Anthocyanin Composition of the Purple Sweet Potato Cultivar ‘Churakanasa’

Abstract

We investigated the anthocyanin composition of the purple sweet potato cultivar ‘Churakanasa’. The color tone of the paste was compared by L*, a*, and b* values and ‘Churakanasa’ exhibited a lower b* value, which indicates a bluish tint, than ‘Churakoibeni’, a popular cultivar for paste processing in Okinawa. High performance liquid chromatography (HPLC) analysis of the pigment extract showed that the anthocyanin composition of ‘Churakanasa’ was quite different from that of ‘Ayamurasaki’ and ‘Churakoibeni’. The analysis of the aglycone composition revealed that the cyanidin content (%) of ‘Ayamurasaki’ and ‘Churakoibeni’ contained 19.2% and 22.6% of cyanidins and 80.8% and 77.4% of peonidins, respectively. These findings indicate that these cultivars belong to the peonidin type. In contrast, ‘Churakanasa’ contained 86.4% cyanidin, indicating it to be a cyanidin-type cultivar. The steamed sweet potato paste made from ‘Churakanasa’ (cyanidin type cultivar) exhibited a bluer color compared to the peonidin-type cultivars. This observation suggests a direct correlation between the higher cyanidin content and the blue color intensity of the paste. HPLC-MS analysis of the two major HPLC peaks (peak I and II) of ‘Churakanasa’ suggested that the substance in peak I was YGM-0c; cyanidin-3-p-hydroxybenzoyl sophoroside-5-glucoside by mass, whereas peak II was YGM-1a; cyanidin-3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside. ‘Churakanasa’ exhibits unique color and pigment characteristics as it is the only purple sweet potato cultivar that has YGM-0c and -1a as its primary pigments.

Introduction

Purple-fleshed sweet potatoes are rich in anthocyanins and purple pigments that are found in their tuberous roots. In Japan, various cultivars of purple sweet potatoes have been developed and utilized extensively, including table-use cultivars like ‘Tanegashima-murasaki’ and ‘Fukumurasaki’, with lower anthocyanin content and sweetness, highly pigmented cultivars such as ‘Ayamurasaki’ used in food dye production, and ‘Murasakimasari’ for shochu (distilled spirit) production. In recent years, the cultivar ‘Churakoibeni’ has been grown for confectionery processing, mainly as a raw material for paste, and the uses for purple sweet potatoes are expanding (MAFF, 2021).

Many purple sweet potatoes have been reported to contain eight major anthocyanidins; YGM-1a, -1b, -2, -3, -4b, -5a, -5b, and -6, identified from the ‘Yamagawamurasaki’ (YGM) (Oki et al., 2017; Terahara et al., 1999) (Fig. 1). The anthocyanin aglycones (anthocyanidins) of YGM-1a, -1b, -2 and -3 are cyanidins with a blueish purple color, whereas those of YGM-4b, -5a, -5b and -6 are reddish-purple peonidins. Yoshinaga et al. (1999) suggested that the color tone of purple sweet potatoes varies with the proportion of aglycones. During the initial stages of purple sweet potato breeding, the peonidin type was prevalent as it offered a reddish-purple hue, which was favored over a bluish hue. However, with advancements in storage techniques and logistics in recent years, differently colored cultivars have become available in the market. As a result, a strict emphasis on anthocyanin coloration in crops is no longer necessary.

Fig. 1

Chemical structure of anthocyanins in purple sweet potatoes. Cy; cyanidin, Pn; peonidin, soph; sophoroside, glc; glucoside, RRF; relative response factor.

Many functional studies have been conducted on anthocyanins. For example, the reduction of eye strain by blueberry and bilberry pigments (Tsuda, 2012) and the hepatoprotective effects of purple sweet potato pigments (Cai et al., 2018; Suda et al., 2003; Wang et al., 2017) have been reported. These findings have contributed to the advancement of functional food development in Japan by highlighting the beneficial effects of anthocyanins. Explanations and advertising of these products have further increased public awareness of anthocyanins as healthy substances, as well as increasing the health value of many vegetables containing anthocyanins.

‘Churakanasa’ was released by NARO/KARC in 2020. This cultivar is derived from the progeny of a cross between ‘Purple Sweet Lord’ and ‘90IDN-47’. The female parent, ‘Purple Sweet Lord’, is a cultivar for table use with purple flesh color containing anthocyanin. On the other hand, the male parent ‘90IDN-47’ is a breeding material with genetic resistance against soil rot disease (Okada et al., 2019). ‘Churakanasa’ is a high-yielding cultivar grown in the Okinawa region for use as a paste. The purple color of its paste is slightly different compared to previous purple sweet potato cultivars. In general, the flesh color of raw purple sweet potatoes depends on the pigment distribution and is often not uniform. The raw flesh color of ‘Churakanasa’ and ‘Churakoibeni’ is also whitish around the cortex. Therefore, in order to make comparisons between cultivars, the flesh color was made uniform by steaming and forming a paste. Here, we report a detailed investigation of anthocyanins contained in ‘Churakanasa’.

