Polyphenols in Naruto Kintoki sweet potato enhanced antiallergic activity after baking and microwave cooking

Abstract

The antiallergic activity of extracts of Naruto Kintoki sweet potato peel was enhanced after cooking the peel using three conventional methods. Using 400 µg/mL extracts, baking showed the highest suppression of β-hexosaminidase release (36.9%), followed by microwaving (41.9%) and boiling (64.2%). Significant suppression of β-hexosaminidase release from RBL-2H3 cells in response to Naruto Kintoki peel extract was attributed to compounds 9 (r = 0.91, p < 0.01) and 11 (r = 0.76, p < 0.05). UPLC/ESI-Q-TOF-MS analyses of compounds 5 and 8 clarified the existence of two sulfates of flavonol aglycones (3, 5, 3′-trihydroxy-7, 4′-dimethoxyflavone 3-O-sulfate and 3, 5-dihydroxy-7, 4′-dimethoxyflavone 3-O-sulfate). Baking the sweet potato peel changed compounds 5 and 8 to compounds 9 and 11 with enhanced antiallergic activity. Specifically, IC₅₀ values changed from 11.0 µg/mL (5) to 4.1 µg/mL (9) and from 12.1 µg/mL (8) to 4.4 µg/mL (11). We found those four chemicals contributed to the observed antiallergic activity of Naruto Kintoki sweet potato peel extracts.

Introduction

Naruto Kintoki is a well-known Japanese sweet potato produced on Shikoku Island, primarily near Naruto City (Tokushima Prefecture). Compared to other sweet potato varieties, Naruto Kintoki has a unique golden flesh with a fluffy texture and sweeter taste. Recently, Naruto Kintoki has become a popular variety that can be served for consumption in homes, in confectioneries, and can also be used for alcoholic beverage production.

Cooking sweet potatoes generally changes its physical characteristics and chemical composition leading to a highly nutritious food. However, studies have shown that baking, steaming, and microwaving can decompose phenolic compounds, carotenoids, and flavonoids, as well as vitamin C (Chen et al., 2017; Kourouma et al., 2019; Musilova et al., 2020). Boiling, baking, and drying have been reported to be effective for enhancing extractable phenolics and flavonoids due to the improvement in the solubility of compounds due to disruption of the cell matrix during cooking (Xu et al., 2015; Phomkaivon et al., 2021). However, Dincer et al. (2011) reported that several phenolic derivatives, such as caffeic acid, chlorogenic acid and vanillic acid, all decrease during cooking. It is considered that changes in phytochemicals during cooking could contribute to differences in the biological activity of sweet potato.

The spread of type I allergies has become a serious problem globally, especially food allergies (Loh and Tang, 2018). A food allergy is a reaction to food components by the body's immune system. The immune system reacts to allergens and can lead to a massive secretion of allergy-related mediators, such as histamine, prostaglandins, and leukotrienes that cause allergic symptoms (Singh et al., 2011). Recently, plants containing antiallergic compounds, phenolics and flavonoids, have been shown to have potential immunomodulatory and anti-inflammatory properties without side effects. Moreover, the daily intake of polyphenol-rich ingredients in food may suppress the development of modern diseases.

Sweet potatoes are rich in flavonoids and phenolics (Wang et al., 2018), which exhibit strong antioxidant activity (Tamura and Yamagami, 1994), antiallergic activity (Sato et al., 2012), and have been shown to have anticancer, anti-inflammatory, and anti-obesity effects (Suda et al., 2003). Antiallergic activity associated with the consumption of fruits, vegetables, and tubers, as well as their by-products, has been demonstrated in several studies. For example, flavonoids and phenolics, such as 5, 6, 7-trihydroxyflavone and 5, 7-dihydroxyflavone (Sato and Tamura, 2015), quercetin 4′-glucoside (Sato et al., 2015), and rosmarinic acid (Zhu et al., 2014), can all suppress type I allergic reactions. Sato et al. (2014) reported that Naruto Kintoki, the most popular variety, showed the greatest antiallergic activity among eight Japanese and six Thai sweet potatoes tested. However, there is limited information on the effects of cooking on phytochemicals and the antiallergic activity of sweet potatoes.

