Glycosidic Linkage from Green Mature Acerola (Malpighia Emarginata DC.) Fruit

60513 (161)

Biosci. Biotechnol. Biochem., 71, 60513-1–6, 2007

Isolation and Characterization of a Novel Flavonoid Possessing a 4,200-Glycosidic Linkage from Green Mature Acerola (Malpighia emarginata DC.) Fruit

y Masakazu KAWAGUCHI, Hideya TANABE, and Kenichi NAGAMINE

Research & Development Division, Research & Development Center, Nichirei Biosciences Inc., 1-52-14 Kumegawa-cho, Higashimurayama-shi, Tokyo 189-0003, Japan

Received September 21, 2006; Accepted February 10, 2007; Online Publication, May 7, 2007 [doi:10.1271/bbb.60513]

The novel flavonoid, leucocyanidin-3-O- -D-glucoside, analyzed the volatile flavor constituents of acerola fruit possessing a 4,200-glycosidic linkage was isolated from by GC–MS, and elucidated the components of acerola green mature acerola (Malpighia emarginata DC.) puree fruit.5) In terms of function, it has been reported that an and given the trivial name ‘‘aceronidin.’’ To examine acerola extract had an inhibitory effect on NO produc- theAdvance functions of aceronidin, its antioxidative activity View and tion in mouse macrophage-like cells and an antitumor both its -glucosidase and -amylase inhibition activ- effect against lung cancer.6,7) Hanamura et al. have also ities, as a potential inhibitor of the sugar catabolic reported the isolation of several flavonoids from red enzyme, were evaluated against those of taxifolin, mature acerola fruit and their functional characteriza- catechin, isoquercitrin and quercitrin which each have tion.8) a similar structure. The 1,1-diphenyl-2-picrylhydrazyl After acerola fruit has completely matured to about (DPPH) radical quenching activity of aceronidin was 2 cm in diameter, its color gradually changes from green stronger than that of -tocopherol and comparable to to bright red in about 4 days. It has been confirmed that that of flavonoids. In the yeast -glucosidase inhibitory this change in color is caused by an increase in the assay, aceronidin showed significantly greater inhibition flavonoids, cyanidin-3-rhamnosideProofs (C3R) and pelargo- than the other flavonoids tested. In the human salivary nidin-3-rhamnoside (P3R), that are not detectable in -amylase inhibitory assay, aceronidin showed inhib- green mature acerola fruit. On the basis of these results, ition activity. Taken together, these results indicate it has been clarified that an alteration in part of the poly- aceronidin to be a potent antioxidant that may be phenol constituents takes place as acerola fruit ripens. valuable as an inhibitor of sugar catabolic enzymes. Green mature acerola fruit contains a higher amount of vitamin C than red mature fruit (data not shown). Key words: acerola; flavonoid; glycosidic linkage; anti- Although there are few studies on its polyphenol com- oxidant; sugar catabolic enzyme inhibition position, we have confirmed some polyphenols, querci- trin (quercetin-3-rhamnoside) and p-coumaric acid by Polyphenols, which are ubiquitous components of HPLC, its absorption spectrum and TLC analysis. We plants, are well known to have antioxidative properties. isolated in this present study a polyphenol to investigate Many plant constituents have recently been evaluated other polyphenols contained in green mature acerola for such predicted functions as antioxidative activity, fruit and clarified a novel flavonoid from the result of apoptosis induction and some enzyme inhibition abil- an NMR analysis. We report here the isolation of this ities.1–4) distinctive flavonoid from a puree of green mature Acerola, which originally comes from a West Indian acerola fruit and the evaluation of its functions in vitro, island in the Atlantic ocean, is a fruit containing a large including its antioxidative activity against both DPPH amount of vitamin C and abundant fructose and malic radicals and linoleic acid, and its inhibitory activity acid, and has high nutritional value, making acerola fruit against both -glucosidase and -amylase. very beneficial to human health. Pino and Marbot have

