α-Glucosidase inhibitory flavonoids of Actinidia arguta fruits: Comparison of different cultivars

Four cultivars of A. arguta (Siebold & Zucc.) Planch. ex Miq., namely, 'Saehan', 'Daesung', 'Chilbo' and 'Autumn sense' (Figure 1), were obtained from the National Institute of Forest Science (Suwon, Korea) in April 2016. They were identified by the herbarium of College of Pharmacy at Chungbuk National University (Cheongju, Korea), where a voucher specimen (CBNU2016-AAS, AAD, AAC and AAA) was deposited.

Figure 1.

Images of four cultivars, namely, 'Saehan', 'Daesung', 'Chilbo' and 'Autumn sense', of Actinidia arguta fruits.

The dried powder of A. arguta fruits (12.0 kg) was extracted twice with 80% MeOH at room temperature, which yielded the methanol extract (1.0 kg). The methanol extract was suspended in H₂O and partitioned successively with n-hexane, CH₂Cl₂, EtOAc and n-BuOH.

The CH₂Cl₂ fraction (AAM, 26.8 g) was chromatographed on silica gel eluting with a mixture of n-hexane-EtOAc by step gradient to give 10 subfractions (AAM1–AAM10). Compound 1 was purified from AAM8 by Sephadex LH-20 (Cytiva, MA, USA) eluting with n-hexane-CH₂Cl₂-MeOH (5:5:1).

The EtOAc fraction (AAE, 31.3 g) was chromatographed on silica gel eluting with a mixture of CH₂Cl₂-MeOH by step gradient to give 12 subfractions (AAE1–AAE12). AAE5 was subjected to MPLC on reverse phase (RP)-silica gel and eluted with mixtures of MeOH–H₂O to obtain six subfractions (AAE5A– AAE5F). Compound 19 was purified from AAE5F by Sephadex LH-20 eluting with MeOH. AAE6 was subjected to MPLC on RP-silica gel and eluted with mixtures of MeOH–H₂O to obtain four subfractions (AAE6A–AAE6D). Compound 4 was purified from AAE6B by Sephadex LH-20 eluting with MeOH, followed by semi-preparative HPLC eluting with MeCN–H₂O (40:60). AAE9 was subjected to MPLC on RP-silica gel and eluted with mixtures of MeOH–H₂O to obtain six subfractions (AAE9A–AAE9F). Compounds 20 and 22 were purified from AAE9D by Sephadex LH-20 eluting with CH₂Cl₂-MeOH (1:1), followed by semi-preparative HPLC eluting with MeCN–H₂O (20:80). AAE9F was subjected to Sephadex LH-20 eluting with CH₂Cl₂-MeOH (1:1) to yield two subfractions (AAE9F1–AAE9F2). Semi-preparative HPLC eluting with MeCN–H₂O (17:83) yielded compounds 2, 3, 5, 7, 8, 9 and 10. Compound 21 was purified from AAE9F2 by MPLC on RP-silica gel and eluted with mixtures of MeOH–H₂O. AAE10 was subjected to MPLC on RP-silica gel and eluted with mixtures of MeOH–H₂O to yield seven subfractions (AAE10A–AAE10G). Compounds 12 and 16 were purified from AAE10G and AAE10E, respectively, by Sephadex LH-20 eluting with CH₂Cl₂-MeOH (1:1), followed by semi-preparative HPLC eluting with MeCN–H₂O (18:82). AAE11 was subjected to MPLC on RP-silica gel and eluted with mixtures of MeOH–H₂O to yield seven subfractions (AAE11A– AAE11G). AAE11E was subjected to Sephadex LH-20 eluting with CH₂Cl₂-MeOH (1:1) to yield two subfractions (AAE11E1–AAE11E2). Compounds 11, 13 and 17 were purified from AAE11E1 by semi-preparative HPLC eluting with MeCN–H₂O (19:81). Compounds 6 and 15 were purified from AAE11E2 and AAE11D, respectively, by semi-preparative HPLC eluting with MeCN–H₂O (23:77). Compounds 14 and 18 were isolated from AAE12 MPLC on RP-silica gel and eluted with mixtures of MeOH–H₂O, followed by Sephadex LH-20 eluting with CH₂Cl₂-MeOH (1:1) and semi-preparative HPLC eluting with MeCN–H₂O (14:86).

