Strychnovanosides A - C,Three New Lignan Glycosides from Strychnos vanprukii

Abstract

Phytochemical study of the methanolic extract of the stems and leaves of Strychnos vanprukii led to the isolation of three new (1-3) and four known (4-7) compounds. Their chemical structures were elucidated to be (+)-lyoniresinol 3α-O-β-D-allopyranoside (1), (-)-lyoniresinol 3α-O-β-D-allopyranoside (2), 2α-[(3,5-dimethoxy-4-hydroxy)-benzoyl]-(-)-lyoniresinol-3α-O-β-D-glucopyranoside (3), (+)-lyoniresinol 3α-O-β-D-glucopyranoside (4), (-)-lyoniresinol 3α-O-β-D-glucopyranoside (5), palicoside (6), and rutin (7) by means of detailed HR-ESI-MS, 1D and 2D NMR spectroscopic analysis. In addition, the absolute configurations of 1–3 were determined by experimental Circular Dichroism (CD) spectra. This is the first report of compounds 5 and 7 from S. vanprukii. Compounds 1 - 7 showed inhibitory effects on NO production in LPS stimulated RAW 264.7 cells with IC₅₀ values of 58.50 ± 2.30, 67.70 ± 2.23, 54.44 ± 1.26, 71.58 ± 3.59, 74.47 ± 2.61, 83.62 ± 2.25, and 47.57 ± 4.01 µM, respectively, compared to a value of 43.24 ± 2.41 µM for the positive control compound, N^G-monomethyl-L-arginine (L-NMMA).

Introduction

Strychnos vanprukii Craib (Loganiaceae) grows in India, Borneo, the Malay peninsula, Lao, Thailand and Vietnam. The leaves and stems of this plant have been used in Vietnam as a muscle relaxant, and to treat muscle aches, osteoarthritis, and poliomyelitis. ¹ Previous phytochemical studies of this plant reported that the stem and leaves contained the alkaloids 3,4-dehydropalicoside, palicoside, 3,4,5,6-tetradehydropalicoside, akagerine and 17-O-methylakagerine ² ; triterpenoids, 3β-O-trans-feruloylbetulinic acid, 3β-O-cis-coumaroylbetulinic acid, 3β-O-trans-coumaroylbetulinic acid, and ursolic acid ³ ; lignan glucosides, vanprukoside, strychnoside, (+)-lyoniresinol-3α-O-β-glucopyranoside ⁴ ; diterpenoids, 7β-hydroxypimara-8,15-dien-14-one, ² 7β,12β-dihydroxypimara-8,15-dien-14-one, and 14-hydroxy-15,16-dinorpimara-8,11,13-trien-7-one. ⁵ Of these compounds, betulinic acid derivatives and ursolic acid showed moderate anti-HIV activity ³ and all the lignans exhibited antioxidant activity. ⁴ In the course of our ongoing study on bioactive compounds from Vietnamese folk medicinal plants, we report herein the isolation and chemical structure determinations of three new lignan glycosides, together with four known compounds, and the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS) stimulated RAW 264.7 cells by these compounds.

Results and Discussion

Compounds 4 and 5 were identified to be (+)-lyoniresinol 3α-O-β-D-glucopyranoside (4) and (-)-lyoniresinol 3α-O-β-D-glucopyranoside (5) by analyzing their NMR ⁶ and CD ⁷ spectra [λ (θ: mdeg): 244 (+4.10), 274 (+1.26) nm for 4 and 245 (−4.53), 274 (−1.67) nm for 5] and comparison with those reported in the literature. Compound 4 ⁴ and palicoside (6) ² have been isolated from S. vanprukii, but 5 and rutin (7) ⁸ are reported from this species for the first time.

