2025 Volume 101 Issue 10 Pages 648-656
Red adzuki beans (Vigna angularis) are primarily used in producing sweet an-paste for use in Japanese confectionery, where the quality of the an-paste is evaluated based on its purple color. Catechinopyranocyanidins A and B (cpcA and cpcB) are seed coat pigments and might be responsible for the purple color of an-paste, thus, analyzing them in an-paste is crucial. However, no quantitative analyses of these compounds in an-paste have been reported. We examined extraction conditions for cpcA and cpcB from an-paste and established a quantitative analytical method. Additionally, we developed a pre-vacuum processing procedure for preparing purple an-paste. The more purple the an-paste was, the higher the content of cpcA and cpcB extracted from it. The purple color of an-paste originated from the catechinopyranocyanidins eluted from the seed coats upon boiling. Using the proposed procedure, we prepared more intensely purple wet sarashi-an compared with that generated via a conventional procedure.
The purple color of an-paste is due to catechinopyranocyanidins in seed coat
The major industrial use of red adzuki beans (V. angularis) in Japan is for producing reddish purple ‘an-paste,’ which is a main ingredient in traditional Japanese confectionery—wagashi.1)–6) A previous report by Kuroda and Wada in 1934 claimed that seed-coat pigments of red adzuki beans dissolved in hot water to produce the purple-colored an-paste1); however, detailed chemical characteristics of the pigment were not elucidated. In 2019, we reported that the red seed coat of adzuki beans contains two purple pigments—catechinopyranocyanidin A (cpcA; 1) and catechinopyranocyanidin B (cpcB; 2) (Fig. 1).7)–9) These are non-anthocyanin pigments with characteristics differing from those of anthocyanins; 1 and 2 are almost insoluble in water, exhibit purple color in strongly acidic to neutral media and readily decompose to produce colorless photo-degraded cpcs upon light irradiation.
Structure of catechinopyranocyanidin A (cpcA, 1) and catechinopyranocyanidin B (cpcB, 2).
An-paste is composed of an-particles (an-ryushi in Japanese), which are colorless seed cells (cotyledon cells) covered by thermally denatured cell walls and cytoplasmic membranes and filled with heat-alpha starch. For preparing an-paste, the red seed coat of boiled beans are removed and the obtained an-particles are washed with water several times to give wet sarashi-an (nama-an), unsweetened an-paste. Sugar is then added to this, followed by heating to obtained a sweetened an-paste—koshi-an.3)–6) It has been considered that during boiling of the beans, 1 and 2 in seed coats may transferred to give distinct purple-colored an-particles (Fig. 2).1),10) However, this coloring mechanism has not been clarified until now.
Photos of adzuki beans and wet sarashi-an. (A) Dried red adzuki beans (V. angularis cv. Erimo-shozu). (B) Split beans. The seed coat is pigmented, whereas the cotyledon cells are colorless. (C) Wet sarashi-an processed from adzuki beans. Scale bars: 1 cm.
Typically, an-paste is evaluated based on its color, flavor, and taste. Among these, color is one of the most important factors, particularly in high-quality handmade wagashi. Purple-colored an-paste is highly valued in this context.3)–5) Therefore, analysis of the purple pigments in an-paste is crucial for understanding the purple color. We previously reported a quantitative analytical method for 1 and 2 in dried adzuki beans.9) However, procedures for extracting and quantifying purple pigments 1 and 2 from an-paste have not been clarified. In this study, we examined the extraction procedure of 1 and 2 from sarashi-an and carried out a quantitative analysis of them. Additionally, the processing procedures and conditions for obtaining purple-colored sarashi-an have been experimented. Finally, a pre-vacuum processing procedure was developed for the desired purple-colored sarashi-an. By quantitatively evaluating sarashi-an, a correlation has been observed between the content of cpcA and cpcB and the purple color.
1 and 2 are highly sensitive to light irradiation, including indoor fluorescent light, therefore, all the analytical experiments, including extraction of pigments, evaporation, sample preparation for high-performance liquid chromatography (HPLC), and HPLC analysis were conducted in dark conditions or under red light.
2.2. Plant materials and chemicals. 2.2.1. Plant materials.Three cultivars of red adzuki beans (V. angularis), Erimo-shozu, Shumari, and Kita-roman, were donated by the Agricultural Research Department of the Hokkaido Research Organization, Tokachi Experimental Station, Japan. All the beans were stored at 5 °C prior to use.