In this study, we report the effect of three conventional cooking methods (boiling, microwaving, and baking) on the antiallergic activity of Naruto Kintoki. Antiallergic substances were determined using the correlation between antiallergic effect and the amount of chemicals. In addition, the most antiallergic substances in Naruto Kintoki were identified.

Materials and Methods

Sweet potatoes Naruto Kintoki, Japanese sweet potato, was purchased from a local supermarket (Miki-cho, Kagawa, Japan) in 2019. The tubers were cleaned with tap water and air-dried at room temperature for 30 min. The peel of the Naruto Kintoki sweet potatoes (1 mm thick) was removed from a fleshy area using a vegetable hand-peeler and then stored at −20 °C.

Cooking The Naruto Kintoki peel was cooked using the following three conventional methods: (1) boiling: the peel (50 g) was boiled in 200 mL water at 100 °C for 15 min and drained for 1 min; (2) microwaving: the peel (50 g) was placed in a microwave-safe bowl and cooked in a microwave oven (Sharp RE-CE 30-KB, Chachoengsao, Thailand) at 500 W for 6 min; and (3) baking: the peel (50 g) was baked in an air fryer (Innsky IS-AF004, Shenzhen, China) at 200 °C for 12 min. All of the cooked samples were stored at −20 °C until further analysis.

Extraction using the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method Extracts from raw and cooked sweet potato peel were prepared using the QuEChERS method, as described by Sato et al. (2014). Chopped Naruto Kintoki peel (10 g) was placed in a centrifuge tube (50 mL) and then mixed with water (6.5 mL) and acetonitrile (10 mL) to homogenize for 3 min. Sodium chloride (1 g), trisodium citrate dihydrate (1 g), disodium hydrogen citrate sesquihydrate (0.5 g), and anhydrous magnesium sulfate (4 g) were mixed with the solution, shaken by hand for 1 min, and centrifuged at 3 000 rpm (1 946 × g) for 5 min. The supernatants (acetonitrile layers) of the raw peel and the peel cooked using the three cooking methods were individually evaporated to dryness under a vacuum at 35 °C. QuEChERS crude extracts measuring 15.9, 13.4, 13.7, and 67.2 mg) were obtained from raw, boiled, microwaved, and baked Naruto Kintoki sweet potatoes, respectively. When higher amounts of extract were required, we repeated the QuEChERS extraction using 10 g of raw material.

Liquid-liquid partition of Naruto Kintoki QuEChERS extract The QuEChERS crude extract from the raw Naruto Kintoki peel was partitioned using three different combinations of liquid-liquid partition: ethyl acetate (EtOAc)-water, dichloromethane (DCM)-water, and butanol (BuOH)-water. For example, the QuEChERS crude extract (15.9 mg) was partitioned using an EtOAc (4 mL)-water (4 mL) mixture in triplicate. The EtOAc layer and the water layer were concentrated using an evaporator for further analysis, as shown in Table 1. The DCM-water partition and BuOH-water partition were prepared individually, as described for the EtOAc-water partition.

Table 1. Correlation coefficient between the peak areas of compounds and antiallergic activity of extracts obtained from liquid-liquid partition

Sample		Yield (mg/10 g)	% Inhibition1	Peak number and peak area (mAU)2						Total peak area (mAU)
				4	5	7	8	9	11
QuEChERS		15.9	38.1 ± 0.4	1 406.7	1 892.2	507.9	1 202.9	n.d.	32.5	9 481.7
Ethyl acetate	EtOAc layer	5.9	59.7 ± 0.6	1 956.6	33.4	649.8	170.5	916.2	980	8 891.1
(EtOAc)-water	Water layer	9.6	63.1 ± 1.7	752.5	364	202.3	159.7	797.9	307.1	3 824.3
Dichloromethane	DCM layer	6.2	47.1 ± 2.7	896.8	117.7	104.8	139.7	46.2	173.6	4 497.2
(DCM)-water	Water layer	9.3	32.6 ± 1.5	229.5	180.4	n.d.	32.1	n.d.	n.d.	7 137.3
Butanol (BuOH)-	BuOH layer	9.6	44.9 ± 1.6	2 393.8	2 411.4	732.6	1 531.2	108.2	158.8	13 973.3
water	Water layer	5.0	33.0 ± 2.1	137.5	247.2	n.d.	72.5	17.2	n.d.	1 770.8
	Correlation coefficient3			0.42	−0.14	0.39	−0.13	0.91**	0.76*