Compound 1 was obtained as a colorless solid. The high-resolution electrospray ionization mass spectrum (HR-ESI-MS) of 1 exhibited quasi-molecular ion peaks at m/z 600.2661 [M + NH₄]⁺ (calcd. for [C₂₈H₄₂NO₁₃]⁺: 600.2651, Δ = + 1.7 ppm); m/z 605.2207 [M + Na]⁺ (calcd. for [C₂₈H₃₈O₁₃Na]⁺: 605.2205, Δ = -0.3 ppm); and m/z 583.2377 [M + H]⁺ (calcd. for [C₂₈H₃₉O₁₃]⁺: 583.2385, Δ = -1.4 ppm) indicating its molecular formula to be C₂₈H₃₈O₁₃. The nuclear magnetic resonance (NMR) spectra of 1 were very similar to those of lignan glycoside 4 with two aromatic rings, four methoxy groups and one sugar moiety (Table 1), ⁴ suggesting that they have the same aglycone planar structure (Figure 1). The chemical structure of 1 was further determined based on the HSQC, HMBC, ¹H-¹H COSY, and NOESY spectra (Figure 2). The HMBC correlations of H-3/C-1′; H-4/C-2′, C-6′; H-3α/C-4, C-2; H-2α/C-1, C-2, C-3; H-1/C-8, C-9, C-10; H-1″ (allose)/C-3α clearly confirmed the free hydroxy group at C-2α, and the glycosyl linkage was formed at C-3α. In addition, the methoxy protons at 3.37, 3.87, and 3.77 (6H) had HMBC interactions with C-5 (δ 147.6), C-7 (δ 148.6), and C-3′/C-5′ (δ 149.0), respectively, indicating four methoxy groups at C-5, C-7, C-3′, and C-5′. The HSQC cross peaks of H-1″/C-1″, H-2″/C-2″, H-3″/C-3″, H-4″/C-4″, H-5″/C-5″, and H-6″/C-6″ and the ¹H-¹H COSY cross peaks of H-1″/ H-2″/ H-3″/ H-4″/ H-5″/ H-6″ were observed clearly confirming the NMR data of the sugar moiety (Table 1). In addition, a set of carbon chemical shifts [102.3, 72.4, 73.1, 68.9, 75.4 (5 x CH), and 63.2 (CH₂) (C-1″ to C-6″)] together with the appearance of the H-3″ proton as a doublet of doublets with J values of 3.0 and 3.0 Hz indicated the equatorial orientation of H-3″, corresponding to the allopyranose moiety.^9,10 The large value J_{H−1″/H−2″} (7.8 Hz) suggested the β-form of the glycosyl linkage of 1. Acid hydrolysis of 1 gave D-allose, which was identified by the positive sign of its optical rotation and TLC analysis in comparison with the authentic monosaccharide.^11,12 In the NOESY spectrum, the cross peaks of H-2α/H-3, H-3/H-2′, and H-3α/H-2 were clearly observed confirming that H-2 and H-3α were on one side, and H-2α, H-3, and H-2′ on the other side of the molecule, similar to 4. Furthermore, the CD spectrum of 1 exhibited positive Cotton effects at 244 and 274 nm indicating the (2R,3R,4S)-configuration (Figure 3). ⁷ Thus, 1 was determined to be (+)-lyoniresinol 3α-O-β-D-allopyranoside, a new compound, named strychnovanoside A (Supplemental Figures S1-S8).

Figure 1.

Chemical structures of 1-7.

Figure 2.

Cd spectra of 1-3.

Figure 3.

Key ¹H-¹H COSY and HMBC correlations of 1-3.

Table 1.

¹H and ¹³C NMR Spectroscopic Data for 1 and 2.

Pos.	1		2
	a,b δ C	a,cδH (mult, J in Hz)	a,b δ C	a,cδH (mult, J in Hz)
1	33.8	2.63 (dd, 15.0, 10.8)	33.9	2.67 (dd, 15.0, 10.8)
		2.74 (dd, 15.0, 5.4)		2.72 (dd, 15.0, 5.4)
2	40.7	1.73 (m)	41.2	1.71 (m)
2α	66.3	3.56 (dd, 10.8, 6.6)	66.2	3.63 (2H, d, 4.8)
		3.66 (dd, 10.8, 4.2)
3	46.8	2.10 (m)	46.6	2.13 (m)
3α	71.5	3.46 (dd, 9.6, 4.2)	71.8	3.60 (dd, 10.2, 5.4)
		3.91 (dd, 9.6, 4.2)		3.88 (dd, 10.2, 3.0)
4	42.8	4.42 (d, 6.6)	43.2	4.26 (d, 6.6)
5	147.6	-	147.5	-
6	138.9	-	138.9	-
7	148.6	-	148.7	-
8	107.8	6.60 (s)	107.8	6.60 (s)
9	130.2	-	130.2	-
10	126.4	-	126.4	-
1′	139.4	-	139.5	-
2′	106.9	6.44 s	107.1	6.43 (s)
3′	149.0	-	149.0	-
4′	134.5	-	134.6	-
5′	149.0	-	149.0	-
6′	106.9	6.44 s	107.1	6.43 (s)
1″	102.3	4.68 d (7.8)	101.8	4.54 d (7.8)
2″	72.4	3.38 (dd, 7.8, 3.0)	72.4	3.35 (dd, 3.0, 7.8)
3″	73.1	4.08 (dd, 3.0, 3.0)	72.9	4.05 (dd, 3.0, 3.0)
4″	68.9	3.50 (dd, 9.0, 3.0)	68.9	3.53 (dd, 9.0, 3.0)
5″	75.4	3.69 (m)	75.4	3.62 (m)
6″	63.2	3.50 dd (12.0, 4.4)	63.1	3.83 dd (12.0, 2.0)
		3.83 dd (12.0, 1.8)		3.70 dd (12.0, 5.4)
5-OCH3	60.2	3.37 (s)	60.1	3.37 (s)
7-OCH3	56.6	3.87 (s)	56.6	3.88 (s)
3′- OCH3	56.9	3.77 (s)	56.9	3.77 (s)
5′- OCH3	56.9	3.77 (s)	56.9	3.77 (s)