Argutiflavone (13): yellow syrup; IR (KBr)_max 3403, 1658 cm^−1., ¹H NMR (400 MHz, CD₃OD) δ_H 6.23 (1H, d, J = 1.6 Hz, H-6), 6.41 (1H, d, J = 1.6 Hz, H-8), 7.64 (1H, d, J = 2.0 Hz, H-2′), 6.90 (1H, d, J = 8.4 Hz, H-5′), 7.66 (1H, dd, J = 8.4, 2.0 Hz, H-6′), 5.34 (1H, d, J = 7.6 Hz, H-1″), 4.59 (1H, d, J = 1.2 Hz, H-1″′), 4.81 (1H, t, J = 9.6 Hz, H-4″′), 0.87 (1H, d, J = 6.0 Hz, H-6″′), 2.02 (3H, s, OAc) ppm; ¹³C NMR (100 MHz, CD₃OD) δ_C 157.5 (C-2), 133.9 (C-3), 178.1 (C-4), 161.7 (C-5), 98.5 (C-6), 164.6 (C-7), 93.3 (C-8), 157.1 (C-9), 104.2 (C-10), 122.1 (C-1′), 114.6 (C-2′), 144.6 (C-3′), 148.4 (C-4′), 116.0 (C-5′), 121.7 (C-6′), 102.4 (C-1″), 74.3 (C-2″), 75.5 (C-3″), 69.8 (C-4″), 76.8 (C-5″), 66.9 (C-6″), 100.8 (C-1″′), 68.9 (C-2″′), 70.8 (C-3″′), 74.0 (C-4″′), 66.1 (C-1″′), 16.2 (C-2″′), 171.3 (COCH₃), 19.6 (COCH₃) ppm; ESIMS m/z 675 [M + Na]⁺; HRESIMS m/z 675.1532 ([M + Na]⁺ calcd. for C₂₉H₃₂NaO₁₇ 675.1537).

The inhibitory effect on α-glucosidase was measured using α-glucosidase extracted from Saccharomyces cerevisiae (EC 3.2.1.20) (Indrianingsih et al., 2015). The amount of p-nitrophenol that was cleaved by the enzyme was determined by measuring the absorbance at 405 nm. Acarbose was used as a positive control.

The antioxidant activity was evaluated by measuring the free radical scavenging activity using DPPH (Indrianingsih et al., 2015). The mixture of freshly prepared DPPH solution and sample was allowed to react at room temperature for 10 min, and the absorbance was measured at 550 nm. Ascorbic acid was used as a positive control.

An aluminium chloride colorimetric test was used to quantify the total flavonoid concentration in the samples (An et al., 2023). Briefly, 10% AlCl₃ was added to the mixture of samples and 5% concentration of NaNO₃ After being incubated with mild stirring for 5 min, 1 N NaOH solution was added to the reaction mixture. A spectrophotometric analysis was performed by measuring the absorbance at a wavelength of 510 nm. The flavonoid content in each specimen was quantified by expressing it as catechin equivalent (CE), using catechin as the reference standard.

For the characterisation of constituents of A. arguta fruits, the total extract was fractionated and isolated by extensive chromatographic analysis to obtain 22 compounds including one new compound (Figure 2A).

Figure 2.

(A) Chemical structures of compounds 1–22 isolated from the fruits of Actinidia arguta and (B) key HMBC correlation of a new compound (13).

The known compounds were identified as kaempferol (1), kaempferol 3-O-β-glucopyranoside (2), kaempferol 3-O-β-galactopyranoside (3), quercetin (4), quercetin 3-O-β-galactopyranoside (5), quercetin 4′-O-β-glucopyranoside (6), quercetin 3-O-β-glucopyranoside (7), isorhamnetin 3-O-β-glucopyranoside (8), quercetin 3-O-6″-acetyl-β-glucopyranoside (9), quercetin 3-O-6″-methoxy-β-glucopyranoside (10), kaempferol 3-O-(6″-O-α-rhamnosyl)-β-glucopyranoside (11), kaempferol 3-O-(6″-O-4″′-acetylrhamnosyl)-β-glucopyranoside (12), quercetin 3-O-(6″-O-α-rhamnosyl)-β-glucopyranoside (14), quercetin 3-O-(6″-O-α-rhamnosyl)-β-galactopyranoside (15), quercetin 3-O-(6″-O-α-4″′-acetylrhamnosyl)-β-galactopyranoside (16), kaempferol 3-O-(6″-O-α-rhamnosyl)-β-galactopyranoside (17), quercetin 3-O-β-sambubioside (18), naringenin (19), cinchonain Ia (20), cinchonain Ib (21) and cinchonain Ic (22) by the spectroscopic data analysis and comparison with literature values (Nonaka and Nishioka, 1982; Yoshida et al., 1990; Pizzolatti et al., 2002; Han et al., 2004; Lim et al., 2004; Scharbert et al., 2004; Imperato, 2008; Jeon et al., 2008; Itoh et al., 2009; Jaramillo et al., 2011; Sun et al., 2011; Wan et al., 2012; Lee et al., 2013; Zhang et al., 2020).