¹  Inhibition (%) was calculated at 400 µg/mL concentration from the results of β-hexosaminidase release inhibition (n = 4)

²  Peak areas were calculated from HPLC chromatogram (λ = 290 nm, n.d. = not detected)

³  Pearson's correlation coefficient of each peak between antiallergic activity and peak area (**, * shows correlation is significantly different at p < 0.01 and 0.05, respectively)

Isolation and purification of compounds 5, 8, 9, and 11 The QuEChERS crude extract (400.7 mg) was partitioned with ethyl acetate and water to yield an ethyl acetate extract (130.1 mg) and a water extract (202.9 mg).

Compounds 5 and 8 were purified from the water extract using an HPLC Develosil ODS-5 column (10 × 250 mm; Nomura Chemical Co., Ltd., Aichi, Japan) with a 40% (v/v) acetonitrile aqueous solution. The flow rate was set to 1 mL/min and the compounds were detected at 290 nm. The purity of each compound was checked by analytical HPLC at 290 nm. Final purities of compound 5 (1.1 mg) and compound 8 (0.8 mg) were 95.4% and 95.2%, respectively. With 70% (v/v) acetonitrile aqueous solution as an HPLC isocratic solvent, the purity of compounds 9 (0.4 mg) and 11 (0.2 mg) after detection at 290 nm was 95.5% and 96.7%, respectively.

HPLC analysis of flavonoids The target flavonoids were analyzed using a JASCO HPLC system with an Inertsil ODS column (250 × 4.6 mm, 5 µm; GL Sciences, Inc., Tokyo, Japan). The gradient elution of solvent A (10% acetonitrile aqueous solution containing 0.5% TFA) and solvent B (99.5% acetonitrile containing 0.5% TFA) was performed as follows: 0% B to 70% B (0–30 min), 70% B to 100% B (30–40 min), and held at 100% B for 10 min. The column oven was kept at 40 °C, the flow rate was set at 1.0 mL/min, and injection volume was 10 µL. Metabolites from the sweet potato were detected at 290 nm.

