizes. The sizes and the contents of these glycosides should be different, depending on the manufacturing process. In the second part of this paper, three commercial products were analyzed to determine their chemical compositions. LC/MS was used for separation and detection of species of different sizes. Finally, quantitation of the major constituents was attempted with HPLC equipped with a UV detector. Materials and Methods Samples and reagents Commercial enzymatically modified rutin (extract) (two products, designated as A and B) and enzymatically modified isoquercitrin (one product, designated as

2 February 2000 Enzymatically Modified Isoquercitrin and Rutin 55 Fig, 1. Structures of rutin, Isoquercitrin and their a-glucosylated derivatives The structures of IGns were elucidated in this paper. C) were obtained through the Japan Food Additive Association. Rutin of reagent grade was purchased from Katayama Chemical Industries. Isoquercitrin was manufactured by San-Ei Gen F. F. I. Glucoamylase from Rhizopus delemer was from Nagase Seikagaku Kogyo and b-amylase from soybeans was purchased from Wako Pure Chemicals Industries. Amberlite XAD-2 was purchased from Organo. b-cyclodextrin polymer resin was from Ensuiko Sugar Refining. Other chemicals were of reagent or HPLC grade. Ultrapure water (>18 MQcm) prepared with a Milli-Q SP Reagent Water System (Millipore) was used throughout the experiment. Isolation of ICl Twenty grams of sample C and glucoamylase from Rhizopus delemer (0.1 g) were dissolved in 200 ml of water. This solution was incubated at 50C for 3 hr. The reaction mixture was loaded onto an Amberlite XAD-2 column (40 mm i.d, x 400 mm). This column was washed with 1.5 L of water, and the compounds were eluted by stepwise addition of 500 ml each of aq. 25% McOH, aq. 30% McOH and aq. 35% McOH. Fractions (ca. 100 ml each) were analyzed by HPLC. Fractions containing IG1 in higher ratio eluted with aq. 30% McOH and aq. 35% McOH were pooled. These fractions were loaded onto a/3-cyclodextrin polymer column (20 mm i.d. x 300 mm) and eluted with water. Fractions (ca. 50 ml each) were analyzed by HPLC. Fractions containing IG1 in higher ratio were pooled and subjected to preparative HPLC. HPLC conditions were: column, Chemcopak ODS (20 mm i.d. X 250 mm, Chemco Scientific); flow rate, 3 ml/min; temperature, ambient; detection, 351 nm; mobile phase, THF-0.085% phosphoric acid (2: 3, v/v). As the final pure product, 1.10 g was obtained. Isolation of IC2 Twenty grams of sample C and, S-amylase from soybeans (500 units) were dissolved in 200 ml of water. This solution was incubated at 55C for 5 hr. IG2 was purified by a method similar for that used for IG1. As the final pure product, 0.35 g was obtained. 1H and 13C-NMR analysis 1H- and 13C-NMR spectra were recorded with a JML-LA400 (400 MHz) system (JEOL) in methanol-d4 with tetramethylsilane as the internal reference. For IG1, a small portion of DMSO-d4 was added to dissolve the crystals completely. LC/MS analysis For the LC/electrospray ionization (ESI)-MS analysis, an LCQ mass spectrometer (Finnigan MAT) equipped with an ESI interface was utilized. The HPLC system included an L-7100 pump (Hitachi), an L-7300 column oven (Hitachi) and an L-7400 UV detector (Hitachi). Samples were dissolved in water at the concentrations of 4,000, 200 and 2,000 mg/l for samples A, B and C, respectively. ESI parameters were: ion spray voltage, 3.5 kv; capillary temperature, 270C; capillary voltage, -8 kv. Full scan spectra from m/z 150 to 2,000u in the negative mode were obtained. HPLC conditions were: column, Inertsil ODS-3 (1.5 mm i.d, x 150 mm, GL Science); flow rate, 0.05 ml/min; temperature, 40C; injection volume, 5aL; detection, 254nm; mobile phase, 30% methanol containing 3.5% acetic acid. Quantitation by HPLC Samples were dissolved in water at the