The molecular formula of 2 was C₂₈H₃₈O₁₃ determined by HR-ESI-MS results. All the NMR spectra of 2 were very similar to those of 1 (Table 1), including the NOESY spectrum, indicating that they have the same planar structure. The sugar moiety was identified as D-allose by acid hydrolysis, similar to that of 1,^11,12 which linked to C-3α by a β-glycosyl linkage (J_{H−1″/H−2″} = 7.8 Hz), which was confirmed by the observed HMBC correlation from the anomeric proton (δ_H 4.54) to C-3α (δ_C 71.8). Furthermore, the CD spectrum of 2 exhibited negative Cotton effects at 244 and 274 nm, indicating the (2S,3S,4R)-configuration. ⁷ Thus, 2 was determined to be (-)-lyoniresinol 3α-O-β-D-allopyranoside, a new compound, named strychnovanoside B (Supplemental Figures S9-S16).

The NMR spectra of 3 were almost the same as those of 2, but had more signals of the 4-hydroxymesitylenic acid moiety (Table 2). The lower field signal of C-2α (δ_C 68.7), together with the HMBC correlation from H-2α (δ_H 4.44 and 4.32) to C-7″′ (δ_C 168.2) clearly confirmed that the syringyl moiety linked to C-2α by an ester linkage. The HR-ESI-MS of 3 exhibited quasi-molecular ion peaks at m/z 761.2652 [M-H]⁻ (calcd. for [C₃₇H₄₅O₁₇]⁻: 761.2262, Δ = -1.3 ppm); m/z 797.2441 [M + ³⁵Cl]⁻ (calcd. for [C₃₇H₄₅O₁₇³⁵Cl]⁻:797.2429, Δ = + 1.9 ppm); m/z 798.2459 [M + ³⁵⁺³⁷Cl]⁻ (calcd. for [C₃₇H₄₅O₁₇³⁵⁺³⁷Cl]⁻: 798.2462, Δ = -0.4 ppm), and m/z 799.2433 [M + ³⁷Cl]⁻ (calcd. for [C₃₇H₄₅O₁₇³⁷Cl]⁻: 799.2395, Δ = + 4.8 ppm), determining its molecular formula as C₃₇H₄₆O₁₇. Furthermore, the sugar moiety was identified as D-glucose by acid hydrolysis,^11,12 which linked to C-3α by a β-glycosyl linkage (J_{H−1″/H−2″} = 7.8 Hz), confirmed by the observed HMBC correlation from the anomeric proton (δ_H 4.30) to C-3α (δ_C 71.8). In the NOESY spectrum of 3, cross peaks of H-2/H-3α, H-3/H-6′, and H-2/H-4 were observed indicating that H-2 and H-4 were on one side and H-3 on the other side of the molecule. All the above data suggested that the structure of 3 was similar to that of 2α-[(3,5-dimethoxy-4-hydroxy)-benzoyl]-(+)-lyoniresinol-3α-O-β-D-glucopyranoside (vanprukoside). ⁴ However, the CD spectrum of 3 exhibited negative Cotton effects at 245 and 275 nm, indicating the (2S,3S,4R)-configuration. ⁷ Consequently, 3 was determined to be 2α-[(3,5-dimethoxy-4-hydroxy)-benzoyl]-(-)-lyoniresinol-3α-O-β-D-glucopyranoside, a new compound, named strychnovanoside C (Supplemental Figures S17-S23).

Table 2.

¹H and ¹³C NMR Spectroscopic Data for 3.

