Large-Scale Isolation of Three O-Methyl Anthocyanins from Bilberry (Vaccinium myrtillus L.) Extract

Abstract

Three O-methyl anthocyanidin 3-O-β-D-glucopyranosides were isolated from bilberry extract on a large-scale basis together with two non O-methyl analogues. Anthocyanidin 3-O-β-D-galactopyranosides were removed from bilberry extract together with parts of anthocyanidin 3-O-α-L-arabinopyranosides after treatment with β-galactosidase. The remaining arabinopyranosides were removed by applying acid catalytic hydrolysis. The amounts of anthocyanins recovered as flavylium trifluoroacetic acid salt were as follows: 630 mg for petunidin 3-O-β-D-glucopyranoside, 423 mg for peonidin 3-O-β-D-glucopyranoside, 588 mg for malvidin 3-O-β-D-glucopyranoside, 877 mg for delphinidin 3-O-β-D-glucopyranoside, and 742 mg for cyanidin 3-O-β-D-glucopyranoside.

Introduction

Anthocyanins have attracted much attention because of their abundance in a balanced daily diet¹⁾ and their multiple biological functions that have a positive influence on human health.²⁾ Six types of anthocyanidin—delphinidin, cyanidin, pelargonidin, petunidin, peonidin, and malvidin—and several anthocyanins (O-glycosides of anthocyanidin) are normally co-localized in plant materials.³⁾ Although biological functions of typical anthocyanins, 3-O-β-D-glucopyranoside of cyanidin (Cy3G) and delphinidin (Dp3G), were evaluated in an animal model⁴⁾ with authentic samples,⁵⁾ O-methyl anthocyanins have seldom been examined in vivo because of the limited availability of authentic samples. In the present study, we aimed to isolate three typical O-methyl anthocyanins, 3-O-β-D-glucopyranoside of petunidin (Pt3G), peonidin (Pn3G), and malvidin (Mv3G), on a large-scale by applying hydrolysis of anthocyanins co-localized in bilberry extract (BBE).

Results and Discussion

Previous studies have indicated that several plants are useful sources of O-methyl anthocyanin.^6,7) However, constitution of phytochemicals in wild plants varies according to plant strains⁸⁾ and environmental factors.⁹⁾ Bilberry (BB) contains a series of anthocyanins comprised of five types of anthocyanidin, except pelargonidin, and three types of O-glycosyl moieties attaching on the hydroxyl group of the 3-position of the anthocyanidin C ring¹⁰⁾ (Fig. 1). Powdered BBE is available commercially worldwide and its quality is controlled strictly by manufactures,¹¹⁾ thus it was trusted as a convenient and useful source of O-methyl anthocyanin. HPLC analysis revealed 15 separate pigments in BBE (Fig. 2A), although the resolution was too close for large-scale isolation of O-methyl anthocyanidin-glucopyranoside from the extract. The key step of the present method was selective reduction of 10 anthocyanins co-localized in BBE through a combination of enzymatic and acid catalytic hydrolysis to improve the resolution among O-methyl anthocyanidin-glucopyranosides.

Fig. 1. Structures of Anthocyanins in BBE

Fig. 2. Typical HPLC Chromatogram of BBE Detected at 520 nm before and after Treatment

A: BBE, B: One hour after treatment with β-galactosidase, C: GM. Roman numerals above peaks correspond to compounds in Fig. 1. *Indicates cyanidin (anthocyanidin).

The stability of anthocyanin in various pH buffers was evaluated and determined to be pH 3.5, the value at which the enzymatic reaction was able to proceed. After treatment of BBE with β-galactosidase (36400 U) for 1 h, anthocyanidin 3-O-β-D-galactopyranosides (II, V, VIII, XI, and XIV) were removed completely from the reactant (Fig. 2B). The new finding in the present study is that parts of anthocyanidin 3-O-α-L-arabinopyranosides (III, VI, IX, XII, and XV) were also removed by using excess amount of β-galactosidase. However, hydrolysis of arabinopyranosides was insufficient after treatment with 3640 U of β-galactosidase and targeting glucopyranoside decreased after treatment with 72800 U of β-galactosidase. The remaining arabinopyranosides in the reactant were removed by applying acid catalytic hydrolysis.¹²⁾ After the reaction, excess trifluoroacetic acid (TFA) was removed from the reactant together with ring fission products and polymerized products derived from anthocyanidin by passing the reactant through an HP-20 column chromatography (CC). Finally, 31 g of solid matter, which we refer to as “glucosides mixture” (GM), was obtained from 100 g of BBE. The anthocyanin content of the GM was estimated to be 37%, whereas BBE contained 33% anthocyanin.