Compound 13 was isolated and purified as yellow syrup and gave a pseudo-molecular ion [M + Na]⁺ by HRESI-TOF-MS (Thermo Fisher Scientific, MA, USA) at 675.1532 (calcd. for C₂₉H₃₂NaO₁₇ 675.1537), consistent with a molecular formula of C₂₉H₃₂O₁₇. Compound 13 was supposed to be a flavonoid from the signal of meta-coupling aromatic ring at (δ_H 6.23 [1H, d, J = 1.6 Hz, H-6], 6.41 [1H, d, J = 1.6 Hz, H-8]; δ_C 98.5 [C-6], 93.3 [C-8]) and 1,3,4-trisubstituted aromatic ring at (δ_H 7.66 [1H, dd, J = 8.4, 2.0 Hz, H-6′], 7.64 [1H, d, J = 2.0 Hz, H-2′] 6.90 [1H, d, J = 8.4 Hz, H-5′]; δ_C 122.1 [C-1′], 114.6 [C-2′], 144.6 [C-3′], 148.4 [C-4′], 116.0 [C-5′], 121.7 [C-6′]). The ¹H NMR spectrum of compound 13 showed typical signals for a glucosyl anomeric proton in β-configuration at δ_H 5.34 (1H, d, J = 7.6 Hz, H-1″), and the presence of glucosyl moiety was also confirmed by the glucosyl carbon signals at (δ_C 74.3 [C-2″], 75.5 [C-3″], 69.8 [C-4″], 76.8 [C-5″], 66.9 [C-6″]). The presence of a rhamnose in α-configuration was also suggested by the additional anomeric signals at (δ_H 4.59 [1H, d, J = 1.2 Hz, H-1″′]; δ_C 100.8 [C-1″′]) together with signals at (δ_H 3.73 [1H, dd, J = 1.2, 1.6 Hz, H-2″′], 3.36–3.61 [2H, m, H-3″′, 5″′], 4.81 [1H, t, J = 9.6 Hz, H-4″′], 0.87 [3H, d, J = 6.0 Hz, H-6″′]; δ_C 68.9 [C-2″′], 70.8 [C-3″′], 74.0 [C-4″′], 66.1 [C-5″′],16.2 [C-6″′]). Additional signals at (δ_H 2.02 [3H, s]; 171.3 [COCH₃], 19.6 [COCH₃]) revealed the presence of an acetyl group in compound 13. Based on the aforementioned finding, compound 13 was suggested as a flavonoid with a glucose, a rhamnose and an acetyl group. The position of the glucose was determined to be C-3 of the flavonoid skeleton from the HBMC correlation between δ_H 5.34 (H-1″) and δ_C 133.9 (C-3). The position of the rhamnose was assigned to C-6″ of the glucose from the HMBC correlation between δ_H 4.59 (H-1″′) and δ_c 66.9 (C-6″). The position of the acetyl group was assigned at C-4″′ of rhamnose due to the downfield shifted H-4″′ at δ_H 4.81 (1H, t, J = 9.6 Hz) together with HMBC correlation between δ_H 4.81 (H-4″′) and δ_C 171.3 (COCH₃) (Figure 2B). Based on these analysis results, compound 13 was determined as 3-O-(6″-O-4″′-acetylrhamnosyl)-glucoside and named 'argutiflavone' (Figure 2B).

Next, the α-glucosidase inhibitory efficacy and antioxidant activity of the isolated compounds were evaluated. Among the isolated compounds, compounds 1, 2, 4, 5, 7–9, 15 and 19–22 showed >50% decline in the inhibition and antioxidant activities of α-glucosidase at 100 μM (Figure 3). However, a new compound 13 and compound 16 showed antioxidant effects but relatively weak α-glucosidase inhibitory effects in our assay system.

Figure 3.

Antioxidant and α-glucosidase inhibitory activities of compounds 1–22.

All the compounds isolated from A. arguta fruits in this study belong to a class of flavonoids. They can be further subdivided according to the position and numbers of the hydroxy, methoxy, acetyl, phenylpropanoid and sugar moiety. First, compounds can be divided into flavanones (1–18) and flavanone derivatives (19–22) depending on the flavonoid skeleton. Flavanone derivatives are divided into aglycones, glycosides with one sugar and glycosides with two sugars, and they differ in the number and position of OH, OCH₃ and acetyl groups. Flavanone derivatives are divided into simple flavanone (19) and flavolignans (20–22), which have additional phenylpropanoids. Considering the relationship between chemical structure and efficacy, both antidiabetic and antioxidant efficacies were weakened as the number of sugars increased, and the presence of 3,4-dihydroxy moiety in the aromatic ring was important for the antioxidant activity. Consistent with this finding, the flavonolignans, aglycones with two 3,4-dihydroxybenzene rings, had excellent effects on both antidiabetic and antioxidant activities.

Due to the advantages in horticulture and nutrition of A. arguta, new cultivars have been constantly being developed to improve the appearance, stability, resistance to diseases and nutritional composition. In Korea, four cultivars, namely, 'Saehan', 'Daesung', 'Chilbo' and 'Autumn sense', were developed and registered as new cultivars due to their characteristics and advantages. As shown in Figure 1, four cultivars differ in size and shape. Assessment of total flavonoid content showed that all four cultivars showed high flavonoid content, with some differences in amount (Figure 4). Among them, the new cultivar 'Autumn sense' showed the highest flavonoid content in the present study. These results indicate that the new cultivars differ not only in morphology and growth characteristics but also in their composition of beneficial compounds. Therefore, the development of superior varieties has the potential to enhance health benefits such as increased antioxidant properties.

Figure 4.

Total flavonoid contents of four cultivars, namely, 'Saehan', 'Daesung', 'Chilbo' and 'Autumn sense' of Actinidia arguta fruits. CE, catechin equivalent.