Characterization and quantification of flavonoids and saponins in adzuki bean (Vigna angularis L.) by HPLC–DAD–ESI–MSn analysis
BackgroundBioactive activities of adzuki bean have been widely reported, however, the phytochemical information of adzuki bean is incomplete. The aim of this study was to characterize and quantify flavonoids and saponins in adzuki bean. High performance liquid chromatography with diode array detection and electro spray ionization-tandem multi-stage mass spectrometry (HPLC–DAD–ESI–MSn) were applied to do qualitative and quantitative analyses.ResultsA total of 15 compounds from adzuki bean were identified by HPLC–DAD–ESI–MSn. Among 15 compounds identified, four flavonoids (catechin, vitexin-4″-O-glucoside, quercetin-3-O-glucoside, and quercetin-3-O-rutinoside) and six saponins (azukisaponin I, II, III, IV, V, and VI) in adzuki bean were further quantified by external calibration method using HPLC–MS with the program of time segment and extract ion chromatogram (EIC) analysis.ConclusionsCurrent qualitative and quantitative method based on HPLC and MS technique provides a scientific basis for in vitro and in vivo pharmacological study in the future.Graphical abstractIsolation and characterization of flavonoids and saponins from adzuki bean.
<!>Introduction<!>Materials<!>Chemicals and reagents<!><!>High performance liquid chromatography analysis<!>Electro spray ionization-tandem multi-stage mass spectrometry (ESI–MSn) analysis<!>Optimization of sample preparation<!><!>Analysis of flavonoids in adzuki bean by HPLC–ESI–MSn<!><!>Analysis of saponins in adzuki bean by HPLC–ESI–MSn<!><!>Analysis of saponins in adzuki bean by HPLC–ESI–MSn<!><!>Quantification of flavonoids and saponins in adzuki bean<!><!>Conclusions
<p>Isolation and characterization of flavonoids and saponins from adzuki bean.</p><!><p>Adzuki bean is mainly produced and consumed in China and several other countries in East Asia. It has been used as a diuretic, antidote, and remedy for dropsy and beriberi in traditional Chinese medicine and also used as food for thousands of years. Extensive bioactivities of adzuki bean, such as anti-tumor [1, 2], anti-diabetes, [3, 4], antioxidant [5–8], and hepatoprotective effect [9] have been reported. These bioactivities are contributed by chemical constituents in beans, mainly including flavonoids and saponins. The previous articles showed that adzuki bean contained flavonoids such as (+) epicatechin, (+) catechin, quercetin, vitexin or their derivatives; [6, 10, 11] and saponins, such as azukisaponin I, II, III, IV, V, and VI [12] and AZ I [13], II, III, and IV [14]. The information, especially saponin information on adzuki bean, is incomplete, the name and structure of "AZ" and azukisaponin are confused. Moreover, there are limited articles in recent years to systematically and comprehensively investigate flavonoids and saponins of adzuki bean. Therefore, the present study aimed to establish a method to separate individual flavonoids and saponins from adzuki bean, characterize their chemical structures by HPLC–DAD–ESI–MSn, and further quantify them by HPLC–MS.</p><!><p>Adzuki beans (Vigna angularis L.) were purchased from local market in Changchun, Jilin Province, and identified by Prof. Jinming Mu of Faculty of Agronomy in Jilin Agricultural University.</p><!><p>Chromatographic grade acetonitrile and methanol were purchased from Merck (Darmstadt, USA). (+) Catechin, (+) epicatechin, quercetin-3-O-rutinoside, quercetin-3-O-glucoside and vitexin-4″-O-glucoside were purchased from Sigma (St. Louis, MO, USA). Saponin standards of azukisaponin I, II, III, IV, V, and VI were isolated in our lab. Other chemicals, such as ethanol, methanol, and acetone were of analytical grade. Macro porous resins AB-8 were supplied by Nankai University. Polyamide resin was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).