β-Hexosaminidase release assay of RBL-2H3 cells β-Hexosaminidase release by basophilic leukemia cells (RBL-2H3) was determined using a previously described method with minor modifications (Sato et al., 2012; Sato et al., 2014). RBL-2H3 cells were purchased from RIKEN BioResource Center Cell Bank (Ibaraki, Japan) and cultured in D-MEM containing 10% FBS and 1% antibiotic-antimycotic (100×) at 37 °C under a humidified 5% CO₂ atmosphere. For the degranulation assay, cells were seeded into a 24-well plate (2.5 × 10⁵ cells/well) and incubated overnight. The cells were washed with phosphate buffered saline (PBS) 1 mL and then sensitized with 500 µL anti DNP-IgE (mouse monoclonal anti-dinitrophenyl antibody, 50 ng/mL in D-MEM) for 2 h. After washing twice with 500 µL of modified Tyrode's buffer (MT buffer, Tyrode's salt solution containing 1 g/L bovine serum albumin (BSA) and 4.76 g/L HEPES), 490 µL of MT buffer (control) or various concentrations of crude extract were added to each well and the plates were incubated for 10 min. A test sample was prepared with DMSO and diluted with MT buffer to give a final concentration of DMSO of less than 0.1%. The cells were then stimulated with 10 µL of albumin dinitrophenyl (final concentration at 50 ng/mL) for 30 min. The reaction was stopped by cooling in an ice bath for 10 min. The supernatant (50 µL) was transferred to a 96-well plate and incubated with 100 µL substrate (3.3 mM p-nitrophenyl-2-acetamide-2-deoxy-β-D-glucopyranoside) in 0.1 M citrate buffer (pH 4.5) at 37 °C for 25 min. The reaction was stopped by adding 100 µL of stop solution (2 M glycine buffer, pH 10.0) and the absorbance (OD) was measured at 405 nm using a microplate reader (Multiskan FC, Thermo Scientific, Yokohama, Japan). The gain in OD reflects β-hexosaminidase release. The calculation was performed using Equations (1) and (2) below. To obtain a valid value, the factors that are not typically induced by samples need to be excluded. In the “blank”, neither antibody solution nor sample was added to the cells to confirm spontaneous β-hexosaminidase release from the cells. In the “control”, MT buffer instead of samples was added to the cells to confirm β-hexosaminidase release from the cells in conditions without a sample. In the “total” sample, the cells were lysed in 0.1% Triton X-100 in MT buffer to confirm the total amount of β-hexosaminidase contained in the cells. In the “sample”, both antibody solution and samples were added to the cells to confirm β-hexosaminidase release from the cells under these conditions.

Ratio of β-hexosaminidase release (%) =   

β-hexosaminidase release (%) =   

The ratio of β-hexosaminidase release of control should be more than 25%.

Chemical conversion of compounds 5 and 8 to compounds 9 and 11 Compounds 5 and 8 were separately dissolved in test tubes and then heated at 80 °C for 30 min in 0.5% TFA and 10% acetonitrile containing aqueous solution. Chemical conversion was monitored by HPLC (analytical conditions are same with the method described in HPLC analysis of flavonoids).

Mass spectrometry analysis The mass spectra of compounds 5, 8, 9, and 11 were obtained using an UPLC/ESI-Q-TOF-MS system (Acquity UPLC H class and Xevo G2-Xs ToF MS; Waters Corp., Milford, MA, USA) with a TUV detector at 290 nm wavelength, and Acquity UPLC HSS C18 SB (100 × 2.1 mm, 1.8 µm; Waters Corp.). The gradient elution of solvent A (ultra-pure water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid) was performed as follows: 10% B to 100% B (0–10 min). The flow rate and injection volume were individually set at 0.3 mL/min and 5 µL, respectively.

NMR analysis ¹H, ¹³C DEPT-90, DEPT-135, COSY, HMQC, and HMBC NMR spectra of compounds 9 and 11 were recorded with a 500 MHz JNM-ECZ R spectrometer (JEOL Ltd., Tokyo, Japan), dissolving samples in acetone-d₆.

Statistical analysis All analyses were performed in triplicate. The analytical data were expressed as mean ± SD. Duncan's multiple range test was conducted for multiple comparisons using IBM SPSS statistics version 25 software. Pearson's correlation coefficients between the peak areas of individual phytochemicals and antiallergic activity were determined by the using IBM SPSS statistics package (version 25). Significant differences were considered to be at p < 0.05.

Results and Discussion

Influence of cooking methods on antiallergic activity of Naruto Kintoki The significant potential for antiallergic activity of Naruto Kintoki peel has already been studied and compared to other Japanese sweet potatoes (Sato et al., 2014). However, there is limited information on changing or enhancing the antiallergic activity during cooking. In this study, the antiallergic activity following the three conventional cooking methods of boiling, microwaving, and baking Naruto Kintoki were compared with raw peel (Fig. 1). At 400 µg/mL, raw Naruto Kintoki peel had β-hexosaminidase release of 75.4%. Baking exhibited the highest suppression of β-hexosaminidase release of 36.9%, followed by microwaving (41.9%) and boiling (64.2%), with significant differences observed between treatments (p < 0.05).

Fig. 1.

Antiallergic activity of Naruto Kintoki peel after cooking.