y To whom correspondence should be addressed. Tel: +81-42-396-1464; Fax: +81-42-396-1730; E-mail: [email protected] Abbreviations: DPPH, 1,1-diphenyl-2-picrylhydrazyl; HPLC, high-performance liquid chromatography; TLC, thin-layer chromatography; NMR, nuclear magnetic resonance spectroscopy; ESI-MS, electrospray ionization mass spectrometry; IR, infrared spectroscopy; CD, circular dichroism; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; DEPT, distortionless enhancement by polarization transfer; DQF-COSY, double quantum filtered correlation spectroscopy; HSQC, heteronuclear single quantum coherence; NOE, nuclear overhauser effect; HMBC, heteronuclear multiple bond correlation 60513-2 M. KAWAGUCHI et al. Materials and Methods data: see Table 1. IR (KBr): 3402, 3314, 2959, 2883, 1633, 1612, 1123 cm1; CD (MeOH) nm ([]): 300 Samples. The green mature acerola (Malpighia emar- (55:7926), 290 (394.613), 280 (2714.6), 270 20 ginata DC.) puree was provided by Nichirei do Brazil (1366.88), 260 (553.825), 250 (198.409); ½D 46.86 Agricola (Recife, Brazil) and immediately stored at (c 1.03, MeOH). 24 C until needed. Assay for DPPH radical quenching activity of Chemicals. DPPH was purchased from Nacalai flavonoids. The DPPH radical quenching activity was Tesque (Kyoto, Japan). Taxifolin, quercitrin, catechin, measured by the method of Shimada et al.9) with some -glucosidase and -amylase were purchased from modifications. In brief, each reaction solution consisted Sigma (St. Louis, MO, USA). Isoquercitrin was pur- of a 100 mM acetate buffer (pH 5.5), the flavonoid sam- chased from Kanto Chemical Co. (Tokyo, Japan), and - ple in MeOH and 100 mM DPPH in EtOH. The reaction tocopherol was purchased from Wako Pure Chemical was started by adding a DPPH solution and, after in- Industries (Osaka, Japan). cubating for 30 min at 30 C, the absorbance was measured by a UV-2400PC spectrometer (Shimadzu, Isolation and purification of a novel flavonoid from Kyoto, Japan) at 517 nm. The IC50 value was calculated green mature acerola puree. The supernatant of the by using MeOH as a control. green mature acerola puree (44 kg) was concentrated by evaporation and mixed with Sepabeads SP70 (500 g; Assay for linoleic acid autoxidation inhibitory activ- Mitsubishi Chemical Corp., Japan) by the batch method. ity. The antioxidative activity against the autoxidation of The Sepabeads SP70 resin was then extensively washed linoleic acid was measured by the method of Osawa and with distilled water. The adsorbed extract was eluted Namiki10) with some modifications. In brief, each assay withAdvance 4000 ml of 10% (v/v) ethanol (EtOH). View After solution containing 0.5% linoleic acid, 40% (v/v) EtOH evaporation (150 g), the eluted fraction was passed and a 20 mM phosphate buffer (pH 7.0) was divided through an ODS-A column (300 g, 40 mm i.d. 500 between two glass bottles with screw caps, one being mm; YMC, Japan), extensively washed with distilled kept at 40 C and the other of 4 C for 3 weeks. An water and successively eluted with 2500 ml of 10% aliquot of each solution was taken out and