2.2.2. Chemicals.1 was purified from red adzuki beans in accordance with our previous report.7)–9) The purity of 1 for quantitative analysis was determined to be 76.2% by elemental analysis.9) Acetonitrile (MeCN), ethyl acetate (EtOAc), and trifluoroacetic acid (TFA) were purchased from FUJIFILM Wako Pure Chemicals (Osaka, Japan). Methanol (MeOH) was purchased from Kanto Chemical Co. (Tokyo, Japan). Water was distilled from tap water. MeCN and MeOH were distilled before use. Solvents for HPLC analysis were filtered through membrane filters (DISMIC-CP and -HP, nonsterile, ADVANTEC, Tokyo, Japan) with a pore size of 0.45 µm.
2.3. Analytical methods. 2.3.1. HPLC analysis.Analytical HPLC was performed on a JASCO HPLC system (Jasco, Hachioji, Japan) comprising two pumps, along with a mixer, a degasser, a PDA detector, a column oven, and an autosampler, all controlled using ChromNAV 2.0 HPLC software (Jasco, Hachioji, Japan) on a personal computer, as reported previously.9) A reversed-phase column, PRAQUEOUS-AR-3 (2.0 mm i.d. ×150 mm) with an RP-AR-S guard cartridge (1.5 mm i.d. ×10 mm, Nomura Chemical, Seto, Japan) was employed. A linear gradient elution was applied using solvent A (10% (v/v) aqueous (aq.) MeCN with 0.5% (v/v) TFA) and solvent B (90% (v/v) aq. MeCN with 0.5% (v/v) TFA). The gradient conditions were as follows: isocratic elution of 30% (v/v) B in A from 0 to 7 min, followed by linear increase to 85% (v/v) B in A from 7 to 22 min. The flow rate was 0.2 mL/min, and the column temperature was set to 40 °C. All sample solutions were filtered using a DISMIC-13HP cartridges (Advantec, Tokyo, Japan), and 5 µL of each solution was injected. For calibration, dried cpcA (1) was weighed precisely in triplicate, and the individual samples were diluted with MeOH. Each sample was analyzed by HPLC in triplicate, and the obtained peak areas were used to construct a calibration curve. Triplicate or duplicate weighed samples were analyzed twice using HPLC, and the peak areas of 1 and 2 were calculated using ChromNAV 2.0 software. For quantification of 1 and 2, the same calibration curve was used.9)
2.3.2. Color difference.The color of an-paste was measured using a method standardized by the International Commission on Illumination (CIE) in 1976, adopted in Japan as JIS (JIS Z 8781-4, https://kikakurui.com/z8/Z8781-1-2012-01.html, Sept. 30th, 2025, in Japanese), and indicated with L*, a* and b* values. In the L*a*b* color space, lightness is represented by L*, and chromaticity, which indicates hue and saturation, is represented by a* and b*. In the color space, a* indicates red, −a* as green, b* as yellow, and −b* as blue. The larger the value, the more vivid the color. The each wet sarashi-an was settled and the color was measured using a spectrophotometer (SA-4000, Nippon Denshoku Industries Co., LTD, Tokyo, Japan) and expressed as L*, a*, and b* values. Water was added to 5 g of wet sarashi-an sample sufficient to cover the surface. The precipitated wet sarashi-an was placed in 30 mm diameter cells and irradiated using a halogen lamp operating (12 V and 50 W, NA55928).
2.4. Lyophilization.Lyophilization of the seed coat, wet sarashi-an, and extracts of seed-coat was carried out using vacuum freeze-drying equipment (TFD-550-8, Takara ATM Inc., Tokyo, Japan). Each sample was frozen at −40 °C, the frozen sample was placed into the instrument, then lyophilized to a temperature of 30 °C.
2.5. Extraction of cpcs (1 and 2) with H2O from seed-coat.Two hundred grams of dried red adzuki beans (V. angularis, cv. Shumari) and 800 g of water were placed in a container and left to stand at 25 °C for 18 h. After washing with water, the water-adsorbed beans were peeled to separate the seed coats and cotyledons. The wet seed coats, equivalent to 50 g dried beans each, were placed separate beakers filled with 250 g of water each and allowed to stand at a specific temperature and period (25 °C for 18 h, or 60 °C, 80 °C, and 100 °C for 30 min). Subsequently, the mixtures were filtered through strainers (hole diameter: 1.5 mm), and the extracts were lyophilized for HPLC analysis.