Each value represents mean ± SD (n = 4). Means with different letters are significantly different (p < 0.05). The absorbance of β-Hexosaminidase release by basophilic leukemia cells (RBL-2H3) was measured at 405 nm.

HPLC chromatograms of the QuEChERS extracts from raw Naruto Kintoki peel and microwaved peel are shown in Fig. 2; the 12 peaks at 290 nm indicate the main compounds in Naruto Kintoki (compounds 1–12). Changes in the chemicals of the sweet potato samples were clearly observed during cooking. Boiling (13.4 mg dry extract/10 g sample) showed the highest concentration of compound 1 (68.3%) of the QuEChERS extracts. Microwaving increased the concentration of compounds 5, 8, 9, and 11, comprising 35.1, 13.4, 6.0, and 3.6% of the QuEChERS dry extract (13.7 mg dry extract/10 g sample), respectively. These phenomena have been observed in pre-gelatinized sweet potato with increased antioxidant activity (Phomkaivon et al., 2021). Baking resulted in severe degradation of compounds 5 and 8 in the Naruto Kintoki compared to those in the raw peel, accompanying the rise in concentration of compounds 9 and 11, comprising 11.8 and 8.3% of the QuEChERS dry extract (67.2 mg dry extract/10 g sample), respectively.

Fig. 2.

Chromatograms of Naruto Kintoki peel at 5 mg/mL concentration before and after three cooking conditions (at 290 nm). Peaks 1–12 correspond to compound number or peak number in Table 1 to 3 and Figure 3 to 4.

Prediction of antiallergic substances in the Naruto Kintoki peel The Pearson's correlation coefficients among the peak areas of the 12 compounds from the 7 extracts from the liquid-liquid partition and their antiallergic activity are shown in Table 1 and in Supplementary Table S1. Correlations can be used as a tool for the quick selection of the antiallergic substances in Naruto Kintoki, and we have applied this technique in a previous study to determine the antiallergic substances in 11 varieties and lines of onion (Sato et al., 2015). The strongest positive correlation was observed between compounds 9 (r = 0.91, p < 0.01) and 11 (r = 0.76, p < 0.05). However, these two chemicals are not the main compounds in the raw sweet potato. An increase in the concentration of compounds 9 and 11 was noticed after microwaving and baking. Compounds 9 and 11 appear to be putative antiallergic substances. Conversion of pure compounds 5 and 8 to 9 and 11 was examined and shown in acidic solution as a model reaction (Fig. 3 and 4). Therefore, four target compounds (5, 8, 9, and 11) should be tested for antiallergic activity after isolation and purification.

Fig. 3.

The HPLC chromatograms of the conversion of compound 5 to 9 (A) and compound 8 to 11 (B) during heating (0–20 min) at 80 °C in 0.5% TFA and 10% acetonitrile containing aqueous solution.

Fig. 4.

Chemical conversion of compound 5 to 9 and compound 8 to 11 during heating at 80 °C in 0.5% TFA and 10% acetonitrile containing aqueous solution.

