One New Veratramine-Type Alkaloid From Veratrum maackii var. japonicum and Antioxidative Activities of Isolated Compounds

Abstract

A new veratramine-type alkaloid (1), along with 4 known compounds (2-5), was isolated from the roots of Veratrum maackii var. japonicum (Baker) T. Shimizu. Their structures were elucidated on the basis of NMR and mass spectroscopic data. All compounds were evaluated for their antioxidant activities using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS ⁺ ) and 2,2-diphenyl-1-picrylhydrazyl (DPPH^·) radical scavenging assays. Compounds 1 and 3-5 showed ABTS⁺ radical scavenging activity with IC₅₀ values ranging from 15.0 to 85.7 μM.

Veratrum maackii var. japonicum (Baker) T. Shimizu is a traditional medicinal herb belonging to the family Melanthiaceae which comprises about 40 species. ^{1 -3} It is widely distributed in Korea, China, and America. Previous chemical investigations of Veratrum sp. revealed the existence of various types of steroidal alkaloids such as jervanine, veratramine, cevanine, and verazine ⁴ with anti-inflammatory, ⁵ anti-hypotensive, ⁶ anti-oxidative, ⁷ and anti-fungal activities. ⁸ In our continuous investigation of anti-oxidative constituents from herbal plants, we isolated one new steroidal alkaloid (1), along with four known compounds (2-5) from the roots of V. maackii var. japonicum. Herein, we described the isolation, structure elucidation, and anti-oxidative activities of compounds 1-5 (Figure 1).

Figure 1

The structures of compounds 1-5.

Results and Discussion

Compound 1 was acquired as colorless oil with a specific rotation value of −24.9 (c 0.01, MeOH). The HR-ESI-MS showed a pseudomolecular ion at m/z 618.4006 [M+H]⁺ (calcd for C₃₅H₅₆NO₈, 618.4006) and the molecular formula of 1 was established as C₃₅H₅₅NO₈. The ¹H NMR spectrum of 1 (Supplemental Material S1) showed the resonances of one olefinic methine, six oxygenated methines, one acetal methine, one nitrogenated methine, six methines, one oxygenated methylene, one nitrogenated methylene, eight methylenes, and five methyls. The ¹³C NMR spectrum of 1 (Supplemental Material S3) showed the resonances of 35 carbons including one carbonyl carbon, three olefinic quaternary carbons, one quaternary carbon, one olefinic methine carbons, one acetal methine carbon, six oxygenated methine, one nitrogenated methine carbon, six methine carbons, one oxygenated methylene carbon, one nitrogenated methylene carbon, eight methylene carbons, and five methyl carbons (Table 1). These 1D NMR data indicated that compound 1 was a veratramine-type alkaloid. ⁹ One bond correlations from proton (¹H) to carbon (¹³C) were assigned by heteronuclear multiple-quantum correlation (HMQC) experiment (Supplemental Material S5). The steroidal moiety was determined by the HMBC correlations (Supplemental Material S6) from H-19 to C-1, C-5, C-9, and C-10, from H-4 to C-2, C-3, C-5, and C-6, from H-7 to C-6, from H-9 to C-19, from H-11 to C-8, C-9, C-12, and C-13, and from H-18 to C-12, C-13, and C-17 and the ¹H-¹H correlation spectroscopy (COSY) correlations of H-2/H-3/H-4, H-6/H-7, H-8/H-14, and H-15/H-16/H-17. The glucose moiety was determined using ¹H-¹H COSY correlations of H-1′/H-2′/H-3′/H-4′ and H-5′/H-6′ and heteronuclear multiple bond correlation (HMBC) from H-6′ to H-4′ and H-5′. The anomeric proton at H-1′ in 1 was assigned at β-form from the ¹H NMR coupling constant (J = 7.7 Hz). The absolute configuration of d-glucose was elucidated using acid hydrolysis and HPLC. Furthermore, the attachment between steroidal moiety and glucoside moiety was elucidated by HMBC correlation from H-1′ to C-3. The HMBC correlations from H-22 to C-23, from H-24 to C-22, C-23, and C-25, from H-27 to C-24, C-25, and C-26, from H-26 to C-22, C-24, and from H-27 to C-24, C-25, and C-26 and the ¹H-¹H COSY correlations of H-22/H-23/H-24 and H-25/H-26 indicated the presence of piperidine moiety. The linkage between steroidal moiety and piperidine moiety suggested the HMBC correlations from H-21 to C-20 and C-22, and from H-22 to C-17 and C-20. Furthermore, the acetate moiety was elucidated by HMBC correlation from H-29 to C-28 (Figure 2). The downfield proton signal at H-23 (δ 4.63) indicated the connectivity between piperidine moiety and acetate moiety. In addition, it was confirmed by the comparison of ¹H NMR chemical shift at H-23 of veratramine-3-yl acetate in the literature. ⁹ The relative configuration of 1 was determined using ROESY experiments(Supplemental Material S7). The rotating frame nuclear Overhauser effect spectroscopy (ROESY) correlations of H-19/H-8, H-19/H-4b, H-19/2b, and H-19/H-11b suggested the same face. Furthermore, the ROESY correlations of H-1a/H-3, H-1a/H-9, H-9/H-14, and H-9/H-11a indicated the other face. The key ROESY correlations of H-19/H-8 and H-9/H-14 suggested the same stereochemistry of steroidal moiety in 1 with veratramine-3-yl acetate. ⁹ The key ROESY correlations of piperidine moiety were H-23/H-25, H-23/H-24a, H-22/H-26a, and H-22/H-24a. The configuration of C-25 (δ 30.4) was assigned based on a comparison of ¹³C NMR spectroscopic data with jervine-3-yl formate and veratramine-3-yl acetate in the literature. ⁹ Furthermore, the ROESY correlations between H-21/H-23 were assigned to be same orientation. We tentatively proposed it to be in β-orientation at H-17 by spectral comparison with the literature values. ¹⁰ Accordingly, compound 1 was established as 22S,25S,5α-veratramine-5(6), 12(13)-diene-23β-acetyl-3-O-β-d-glucopyranoside, and named as veratramanol A.