concentrations of 4,000, 200 and 2,000mg/L for samples A, B and C, respectively. Rutin was dissolved in ethanol at the concentrations of 20.0, 50.0, 100, 200, 500 and 1,000 timol/l. A 5;uL aliquot was subjected to HPLC (LC-6A, Shimadzu). Duplicate injections were carried out for all samples and standards. HPLC conditions were: column, Inertsil ODS-3V (4.6 mm id. X 250mm, GL Science); flow rate, 0.8mL/min; temperature, 40C; detection, 254nm; mobile phase, 30% methanol containing 3.5% acetic acid. Results and Discussion Isolation and structure elucidation of ICl and IC2 In order to clarify the glucosylation site in IGn molecules, structure elucidation of the major constituents of enzymatically modified Isoquercitrin was attempted. Two major constituents of sample C, which were designated IG1 and IG2, were isolated and subjected to NMR

3 56 J. Food Hyg. Soc. Japan Vol. 41, No. 1 Table 1. 1H-NMR Data of Isoquercitrin, IG1 and IG2 *: Signal assignments may be interchanged. Table 2. 13C-NMR Data of Isoquercitrin, IG1 and IG2 a), b), c), d): Signal assignments may be interchanged in each column. analyses. Two glycolytic enzymes were utilized to increase the content of the desired constituents. Glucoamylase, which hydrolyzes terminal 1,4-linked a-d-glucose residues successively from the non-reducing ends, can form IG1 from all IGns. On the other hand, j3-amylase, which removes successive maltose units from the nonreducing ends of the a-1,4-glucan, should transform IGn (n=even number) and IGn (n=odd number) to IG2 and IG1, respectively. So IG1 was isolated from sample C reacted with glucoamylase, and the reaction mixture of sample C and,-amylase was used for the purification of IG2. IG1, IG2 and authentic Isoquercitrin were analyzed by 1H- and 13C-NMR (in methanol-d4, Tables 1 and 2). HMBC experiments were also carried out. The 1H- and the 13C-NMR spectra of IG1 were compared with those of isoquercitrin. In the 1H-NMR spectrum of IG1, an anomeric proton signal at 5.18 ppm, which was not found in the spectrum of isoquercitrin and was therefore assigned to H-1'", showed an a-glucosidic linkage as judged from its coupling constant. The C-4" of 1G1 (80.6 ppm) showed a much larger chemical shift than that of isoquercitrin (71.2 ppm), indicating glycosylation at this position. In the HMBC spectrum, a correlation was observed between H-1" and C-4". These results, which show an a-1,4-linkage between the 2 glucose moieties, confirmed the expected structure of IG1 and demonstrated that the 4-hydroxyl group of the glucose moiety of isoquercitrin was a-glucosylated by CGTase (Fig. 1). In the 1H-NMR spectrum of IG2, two anomeric proton signals (5.14 and 5.18 ppm) showed a-glucosidic linkages. In the 13C-NMR spectrum, C-4", as well as C-4", showed a large chemical shift. The 13C-1H long-range correlation between H-1" and C-4" and that between H-1 " and C-4" were observed as a pair of signals in the HMBC spectrum. One was observed between the proton signal at 5.14 ppm and the carbon signal at 81.3 ppm, and the other between the proton signal at 5.18 ppm and the carbon signal at 80.6 ppm. These results showed that IG2 was formed by the a-glucosylation of the 4"-hydroxyl group of IG1 (Fig. 1). Based on the results described above, the structures of IGns were elucidated (Fig. 1). LC/ESI MS analysis The chemical compositions of enzymatically modified isoquercitrin and enzymatically modified rutin (extract) are comparably simple. All constituents in these food additives differ from each other only in the length of the a-glucan chain. To analyze their compositions, LC/ MS should provide sufficient information because it should be able to separate each constituent and deter-