Identification of antiallergic substances using UPLC/ESI-Q-TOF-MS analysis Compounds 5, 8, 9, and 11 were isolated using preparative HPLC, and the chemical structures were determined using a UPLC/ESI-Q-TOF-MS system and UV-VIS spectra. Compounds 5, 8, 9, and 11 were finally determined to be flavonols, as follows. The molecular compositions of the four antiallergic substances are presented in Table 2. Compounds 5 and 9 exhibited a common fragment ion at 331.0813, which means that both chemicals have a common partial structure. The calculated molecular formula C₁₇H₁₄O₇ of compound 9 can be assigned as 3, 5, 3′-trihydroxy-7, 4′-dimethoxyflavone (the ¹H-, ¹³C-NMR data of this chemical are provided in Table S2). Compound 5 gave a [M+H]⁺ ion at m/z 411.0393, 353.0620, and 331.0813. In the case of m/z 411.0393, the gain of m/z 79.96 Da from the molecular ion at m/z 331.0813 of compound 9 indicates SO₃ moiety on the molecule of compound 9. The λ_max of band I at 347 nm of compound 5 (λmax at 350 nm) suggested that the OH group at the 3-position should be esterified by SO₃ (Barron et al., 1988). Therefore, compound 5 (C₁₇H₁₄O₁₀S) should be 3, 5, 3′-trihydroxy-7, 4′-dimethoxyflavone 3-O-sulfate (ombuin 3-O-sulfate) (Zhang et al., 2015; Fu et al., 2016). Similarly, compound 11 (C₁₇H₁₄O₆) showed m/z at 315.0858, being closely related to 3, 5-dihydroxy-7, 4′-dimethoxyflavone. Compound 8 had [M+H]⁺ ion at m/z 395.0417, 337.0667, and 315.0858, which has an identical aglycone with compound 11 and a gain of m/z 79.96 Da to the molecular ion at m/z 315.0858 of compound 11 (the ¹H-, ¹³C-NMR data of compound 11 are presented in Table S2). The λ_max of band I at 339 nm of compound 8 (λ_max at 337 nm) suggested that the OH group at the 3-position should be esterified by SO3 (Barron et al., 1988). Therefore, compound 8 was assigned as 3, 5-dihydroxy-7, 4′-dimethoxyflavone 3-O-sulfate (Zhang et al., 2015; Papazian et al., 2019). This study is the first to report two sulfated flavonoids identified in sweet potato peel as potent antiallergic substances.

Table 2. Mass spectra of antiallergic substances found in Naruto Kintoki

Comp.	UV λmax (nm)	Obs. MS [M+1]+	Cal. MS [M+1]+	Error (ppm)	Molecular formula	Fragmention	Identification
5	255, 347	411.0393	411.0386	1.0	C17H14O10S	353.0620, 331.0813	3, 5, 3′-trihydroxy-7, 4′-dimethoxyflavone 3-O-sulfate
8	267, 339	395.0417	395.0437	−4.3	C17H14O9S	337.0667, 315.0858	3, 5-dihydroxy-7, 4′-dimethoxyflavone 3-O-sulfate
9	251, 371	331.0813	331.0818	−1.5	C17H14O7	242.2855	3, 5, 3′ trihydroxy-7, 4′-dimethoxyflavone
11	267, 363	315.0858	315.0869	−3.5	C17H14O6	-	3, 5-dihydroxy-7, 4′-dimethoxyflavone

Synonyms of 3, 5, 3′ trihydroxy-7, 4′-dimethoxyflavone is ombuin

Sulfated flavonoids are an uncommon group of flavonoid derivatives that are produced by aryl sulfotransferase in bacteria or human intestinal bacteria (Koizumi et al., 1990). Sulfated flavonoids are also found in certain plant families such as Polygonaceae, Asteraceae, and Arecaceae (Harborne, 1975; Barron et al., 1988). Approximately 150 types of natural sulfated flavonoid have been reported as flavones or flavonols linked with one or more sulfate groups at position 3 or 7 (Barron et al., 1988; Teles et al., 2018). There are few reports on these chemicals in sweet potatoes. Zhang et al. (2015) reported a minor flavonoid as sulfated flavone in leaves, but the biological activities of these flavonoids and their presence in peel and the edible parts was not mentioned. Therefore, this study is the first to quantify two sulfated flavonoids in raw and cooked Naruto Kintoki peel and to determine their effect on suppressing type I allergies.