2.6. Production of an-paste. 2.6.1. General procedure for an production.Two hundred grams of dried red adzuki beans (V. angularis) and 800 g of water were added to a 5 L stainless steel pot, heated over a gas flame, and boiled for 15 min. After adding 200 g of cold water to each pot, the mixture was allowed to stand at room temperature for 20 min. Following separation from the soaking water (shibukiri treatment), the beans were boiled again in 1600 g of water for 60 min in low heat conditions. Additional water (∼100 g) was added intermittently to prevent the beans from drying. The boiled beans were mashed, and the seed coats were removed from the cotyledons using a strainer (hole diameter: 1.5 mm). The mixture was filtered through a sieve (hole diameter: 250 µm), and the resulting an-ryushi was washed with water several times and pressure-dehydrated to obtain wet sarashi-an.
2.6.2. General procedure for an production using water-soaked beans.Two hundreds grams of dried red adzuki beans (V. angularis) and 800 g of water were placed in a container and allowed to stand at 25 °C for 18 h. The water was discarded, and the beans were washed with water several times. The soaked beans were then processed in accordance with the procedure described in Section 2.6.1.
2.6.3. Pre-vacuum procedure for an production.13)Pre-vacuum cooking was conducted using a low-temperature and pressure-reducing cooker (Vide pro; EVC-221, Espec Inc., Osaka, Japan). The cooker has a gas-tight structure, and using a vacuum pump inside of the cooler can be kept reduced pressure. Using the cooker, cooking can be carried out under reduced pressure, or, by repeated creating a vacuum inside and then releasing it, oxygen contained in the immersion liquid can be removed. Two hundred grams of dried red adzuki beans (V. angularis) and 800 g of water were placed in a container and allowed to stand at 25 °C for 18 h and washed in accordance with the procedure described in Section 2.6.2. The soaked beans and 1600 g of water were added to the cooker. The cooker was evacuated to 10–30 kPa for 5 min, followed by release to atmospheric pressure using air. The treatment cycle was repeated five times, and then the beans were heated at 100 °C for 1 h under airtight conditions, allowing only steam to escape. Subsequently, the beans were mashed and wet sarashi-an was prepared following the procedure described in Section 2.6.1.
2.7. Extraction of cpcs from sarashi-an. 2.7.1. Preliminary trials of extracting cpcs from lyophilized sarashi-an.For preliminary trials for extracting cpcs, lyophilized sarashi-an produced by general procedure using cv. Shumari was used. To lyophilized sarashi-an (5 to 50 g) the desired solvent (H2O, EtOAc, or aq. EtOAc) was added, and the mixture was shaken or sonicated in the dark for the indicated duration. The extract was filtered, then, obtained supernatant was dried under reduced pressure, and the residue was dissolved in MeOH. The solution was analyzed using HPLC.
2.7.2. Established procedure for extracting of cpcs from lyophilized sarashi-an.Lyophilized sarashi-an (3.0 g) was placed in a brown sample tube (No. 7 L, Maruemu Corp., Osaka, Japan), and 10 mL of water was added to the tube. After manually shaking the tube for 1 min, the tube was stood at room temperature for 5 min, followed by addition of 25 mL of EtOAc. Again, the mixture was manually shaken for 1 min, then placed in a shaker (Personal-11, TAITEC Corp., Koshigaya, Japan) and shaken at 25 °C with 180 rpm for 18 h in dark condition. After filtration using a glass filter (G-4, Sibata Scientific Technology Limited, Tokyo, Japan), the filtrate was collected in brown glassware. The precipitate was returned to the sample tube, and 10 mL of EtOAc was added. The sample was shaken at 25 °C with 180 rpm for 1 h and filtered again. The same procedure was repeated two more times, and the combined filtrate was evaporated under reduced pressure. The residue was dissolved in MeOH (1.0 mL) for HPLC analysis.