Five pigments corresponding to anthocyanidin 3-O-β-D-glucopyranosides (I, IV, VII, X, and XIII) were detected in the GM (Fig. 2C). The pigment in the GM was separated into four fractions (fr. 1–fr. 4) using MCI CC. Each fraction except fr. 3 contained a single pigment (I for fr. 1, IV for fr. 2, and XIII for fr. 4). Thus, fr. 3 was separated further in two fractions (VII for fr. 3-1 and X for fr. 3-2) with LH-20 CC. By reducing the anthocyanin component in BBE, O-methyl anthocyanidin-glucopyranoside was almost the sole component present in each fraction after separation with CC. Five pigments were isolated with semi-preparative HPLC and recovered as flavylium TFA salt. The m/z values of molecular and product ion pairs of isolated pigments agreed well with anthocyanidin mono-glucopyranoside (Table 1). ¹H- and ¹³C-NMR analysis was performed for the structure determination of isolated pigments and the summaries are shown in Table 1. HPLC analysis revealed that the purity of anthocyanin isolated in the present study was >99.5%.

Table 1. Summary of m/z and NMR Data for Isolated Pigments^a)

a) Roman numerals in table correspond to compounds in Fig. 1. *Overlapped with trifluoroacetic acid signals.

The amounts of the purified pigments were determined to be the following: 630 mg for Pt3G, 423 mg for Pn3G, 588 mg for Mv3G, 877 mg for Dp3G, and 742 mg for Cy3G. Table 2 summarizes the recovery of each pigment obtained from 100 g of the BBE and from 31 g of the GM. The glucopyranoside content in the GM (11.51 g) decreased 30.9% from that of the BBE (16.66 g) during the hydrolysis. Conditions for enzymatic hydrolysis were optimized as described above, thus, we successfully saved the cost and time to obtain highly purified glucopyranosides compared to the previous reports.^13,14) If we shorten the time for acid catalytic hydrolysis, we may improve the recovery of glucopyranoside. Here, we succeeded to prepare three typical O-methyl anthocyanins (Pt3G, Pn3G, and Mv3G) on a large-scale basis for the first time, together with two non O-methyl analogues (Cy3G and Dp3G) by selective hydrolysis of anthocyanin components in BBE. Precise studies on the beneficial health effects of O-methyl anthocyanin are now in progress.

Table 2. Amount and Recovery of Anthocyanins Isolated from BBE and GM

Experimental

Reagents

β-Galactosidase (from Aspergillus oryzae) was purchased from Sigma-Aldrich Co. (Saint Louis, MO, U.S.A.). Other reagents including TFA were obtained from Wako Pure Chemical Corporation (Osaka, Japan). MCI-gel CHP 20P (70–150 µm) and HP-20 ion exchange resin were purchased from Mitsubishi Chemical (Tokyo, Japan). Sephadex LH-20 (25–100 µm) was obtained from Amersham Biosciences, Inc. (Buckinghamshire, U.K.). Commercially available BBE (Vaccinium myrtillus L.), Mirtoselect, was a kind gift of Indena Co., Ltd. (Milan, Italy). Authentic Cy3G was isolated from purple black rice as described in our previous work.¹⁵⁾

Methods

Analytical Method

Total anthocyanin contents in BBE and GM were estimated as the Cy3G equivalent at 510 nm absorbance (ε = 26300) using a Hitachi spectrophotometric meter U-1900 according to a method described previously.¹²⁾

Analysis of individual anthocyanin in the reactant was performed according to our previous method.¹⁰⁾ Briefly, aliquots of the samples were analyzed with an HPLC system (Hitachi L-7200, Tokyo, Japan) equipped with a Develosil ODS HG-5 column (4.6 × 150 mm). The anthocyanins were eluted with 20% aqueous methanol solution containing 0.5% TFA at a flow rate of 2.0 mL/min. The elution profile was monitored at 520 nm.

Preparation of GM

We dissolved 100 g of powdered BBE in 5 L of 50 mM sodium acetate buffer (pH 3.5), added 4 g (36400 U) of β-galactosidase to the reaction solution, and stirred for 1 h at 40 °C. After addition of 150 mL of TFA, acid catalytic hydrolysis of anthocyanins was carried out according to our previous method.¹²⁾ Briefly, the reaction solution prepared above was heated at 90 °C for 1 h with stirring, cooled to room temperature, passed through a paper filter (Advantech No. 2) and concentrated in vacuo at 40 °C. The reactant cocktail was diluted with distilled water, applied to a HP-20 CC (10 × 150 cm), washed well with distilled water, and pigments were eluted with 50% methanol aqueous solution. The fraction containing pigments was evaporated to dryness in vacuo at 40 °C and the resulting solid matter, GM, was obtained.