</p><!><p>Extraction and isolation scheme of total extract, flavonoids and saponins from adzuki bean</p><!><p>The chemical constituents of adzuki bean total extract (ABTE), adzuki bean flavonoids (ABF) and adzuki bean saponins (ABS) were identified by liquid chromatography-ion trap mass spectrometry. HPLC analysis was performed on an Agilent 1100 series HPLC system equipped with degasser, binary pump, diode array detector and auto-sampler (San Francisco, USA). The separation was performed on a Phenomenex C8 column (150 × 2.0 mm, 5 μm). Gradient elution was performed using water containing 10 mM ammonium acetate (A) and acetonitrile (B). Initial conditions were 10% B for 10 min, changed to 15% B at 30 min and 25% B at 45 min, and then 35% B at 55 min, 45% B at 60 min and 55% B at 70 min. Flow rate was set at 0.2 mL/min, and UV absorption was measured at wavelength of 205 nm and 262 nm for saponins and flavonoids, respectively. The sample injection volume was 10 μL.</p><!><p>ESI–MS analysis was carried out on an Esquire 4000 ion trap mass spectrometer (Bruker–Daltonics, Bremen, Germany) with an electrospray ionization (ESI) interface. The instrument was operated at an ionization voltage of +4000 V and source temperature of 300 °C. Nitrogen was used as nebulizer gas at 30 psi and drying gas at a flow rate of 9 L/min. Collision energy was optimized for each compound. Three time segments were used in mass spectrometric acquisition in order to optimize the instrumental parameters for each compound to increase the peak intensity. The full scan of ions ranging from m/z 100 to m/z 1200 in the negative ion mode was used. Retention times and MS chromatograms of all flavonoids and saponins were confirmed by authentic standards, respectively. The HPLC chromatograms and total ion chromatograms (TIC) were obtained using the above method.</p><!><p>Flavonoids distribute in nature widely, especially in a large number of biologically active natural products. Most flavonoids exhibit two major maximum UV absorption wavelengths, namely the range of 240–285 nm and the range of 300–400 nm. While most saponins exhibit no ultraviolet absorption. The current results revealed the presence of flavonoids mainly in 45% ethanol fractions and the presence of saponins mainly in 80% ethanol fractions by AB-8 resin column. It was reported that AB-8 resin was good at separating chemical constituent according to the polarity [22]. After that, polyamide column was employed to further purify the flavonoids. 40% Ethanol eluent from polyamide column was obtained, and the fraction was rich in flavonoids. Precipitation with methanol–acetone was applied to further separate flavonoids and saponins from adzuki bean. Flavonoids existed in the supernatant fraction, while saponins presented in the precipitate fraction. Finally, adzuki bean total extract (ABTE), adzuki bean flavonoids (ABF) and adzuki bean saponins (ABS) (Fig. 1) were obtained and utilized for HPLC–DAD–ESI–MSn analysis.</p><!