In 2023, we reported a method for the extraction and HPLC analysis of cpcs from red adzuki beans.9) At that time, it was essential to use water-soaked beans, and EtOAc resulted in the highest extraction efficiency. First, in this study, 30 g of lyophilized sarashi-an was extracted using 80 mL of EtOAc as the solvent via sonication for 15 min, followed by standing for 45 min. The obtained extract was yellow in color and no cpcs were detected. Next, water-soaked sarashi-an was tested. To 10 g of lyophilized sarashi-an, 55 mL of water was added, and the mixture was allowed to stand at 25 °C for 18 h in the dark. After removing excess water by decanting, 50 mL of EtOAc was added to pre-soaked sarashi-an, sonicated for 15 min, followed by standing for 45 min. However, the extract was yellow and no cpcs were detected.
Then, we considered whether simultaneous adsorbing water into sarashi-an and pigment extraction might be effective. To 10 g of lyophilized sarashi-an, 50 mL of 50% aq. EtOAc was added, and after briefly shaking the bottle, the mixture was allowed to stand at 25 °C for 18 h. The extract appeared pale purple, indicating the presence of pigments 1 and 2 in the extracts. HPLC analysis revealed that the amount of cpcs was 0.68 µg/g lyophilized sarashi-an.
3.1.2. Establishment of procedure for extracting cpcs from lyophilized sarashi-an.Cpcs could be extracted by directly adding aq. EtOAc to lyophilized sarashi-an without any pre-soaking treatment. Based on these results, we investigated the effects of various extraction conditions, such as the water content of the solvent, ratio of solvent to sarashi-an, extraction temperature and duration, treatment during extraction, and the required number of repeated extraction times. First, the extraction efficiencies with sonication and standing-still conditions were compared. To 10 g of lyophilized sarashi-an 50 mL of 50% aq. EtOAc was added, and the mixture was sonicated for 15 min, followed by standing for 45 min, or left undisturbed for 18 h without any sonication. As shown in Table 1, the amounts of cpcs obtained were found to be considerably higher after the 18 h extraction process than after sonication treatment. Next, the conditions for the second and third extractions were optimized. Comparing the extraction solvents for the second extraction, EtOAc performed better than 50% aq. EtOAc, because the 50% aq. EtOAc yielded an emulsion that made it difficult to separate the supernatants from the residues. Quantitative analysis of cpcs in each extract revealed that, from the second extraction onward, the extraction efficiencies show no notable difference between ultrasonic and standing-still overnight treatments (Table 1).
| Times | Condition | Solvent | Period (h) | cpcs** (µg/g dried an-paste) |
|---|---|---|---|---|
| 1st | sonication*** | 50% EtOAc aq. | 1 | 0.004 ± 0.000 |
| 1st | stood overnight | 50% EtOAc aq. | 18 | 1.73 ± 0.02 |
| 2nd | stood overnight | EtOAc | 18 | 0.66 ± 0.03 |
| 1st | stood overnight | 50% EtOAc aq. | 18 | 1.55 ± 0.02 |
| 2nd | sonication*** | EtOAc | 1 | 1.17 ± 0.01 |
| 3rd | sonication*** | EtOAc | 1 | 0.16 ± 0.00 |
*Data represent averages from duplicate experiments.
**Total content of cpcA (1) and cpcB (2).
***Sonication for 15 min, followed by standing for 45 min.
Finally, the following extraction procedure was established as the optimized procedure for extracting cpcs from lyophilized sarashi-an. To 3.0 g of lyophilized sarashi-an, 10 mL of water was added, followed by the addition of 25 mL of EtOAc. The prepared mixture was shaken at 25 °C overnight. After separating the extract the following day, extraction was repeated three more times using 10 mL of EtOAc in each cycle by shaking for 60 min. The fifth and sixth extracts were analyzed using HPLC and they contained only trace amounts of cpcs. Therefore, a four-fold extraction is considered sufficient in quantitative analysis.
3.2. Elution of cpcs from seed coats of red adzuki beans in boiling water.The purple color of an-particle may be produced during boiling of red adzuki beans in water.3),5),10) Before boiling, the cotyledon cells are colorless (Fig. 2B), and they adsorb water to increase in size to approximately 100 to 200 µm depending on the type of bean and turn purple upon boiling in water (Fig. 2C).3),5),10) This strongly indicated that cpcs within the seed coats of red adzuki beans may eluted into boiling water and adsorbed onto the heat-denatured cell walls of an-particles. However, this procedure cannot be supported by previous studies because the analysis of the colorants in boiling water or an-paste is not reported previously. Therefore, the cpcs extracted from seed-coats into the water were examined.