Figure 2

Key ¹H-¹H COSY (bold lines), HMBC (arrows), and ROESY (dotted arrows) correlations of compound 1.

Table 1

The ¹H (500 MHz) and ¹³C (125 MHz) NMR Data for Veratramanol A in CD₃OD.

No.	1
	δ C	δ H (mult. J in Hz)
1a	38.1	1.19, m
1b		1.71, m
2a	29.1	1.92, m
2b		1.62, m
3	78.6	3.58, m
4a	37.9	2.50, m
4b		2.23, m
5	141.4
6	121.7	5.38, brt (2.5)
7	30.6	1.73, m
		2.19, m
8	42.7	1.15, m
9	52.4	1.44, m
10	36.7
11	27.2	2.25, m
		2.05, m
12	125.7
13	139.1
14	48.6	1.77, m
15	23.0	1.78, m
		1.12, m
16	27.6	1.70, m
		1.54, m
17	41.0	2.09, m
18	19.0	1.61, s
19	17.5	0.95, s
20	41.1	1.78, m
21	15.1	0.93, d (7.7)
22	62.1	2.64, dd (10.0, 3.6)
23	71.8	4.63, ddd (10.0, 10.0, 4.5)
24a	39.3	2.09, m
24b		0.98, m
25	30.4	1.69, m
26a	52.9	2.85, brd (11.9)
26b		2.13, m
27	17.6	0.85, d (6.4)
28	170.3
29	20.1	2.00, s
1′	101.2	4.36, d (7.7)
2′	73.8	3.12, dd (9.1, 7.7)
3′	76.7	3.32, dd (9.1, 8.7)
4′	70.3	3.23, m
5′	76.5	3.23, m
6′	61.4	3.82, dd (12.0, 1.8)
		3.62, dd (12.0, 5.2)

The known compounds were identified as angeloylzygadenine (2), ¹¹ oxyresveratrol-4′-O-β-glucopyranoside (3), ¹² oxyresveratrol-3-O-β-glucopyranoside (4), ¹² and polydatin (5). ¹³

