Two New Acylated Sucroses From the Roots of Canna indica L. and Their Antioxidant Activity

Abstract

Two new acylated sucroses, (6-O-axetoxyl)-β-d-fructofuranosyl-(2→1)-(6-O-feruloyl-α- d-glucopyranoside (1) and (6-O-axetoxyl)-β-d-fructofuranosyl-(2→1)-(6-O-(E)-p-coumaroyl-α- d-glucopyranoside (2), and four known compounds, 2-(3′,4′-dihydroxyphenyl)-1-propanol-4′-O-[4′′′-hydroxy-3′′′,5′′′-dimethoxybenzoyl-(→6′′)-β-d-fructofuranoside (3), tryptophol glucoside (4), corchonoside C (5), and 2-hydroxy-5-(2-hydroxyethyl)phenyl β-d-fructofuranoside (6), were isolated from the roots of Canna indica L. (Cannaceae). Their structures were determined by extensive analysis of HR-ESI-MS, 1D, and 2D NMR spectral data, as well as by comparison of these with those reported in the literature. Antioxidant activity of compounds 1-6 were evaluated by peroxyl radical absorbance capacity assay. Compounds 1 and 3 neutralized high amounts of peroxyl radical. At a concentration of 1 µM, their ORAC_ROO* values were 3.07 ± 0.15 and 4.27 ± 0.30, respectively, many times greater than that of trolox, which was used as an internal standard. At 10 µM, the peroxyl radical absorbance capacity of compounds 1 and 3 exhibited equivalents to 15.60 ± 0.22 and 24.91 ± 0.43 times the net protection by 1 µM of trolox.

Canna indica L. belongs to the Cannaceae family, which consists of about 19 species of flowering plants. In Vietnam, C. indica is an agricultural plant used as a starch source. In addition, Canna species have been used in traditional medicine for the treatment of different ailments such as hepatitis, to clean the coronary lumen, support the treatment of atherosclerosis, and myocardial anemia. ^1,2 Chemical studies of Canna species indicated the presence of carbohydrates, proteins, amino acids, steroids, alkaloids, phenolics, flavonoids, tannins, and terpenoids. ^{2 -4} Some of them exhibited anthelmintic, antioxidant, cytotoxic, antibacterial, antimicrobial, antiviral, antidiabetic, antidiarrheal, anti-inflammatory, analgesic, immunomodulatory, hemostatic, hepatoprotective, molluscicidal, and α-glucosidase inhibition. ^{1 -7} Up until now, there has been no particular evaluation of the chemical composition of Canna indica in Vietnam. Herein, we report the isolation, structural elucidation of six compounds, including two new acylated sucroses, and their antioxidant activity in vitro.