After pre-soaking in water, the beans were peeled and separated into seed coats and cotyledons. The seed coats were soaked in water at 25 °C for 18 h or heated at temperatures ranging from 60 to 100 °C for 30 min. After filtration, the extract was lyophilized, and the cpcs in each extract were analyzed using HPLC (Table 2). The cpcs were successfully extracted using water at 25 °C and the mass of the extract increased with increasing extraction temperature. The total amount of 1 and 2 per gram extract reached a maximum at 80 °C, but the total amount of cpcs continued to increase with increasing temperature. (Table 2). These results demonstrate at the first that cpcs in the seed-coat were eluted into water during boiling.
| Temp. (°C) |
Period (h) |
Weight of extract (mg/100 g dried beans) |
cpcA | cpcB (µg/g dried beans)* |
Total | cpcA ratio (%)** |
|---|---|---|---|---|---|---|
| 25 | 18 | 200 | 0.20 ± 0.01 | 0.02 ± 0.002 | 0.22 ± 0.01 | 91 |
| 60 | 0.5 | 300 | 0.93 ± 0.08 | 0.21 ± 0.02 | 1.14 ± 0.09 | 82 |
| 80 | 0.5 | 360 | 1.41 ± 0.23 | 0.46 ± 0.08 | 1.87 ± 0.30 | 75 |
| 100 | 0.5 | 580 | 1.07 ± 0.02 | 0.46 ± 0.03 | 1.53 ± 0.01 | 70 |
*Data represent averages from duplicate experiments.
**Ratio of cpcA to total cpcs.
We previously reported the cpcA ratio, defined as the amount of 1 to the total amount of cpcs (1 and 2). The ratio of the extract of dried beans of Hokkaido cultivars was approximately 80%, whereas those of Korean and Chinese cultivars were approximately 75%, and thus, the cpcA ratio may be genetically regulated.9) Meanwhile, the cpcA ratio of water extracts decreased with increasing extracting temperature (Table 2), which may be due to differences in the solubilities and/or thermal stabilities of 1 and 2 in hot water.
3.3. Production of purple-colored an-paste and determination of the amount of cpcs. 3.3.1. Production of purple-colored an-paste.The color of an-paste is influenced by several factors, including the quality of the raw material—adzuki beans, chemical properties of the water used, and material of the pot employed during preparation. The traditional procedure for obtaining purple an-paste for Japanese confectionary heavily has relied on the intuition and experience of the craftspeople involved. This is because the chemical mechanisms for development of the purple color of an-paste have not been clarified.
The red seed coat of adzuki beans contains substantial amounts of proanthocyanidins, which are oligomers and polymers of catechin derivatives.1),10)–12) These compounds, upon oxidation, can lead to the formation of yellow-to-brown masses that affect the color of sarashi-an. However, the colorless polyphenols and their oxidized compounds readily dissolve in water, in contrast to purple pigments 1 and 2. Therefore, in general processing procedures, shibukiri treatment, which involves removal of the boiling broth once or twice, is effective in preventing the an-paste from turning brown. We considered that removal and suppression of oxidation of the colorless polyphenols may effective in preventing the an-paste from turning brown. Thus, we designed and adopted a combination of pre-soaking treatment for the removal of polyphenols and pre-vacuum processing to suppress formation of the brown-colored mass.
Red adzuki beans (cv. Shumari) were soaked in water for 18 h, and then washed with water, followed by heating in a cooker (Vide pro EC-221) with water. Before heating, the pressure of the cooker was reduced to 10–30 KPa, then, released to 1 atm with air. This process was repeated five times, followed by heating the water-soaked beans at 100 °C for 1 h.13) To compare the results of the pre-vacuum procedure with those of the general procedure, the same mass of beans was heated in a pot over a gas flame, without any vacuum treatment. To examine the effects of pre-soaking and water washing on the color of sarashi-an, the pre-treated beans were processed in a pot over a gas flame.