The antioxidant activities of 1-5 were evaluated using 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) and 2,2-diphenyl-1-picrylhydrazyl (DPPH^·) radical scavenging assay. ¹⁴ The positive control, ascorbic acid, showed IC₅₀ values of 39.5 ± 1.5 and 55.4 ± 2.1 µM in the ABTS⁺ and DPPH^· radical scavenging assay, respectively. Compounds 3 and 5 showed significant ABTS⁺ radical scavenging activity with IC₅₀ values of 15.0 ± 1.3 and 22.3 ± 4.1 µM, respectively. In addition, compounds 1 and 4 exhibited a moderate ABTS⁺ radical scavenging activity with IC₅₀ values of 64.7 ± 1.1 and 85.6 ± 2.5 µM, respectively. However, compound 2 showed no activity at 200 µM. Furthermore, compounds 1-5 displayed no activity at 200 µM in the DPPH^· radical scavenging assay.

Interestingly, compound 3 showed higher activity than compound 5, suggesting that the attachment of glucoside moiety at C-4′ considerably increased the ABTS⁺ radical scavenging activity. Furthermore, compound 5 exhibited much higher ABTS⁺ radical scavenging activity than compound 4, indicating that the absence of hydroxyl group at C-6′ significantly increased the ABTS⁺ radical scavenging activity.

Experimental

General Experimental Procedures

The optical rotation was measured with JASCO P-2000 Digital Polarimeter (JASCO corporation, Tokyo, Japan). The UV spectrum was recorded on Shimadzu UV-1650 PC UV-Visible spectrophotometer (Shimadzu, Kyoto, Japan). The ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectra were measured on a JNM-ECZ500R FT-NMR spectrometer (JEOL, Tokyo, Japan). The HR-ESI-MS spectrum was measured on SYNAPT G2 QTOF mass spectrometer in positive ion mode (Waters, Miliford, United States). Medium-pressure liquid chromatography (MPLC) (CombiFlash RF, Teledyne Isco, Lincoln, United States) was performed using a C₁₈ column (40 g, Agela Technologies Inc, Tianjin, PR China). Column chromatography was performed with silica gel (0.063-0.200 mm, Merck, Darmstadt, Germany) and Sephadex LH-20 (GE Heathcare, Buckinghamshire, United Kingdom). HPLC (Waters 2685 Separation module, Waters, Miliford, United States) was performed using a C₁₈ column (Waters, 5 µm, 4.6 mm × 250 mm) with photodiode array detector (PAD) (Waters 2996).

Plant Materials

The roots of V. maackii var. japonicum (Baker) T. Shimizu were collected from Pyeongchang, Kangwon province, Korea, and authenticated by Dr Dong-Seon Kim. The voucher specimen was deposited at the Korean herbarium of standard Herbal Resources of Korea Institute of Oriental Medicine (KIOM202001023470, Dajeon, Korea). The roots of V. maackii var. japonicum were dried at 26 °C and powdered before ethanol extraction.

Extraction and Isolation

The dried roots of V. maackii var. japonicum (160 g) were powdered and extracted with ethanol for 1 day at 26 °C. The extracts were acidified with HCl (pH 3) and partitioned with EtOAc. The aqueous solutions were basified with NH₄OH (pH 12) and repartitioned with EtOAc. ^2,15 The EtOAc-soluble portions from basic aqueous solutions (2.1 g) were subjected to silica gel (300 g) column chromatography eluted with increasing polarity of CHCl₃:MeOH (100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1, v/v, 2 L) to afford 8 fractions (A-H). Fraction F (331.0 mg) was fractionated on MPLC using a gradient eluent of H₂O:MeOH (from 1:0 to 0:1) to give 5 fractions (F1-F5). Fraction F4 (25.8 mg) was further separated by Sephadex LH-20 (100 g) column chromatography, using MeOH as the eluent to obtain 2 compounds 1 (4.7 mg, R _f = 0.4, CHCl₃:MeOH = 5:1) and 2 (3.9 mg, R _f = 0.2, CHCl₃:MeOH = 5:1). Fraction G (384.0 mg) was separated by MPLC (40 g C₁₈ column, flow rate: 10 mL/min) using gradient H₂O:MeOH solvent condition (90:10-0:100, 120 minutes) to yield 5 fractions (G1-G5). Fraction G2 (40.0 mg) was further isolated by reversed-phase HPLC with isocratic solvent condition of 35% aq. MeOH to provide 3 compounds: 3 (4.8 mg, t _R = 9.4 minutes), 4 (3.2 mg, t _R = 11.4 minutes), and 5 (2.4 mg, t _R = 17.9 minutes).