Results and Discussion

Compound 1 was obtained as a colorless amorphous powder. Its molecular formula was deduced to be C₂₄H₃₂O₁₅ based on the cluster of quasi-molecular ion peaks in the high resolution electron spray ionization mass spectrum (HR-ESI-MS) at m/z 583.1638 [M + Na]⁺ (Calcd. for [C₂₄H₃₂O₁₅Na]⁺, 583.1633) (positive ion mode), m/z 595.1443 [M+³⁵Cl]^- (Calcd. for [C₄₅H₇₂O₁₉ ³⁵Cl]^-, 595.1430), and at m/z 597.1421 [M+³⁷Cl]^- (Calcd. for [C₄₅H₇₂O₁₉ ³⁷Cl], 597.1400) (negative ion mode), indicating 9 degrees of unsaturation (Supplemental Figure S1). The ¹H NMR spectrum of 1 exhibited three aromatic signals at δ _H 7.10 (1H, dd, J = 8.0, 2.0 Hz), 6.83 (1H, d, J = 8.0 Hz), and 7.21 (1H, d, J = 2.0 Hz), corresponding to a 1,3,4-tri-substituted benzene ring; two doublet signals of one double bond at δ _H 7.65 (1H) and δ _H 6.40 (1H) with trans configuration, confirmed by the relative large coupling constants (J = 16.0 Hz), one singlet of an acetyl group at δ _H 2.11 (3H, s), and one methoxy group at δ _H 3.92 (3H, s). In addition, two sugars were identified by signals of fourteen proton resonances from δ _H 3.27 to 5.41, including one anomeric proton at δ _H 5.41 (d, J = 3.5 Hz), and one singlet of fructose at δ _H 3.65 (2H, s) (Supplemental Figure S2). ^{8 -10} Detailed analyses of the ¹³C NMR and HSQC spectra showed that compound 1 contains 24 carbon atoms comprising 2 ester carbonyl, 6 aromatic, 2 olefinic, 12 oxygenated, 1 methyl and 1 methoxy (Supplemental Figures S3 and S4). In the HSQC spectrum, protons at δ _H 7.10, 6.83, 7.21, 7.65, 6.40 had cross peaks to carbons at δ _C 124.2, 116.5, 111.8, 147.0, and 115.3, respectively, while protons at δ _H 2.11, 3.92, 5.41, and 3.65 had HSQC cross peaks to carbons at δ _C 20.9, 56.5, 93.2, and 64.1, respectively. One acetyl group was further confirmed by two signals at δ _C 20.9 and 173.0. A set of carbon signals at δ _C 93.2, 73.2, 74.7, 72.0, 71.9 (5 CH), 66.7 (CH₂) were assigned for the glucopyranosyl sugar, while signals at δ _C 64.0/δ _H 3.65 (2H, s), δ _C 105.4 (C), δ _C 78.9/ δ _H 4.12, δ _C 76.9/δ _H 4.09, δ _C 80.7/δ _H 4.03, δ _C 65.6/ δ _H 4.45 and 4.12 were assigned for the fructofuranosyl unit, which matched well with a sucrose unit. ^{8 -10} The sugar linkage must be in the α-form, indicated from the small coupling constant of the anomeric proton of the glucose unit (δ _H 5.38, d, J = 3.5 Hz). The presence of the sucrose moiety was further confirmed by TLC analysis of the alkaline hydrolysis product of 1 in comparison with authentic sucrose. ¹¹ This evidence suggested that the chemical structure of 1 was similar to that of arillatose B⁸, but 1 had one more acetyl group. A detailed structural elucidation of compound 1 was further performed using ¹H-¹H COSY and HMBC experiments, as shown in Figure 1 (Supplemental Figures S5 and S6). HMBC correlations from the protons at δ _H 4.50 and 4.47 of the oxymethylene group to the carbonyl carbon at δ _C 168.9, from anomeric proton H-1′ (δ _H 5.41) to anomeric carbon C-2 (δ _C 105.4), and from the other oxymethylene protons at δ _H 4.45/4.12 to the carbonyl carbon at δ _C 173.0 confirmed that the feruoyl group was attached to C-6′ of the glucosyl sugar, which linked to C-2 of the fructofuranosyl unit, and the acetyl group attached to C-6 of the fructofuranosyl unit. Furthermore, the lower field signal of C-6 (δ _C 65.6) confirmed the ester linkage between the acetyl group and C-6. Consequently, the new chemical structure of compound 1 was established as (6-O-acetoxyl)-β-d-fructofuranosyl-(2→1′)−6′-O-feruoyl-α-d-glucopyranoside (Figure 2).

Figure 1

Important HMBC and COSY correlations of compounds 1 and 2.

Figure 2

Chemical structures of compounds 1-6 isolated from the roots of Canna indica.