The qualitative color assessment of the prepared wet sarashi-an samples is shown in Fig. 3 and quantitative color data is presented in Table 3. In the color space, a high a* value indicates redness, and a low b* value indicates blueness; therefore, a high a* value with a low b* value indicates a purple color. Among the samples prepared using three different procedures, wet sarashi-an obtained via pre-vacuum processing exhibited the highest a* value of 9.7 and lowest b* value of −0.3, exhibiting the most intense purple coloration (Fig. 3C, Table 3). The wet sarashi-an prepared using the general procedure, with or without pre-soaking treatment, appeared brownish red in color (Figs. 3A, B), exhibited lower a* values of 5.9 and 7.6 and higher b* values of 6.4 and 2.9 (Table 3). This result indicated that the combination of pre-soaking and pre-vacuum treatment may be effective in reducing the brown-colored mass and obtaining high-quality purple-colored an-paste.
Wet sarashi-an processed using different procedures: (A) General procedure involving heating in a pot without pre-soaking in water. (B) General procedure involving heating in a pot with pre-soaking in water. (C) Pre-vacuum processing procedure.
| Proc.* | Pre-soaking in water |
Color difference | ||
|---|---|---|---|---|
| L* | a* | b* | ||
| G | No | 45.0 | 7.6 | 6.4 |
| G | Yes | 51.0 | 5.9 | 2.9 |
| V | Yes | 44.1 | 9.7 | −0.3 |
*G: general processing procedure with/without pre-soaking in water (see Section 2.6.1 and 2.6.2); V: pre-vacuum processing procedure.
To highlight the advantages of pre-vacuum processing to obtain purple an-paste, adzuki beans from three different cultivars, Erimo-shozu, Shumari, and Kita-roman from Hokkaido Prefecture, were processed in accordance with the general and pre-vacuum processing procedures (Fig. 4). The color differences among wet sarashi-an and the contents of 1 and 2 in lyophilized sarashi-an quantified using HPLC are shown in Table 4. The superiority of pre-vacuum processing in producing purple an-paste was demonstrated. Across all cultivars, pre-vacuum processing yielded wet sarashi-an with a more intense purple color compared with that obtained via general processing (Fig. 4). Regardless of the cultivar, the b* values (yellowness) of wet sarashi-an samples prepared via pre-vacuum processing were lower than those of samples prepared using general processing (Table 4). In addition, the content of cpcs in lyophilized sarashi-an prepared via pre-vacuum processing exceeded 10 µg/g, whereas those prepared via general processing were below 8.5 µg/g (Table 4). These data strongly indicate that the stronger the purple coloration of wet sarashi-an, the higher the content of cpcA and cpcB.
Wet sarashi-an obtained from different red adzuki bean cultivars (V. angularis) using general or pre-vacuum processing procedures: (A) Erimo-shozu (general); (B) Shumari (general); (C) Kita-roman (general); (D) Erimo-shozu (pre-vacuum); (E) Shumari (pre-vacuum); and (F) Kita-roman (pre-vacuum).
| Cultivar | Proc.* | Color difference | cpcA | cpcB | Total | cpcA ratio (%)*** |
||
|---|---|---|---|---|---|---|---|---|
| L* | a* | b* | (µg/g dried sarashi-an)** | |||||
| Erimo-shozu | G | 40.9 | 9.3 | 8.4 | 5.35 ± 0.13 | 2.22 ± 0.06 | 7.58 ± 0.19 | 71 |
| Shumari | G | 41.9 | 10.6 | 7.1 | 5.95 ± 0.32 | 2.46 ± 0.13 | 8.42 ± 0.45 | 71 |
| Kita-roman | G | 40.8 | 9.8 | 8.0 | 5.32 ± 0.19 | 2.17 ± 0.10 | 7.49 ± 0.48 | 71 |
| Erimo-shozu | V | 46.1 | 9.9 | 1.6 | 9.97 ± 0.56 | 3.96 ± 0.21 | 13.93 ± 0.76 | 72 |
| Shumari | V | 42.7 | 9.8 | 1.3 | 7.67 ± 0.49 | 3.11 ± 0.25 | 10.79 ± 0.74 | 71 |
| Kita-roman | V | 45.9 | 10.2 | 0.4 | 8.34 ± 0.12 | 3.33 ± 0.04 | 11.67 ± 0.15 | 71 |
*G: general processing procedure without pre-soaking in water (see Section 2.6.1); V: pre-vacuum processing procedure (see Section 2.6.3).