Materials and Methods

Plant materials

Purple sweet potato cultivars ‘Churakanasa’, ‘Churakoibeni’, ‘Ayamurasaki’ were cultivated at Miyakonojo research station (Miyakonojo, Miyazaki, Japan; 31°45'N, 131°00'E) in 2018, 2019, and 2020, and at Itoman research station (Itoman, Okinawa, Japan; 26°06'N, 127°41'E), Kyushu-Okinawa Agricultural Research Center, NARO in 2018, following each sites’ standard cultivation practices. At Miyakonojo, nitrogen: phosphoric acid: potassium accounted for 4.8:7.2:12.0 g·m⁻² and black mulch was applied in 2019 and 2020. Stem cuttings with seven nodes were planted in May with a ridge width of 75 cm and 35 cm between plants and harvested in October. In Itoman, nitrogen: phosphoric acid: potassium accounted for 4.5:4.5:9.0 g·m⁻² and no mulch was applied. Vines (leaves and stem) were planted in May, with 80 cm ridges and 20 cm between plants and harvest was in late September (120 days) and early October (150 days). The harvested tuberous roots samples showed no significant morphological differences by cultivation site. Approximately 80–130 g of tuberous roots stored for one week after harvest were washed and used for paste processing and anthocyanin extraction analysis.

Paste color measurement

Each purple sweet potato was processed into a paste to measure differences in color values. Samples were steamed for 50 min, sieved through a 1 mm mesh and packed in a 35 mm diameter dish. L*, a*, and b* values were measured using a spectrophotometer CM-2600d (Konica Minolta, Inc., Tokyo, Japan).

Anthocyanin extraction, acid hydrolysis, and quantitative analysis

Five grams of a fresh weight sample sliced from a purple sweet potato strain were extracted in 100 mL of 0.5% sulfuric acid solution at 4°C overnight. The extract supernatant was used to determine the anthocyanin composition. The supernatant was hydrolyzed to determine the ratio of anthocyanin aglycones (anthocyanidins). Acid hydrolysis was performed by adding 30% hydrochloric acid to the extract and then heating at 90°C for 120 min. The dehydrochlorinated extract was obtained through solid-phase extraction using MonoSpin C18 (GL Science, Tokyo, Japan).

The composition of anthocyanins and their aglycones was measured using high-performance liquid chromatography (HPLC) following the methods described by Oki et al. (2017) and calculated as YGM-6 equivalents (equiv.) using YGM-6 as the standard. YGM-6 was isolated by Oki et al. Because the relative response factors (RRF) for YGM-0a–0i are unknown, these coefficients were calculated as 1.0, as in YGM-6.

Briefly, the extract was filtered using a cellulose acetate membrane (0.20 μm; Advantec, Tokyo, Japan), a 10-μL aliquot of the filtrate was injected into the HPLC system and this was subsequently eluted as described below. The HPLC system consisted of a DUG-14A degasser, SIL-10A XL auto-injector, a CTO-10A column oven, an SPD-M10AVP diode array detector, a CBM-20A communications bus module and two LC-10AT pumps (Shimadzu Co., Kyoto, Japan). The system was controlled using a Lab Solution (version 5.73) workstation (Shimadzu Co.). A Cadenza CD-C18 (250 mm × 4.6 mm i.d., 3 μm particles; Imtakt, Kyoto, Japan) was used and the temperature of the column oven was set to 35°C. Anthocyanins were detected by measuring the absorbance at 520 nm. The mobile phase comprised water containing 0.6% (v/v) formic acid (A) and 50% (v/v) acetonitrile containing 0.6% (v/v) formic acid (B). Elution was performed using a linear gradient of B as follows: 20–50% from 0 to 50 min, with a flow rate of 0.6 mL·min⁻¹. The anthocyanins obtained were compared with YGM-6, a standard substance.

HPLC-TOF/MS and MS/MS analysis for characterization

HPLC-TOF/MS and MS/MS analyses were performed using an L-2000U series HPLC system (Hitachi High-Tech Co., Tokyo, Japan) with an electrospray ionization-quadrupole-time-of-flight mass spectrometer, micrOTOF-Q III (Bruker Daltonics, Bremen, Germany). The column was an Inertsustain Swift C18 (4.6 × 150 mm i.d., 5 μm particles; GL Science). The injection volume was 20-μL and the other conditions were the same as those used for the quantitative analysis. Mass spectra in the m/z range of 100–1,500 were obtained by electrospray ionization in the positive-ion mode. The mass spectrometric conditions were optimized as follows: dry heater 180°C, drying gas flow rate 8.0 L·min⁻¹, nebulizer gas pressure 1.6 bar. The MS/MS conditions were identical to those used for TOF/MS analysis. Argon was used as the collision gas to obtain MS/MS fragment ions. The collision energies were optimized for individual analytes using repeated analyses.