Compound 2 was also obtained as a colorless amorphous powder. Its molecular formula was deduced to be C₂₃H₃₀O₁₄ based on the cluster of quasi-molecular ion peaks in the HR-ESI-MS at m/z 553.1521 [M + Na]⁺ (Calcd. for [C₂₃H₃₀O₁₄Na]⁺, 553.1528) (positive ion mode), and m/z 565.1340 [M+³⁵Cl]^- (Calcd. for [C₂₃H₃₀O₁₄ ³⁵Cl]^-, 565.1330) (negative ion mode) indicating 9 degrees of unsaturation (Supplemental Figure S7). The NMR spectra of 2 were similar to those of 1 suggested that compound 2 was a derivative of 1 with the same chemical skeleton, including one sucrose, one coumaroyl and one acetyl group. ⁸ The coumaroyl group was identified from the observation of a para-substituted benzene ring [(δ _H 7.48 (2H, d, 8.0 Hz)/ δ _C 131.2), δ _H 6.83 (2H, d, 8.0 Hz)/ δ _C 116.8)] and a trans double bond [(δ _H 7.65 (1H, d, 16.0)/ δ _C 146.8), δ _H 6.36 (1H, d, 16.0)/ δ _C 115.0)], and the acetyl group was identified at δ _H 2.11/δ _C 20.9 and δ _C 173.0. The NMR data of the sugar moiety of 2 were very similar to those of 1 measured in the same solvent, suggesting the presence of a sucrose (Table 1) (Supplemental Figures S8-S12), which was further confirmed by the small coupling constant of the anomeric proton of the glucose unit (δ _H 5.38, d, J = 3.5 Hz), and by TLC analysis of the alkaline hydrolysis product of 2 in comparison with authentic sucrose. ¹¹ In the HMBC spectrum of 2, H-6′ (δ _H 4.43/4.48) correlated with C-9′′ (δ _C 169.0), H-1′ (δ _H 5.42) with C-2 (δ _C 105.4), and H-6 (δ _H 4.13/4/46) with the carbonyl carbon at δ _C 173.0. The above evidence confirmed that the p-coumaroyl group linked to C-6′ of the glucose, C-1′ of this glucose linked to C-2 of the fructose, and the acetyl group attached to C-6 of the fructose. Consequently, the new chemical structure of compound 2 was established as (6-O-axetoxyl)-β-d-fructofuranosyl-(2→1)-(6-O-(E)-p-coumaroyl-α- d-glucopyranoside (Figure 2).

Table 1

¹H-NMR and ¹³C-NMR Spectroscopic Data for Compounds 1 and 2 in Deuterated Methanol.

Pos.	δ Ca	δ Hb (mult., J in Hz)	δ Ca	δ Hb (mult., J in Hz)
1	64.0	3.65 (2H, s)	64.0	3.65 (2H, s)
2	105.4	-	105.4	-
3	78.9	4.12 (1H, d, 9.0)	78.8	4.12 (1H, d, 9.0)
4	76.7	4.09 (1H, t, 9.0)	76.7	4.10 (1H, t, 9.0)
5	80.9	4.03 (1H, m)	80.8	4.02 (1H, m)
6	65.6	4.12 (1H, d, 12.0) 4.45 (1H, d, 12.0)	65.6	4.13 (1H, d, 12.0) 4.46 (1H, d, 12.0)
1′	93.2	5.41 (1H, d, 3.5)	93.1	5.42 (1H, d, 3.5)
2′	73.2	3.43 (1H, dd, 9.0, 3.5)	73.2	3.46 (1H, dd, 9.0, 3.5)
3′	74.7	3.74 (1H, t, 9.0)	74.7	3.74 (1H, t, 9.0)
4′	72.0	3.27 (1H, t, 9.0)	72.0	3.27 (1H, t, 9.0)
5′	71.9	4.10 (1H, m)	71.9	4.08 (1H, m)
6′	66.7	4.47 (1H, m)*	66.8	4.43 (1H, m)*
		4.50 (1H, m)*		4.48 (1H, m)*
1′′	127.7	-	127.2	-
2′′	111.8	7.21 (1H, d, 2.0)	131.2	7.48 (1H, d, 8.0)
3′′	149.4	-	116.8	6.83 (1H, d, 8.0)
4′′	150.7	-	161.3	-
5′′	116.5	6.83 (1H, d, 8.0)	116.8	6.83 (1H, d, 8.0)
6′′	124.2	7.10 (1H, dd, 8.0, 2.0)	131.2	7.48 (1H, d, 8.0)
7′′	147.0	7.65 (1H, d, 16.0)	146.8	7.65 (1H, d, 16.0)
8′′	115.3	6.40 (1H, d, 16.0)	115.0	6.36 (1H, d, 16.0)
9′′	168.9	-	169.0	-
CH3 CO	173.0	-
CH3CO	20.9	2.11 (3H, s)

Abbreviation: NMR, Nuclear magnetic resonance.

*Overlapped signals.

^aMeasured at 125 MHz.

^bMeasured at 500 MHz.