Determination of antiallergic substances in the Naruto Kintoki sweet potato The antiallergic activity of the isolated compounds are compared in Table 3. Ombuin (9) and 3, 5-dihydroxy-7, 4′-dimethoxyflavone (11) showed the lowest IC₅₀ values at 4.1 and 4.4 µg/mL, respectively. Two sulfated flavonoids, 3, 5, 3′-trihydroxy-7, 4′-dimethoxyflavone 3-O-sulfate (5) and 3, 5-dihydroxy-7, 4′-dimethoxyflavone 3-O-sulfate (8), also showed rather low antiallergic activity (compounds 5 and 8, IC₅₀ 11.0 and 12.1 µg/mL, respectively). The 3- or 7-glucoside moiety reduces the antiallergic activity (Matsuda et al., 2002). In addition, the absence of a hydroxyl group on the C rings did not affect antiallergic activity. The antiallergic activity of these four compounds was higher than luteolin 7-O-glucoside, apiginin7-O-glucoside, and rosmarinic acid (Sato and Tamura, 2015). Even though polar flavonol sulfate slightly reduces the antiallergic activity, using them in processed foods should be considerably more useful than using aglycones in solution during processing.

Table 3. Antiallergic activity of four substances isolated from Naruto Kintoki peel.

Compound	IC50 (µg/mL)	Cooking method for isolation2	Amount3		Total activity4
			mg/100 mg extract	mg/kg of peel
3, 5, 3′-trihydroxy-7, 4′-dimethoxyflavone 3-O-sulfate (5)	11.0 ± 1.5	Microwaved	35.1 ± 0.9	480.9 ± 1.3	43 715
3, 5-dihydroxy-7, 4′-dimethoxyflavone 3-O-sulfate (8)	12.1 ± 0.7	Microwaved	13.4 ± 0.3	183.6 ± 4.0	15 298
3, 5, 3′-trihydroxy-7, 4′-dimethoxyflavone (9)	4.1 ± 1.5	Baked	11.8 ± 0.4	793.0 ± 1.7	193 404
3, 5-dihydroxy-7, 4′-dimethoxyflavone (11)	4.4 ± 0.2	Baked	8.3 ± 0.1	557.8 ± 2.5	126 763
Quercetin1	1.1 ± 0.1	-	-	-	-

Each value represents mean ± SD (n = 3)

¹  The authentic compounds

²  Different cooked peel was used to isolate the four pure chemicals (compounds 5 and 8 were isolated from microwaved peel and compounds 9 and 11 were isolated from baked peel)

³  Amount was calculated from the chromatograms of the microwaved and baked Naruto Kintoki

⁴  Total activity was estimated using the following formula: total activity = amount (mg/kg of peel)/IC₅₀ (mg/mL) Synonyms of 3, 5, 3′ trihydroxy-7, 4′-dimethoxyflavone is ombuin

The results of this study showed the importance of flavonol sulfates in antiallergic activity. Moreover, microwaving and baking enhanced the amount of ombuin (9) and 3, 5-dihydroxy-7, 4′-dimethoxyflavone (11), as well as their antiallergic activity. The chemical change that occurs during the processing of sweet potatoes is shown in Fig. 3.

Conclusion

Conventional cooking methods showed a positive effect on the antiallergic activity of Naruto Kintoki peel, with microwaving and baking significantly increasing the activity. Pearson's correlation coefficient was used to select four target compounds. The antiallergic activities of ombuin (9) and 3, 5-dihydroxy-7, 4′-dimethoxyflavone (11) were higher than 3, 5, 3′-trihydroxy-7, 4′-dimethoxyflavone 3-O-sulfate (5) and 3, 5-dihydroxy-7, 4′-dimethoxyflavone 3-O-sulfate (8). Baking appeared to be a suitable method for enhancing effective substances, such as ombuin (9) and 3, 5-dihydroxy-7, 4′-dimethoxyflavone (11). Cooked Naruto Kintoki peel is an excellent source of flavonols and sulfated flavonoids for suppressing allergies (type I).

Acknowledgements This research was financially supported by a scholarship to N. Phomkaivon from the Japanese Ministry of Education, Culture, Sports, Science and Technology. We are very grateful to Professor Masahiro Sato for valuable suggestions and assistance with instrumental analysis.

Conflict of interest There are no conflicts of interest to declare.

Supplementary data

Fig. S1.

Change of Compounds 5, 8, 9, and 11 in Naruto Kintoki peel during cooking (0–30min).

Table S1. Correlation coefficient between the peak areas of compounds and antiallergic activity of extracts obtained from liquid-liquid partition.