Statistical analysis

Analysis of variance (ANOVA) was performed for L*, a*, and b* values between cultivars, after which a multiple comparison test used the Tukey-Kramer method as a significant difference test between groups. The anthocyanidin percentage values were validated by the Kruskal-Wallis test, followed by a multiple comparison test using the Steel-Dwass method as a significant difference test between groups. Anthocyanin content and L*, a* and b* values were compared between the two growing regions using Welch’s t-test. The significance level between groups was set at 5% or 1%.

Results

The paste made from ‘Churakanasa’ exhibited a more pronounced bluish color compared to ‘Ayamurasaki’ or ‘Churakoibeni’ (Fig. 2). Colorimetry results demonstrated a significant difference in the b* value for ‘Churakanasa’ only, indicating that a lower b* value in the negative direction corresponds to a more intense bluish appearance of the paste.

Fig. 2

Comparison of the difference in paste color of the purple sweet potato cultivars ‘Churakanasa’, ‘Churakoibeni’ and ‘Ayamurasaki’, and L*a*b* values. Different letters in the same line indicate significant difference at P < 0.05 according to the Tukey-Kramer test. Values are mean ± standard deviation (‘Churakanasa’; n = 11, ‘Churakoibeni’; n = 11, ‘Ayamurasaki’; n = 5).

As shown in Figure 3A, HPLC analysis of anthocyanins in the extract showed similar HPLC chromatograms for ‘Ayamurasaki’ and ‘Churakoibeni’, while ‘Churakanasa’ exhibited a completely different chromatogram, characterized by two main peaks, I and II. Furthermore, HPLC analysis of the aglycones (anthocyanidins) after acid hydrolysis revealed that ‘Ayamurasaki’ and ‘Churakoibeni’ were the peonidin type (cyanidin < peonidin), while ‘Churakanasa’ was the cyanidin type (cyanidins > peonidin) (Fig. 3B). As shown in Figure 4 and Table S1, ‘Ayamurasaki’ and ‘Churakoibeni’ had peonidin contents of 80.8% and 77.4% respectively, whereas ‘Churakanasa’ demonstrated a cyanidin content of 86.4%.

Fig. 3

HPLC chromatogram of anthocyanin in purple sweet potato cultivated in Miyakonojo. A: 0.5% sulfuric acid extract, B: After acid hydrolysis of A. a–i; YGM-0a–0i, 1–8; YGM-1–8, Cy; Cyanidin, Pn; Peonidin. I and II are the major peaks of ‘Churakanasa’.

Fig. 4

Percentage of anthocyanidins after acid hydrolysis treatment of purple sweet potato cultivars ‘Churakanasa’, ‘Churakoibeni’, and ‘Ayamurasaki’. Values are mean ± standard deviation (‘Churakanasa’; n = 16, ‘Churakoibeni’; n = 14, ‘Ayamurasaki’; n = 12).

The major pigments (I and II) of ‘Churakanasa’ were analyzed using HPLC-MS and MS/MS (Fig. 5). Based on the total mass (m/z) and fragment ion mass (m/z), peak I was identified as cyanidin-3-p-hydroxybenzoyl sophoroside-5-glcoside (YGM-0c) with p-hydroxybenzoyl at the R2 position, whereas peak II was cyanidin-3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside (YGM-1a) diacylated with caffeoyl at the R2 position and p-hydroxybenzoyl at the R3 position. The other anthocyanins contained in ‘Churakanasa’ were analyzed by HPLC co-chromatography. As a result, previously identified sweet potato anthocyanins, except for YGM-0b and YGM-0f, were identified (Fig. 1; Table 1). Moreover, the anthocyanin composition differed depending on the cultivation site (Miyakonojo and Itoman). The samples harvested from Miyakonojo contained the highest amounts of YGM-1a, followed by YGM-0c, whereas those harvested from Itoman contained the highest amounts of YGM-0c, followed by YGM-1a (Table 1). In particular, for YGM-0c, the content in Itoman was more than twice that in Miyakonojo. In addition, samples harvested from Miyakonojo did not contain YGM-1b, whereas those harvested from Itoman did not contain YGM-4b, -5b, or -6. The contents of YGM-0d and YGM-0e were significantly lower in Miyakonojo, whereas those of YGM-2, -3 were significantly higher in Miyakonojo (P < 0.01). However, no significant differences were observed in the total anthocyanin content, total cyanidin content, or percentage of cyanidin among the cultivation sites (Table 1). There were no significant changes in color by L*, a*, and b* values measured for each paste among the cultivation sites (Table S2).