Veratramanol A (1)

Colorless oil.

$[\alpha {]}_{\mathrm{D}}^{21}$ : −24.9 (c 0.01, MeOH).

UV (MeOH): λ _max (log ε) 218 nm (3.6).

¹H (500 MHz) and ¹³C (125 MHz) NMR data: see Table 1.

HR-ESI-MS (+): m/z 618.4006 [M+H]⁺ (calcd for C₃₅H₅₆NO₈, 618.4006). (Supplemental Material S8)

Acid Hydrolysis of 1 and Determination of Sugar

Compound 1 (1.0 mg) was heated at 60 °C for 2 hours in 2 mL of 2 N trifluoroacetic acid, and the resulting product was extracted with EtOAc. The water-soluble layer was concentrated to dryness in vacuo. The residue was dissolved in pyridine (0.1 mL) and added by l-cysteine methyl ester hydrochloride (2.0 mg) at 60 °C for 1 hour. The o-tolyl isothiocyanate (10 µL) was added to the mixture and the mixture was heated at 60 °C for 1 hour. The reaction mixture was analyzed with HPLC using C₁₈ column (5 µm, 4.6 mm × 250 mm, Waters), UV detection at 250 nm, and isocratic condition of 23% aq. ACN in 0.1% formic acid (1 mL/min, 25 minutes). ^16,17 Under same condition, the retention time of authentic samples was 17.3 minutes (d-glucose) and 16.1 minutes (l-glucose), respectively. The peak of 1 was coincided with the derivative of authentic d-glucose (17.3 minutes).

ABTS⁺ Radical Scavenging Assay

The ABTS ⁺ radical scavenging activity was evaluated using the modified methods. ¹⁴ Breifly, the ABTS ⁺ was dissolved in water (7 mM) and 2.45 mM potassium persulfate was added. The mixture was incubated for 12 hours in dark condition. The sample (10 µL) was reacted with ABTS ⁺ solution (190 µL) in a 96-well plate for 10 minutes. The absorbance was measured at 734 nm in microplate. The ascorbic acid was used as positive control.

$$\mathrm{A}\mathrm{B}\mathrm{T}{\mathrm{S}}^{+}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{\%})=\frac{\mathrm{A}\mathrm{b}\mathrm{s}\left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)-\mathrm{A}\mathrm{b}\mathrm{s}\left(\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\right)}{\mathrm{A}\mathrm{b}\mathrm{s}\left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}\times 100$$

DPPH^· Radical Scavenging Assay

The DPPH^· radical scavenging activity was determined by minor modified methods. ¹⁴ The DPPH^· was dissolved in ethanol. The sample (10 µL) was mixed with 0.45 mM DPPH^· solution (90 µL) in a 96-well plate. The mixture was reacted for 10 minutes at room temperature (26 °C) and measured at 517 nm. The ascorbic acid was used as positive control.

$${\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}}^{\cdot }\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{\%})=\frac{\mathrm{A}\mathrm{b}\mathrm{s}\left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)-\mathrm{A}\mathrm{b}\mathrm{s}\left(\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\right)}{\mathrm{A}\mathrm{b}\mathrm{s}\left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}\times 100$$

Supplemental Material

Supplementary Material 1 - Supplemental material for One New Veratramine-Type Alkaloid From Veratrum maackii var. japonicum and Antioxidative Activities of Isolated Compounds

Supplemental material, Supplementary Material 1, for One New Veratramine-Type Alkaloid From Veratrum maackii var. japonicum and Antioxidative Activities of Isolated Compounds by Ji-Yul Kim, Eunjung Son and Dong-Seon Kim in Natural Product Communications