><p>HPLC–DAD chromatograms of adzuki bean extracts. a HPLC–DAD chromatogram of adzuki bean total extract at 205 and 262 nm, respectively; b HPLC–DAD chromatogram of adzuki bean flavonoids at 262 nm; c HPLC–DAD chromatogram of adzuki bean saponins at 205 nm</p><p>HPLC–ESI–MS total ion chromatograms of adzuki bean extracts. a HPLC–ESI–MS total ion chromatogram of adzuki bean total extract; b HPLC–ESI–MS total ion chromatogram of adzuki bean flavonoids; c HPLC–ESI–MS total ion chromatogram of adzuki bean saponins</p><p>HPLC–ESI–MSn data of identified flavonoids and saponins in adzuki bean</p><p>Chemical structures of flavonoids in adzuki bean identified by HPLC–ESI–MSn</p><p>ESI (−) MS, MS2, and MS3 spectra of identified flavonoids in adzuki bean. Peak 1 (A): A-1 MS spectrum of peak 1 ([M−H]−), A-2 MS2 spectrum of the ion at m/z 451. Peak 2 (B): B-1 MS spectrum of peak 2 ([M−H]−). Peak 3 (C): C-1 MS spectrum of peak 3 ([M−H]−), C-2 MS2 spectrum of the ion at m/z 613, C-3 MS3 spectrum of the ion at m/z 451. Peak 4 (D): D-1 MS spectrum of peak 4 ([M−H]−), D-2 MS2 spectrum of the ion at m/z 451</p><p>ESI (−) MS, MS2, and MS3 spectra of identified flavonoids in adzuki bean. Peak 5 (E): E-1 MS spectrum of peak 5 ([M−H]−), E-2 MS2 spectrum of the ion at m/z 625, E-3 MS3 spectrum of the ion at m/z 463. Peak 6 (F): F-1 MS spectrum of peak 6 ([M−H]−), F-2 MS2 spectrum of the ion at m/z 739, F-3 MS3 spectrum of the ion at m/z 593. Peak 7 (G): G-1 MS spectrum of peak 7 ([M−H]−)</p><p>ESI (−) MS, MS2, and MS3 spectra of identified flavonoids in adzuki bean. Peak 7 (G): G-2 MS2 spectrum of the ion at m/z 609, G-3 MS3 spectrum of the ion at m/z 463. Peak 8 (H): H-1 MS spectrum of peak 8 ([M−H]−), H-2 MS2 spectrum of the ion at m/z 463. Peak 9 (I): I-1 MS spectrum of peak 9 ([M−H]−), I-2 MS2 spectrum of the ion at m/z 593</p><!><p>For peak 9, the de-protonated molecular ion [M−H]− was at m/z 593(Fig. 7I-1), which molecular weight can be 594. In the ESI–MS2 spectrum (Fig. 7I-2), the fragment ions at m/z 431 and m/z 413 were the daughter ions from the precursor ion m/z 593. Based on the above information and the retention time of the standard vitexin and vitexin-4″-O-glucoside, peak 9 was finally confirmed to be vitexin-4″-O-glucoside. Similar to peak 6, the daughter ions m/z 431 and m/z 413 were also observed in the ESI–MS2 spectrum (Fig. 6F-2).</p><!><p>Chemical structures of saponins in adzuki bean identified by HPLC–ESI–MSn</p><p>ESI (−) MS, MS2, and MS3 spectra of identified saponins in adzuki bean. Peak 10 (A): A-1 MS spectrum of peak 10 ([M−H]−), A-2 MS2 spectrum of the ion at m/z 971, A-3 MS3 spectrum of the ion at m/z 809. Peak 11 (B): B-1 MS spectrum of peak 11 ([M−H]−), B-2 MS2 spectrum of the ion at m/z 1133, B-3 MS3 spectrum of the ion at m/z 971; B-4 MS3 spectrum of the ion at m/z 809</p><p>ESI (−) MS, MS2, and MS3 spectra of identified saponins in adzuki bean. Peak 11 (B): B-5 MS3 spectrum of the ion at m/z 795. Peak 12 (C): C-1 MS spectrum of peak 3 ([M−H]−), C-2 MS2 spectrum of the ion at m/z 941, C-3 MS3 spectrum of the ion at m/z 795; C-4 MS3 spectrum of the ion at m/z 633. Peak 13 (D): D-1 MS spectrum of peak 13 ([M−H]−), D-2 MS2 spectrum of the ion at m/z 795, D-3 MS3 spectrum of the ion at m/z 633</p><!><p>The retention time of peak 12 was 63.2 min and the molecular ion was at m/z 941 (Fig. 10C-1). CID of the molecular ion of peak 12 produced three predominant fragments at m/z 795 ([M−H-Rha]−), m/z 633 ([M−H-Rha-Glc]−), and m/z 457 ([M−H-Rha-Glc]−) (Fig. 10C-2). Its MS3 spectrum at m/z 795 exhibited the fragment ions at m/z 633 and m/z 457, in which the ion m/z 795 lost a glucosyl, and a glucosyl with a glucuronic residue (Fig. 10C-3). MS3 spectrum at m/z 633 also produced the fragment ion m/z 457, which lost a glucuronic residue from the m/z 633 (Fig. 10C-4). It was identified to be azukisaponin V, which was consistent with the previous articles [24, 28, 29].