Compounds 3-6 were identified as 2-(3′,4′-dihydroxyphenyl)−1-propanol-4′-O-[4′′′-hydroxy-3′′′,5′′′-dimethoxybenzoyl-(→6′′)-β-d-fructofuranoside (3), ¹² tryptopholglucoside (4), ¹³ corchonoside C (5), ¹⁴ and 2-hydroxy-5-(2-hydroxyethyl)phenyl β-d-fructofuranoside (6), ¹⁵ The NMR spectral data of compounds 3-6 were consistent with those previously reported in the literature (Supplemental Figures S13-S20). Antioxidant activity of compounds 1-6 was evaluated by peroxyl radical absorbance capacity assay. Each compound was examined at two concentrations (1 and 10 µM). Compounds 4 and 5 were inactive. However, at both tested concentrations (1 and 10 µM), compounds 1-3 and 6 exhibited ORAC_ROO* values greater than those of Trolox equivalents, indicating that those compounds could be potential peroxyl radical scavenging agents (Supplemental Table S1).

Material and Methods

General Experimental Procedures

Optical rotation was measured on a Jasco P-2000 polarimeter. The NMR spectra were recorded on a Bruker 500 MHz spectrometer and HR-ESI-MS on an Agilent 6530 Accurate Mass Q-TOF LC/MS. The QTOF instrument was set at 2 GHz extended dynamic range resolution mode, negative ESI capillary voltage of 3500 V, fragmentor voltage of 175 V, MS scan ranging from m/z 100‐1700, and an MS acquisition rate of 1.0 spectra/s. Flash column chromatography was performed using either silica gel or reversed phase (RP-18) resins as adsorbent. Thin layer chromatography was carried out on pre-coated silica gel 60 F₂₅₄ and/or RP-18 F_254S plates. Traces of compounds were visualized under UV irradiation (254 and 365 nm) and by spraying with H₂SO₄ solution (5%), followed by heating with a heat gun. HPLC was carried out using an AGILENT 1100 HPLC system.

Plant Material

The roots of Canna indica L. were collected at Bach Thong District, Backan Province, Vietnam in May 2020 and taxonomically identified by Dr Nguyen The Cuong at the Institute of Ecology and Biological Resources, VAST. A voucher specimen (NCCT-P95) was deposited at the Institute of Marine Biochemistry, VAST.

Extraction and Isolation

The roots of C. indica (fresh sample, 13 kg) were cut, dried, and powdered to obtain 3.5 kg, which was ultrasonically extracted with methanol at room temperature, three times (each, 10 L of methanol for 2 hours), and then filtered through filter paper. The solvent was removed to yield 150 g of a dark solid extract. This was suspended in water and successively partitioned with dichloromethane then ethyl acetate giving dichloromethane (35 g) and ethyl acetate extracts (2.5 g), and a water layer. The water layer was chromatographed on a Diaion HP-20 column eluting with water to remove sugar, then with an increasing concentration of methanol in water (25% and 100%) to obtain four fractions, F1 (25% MeOH, 25.0 g), F2 (50% MeOH, 17.0 g), F3 (75% MeOH, 14.0 g), and F4 (100% MeOH, 23.5 g). The F3 and F4 fractions were combined to yield one fraction (27.5 g), which was chromatographed on a silica gel column eluting with dichloromethane/methanol (1/3, v/v) to give five sub-fractions F5A-F5E. F5E (10.2 g) was chromatographed on a silica gel column eluting with dichloromethane/acetone/water (1/1/0.04, v/v/v) to give nine smaller fractions (F5E1-F5E9). F5E9 was further chromatographed by HPLC using a J’sphere ODS H-80, 250 mm × 20 mm column, and ACN in H₂O (15 %) to yield compounds 1 (4.5 mg) and 2 (15.0 mg). F5E6 was further purified by HPLC using a J’sphere ODS H-80, 250 mm × 20 mm column, and ACN in H₂O (15 %) to yield compound 4 (4.4 mg). Compound 3 (3.4 mg) was obtained from F5E7 by purifying by HPLC using the same conditions. The F5B fraction was chromatographed on a silica gel column eluting with dichloromethane/acetone/water (1/2/0.1, v/v/v) to obtain 5 smaller fractions (F5B1-F5B5). F5B4 was further purified by HPLC using a J’sphere ODS H-80, 250 mm × 20 mm column, and ACN in H₂O (5 %) to yield compound 6 (20.4 mg). F5B5 was further purified on HPLC using a J’sphere ODS H-80, 250 mm × 20 mm column, and ACN in H₂O (15 %) to yield compound 5 (4.0 mg).