Fig. 5

HPLC-MS and MS/MS analysis chromatograms and estimated chemical structures of the major peaks of ‘Churakanasa’. Cy; cyanidin, Pn; peonidin, soph; sophoroside, glc; glucoside.

Table 1

Anthocyanin composition of the purple sweet potato cultivar ‘Churakanasa’ cultivated in Miyakonojo and Itoman.

Discussion

Extensive research has been conducted on the functional properties of purple sweet potato anthocyanins, primarily focusing on cultivars utilized for dye production, such as ‘Ayamurasaki’ (Suda et al., 2003). The preference for reddish-purple pigmentation has led to the selection of cultivars with higher peonidin content (Yoshinaga et al., 1999). Additionally, because peonidin has an advantage in terms of high pigment stability (Cevallos-Casals and Cisneros-Zevallos, 2004; Yuan et al., 2023), cultivars that accumulate high levels of peonidin are favored. The breeding of the cyanidin-type purple sweet potato cultivar ‘Churakanasa’ was motivated, in part, by its potential for easy color adjustment during the processing of confectionery paste by incorporating additives like pH adjusters. In contrast, in the paste without additives, the bluish-purple color of cyanidin appeared as a color tone. Moreover, cyanidins have been reported to have higher antioxidant capacity and antimutagenic properties than peonidins (Fraisse et al., 2020; Yoshimoto et al., 2001). We aspire to find processed products that take advantage of the characteristics of cyanidin-type purple sweet potatoes in the future.

Anthocyanin biosynthesis involves the formation of peonidin through the methylation of the 3'-hydroxyl group of cyanidin, catalyzed by the enzyme anthocyanin methyltransferase (AOMT) (Provenzano et al., 2014; Tanaka et al., 2019). It is likely that peonidin synthesis, including the AOMT enzyme, is suppressed in cyanidin-type purple sweet potato cultivars. In the future, we intend to use ‘Churakanasa’ to investigate the genetic and biochemical mechanisms underlying the accumulation of cyanidin-type anthocyanins.

Furthermore, the anthocyanin composition of ‘Churakanasa’ is characterized by particularly high contents of YGM-0c and -1a. Since these two anthocyanins are acylated with p-hydroxybenzoic acid, ‘Churakanasa’ would be a good material to investigate the accumulation mechanism of p-hydroxybenzoyl-acylated anthocyanin glycosides.

YGM-0a, -0b are non-acylated and YGM-0c–0i are mono-acylated anthocyanins, which have been reported as trace pigments in ‘Ayamurasaki’ and its cultured cells (Konczak-Islam et al., 2003; Truong et al., 2010). ‘Churakanasa’ is a cultivar containing high amounts of mono-acylated anthocyanins YGM-0c and YGM-2. YGM-1a, -1b, -3, -4b, -5a, and -6 are diacyl anthocyanins, suggesting that the mechanism of acylation differs from that of the previous cultivars. In addition, the total diacyl anthocyanin (YGM-1a, -1b, -3, -4b, -5a, and -6) content was higher than that of the total mono-acylated anthocyanins (YGM-0c–0i, -2, and -5b) in Miyakonojo, and the total mono-acylated anthocyanin content was higher than the total diacyl anthocyanin contents in Itoman. In brief, diacyl anthocyanin contents were higher in Miyakonojo, whereas mono-acylated anthocyanin contents were higher in Itoman. It has also been reported that anthocyanin content is easily affected by the cultivation soil temperature (Kurata and Kobayashi, 2023), suggesting that differences in major acylated anthocyanin contents (mono- or di-acylated type) in ‘Churakanasa’ cultivation sites may also be influenced by the soil temperature. However, differences in the growing environments between Miyakonojo and Itoman include not only temperature, but also the soil environment and cultivar characteristics. These environmental factors affecting anthocyanin acylation will be investigated in more detail in the future.

In recent years, food products have become even more colorful and there has been a growing acceptance of the colors of natural fruits and vegetables. Anthocyanin colors are diverse, as observed in flowers. Hence, our objective is to expand the range of colors available in purple sweet potatoes and develop visually appealing cultivars that combine vibrant colors with desirable functional properties, including a high anthocyanin content.

Acknowledgements

The authors thank Dr. Yoichi Nishiba for kindly providing the YGM-6 standards, Dr. Masaru Tanaka and Mr. Toru Kobayashi for helpful suggestions on the manuscript, and Ms. Kumiko Tokudome for technical assistance from KARC/NARO.

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