4 February 2000 Enzymatically Modified Isoquercitrin and Rutin 57 UV chromatogram Fig. 2. LC/MS analysis of the enzymatically modified isoquercitrin C (A) UV chromatogram; (B), (C) Mass spectra at the time points indicated with arrows in panel A mine its molecular weight. Therefore, LC/MS analysis of the three samples was attempted. From the results of preliminary experiments, the combination of an ODS column as the stationary phase and an aqueous methanol solution containing acetic acid as the mobile phase was selected for LC. For MS, ESI in the negative mode showed the best sensitivity. Negative mode ESI-MS was also reported to be effective for rutin4) and maltodextrin5). Ten micrograms of sample C, twenty micrograms of sample A and 1 microgram of sample B were analyzed. Figure 2 shows the results of analysis of sample C. The LC chromatogram monitored with a UV detector (254 nm) and the mass spectra of the major chromatopeaks are presented. The peak at RT=16.5 min was identified as isoquercitrin, because its mass spectrum had a base peak with m/z=463.2, which corresponds to the [M- H]-of isoquercitrin. An adjacent peak (RT=15.5 min, m/z=625.3) was identified as IG1. IGns with larger n were detected in the same way. Thus, it was suggested that the major constituents of sample C were isoquercitrin and IGns, as expected. In the mass spectra, singly charged deprotonated species [M-H]-were prominent. Dimeric species [2M-H]-were also observed, although their intensities were weak for IGns with higher molecular weight. IGns with n>7 could not be detected. Glycosides with a longer glucan chain were detected in sample A (Fig. 3). Besides rutin and RG1.8, which were detected as [M,-H]-, RG9.20 and RG21-32 were detected as multiply charged species [M-2H]2-and [M- 3H]3-, respectively. Figs. 3B and 3C show the regions of the mass spectra containing RG11-17 and RG25-32, respectively. Isoquercitrin and RG1 were prominently detected in sample B (Fig. 4). Rutin was also detected as a minor component. These results are summarized in Table 3. Based on the LC/MS experiments described above, the compositions of the analyzed samples were clarified. Differences in their compositions should reflect the processes of their manufacture. Sample C does not contain molecules with more than 7 additional glucose moieties, which are present in sample A. This difference may be related to the characteristics of CGTase used, the conditions of the transglycosylation reaction, the purification method, and so on. Differences between samples A and B, both of which are classified as enzymatically modified rutin (extract), are also evident. Sample B contained no RGns with n>1 and a considerable amount of isoquercitrin. It is probable that the manufacture of this product involved additional enzyme reactions after transglycosylation by CGTase (Fig. 5). Namely, first, RGns (n>1) are transformed to RG1 by glucoamylase, and next, rutin is hydrolyzed to isoquercitrin by a-1,6- rhamnosidase6),7). Further variations in manufacture

5 58 J. Food Hyg. Soc. Japan Vol. 41, No. 1 uv chromatogram Fig. 3. LC/MS analysis of the enzymatically modified rutin A (A) UV chromatogram; (B), (C) Mass spectra at the time points indicated with arrows in panel A uv chromatogram Fig. 4. LC/MS analysis of the enzymatically modified rutin B (A) UV chromatogram; (B) The mass spectrum at the time point indicated with an arrow in panel A may be possible. LC/MS, which can reveal the composition of a product only from a single injection, should be a powerful tool for their analysis. Quantitation by HPLC Solubility and antioxidative activity are different between glycosides with different numbers of additional glucose moieties. So it is important to know the content of each constituent of a product when its quality is discussed. For this reason, the content of each glycoside was quantitated by an absolute calibration method from the peak areas of the HPLC chromatogram. Twenty micrograms of sample A, 1 microgram of sample B and ten micrograms of sample C were analyz-