mixed with (v/v) methanol (MeOH) and 2000 ml of 20% (v/v) 75% (v/v) EtOH, a 30% (w/v) ammonium thiocyanate MeOH. The resulting evaporated fraction (2.47 g) was solution and 20 mM FeCl2 solution containing 3.5% passed through a Sephadex LH-20 column (130 g, 2.5 (v/v) HCl. After precisely 3 min, the absorbance at 500 cm i.d. 100 cm; GE healthcare, USA), eluting with nm was measured with a spectrometer. The OD500 100% MeOH at a flow rate of 1.0 ml/min. Each fraction value was calculated from the difference between OD500 Proofs (15 ml) was analyzed by TLC (1-butanol:AcOH:H2O= of the sample kept at 40 C and that kept at 4 C. 4:1:5) and reversed-phase HPLC (Inertsil ODS-3 col- umn, 4.6 mm i.d. 250 mm; GL Sciences, Japan) to Assay for -glucosidase inhibitory activity. The - ascertain those containing the target flavonoid. HPLC glucosidase inhibition assay was done according to the was run at 35 C and at a flow rate of 1.0 ml/min with a method of Kim et al.11) with some modifications. Each linear gradient of MeOH for 55 min from 5% to 100% reaction solution consisted of 0.1 units/ml of -gluco- MeOH with monitoring at 280 nm. The relevant frac- sidase from Saccharomyces cerevisiae in a 10 mM tions were pooled and freeze-dried (0.14 g). The target phosphate buffer containing 0.2% BSA (pH 7.0), a 100 flavonoid was finally crystallized by leaving it to stand mM flavonoid sample in 10% (v/v) DMSO and 5.0 mM at 4 CinanH2O solution (8.8 mg). p-nitrophenyl--D-glucopyranoside in a 100 mM phos- phate buffer (pH 7.0). The enzyme reaction was started Structural determination. The high-resolution ESI- by adding the substrate solution and, after incubating for MS data was recorded with an LCT instrument (Waters, 10 min at 30 C, the absorbance at 405 nm was measured USA). Leucine-enkephalin was used as an internal with a microplate reader (model 550, Bio-Rad, USA). standard. The NMR spectrum was obtained with a The inhibition rate of -glucosidase was calculated by UNITY INOVA 500 instrument (Varian, USA). TMS using a 10% DMSO solution as the control. was used as an internal standard, and the isolated flavonoid was measured in CD3OD. The IR spectrum Assay for -amylase inhibitory activity. The -am- was determined with an FTS-135 spectrometer (Bio- ylase inhibition assay was carried out according to the Rad, USA), and the CD spectrum was measured with a method of Kim et al.11) with some modifications. Each JASCO J-820 circular dichroism spectrometer. The reaction solution consisted of 0.3 units/mlof-amylase specific rotation was measured with a SEPA-300 instru- from human saliva in a 10 mM phosphate buffer con- ment (Horiba, Kyoto, Japan). taining 0.2% BSA (pH 7.0), a 333 or 167 mM flavonoid Isolated flavonoid. UV–vis max 278 nm (MeOH); sample in 20% (v/v) DMSO and 1.67 mM 4-nitrophenyl- þ high-resolution ESI-MS: m=z 473.1064 (½M þ Na , -D-penta-(1 ! 4)-glucopyranoside in a 100 mM phos- 1 13 473.1060 calcd. for C21H22O11Na); H- and C-NMR phate buffer (pH 7.0). The enzyme reaction was started A Novel Acerola Flavonoid 60513-3 Table 1. 1H- and 13C-NMR Spectral Data for Aceronidin