</p><p>The retention time of peak 13 was 64.9 min, and the precursor ion was at m/z 795 (Fig. 10D-1), which suggested its molecular weight was 796. Its MS2 spectrum at m/z 795 and MS3 spectrum at m/z 633 were shown in Fig. 10D-2 and D-3, respectively. The identification of azukisaponin II of peak 13 was based on the above information and the similarity of MS2 with those reported by Kinjo et al. [24, 30].</p><!><p>ESI (−) MS, MS2, and MS3 spectra of identified saponins in adzuki bean. Peak 14 (E): E-1 MS spectrum of peak 14 ([M−H]−), E-2 MS2 spectrum of the ion at m/z 779, E-3 MS3 spectrum of the ion at m/z 617. Peak 15 (F): F-1 MS spectrum of peak 15 ([M−H]−), F-2 MS2 spectrum of the ion at m/z 809, F-3 MS3 spectrum of the ion at m/z 647</p><!><p>The molecular ion of peak 15 was at m/z 809 ([M−H]−) (Fig. 11F-1), and the molecular weight of peak 15 was 810. The fragment ion of m/z 647 indicated the loss of a glucose residue and m/z 471 indicated the losses of a glucose residue and a glucuronic residue. The detailed results were found in the MS2 spectrum at m/z 809 (Fig. 11F-2). In its MS3 spectrum, the main daughter ion at m/z 471 ([M−H-Glc-GlcA]−) was found from the fragment ion at m/z 647 (Fig. 11F-3). Moreover, it was consistent with the results reported by Kitagawa et al. [12]. Finally, peak 15 was identified as azukisaponin III.</p><!><p>Chemical structures of Az saponins from adzuki bean</p><p>(Adopted from Iida et al. [13, 14])</p><!><p>The program of time segments of MS analysis was employed to enhance sensitivity for flavonoids and saponins analysis. Among 15 compounds identified, four flavonoids (catechin, vitexin-4″-O-glucoside, quercetin-3-O-glucoside, and quercetin-3-O-rutinoside) and six saponins (azukisaponin I, II, III, IV, V, and VI) in adzuki bean samples were further quantified by external calibration using HPLC–MS methods with the program of "time segments" and extract ion chromatogram (EIC) analysis.</p><p>For making a standard curve of flavonoids, seven standard working solutions with 1, 2, 5, 20, 50 and 80, and 100 ng/mL were made by diluting from high concentration stock solutions, and analyzed by HPLC–DAD–MSn using the above conditions, sequentially. Seven levels of saponins standard working solutions with 2, 5, 10, 40, and 60, 80, and 100 ng/mL were used to construct the standard curves. The curve of peak area (Y) versus flavonoid standard concentration (X) was plotted. The linear regression equation were Y = 42,769 X + 3 × 106, (catechin, R2 = 0.994), Y = 2 × 106 X + 5 × 106, (quercetin-3-O-rutinoside, R2 = 0.9976), Y = 2 × 106 X + 2 × 107, (quercetin 3-glucoside, R2 = 0.9943), Y = 3 × 106 X + 7 × 107, (vitexin-4″-O-glucoside, R2 = 0.9969), Y = 2 × 106 X + 4 × 107, (azukisaponin IV, R2 = 0.9916), Y = 3 × 106 X + 2 × 107, (azukisaponin VI, R2 = 0.9906), Y = 2 × 106 X + 3 × 107, (azukisaponin V, R2 = 0.9985), Y = 4 × 106 X + 2 × 107, (azukisaponin II, R2 = 0.9923), Y = 1 × 106 X + 4 × 107, (azukisaponin I, R2 = 0.9911), and Y = 1 × 106 X − 1 × 107, (azukisaponin III, R2 = 0.9909), respectively.</p><!><p>Flavonoids and saponins contents in extracts from Adzuki Bean</p><p>ABTE adzuki bean total extract, ABF adzuki bean flavonoids, ABS adzuki bean saponins, ND not detected</p><!><p>Flavonoids and saponins of adzuki bean have been produced by column chromatography and solvent precipitation. The present study has established a powerful method using HPLC–DAD–ESI–MSn in electro spray negative mode to separate and characterize nine flavonoids and six saponins in adzuki bean rapidly. A simple and sensitive method has been established for quantification of flavonoids and saponins in adzuki bean samples. Current preparation and analysis of flavonoids and saponins from adzuki bean could promote pharmacological experiments and attain much more reasonable experimental results.</p>