(6-O-axetoxyl)-β-d-fructofuranosyl-(2→1)-(6-O-feruloyl-α-d-glucopyranoside (1)

Colorless amorphous powder, $[\alpha {]}_{\mathrm{D}}^{25}:$ − 51.0° (c 0.1, MeOH); HR-ESI-MS m/z 583.1638 [M + Na]⁺ (Calcd. for [C₂₄H₃₂O₁₅Na]⁺, 583.1633), m/z 595.1443 [M+³⁵Cl]^- (Calcd. for [C₄₅H₇₂O₁₉ ³⁵Cl]^-, 595.1430), and m/z 597.1421 [M+³⁷Cl]^- (Calcd. for [C₄₅H₇₂O₁₉ ³⁷Cl], 597.1400); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) data are given in Table 1.

(6-O-axetoxyl)-β-d-fructofuranosyl-(2→1)-(6-O-(E)-p-coumaroyl-α-d-glucopyranoside (2)

Colorless amorphous powder, $[\alpha {]}_{\mathrm{D}}^{25}:$ − 48.5° (c 0.1, MeOH); HR-ESI-MS m/z 553.1521 [M + Na]⁺ (Calcd. for [C₂₃H₃₀O₁₄Na]⁺, 553.1528), m/z 565.1340 [M+³⁵Cl]^- (Calcd. for [C₂₃H₃₀O₁₄ ³⁵Cl]^-, 565.1330). ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) data are given in Table 1.

Alkaline hydrolysis

Compounds 1 and 2 were (each 0.5 mg) dissolved in 1.0 ml solution of KOH (1.0 M) in methanol and heated at 60 °C for 2 hours. The reaction was cooled to room temperature and the solution carefully neutralized with a solution of HCl (1.0 M). The solvent was then driven off under a nitrogen flow. The residue was re-dissolved in 1.0 ml of water and extracted twice with equal volumes of ethyl acetate. Sucrose in the water layer was confirmed by co-TLC analysis in comparison with authentic sucrose (CHCl₃: MeOH : Water = 1.0 : 1.0 : 0.15, R _f = 0.35).

Oxygen radical absorbance capacity assay

Refer to Supplemental Material.

Conclusions

Two new acylated sucroses, (6-O-axetoxyl)-β-d-fructofuranosyl-(2→1)-(6-O-feruloyl- α- d-glucopyranoside (1) and (6-O-axetoxyl)-β-d-fructofuranosyl-(2→1)-(6-O-(E)-p-coumaroyl- α- d-glucopyranoside (2), and four known compounds, 2-(3′,4′-dihydroxyphenyl)-1-propanol-4′-O-[4′′′-hydroxy-3′′′,5′′′-dimethoxybenzoyl-(→6′′)-β-d-fructofuranoside (3), tryptophol glucoside (4), corchonoside C (5), and 2-hydroxy-5-(2-hydroxyethyl)phenyl β-d-fructofuranoside (6), were isolated from the roots of Canna indica L. (Cannaceae). Their structures were determined by extensive analysis of HR-ESI-MS and NMR spectral data, as well as by comparison of the spectral data with those reported in the literature. Compounds 1 and 3 exhibited high peroxyl radical absorbance capacity. At a concentration of 1 µM, their ORAC_ROO* values were 3.07 ± 0.15 and 4.27 ± 0.30, respectively, folds-up to that of Trolox. At the high concentration of 10 µM, their ORAC_ROO* values were equivalent to 15.60 ± 0.22 and 24.91 ± 0.43 times the net protection by 1 µM of Trolox.

Supplemental Material

Supplementary material - Supplemental material for Two New Acylated Sucroses From the Roots of Canna indica L. and Their Antioxidant Activity

Supplemental material, Supplementary material, for Two New Acylated Sucroses From the Roots of Canna indica L. and Their Antioxidant Activity by Luu The Anh, Nguyen Hieu, Do Thi Trang, Bui Huu Tai and Phan Van Kiem in Natural Product Communications