Position 13C 1H

2 74.8 CH 5.31, J2;3 ¼ 11:0 Hz (d) 3 76.3 CH 4.25, J2;3 ¼ 11:0 Hz, J3;4 ¼ 3:4 Hz (dd) 4 68.6 CH 4.87, J3;4 ¼ 3:4 Hz (d) 4a 101.1 C 5 159.4 C 6 97.3 CH 5.99, J6;8 ¼ 2:3 Hz (d) 7 161.1 C 8 95.7 CH 5.80, J6;8 ¼ 2:3 Hz (d) 8a 157.9 C 10 130.0 C 0 2 116.1 CH 6.92, J20;60 ¼ 2:0 Hz (d) 30 146.5 C 40 147.1 C 0 5 116.3 CH 6.79, J50;60 ¼ 8:1 Hz (d) 0 6 121.2 CH 6.84, J20;60 ¼ 2:0 Hz, J50;60 ¼ 8:1 Hz (dd) 00 1 94.6 CH 4.63, J100;200 ¼ 7:9 Hz (d) 00 2 81.4 CH 3.27, J100;200 ¼ 7:9 Hz, J200;300 ¼ 9:5 Hz (dd) 00 3 74.9 CH 3.58, J200;300 ¼ 9:5 Hz, J300;400 ¼ 8:4 Hz (dd) 400 72.1 CH 3.35 (m) 500 79.9 CH 3.36 (m) 00 6 62.6 CH2 3.62, 3.81, J500;600 ¼ 5:3, 1.6 Hz, J600;600 ¼ 12:1 Hz (dd, dd)

1 13 Aceronidin was dissolved in CD3OD, and the H-NMR (499.8 MHz) and C-NMR (125 MHz) data were measured. AdvanceAbbreviations: d, doublet; dd, double doublet; m, multiplet View by adding the substrate solution and, after incubating for 60 min at 30 C, the absorbance at 405 nm was measured with a microplate reader. The inhibition rate of - 473.1064 [M+Na]+ amylase was calculated by using a 20% DMSO solution as the control. Results Proofs Isolation and structural determination of a novel 923.2102 [2M+Na]+ flavonoid from green mature acerola puree We have speculated that some flavonoids are present in green mature acerola from the results of HPLC and TLC. By treating the puree with Sepabeads SP70 and an ODS-A column, the component flavonoids were sub- stantially purified and a proportion of these flavonoids Fig. 1. High Resolution ESI-MS Data for the Isolated Flavonoid. with UV absorption at 280 nm was subsequently frac- The positive ion was observed under the following conditions: tionated by stepwise elution. Sephadex LH-20 separa- cone voltage, 30 V; desolvation temperature, 150 C; ionization tion with MeOH at a flow rate of 1.0 ml/min then led to temperature, 120 C. the selective isolation of the target flavonoid. As a result, 8.8 mg of a colorless flavonoid was isolated from 44 kg of the green mature acerola puree. MS data measurements. Twelve signals appeared in the In the high-resolution ESI-MS data, the ½M þ Naþ benzene ring region, six signals in the sugar region and signal was observed at m=z 473.1064 (Fig. 1). It was three signals in another region. The DQF-COSY spec- thus suggested that the molecular formula was C21H22- trum led to the partial structure of –CH (5.31 ppm)–CH O11. A signal observed at m=z 923.2102 was speculated (4.25 ppm)–CH (4.87 ppm)–. Furthermore, 4.25 ppm þ to be ½2M þ Na . Details of the chemical structure of (J2;3 ¼ 11:0 Hz, J3;4 ¼ 3:4 Hz) and 4.87 ppm (J3;4 ¼ this flavonoid were established from 1H- and 13C-NMR 3:4 Hz) 1H-NMR chemical shifts and the HSQC spectral measurements (Table 1). The 1H-NMR spectrum indi- data indicated a flavan-3,4-diol skeleton. cated that the isolated compound contained a 1, 2, 4- Data in the 1H-NMR spectrum show that sugar trisubstituted (6.79, 6.84 and 6.92 ppm) and a 1, 2, 3, 5- protons were in a high magnetic field (3.27–4.63 ppm). tetrasubstituted (5.8 and 5.99 ppm) benzene. In the 13C- The signal at 4.63 ppm was attributed to C-100, and its NMR spectrum, twenty-one signals were observed, this coupling constant (J100;200 ¼ 7:9 Hz) determined the axial- agreeing with the result from the high-resolution ESI- axial form between H-100 and H-200. The signal at 3.27 60513-4 M. KAWAGUCHI et al.