Purification of Anthocyanidin 3-O-β-D-Glucopyranoside

GM dissolved in distilled water was applied to MCI CC (7 × 100 cm) and anthocyanins were eluted by stepwise elution under gravity flow with increasing concentrations of methanol in distilled water (0 : 1→1 : 0). Fractions containing anthocyanin were collected, evaporated to dryness in vacuo at 40 °C, and stored in the refrigerator. The fraction containing compounds VII and X was dissolved in distilled water and separated with LH-20 CC (4.0 × 45 cm) in the same manner as described above. Anthocyanin fractions obtained above were dissolved in 0.1% TFA aqueous solution and aliquots of the solution were injected in a semi-preparative HPLC (Hitachi L-7200) using a Develosil ODS HG-5 column (20 × 250 mm). Anthocyanin was eluted with 29% aqueous methanol containing 0.1% TFA as elution solvent at a flow rate of 13 mL/min. The elution profile was monitored at 520 nm and the peak fractions containing pigment were collected, evaporated to dryness in vacuo at 40 °C, and re-dissolved in a small amount of TFA. The pigment solution was carefully dropped in an excess amount of diethyl ether (>10-fold by volume) to recover anthocyanin in stable flavylium TFA salt.

Identification of Anthocyanin

Identification of anthocyanin was performed by tandem time-of-flight (TOF) MS and NMR spectroscopy according to our method described previously.¹⁶⁾ Briefly, anthocyanin dissolved in methanol was subjected to MS performed with a Q–Tof Ultima (Micromass, Manchester, U.K.). Conditions for tandem TOF MS were as follows: A syringe pump (KD Scientific Inc., Holliston, MA, U.S.A.) was used for a constant infusion (300 µL) of the sample into the MS ion source. MS parameters used were as follows: 3.2 kV for capillary; 9.1 kV for reflection. Argon gas was used for collision at a pressure of 11 psi and applied voltage was 13 V. ¹H-NMR spectra of the anthocyanin in TFA-d₁ : CD₃OD-d₄ (1 : 10) was recorded on a JEOL-500 NMR spectrometer (500 MHz, JEOL, Tokyo, Japan) using tetramethylsilane as an internal reference. ¹³C-NMR spectra were run at 125 MHz.

Acknowledgments

The authors acknowledge Dr. Yasuo Shida, Yamanashi University, for measuring and analysis of tandem TOF MS for anthocyanins.

The authors also thank Indena Co., Ltd. for providing Mirtoselect.

Conflict of Interest

The authors declare no conflict of interest.

References

• 1) Scalbert A., Williamson G., J. Nutr., 130 (Suppl.), 2073S–2085S (2000).
• 2) Blesso C. N., Nutrients, 11, 2107–2110 (2019).
• 3) Strack D., Wray V., “The Flavonoids advances in research since 1986,” ed. by Harborne J. B., Chapman and Hall/CRC, London, 1994.
• 4) Iida H., Nakamura Y., Matsumoto H., Kawahata K., Koga J., Katsumi O., J. Ocul. Pharmacol. Ther., 29, 604–609 (2013).
• 5) Matsumoto H., Hanamura S., Kawakami T., Sato Y., Hirayama M., J. Agric. Food Chem., 49, 1541–1545 (2001).
• 6) Ogawa K., Sakakibara H., Iwata R., Ishii T., Sato T., Goda T., Shimoi K., Kumazawa S., J. Agric. Food Chem., 56, 4457–4462 (2008).
• 7) Brito A., Areche C., Sepulveda B., Kennelly E. J., Simirgiotis M. J., Molecules, 19, 10936–10955 (2014).
• 8) Gholizadeh-Moghadam N., Hosseini B., Alirezalu A., Phytochem. Anal., 30, 385–394 (2019).
• 9) Cheng G., He Y. N., Yue T. X., Wang J., Zhang Z. W., Molecules, 19, 13683–13703 (2014).
• 10) Ichiyanagi T., Hatano Y., Matsugo S., Konishi T., Chem. Pharm. Bull., 52, 628–630 (2004).
• 11) Gizzi C., Belcaro G., Gizzi G., Feragalli B., Dugall M., Luzzi R., Cornelli U., Eur. Rev. Med. Pharmacol. Sci., 20, 2418–2424 (2016).
• 12) Ichiyanagi T., Oikawa K., Tateyama C., Konishi T., Chem. Pharm. Bull., 49, 114–117 (2001).
• 13) Wang E., Yin Y., Xu C., Liu J., J. Chromatogr. A, 1327, 39–48 (2014).
• 14) Chorfa N., Savard S., Belkacemi K., Food Chem., 197 (Pt B), 1226–1234 (2016).
• 15) Ichikawa H., Ichiyanagi T., Xu B., Yoshii Y., Nakajima M., Konishi T., J. Med. Food, 4, 211–218 (2001).
• 16) Ichiyanagi T., Kashiwada Y., Shida Y., Sekiya M., Hatano Y., Takaishi Y., Ikeshiro Y., J. Agric. Food Chem., 61, 569–578 (2013).