**Average ± standard deviation from triplicate experiments.
***Ratio of cpcA to total cpcs.
The HPLC chromatograms of the extracts of dried beans and lyophilized sarashi-an are shown in Fig. 5. The extracts of dried beans were obtained in accordance with the procedure described in a previous report.9) HPLC chromatograms of lyophilized sarashi-an samples prepared using the general processing and pre-vacuum processing procedures with cv. Shumari are shown in Figs. 5B and 5C, respectively. In the HPLC chromatogram of the extract of adzuki beans detected at 570 nm, several purple pigments with retention times ranging from 12 to 15 min are indicated with * (Fig. 5A). However, these peaks were absent in sarashi-an (Figs. 5B, 5C). Our preliminary experiments showed that the purple pigments indicated with * in adzuki bean extracts are significantly less stable than cpcs 1 and 2. Therefore, these pigments may decompose during processing and subsequently disappear. In the HPLC chromatograms detected at 280 nm, an obvious difference was observed between the bean extract and the sarashi-an extracts. In the chromatogram of bean extract (Fig. 5A) many peaks attributable to colorless proanthocyanidins were observed with retention times from 8 to 12 min, and a baseline rise attributable to a mixture of oxidized proanthocyanidins with reddish brown color was observed at retention times ranging from 10 to 15 min, (Fig. 5A). In contrast, in the both sarashi-an extracts of the general (Fig. 5B) and pre-vacuum procedures (Fig. 5C), the corresponding colorless peaks and the baseline rise attributable to a mixture of oxidized proanthocyanidins were weaker in intensity, indicating that the processing procedure of preparing sarashi-an decreased the amounts of these components.
HPLC chromatograms of extracts of dried adzuki bean (cv. Shumari) and lyophilized sarashi-an obtained by beans cv. Shumari. Black lines: absorbance at 280 nm; purple lines: absorbance at 570 nm. * indicates purple pigment of different structures from 1 and 2. (A) Dried adzuki bean extract. (B) Lyophilized sarashi-an with general processing. (C) Lyophilized sarashi-an with pre-vacuum processing.
All cpcA ratios in sarashi-an samples examined in this study were consistently in the range of 71–72% (Table 4); these values are lower than those observed in dried beans (Erimo-shozu: 78%; Shumari and Kita-roman: 79%).9) Investigating the differences in the chemical characteristics of cpcA and cpcB will be the theme of future research on adzuki bean pigments.
In conclusion, we established an analytical method of quantifying cpcs in an-paste. First, water was added to lyophilized sarashi-an, followed by the addition of EtOAc after a few minutes was effective for extracting cpcs. In addition, the purple color of an-paste originated from cpcs eluted from the seed coats during boiling. Sequential operations—including pre-soaking and washing with water, and pre-vacuum treatment prior to boiling—proved effective in preparing purple an-paste. Using the pre-vacuum processing procedure, we produced more intensely purple an-paste compared with that generated via conventional boiling in air. HPLC analysis of the extracts of lyophilized sarashi-an revealed that an-paste with a more intense purple color contained a higher content of cpcA and cpcB. Further studies regarding the mechanisms of purple pigment absorption into an-particle are underway.
The authors declare no conflicts of interest.
We are grateful to Ms. Yuki Horiuchi of the Hokkaido Research Organization, Agricultural Research Department of the Tokachi Experimental Station, for providing the adzuki beans. This research was financially supported by JSPS KAKENHI JP18032037 and JP18H02146, Nihon Mamerui Kyokai (Nos. 31B2 and 4B1), and Aichi Shukutoku University Research Fund (No. 25KD06).
Edited by Akira ISOGAI, M.J.A.
Correspondence should be addressed to: Faculty of Food and Health Sciences, Aichi Shukutoku University, 2-9 Katahira, Nagakute, Aichi 480-1197, Japan (e-mail: yoshidak@asu.aasa.ac.jp).
aqueous
cpccatechinopyranocyanidin
MeCNacetonitrile
EtOAcethyl acetate
HPLChigh-performance liquid chromatography
MeOHmethanol
TFAtrifluoroacetic acid