HO OH HO OH HO OH

2’ 5’ O O OH OH HO HO H O O H H H OH OH 1’’ 3 A B H O O CH2OH O HO OH 4 2’’ HO OH OH H H H OH O Fig. 2. Structure of Aceronidin. R HO C: R=Glucose O D: R=Rhamnose OH ppm was attributed to C-200, and one of its coupling Fig. 3. Structures of the Flavonoids Tested. constants (J200;300 ¼ 9:5 Hz) also indicated the axial-axial form between H-200 and H-300. Furthermore, NOE signals A, taxifolin; B, catechin; C, isoquercitrin; D, quercitrin were observed between H-100 and H-300 and between H- 100 and H-500. In addition, the hydrolysis of this flavonoid Table 2. DPPH Radical Quenching Activity of Aceronidin, the Other withAdvance trifluoroacetic acid produced D-glucose. View These Flavonoids and -Tocopherol results enabled the glycoside to be determined as -D- glucose. Compound EC50 value (mM) 00 00 The HMBC signals showed C-1 , C-2 and C-4 to be Aceronidin 17:47 0:06 00 respectively correlated with H-3, H-4 and H-2 . It was Taxifolin 11:08 0:05 thus suggested that there were two glycosidic linkages Catechin 7:76 0:21 between C-3 and C-100 and between C-4 and C-200. NOEs Isoquercitrin 8:79 0:03 were observed between H-200 and H-4, between H-3 and Quercitrin 8:31 0:05 -TOC 25:55 1:98 H-4 and between H-2 and H-100. The molecular structure of the compound shown in Fig. 2 was determined from Each reaction solution consisted of a 100 mM acetate buffer (pH 5.5), flavonoid and 100 mMProofsDPPH. After incubating for 30 min at 30 C, the these results. This flavonoid, possessing two glycosidic absorbance at 517 nm was measured. Each value in the table is the linkages between C-3 and C-100 and between C-4 and C- mean SD (n ¼ 3). 200 was identified as a novel flavonoid by this inves- tigation and we have therefore named it ‘‘aceronidin.’’ Inhibitory effect of aceronidin on -glucosidase DPPH radical quenching activity of aceronidin The inhibitory effect of aceronidin on yeast -glu- The antioxidative activity of aceronidin, -tocopherol cosidase is shown in Fig. 5. At a concentration of 100 natural antioxidant, and the related flavonoids, taxifolin, mM, aceronidin exhibited significant inhibitory activity catechin, isoquercitrin and quercitrin, was examined by as compared with the other flavonoids and was thus a DPPH radical quenching system (Fig. 3). The DPPH inferred to be an effective -glucosidase inhibitor. radical quenching activity is shown in Table 2. The EC50 values for aceronidin and -tocopherol were 17.47 Inhibitory effect on -amylase and 25.55 mM, respectively. Thus, the antioxidative The inhibitory activity of aceronidin on human activity of aceronidin was found to be stronger than salivary -amylase is shown in Fig. 6. The inhibition that of -tocopherol. Among the other flavonoids tested, rates for aceronidin were 24.5% and 34.0% at 167 mM however, the EC50 value for aceronidin was comparable and 333 mM, respectively (IC50 value of 820 mM). These to that of taxifolin, catechin, isoquercitrin and quercitrin. results confirm that aceronidin had -amylase inhibitory properties. However, it showed weak inhibitory activity Antioxidative activity of aceronidin for the autoxida- as compared with isoquercitrin and quercitrin. tion of linoleic acid The antioxidative activity of aceronidin towards the Discussion autoxidation of linoleic acid was measured by the ferric thiocyanate method with the results shown in Fig. 4. We isolated in this study a novel flavonoid (named Aceronidin exhibited significant antioxidative activity as aceronidin) from green mature acerola puree and compared with the control, its inhibition intensity being evaluated its functions of antioxidative activity and almost equal to that of the other flavonoids tested. sugar catabolic enzyme inhibitory activity. Aceronidin, A Novel Acerola Flavonoid 60513-5

