Argutinic acid, A New Triterpenoid from the Fruits of Actinidia arguta

Introduction

The fruits of the genus Actinidia is well known as kiwi fruit and widely consumed as fresh fruits.¹ Among diverse species of Actinidia, the fruit of A. arguta, which also called as kiwiberry or hardy kiwifruit, has distinct characteristics such as small and smooth-fruited and tolerance to low temperatures.² It is rich in beneficial nutrients including organic acids, sugars, vitamins and minerals as well as bioactive compounds.^3–4 In our previous research, we isolated diverse phenolic compounds, especially phenolic derivatives conjugated with different organic acids and evaluated the anti-inflammatory and antioxidant activities.^5–6 In a continuation of our research on hardy kiwifruit, here we report the isolation and structural elucidation of seven compounds including a new triterpene and their anti-diabetic effect.

Experimental

Plant material – The fruits of A. arguta was collected at the National Institute of Forest Science in August 2016. After identification by the herbarium of the College of Pharmacy, Chungbuk National University, voucher specimens (CBNU2016-AA) were deposited in a specimen room of the herbarium.

General experimental – A Jasco UV-550 and Perkin-Elmer model LE599 spectrometer were used for the measurement of UV and IR spectra, respectively. NMR spectra were recorded on a Bruker DRX 400 or 500 MHz spectrometer using pyridine-d₆ as a solvent. ESI-MS data were obtained on VG Autospec Ultima mass spectrometers using H₂ as carrier gas with a Hypersil Gold column (flow rate 0.2 mL/min). HR-ESI-MS data was obtained using a maXis 4G (Bruker, Germany). Semipreparative HPLC was performed using a Waters HPLC A system equipped with Waters 600 Q-pumps, a 996 photodiode array detector, and Waters Empower software using a Gemini-NX ODS-column (5 μm, 10 × 150 mm).

Isolation of compounds – The dried powder of A. arguta fruits (12.0 kg) was extracted 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). Subfraction AAE1 was subjected to Sephadex LH-20 eluting with n-hexane-CH₂Cl₂-MeOH (5:5:1) to give 10 subfractions (AAE1A–AAE1J). Compound 1 (2.6 mg) was obtained from AAE1A by MPLC on silica gel eluted with mixtures of n-hexane-EtOAc (5:1). Compounds 6 (4.6 mg) and 7 (3.9 mg) were purified from AAE1J by semi-preparative HPLC (Gemini-NX ODS-column, 5 μm, 10 × 150 mm) eluting with MeOH-water (20:80). Subfraction AAE110 was subjected to MPLC on RP-silica gel and eluted with mixtures of MeOH-H₂O to obtain 7 subfractions (AAE10A-AAE10G). Further purification of AAE10A by Sephadex LH-20 eluting with CH₂Cl₂-MeOH (1:1) yielded compound 5 (6.1 mg). Subfraction AAE11 was subjected to MPLC on RP-silica gel and eluted with mixtures of MeOH-H₂O to obtain 11 subfractions (AAE11A–AAE11K). Subfraction AAE11K was subjected to Sephadex LH-20 eluting with CH₂Cl₂-MeOH (1:1) and compounds 2 (2.6 mg), 3 (4.4 mg) and 4 (12.9 mg) were obtained by semi-preparative HPLC (Gemini-NX ODS-column, 5 μm, 10 × 150 mm) eluting with acetonitrile-water (23:77).

Argutinic acid (1) – brown syrup; $$ {\left[\alpha \right]}_{\mathrm{D}}^{25}$$ −12.7 (c 0.1, MeOH); IR (KBr) ν_max 3430, 1666 cm^-1; ESIMS m/z 543 [M+Na]⁺; HRESIMS m/z 543.3292 ([M+Na]⁺ calcd. for C₃₀H₄₈NaO₇ 543.3298); ¹H-NMR (500 MHz, pyridine-d₅); δ_H 2.31 (1H, dd, J = 12.5, 4.0 Hz, H-1a), 1.39 (1H, m, H-1b), 4.25 (1H, m, H-2), 4.24 (1H, d, J = 10.5 Hz, H-3), 1.84 (1H, dd, J = 10.5, 1.5 Hz, H-5), 1.77 (1H, m, H-6), 1.38 (1H, m, H-7a), 1.75 (1H, m, H-7b), 1.91 (1H, dd, J = 10.5, 7.0 Hz, H-9), 1.72 (1H, m, H-11a), 2.07 (1H, m, H-11b), 2.06 (1H, m, H-15a), 2.85 (1H, ddd, J = 13.5, 10.0, 3.5 Hz, H-15b), 1.22 (1H, m, H-16a), 2.40 (1H, m, H-16b), 3.56 (1H, d, J = 12.0 Hz, H-18), 2.49 (1H, m, H-19), 2.48 (1H, m, H-21), 2.07 (1H, m, H-22a), 2.41 (1H, m, H-22b), 1.10 (3H, s, CH₃-23), 1.09 (3H, s, CH₃-25), 1.14 (3H, s, CH₃-26), 1.28 (3H, s, CH₃-27), 4.24 (1H, d, J = 10.5 Hz, H-24a), 3.76 (1H, d, J = 10.5 Hz, H-24b), 1.39 (3H, d, J = 7.5 Hz, CH₃-29), 3.99 (1H, d, J = 10.5 Hz, H-30a), 4.18 (1H, d, J = 10.5 Hz, H-30b); ¹³C-NMR (125 MHz, pyridine-d₅); δ_C 47.6 (C-1), 68.7 (C-2), 78.0 (C-3), 43.4 (C-4), 47.9 (C-5), 18.3 (C-6), 33.0 (C-7), 39.9 (C-8), 47.9 (C-9), 38.1 (C-10), 23.6 (C-11), 125.4 (C-12), 139.2 (C-13), 42.4 (C-14), 32.2 (C-15), 28.5 (C-16), 47.7 (C-17), 47.7 (C-18), 35.7 (C-19), 72.9 (C-20), 31.1 (C-21), 24.2 (C-22), 14.1 (C-23), 66.4 (C-24), 17.3 (C-25), 17.4 (C-26), 23.6 (C-27), 179.9 (C-28), 12.6 (C-29), 69.6 (C-30).

