constituents of enzymatically modified isoquercitrin and

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: 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan; *2San-Ei Gen F. F. I., Inc.: 1-1-11, Sanwa -cho, Toyonaka-shi, Osaka 561-8588, Japan)

Enzymatically modified isoquercitrin and enzymatically modified rutin (extract) were analy-zed 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 gly-cosides, isoquercitrin and rutin (Fig. 1), have strong antioxidative activity, but their use is limited because of their poor solubility in water. Enzymatically mod-ified isoquercitrin and enzymatically modified rutin (ex-tract) are water-soluble modifications of these antioxi-

dants, which are manufactured by transglycosylation with cyclodextrin glucanotransferase (CGTase)'. These natural food additives are included in the List of Exist-ing Food Additives2). In the list, however, enzymat-ically modified isoquercitrin and enzymatically modi-fied rutin (extract) are merely described as being com-

posed mainly of a-glucosylisoquercitrin and a-glu-cosylrutin, 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 enzymat-

ically 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 commer-cial products were analyzed to determine their chemical compositions. LC/MS was used for separation and detection of species of different sizes. Finally, quantita-tion 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 enzymat-ically modified isoquercitrin (one product, designated as

February 2000 Enzymatically Modified Isoquercitrin and Rutin 55

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 chemi-cals were of reagent or HPLC grade. Ultrapure water

(>18 MQcm) prepared with a Milli-Q SP Reagent Water System (Millipore) was used throughout the experi-ment.

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 con-taining 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 Scien-tific); flow rate, 3 mL/min; temperature, ambient; detec-tion, 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 soy-

beans (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 Sci-

ence); flow rate, 0.05 mL/min; temperature, 40C; injec-tion volume, 5aL; detection, 254nm; mobile phase, 30% methanol containing 3.5% acetic acid.

Quantitation by HPLC Samples were dissolved in water at the concentra-

tions 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 mole-

cules, structure elucidation of the major constituents of enzymatically modified Isoquercitrin was attempted. Two major constituents of sample C, which were desig-nated IG1 and IG2, were isolated and subjected to NMR

Fig, 1. Structures of rutin, Isoquercitrin and their a-glucosylated derivatives

The structures of IGns were elucidated in this paper.


analyses.

Two glycolytic enzymes were utilized to increase the

content of the desired constituents. Glucoamylase,

which hydrolyzes terminal 1,4-linked a-D-glucose resi-

dues 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 non-

reducing 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 ano-meric proton signal at 5.18 ppm, which was not found in the spectrum of isoquercitrin and was therefore as-signed 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 demonstrat-ed 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 sig-nals (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 correla-tion 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-

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.


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 isoquerci-trin 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 molec-ular 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 detect-ed 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, re-spectively. 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 proc-esses 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 condi-tions of the transglycosylation reaction, the purification method, and so on. Differences between samples A and B, both of which are classified as enzymatically mod-ified rutin (extract), are also evident. Sample B con-tained 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

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

UV chromatogram


may be possible. LC/MS, which can reveal the compo-

sition 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 be-

tween 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 glyco-

side 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-

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

uV chromatogram


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 isoquer-citrin. The sum of the molar amounts of the constitu-

ents represents the molar amount of quercetin agly-

con, which probably determines the antioxidative activ-

ity of the whole material. Sample A turned out to have

a lesser amount of aglycon than the other two samples.

Table 3. Detected Ions in LC/ESI-MS

* Molecular weight was calculated for the most abun-

dant isomer determined from the natural abundance of 13C.

Fig. 5. Preparation of enzymatically modified rutin and

enzymatically modified isoquercitrin

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


In summary, the chemical compositions of en-zymatically modified isoquercitrin and enzymatically modified rutin (extract) were analyzed. First, the struc-tures of IG1 and IG2, two major constituents of en-zymatically 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

sample C were restricted to glycosides with compara-

tively short sugar chains. Sample B contained a consid-

erable 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 sam-

ples 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.

Table 4. Results of Quantitative Analyses

References

1) Suzuki, Y., Suzuki, K., Enzymatic formation of 4G-a-D-

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glycosides by cyclodextrin glucanotransferase from an alkalophilic Bacillus species. Biosci. Biotech. Biochem., 60, 645-649 (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

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5) Tinke, A. P., van der Hoeven, R. A. M., Niessen, W. M. A., van der Greef, J., Vincken, J.-P., Schols, H. A., Electro- spray mass spectrometry of neutral and acidic oligosac- charides: methylated cyclodextrins and identification of unknowns derived from fruit material. J. Chromatogr., 647, 279-287 (1993).

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constituents of enzymatically modified isoquercitrin and

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