2.5 Inhibition rate (%) 0 50 100

2.0 Aceronidin

333 µM 1.5 Taxifolin 167 µM 500 OD 1.0 Catechin

0.5 Isoquercitrin

0 Quercitrin 0 51015 2025 days Fig. 6. -Amylase Inhibition Activity of Aceronidin and the Other Fig. 4. Antioxidative Activity of Aceronidin and Other Flavonoids Flavonoids. Measured by the Inhibition of Linoleic Acid Peroxidation. Control ( ), aceronidin ( ), quercitrin ( ), isoquercitrin ( ), Each solution consisted of 0.3 units/mlof -amylase from human catechin ( ), taxifolin ( ). The linoleic acid autoxidation was saliva in a 10 mM phosphate buffer containing 0.2% BSA (pH 7.0), a flavonoid sample and 1.67 mM 4-nitrophenyl--D-penta-(1 ! 4)- measured by the ferric thiocyanate method. An aliquot of each glucopyranoside. After incubating for 60 min at 30 C, the absorb- solution kept at 40 C and 4 C, 75% (v/v) EtOH, a 30% (w/v) ance at 405 nm was measured. Each value in the figure is the Advanceammonium thiocyanate solution and 20 mM FeCl2 solution View contain- ¼ ing 3.5% (v/v) HCl were mixed. The absorbance at 500 nm was mean SD (n 3). measured after precisely 3 min. Each value in the figure is the mean SD (n ¼ 3). stances. The antioxidative intensity of a flavonoid is attributed to a 2,3-double bond, which possesses a catechol group in the B-ring and a 3-hydroxyl group and Inhibition rate (%) glycoside.12) Due to its glycosidic moiety and its 2,3- 050 100 single bond, we expected that aceronidin would not have strong antioxidative activity in comparison to the other Aceronidin flavonoids tested.Proofs Taxifolin In our -glucosidase assay, aceronidin showed the ** strongest inhibitory effect among the five flavonoids tested (Fig. 5). -Glucosidase inhibition activity has Catechin ** been reported for several polyphenols.13–15) It was assumed from these findings that both the glycoside Isoquercitrin * site and the aglycon site of the flavonoid would function in some roles of the enzyme. It is therefore postulated Quercitrin ** for aceronidin that a specific structure must participate in -glucosidase inhibition and additionally enhance its Fig. 5. -Glucosidase Inhibition Activity of 100 mM Aceronidin and inhibition activity. the Other Flavonoids. Concerning its inhibitory effect against -amylase, Each solution consisted of 0.1 units/ml of -glucosidase from the activities of luteolin and ellagitannin have been Saccharomyces Cerevisiae in a 10 mM phosphate buffer containing 0.2% BSA (pH 7.0), 100 mM of a flavonoid and 5.0 mM of p- reported, although the details remain to be elucidat- 11,16) nitrophenyl--D-glucopyranoside. After incubating for 10 min at ed. The result of this study showed that aceronidin 30 C, the absorbance at 405 nm was measured. Each value in the had low inhibitory activity (Fig. 6), suggesting that the figure is the mean SD (n ¼ 4). P < 0:05, P < 0:01. discriminative structure of aceronidin was not involved in the level of its inhibitory effect against -amylase. In conclusion, we isolated the novel flavonoid, whose structure was determined for the first time, is a aceronidin, from green mature acerola puree. Aceronidin relatively minor component in green acerola fruit. showed antioxidative activity, as well as inhibitory We confirmed that aceronidin had antioxidative activity against both -glucosidase and -amylase. The activity equivalent to that of the other flavonoids tested inhibition of -glucosidase and -amylase would lead to in assays for DPPH radical quenching and inhibition of a retardation of carbohydrate digestion. These results linoleic acid autoxidation (Table 2 and Fig. 4). 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