Table 1.

α-Glucosidase inhibitory activities of compounds 1–7

Measurement of α-glucosidase inhibitory activity – The inhibitory effect on α-glucosidase was measured using α-glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20). A test sample was mixed with 80 μL enzyme buffer and 10 μL α-glucosidase and incubated for 15 min at 37^oC. Then, after addition of 10 μL p-nitrophenyl α-D-glucopyranoside solution for enzyme reaction, the amount of p-nitrophenol cleaved by the enzyme was determined by measuring the absorbance at 405 nm in a 96-well microplate reader. Acarbose was used as a positive control.

Results and Discussion

Seven compounds were isolated from the fruits of A. arguta and the structures of known compounds were identified as three triterpenes, niga-ichigoside F2 (2), and niga-ichigoside F1 (3) and ursolic acid (4), and three magastigmanes, roseoside (5), vomifoliol (6) and loliolide (7), respectively, by comparing them with the literature (Fig. 1).^6–9

Fig. 1.

(A) Structures of compounds 1–7 from the fruits of A. arguta and (B) Key HMBC (→) and NOESY (↔) correlations of compound 1

Compound 1 was isolated as a brown amorphous powder, and the molecular formula was determined as C₃₀H₄₈O₇ by HRESI-TOF-MS of molecular ion peak at m/z 543.3292 [M+Na]⁺ (calcd. for C₃₀H₄₈NaO₇, 543.3298). Compound 1 was suggested as an ursane triterpenoid 6 from the one carboxyl group [δ_C 179.9 (C-28)], four tertiary methyl groups [δ_H 1.28 (3H, s, H-27), 1.14 (3H, s, H-26), 1.10 (3H, s, H-23), 1.09 (3H, s, H-25); δ_C 23.6 (C-27), 17.4 (C-26), 17.3 (C-25), 14.1 (C-23)], one secondary methyl group [δ_H 1.39 (3H, d, J = 7.0 Hz, H-29); δ_C 12.6 (C-29)], two oxymethine groups [δ_H 4.25 (1H, m, H-2), 4.24 (1H, d, J = 10.5 Hz, H-3); δ_C 68.7 (C-2), 78.0 (C-3)], one olefin group [δ_H 5.61 (1H, br s, H-12); δ_C 139.2 (C-13), 125.4 (C-12)] in ¹H-NMR, ¹³C-NMR and HSQC spectra. Additionally, presence of two hydroxymethyl groups was suggested from the signals at [δ_H 4.24 (1H, d, J = 10.5 Hz, H-24a), 3.76 (1H, d, J = 10.5 Hz, H-24b); δ_C 66.4 (C-24)] and [δ_H 4.18 (1H, d, J = 10.5 Hz, H-30a), 3.99 (1H, d, J = 10.5 Hz, H-30b); δ_C 69.6 (C-30)]. These data are quite similar to those of compounds 2–4, suggesting that it is a ursolic acid derivatives. Compared to those of compound 2, additional presence of a hydroxymethyl group and absence of a glucose were deduced. The positions of these two hydroxymethyl groups were determined as C-24 and C-30 by the HMBC correlations of H24/C4 and H30/C20, respectively. The stereochemistry of 2-OH, 3-OH, 24-hydroxymethyl and 30- hydroxymethyl groups were determined as 2α, 3β, 24α and 30α by the J value (J_2–3 = 10.5 Hz) and NOESY correlations between H2/H23, H3/H24 and H19/H30. Based on the above analysis results, the structure of compound 1 was determined as shown and named argutinic acid as it is first reported in nature.

As α-glucosidase is an important therapeutic in diabetes, ^10–12 we measured the inhibitory effects of compounds from A. arguta fruits against α-glucosidase. Ursolic acid (4) exists in many plants and has already been reported to have α-glucosidase inhibitory activity. ¹³ Our results also showed an inhibitory effect of 60.2% inhibition at the concentration of 100 μM. The new triterpene, argutinic acid (1) also showed 44.2% inhibition against α-glucosidase at the concentration of 100μM. However, two triterpene glycosides (2 and 3) and three megastigmanes (5–7) exerted little effects.

Kiwi fruits, the fruits of the genus Actinidia, are widely consumed as fruits. It is rich in diverse bioactive ingredients especially phenolic compounds, and it is known to have excellent antioxidant effects. In this study, we conducted a study on the composition of the fruits of A. arguta and isolated one new triterpene, argutinic acid along with three triterpenes and three megastigmines. Triterpenes of A. arguta showed α-glucosidase inhibitory effects. Therefore, this study will contribute to the utilization of A. arguta fruits.

Acknowledgments

This work was supported by the Research grant (2022R1A2C1008081) of the National Research Foundation of Korea (NFR) funded by the Korean government (MSIT).

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