Sample		Yield (mg/10 g)	% Inhibition1	Peak number and peak area (mAU) 2
				1	2	3	4	5	6	7	8	9	10	11	12
QuEChERS		15.9	38.1 ± 0.4	1 764.8	567.8	756.8	1 406.7	1 892.2	513.9	507.9	1 202.9	n.d.	490.4	32.5	345.8
Ethyl acetate (EtOAc)-water	EtOAc layer	5.9	59.7 ± 0.6	n.d.	n.d.	1 092.5	1 956.6	33.4	n.d.	649.8	170.5	916.2	768.2	980	2 323.9
	Water layer	9.6	63.1 ± 1.7	1 076.5	164.3	n.d.	752.5	364	n.d.	202.3	159.7	797.9	n.d.	307.1	n.d.
Dichloromethane (DCM)-water	DCM layer	6.2	47.1 ± 2.7	n.d.	n.d.	n.d.	896.8	117.7	n.d.	104.8	139.7	46.2	n.d.	173.6	3 018.4
	Water layer	9.3	32.6 ± 1.5	1 552.9	710.5	181.5	229.5	180.4	n.d.	n.d.	32.1	n.d.	n.d.	n.d.	4 250.4
Butanol (BuOH)-water	BuOH layer	9.6	44.9 ± 1.6	1 262.8	565.1	1 054.3	2 393.8	2 411.4	596.4	732.6	1 531.2	108.2	759.2	158.8	2 399.5
	Water layer	5.0	33.0 ± 2.1	1 021	275.4	n.d.	137.5	247.2	n.d.	n.d.	72.5	17.2	n.d.	n.d.	n.d.
Correlation coefficient3				−0.54	−0.67	0.19	0.42	−0.14	−0.21	0.39	−0.13	0.91**	0.25	0.76*	−0.17

¹  Inhibition (%) was calculated at 400 µg/mL concentration from the results of β-hexosaminidase release inhibition (n = 4)

²  Peak areas were calculated from HPLC chromatogram (λ = 290 nm, n.d. = not detected)

³  Pearson's correlation coefficient of each peak between antiallergic activity and peak area (**, * shows correlation is significantly different at p < 0.01 and 0.05, respectively)

Table S2. Chemical shifts (δ) and functional groups of compounds 9 and 11 in Naruto Kintoki peel.

Atom	Compound 9			Atom	Compound 11
	δ13C [ppm]	δ1H [ppm]	Functional group		δ13C [ppm]	δ1H [ppm]	Functional group
2	146.94		-C=	2	146.93		-C=
3	137.30		-C=	3	137.14		-C=
4	176.71		-C=O	4	176.71		-C=O
5	161.79		-C=	5	161.73		-C=
6	98.41	6.33 d (J = 2.04 Hz)	-CH=	6	98.39	6.34	-CH=
7	166.74		-C=	7	166.71		-C=
8	92.80	6.74 d (J = 2.04 Hz)	-CH=	8	92.84	6.72	-CH=
9	157.77		-C=	9	157.79		-C=
10	104.86		-C=	10	104.87		-C=
1′	124.81		-C=	1′	124.31		-C=
2′	115.20	7.81 d (J = 2.04 Hz)	-CH=	2′, 6′	130.35	8.26 d (J = 8.59 Hz)	-CH=
3′	147.34		-C=	3′, 5′	114.90	7.13 d (J = 8.59 Hz)	-CH=
4′	150.27		-C=	4′	162.17		-C=
5′	112.16	7.14 d (J = 8.25 Hz)	-CH=
6′	121.25	7.84 dd (J = 2.04 Hz, J = 8.25 Hz)	-CH=
7-OCH3	56.48	3.95 s	-OCH3	7-OCH3	56.48	3.94 s	-OCH3
4′-OCH3	56.32	3.95 s	-OCH3	4′-OCH3	55.84	3.91 s	-OCH3

500 MHz ¹H and ¹³C-NMR were measured in acetone-d₆

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