6 February 2000 Enzymatically Modified Isoquercitrin and Rutin 59 Table 3. Detected Ions in LC/ESI-MS Fig. 5. Preparation of enzymatically modified rutin and enzymatically modified isoquercitrin * Molecular weight was calculated for the most abundant isomer determined from the natural abundance of 13C. Fig. 6. HPLC analyses of the enzymatically modified rutins A and B and the enzymatically modified isoquercitrin C (A)-(C) Typical chromatograms of samples A-C. R=rutin. I=isoquercitrin. (D) The calibration curve for rutin ed. Rutin ethanol solution was used as the standard because it is probable that all the glycosides in question have the same molar absorption coefficient as that of rutin. The calibration curve for rutin showed linearity in the range of 0-5 nmol (Fig. 6D), which was enough for the analyses of all peaks of interest. The results are shown in Fig. 6 and Table 4. Rutin was the most abundant in sample A, and the content decreased with increasing length of the sugar chain. In the case of sample C, IG1 and IG2 were more abundant than isoquercitrin. The sum of the molar amounts of the constituents represents the molar amount of quercetin aglycon, which probably determines the antioxidative activity of the whole material. Sample A turned out to have a lesser amount of aglycon than the other two samples.

7 60 J. Food Hyg. Soc. Japan Vol. 41, No. 1 Table 4. Results of Quantitative Analyses sample C were restricted to glycosides with comparatively short sugar chains. Sample B contained a considerable amount of isoquercitrin and no RGns with n>1, which suggested the use of additional enzyme reactions in its manufacture. Finally, HPLC revealed that sample A had a lesser amount of quercetin aglycon than samples B and C. Analytical techniques used in this work, especially LC/MS, should be effective for other natural food additives manufactured by the use of similar enzyme reactions. Acknowledgement This work was supported by a grant from the Japan Health Sciences Foundation. References In summary, the chemical compositions of enzymatically modified isoquercitrin and enzymatically modified rutin (extract) were analyzed. First, the structures of IG1 and IG2, two major constituents of enzymatically modified isoquercitrin, were elucidated by NMR analysis for the first time. It was demonstrated that the 4-hydroxyl group of the glucose moiety of isoquercitrin was a-glucosylated by CGTase. Next, LC/ MS clarified the compositions of the three samples. It was revealed that sample A contains glycosides with very long sugar chains, whereas the constituents of 1) Suzuki, Y., Suzuki, K., Enzymatic formation of 4G-a-Dglucopyranosyl-rutin. Agric. Biol. Chem., 55, (1991). 2) Notification No. 120 (Apr. 16, 1996), Ministry of Health and Welfare, Japan. 3) Kometani, T., Nishimura, T., Nakae, T., Takii, H., Okada, S., Synthesis of neohesperidin glycosides and naringin glycosides by cyclodextrin glucanotransferase from an alkalophilic Bacillus species. Biosci. Biotech. Biochem., 60, (1996). 4) Constant, H. L., Slowing, K., Graham, J. G., Pezzuto, J. M., Cordell, G. A., Beecher, C. W. W., A general method for the dereplication of fiavonoid glycosides utilizing high performance liquid chromatography/mass spectrometric analysis. Phytochem. Anal., 8, (1997). 5) Tinke, A. P., van der Hoeven, R. A. M., Niessen, W. M. A., van der Greef, J., Vincken, J.-P., Schols, H. A., Electrospray mass spectrometry of neutral and acidic oligosaccharides: methylated cyclodextrins and identification of unknowns derived from fruit material. J. Chromatogr., 647, (1993). 6) Iida, S., Yumoto, T., Gunji, Y., Takaya, I., Japan Kokai Tokkyo Koho, (Aug. 10, 1993). 7) Iida, S., Yumoto, T., Gunji, Y., Takaya, I., Japan Kokai Tokkyo Koho, (Jan. 28, 1997).