Pos.	a,b δ C	a,cδH (mult, J in Hz)	Pos.	a,b δ C	a,cδH (mult, J in Hz)
1	33.5	2.80 (dd, 15.0, 4.5)	1″	104.7	4.30 d (7.8)
		2.75 (dd, 15.0, 10.8)
2	38.0	2.12 (m)	2″	75.2	3.24 (dd, 7.8, 9.0)
2α	68.7	4.44 (dd, 10.8, 4.2)	3″	78.2	3.35 (dd, 9.0, 9.0)
		4.32 (dd, 10.8, 6.6)
3	46.7	2.33 (m)	4″	71.6	3.34 (dd, 9.0, 9.0)
3α	71.8	3.60 (dd, 9.6, 5.4)	5″	75.4	3.25 (m)
		4.00 (dd, 9.6, 4.8)
4	42.6	4.42 (d, 5.4)	6″	62.8	3.72 dd (12.0, 5.4)
					3.88 dd (12.0, 1.8)
5	147.6	-	1″′	121.5	-
6	139.1	-	2″′, 6″′	108.2	7.26 (s)
7	148.8	-	3″′, 5″′	149.0	-
8	107.9	6.63 (s)	4″′	142.2	-
9	129.5	-	7″′	168.2	-
10	125.9	-	5-OCH3	60.3	3.40 (s)
1′	139.3	-	7-OCH3	56.6	3.88 (s)
2′, 5′	106.9	6.42 s	3′- OCH3	56.9	3.73 (s)
3′, 6′	149.0	-	5′- OCH3	56.9	3.73 (s)
4′	134.7	-	3″′, 5″′- OCH3	56.9	3.85 (s)

The NMR spectroscopic data for the known compounds 4-7 are shown in Tables S1-S3 (Supplemental Figures S14-S35).

In continuation of our screening program for bioactive components from plants, the isolated compounds were evaluated for anti-inflammatory activity by their ability to inhibit NO production in LPS stimulated RAW 264.7 cells (Supplemental Material). ¹³ Compounds 1 - 7 showed inhibitory effects on NO production with IC₅₀ values of 58.50 ± 2.30, 67.70 ± 2.23, 54.44 ± 1.26, 71.58 ± 3.59, 74.47 ± 2.61, 83.62 ± 2.25, and 47.57 ± 4.01 µM, respectively, compared to a value of 43.24 ± 2.41 µM for the positive control compound, L-NMMA (Supplemental Table S4).

Materials and Methods

General Experimental Procedures

The used characterization equipment is the same as that described in our previous work.^14,15

Refer to Supplemental Material.

Plant Materials

The stems and leaves of Strychnos vanprukii Craib were collected from the Dakrong Nature Reserve, Dakrong district, Quang Tri province, Vietnam during April 2021 and identified by one of our authors, Prof. Ninh Khac Ban. The voucher specimen (NCCT-P145) has been deposited at IMBC, VAST.

Extraction and Isolation

Refer to Supplemental Material.

Strychnovanoside A (1)

Colorless solid, $[\alpha {]}_{\mathrm{D}}^{25}$ : + 7.5 (c 0.1, MeOH); CD (MeOH, c 0.2 mg/mL) λ (θ: mdeg): 244 ( + 5.86), 274 ( + 1.76) nm. HRESIMS m/z 600.2661 [M + NH₄]⁺ (calcd. for [C₂₈H₄₂NO₁₃]⁺: 600.2651, Δ = + 1.7 ppm); m/z 605.2207 [M + Na]⁺ (calcd. for [C₂₈H₃₈O₁₃Na]⁺: 605.2205, Δ = -0.3 ppm); m/z 583.2377 [M + H]⁺ (calcd. for [C₂₈H₃₉O₁₃]⁺: 583.2385, Δ = -1.4 ppm); ¹H NMR (CD₃OD, 600 MHz) δ (ppm), ¹³C NMR (CD₃OD, 150 MHz) δ (ppm) data: see Table 1.

Strychnovanoside B (2)

Colorless solid, $[\alpha {]}_{\mathrm{D}}^{25}$ : −9.0 (c 0.1, MeOH); CD (MeOH, c 0.2 mg/mL) λ (θ: mdeg): 244 (−6.71), 274 (−2.29) nm. HRESIMS m/z 600.2655 [M + NH₄]⁺ (calcd. for [C₂₈H₄₂NO₁₃]⁺: 600.2651, Δ = + 0.7 ppm); m/z 605.2204 [M + Na]⁺ (calcd. for [C₂₈H₃₈O₁₃Na]⁺: 605.2205, Δ = -0.2 ppm); m/z 583.2377 [M + H]⁺ (calcd. for [C₂₈H₃₉O₁₃]⁺: 583.2385, Δ = -1.4 ppm); ¹H NMR (CD₃OD, 600 MHz) δ (ppm), ¹³C NMR (CD₃OD, 150 MHz) δ (ppm) data: see Table 1.

Strychnovanoside C (3)

Colorless solid, $[\alpha {]}_{\mathrm{D}}^{25}$ : −10.5 (c 0.1, MeOH); CD (MeOH, c 0.2 mg/mL) λ (θ: mdeg): 245 (−2.53), 275 (−0.88) nm. HRESIMS m/z 761.2652 [M-H]⁻ (calcd. for [C₃₇H₄₅O₁₇]⁻: 761.2262, Δ = -1.3 ppm); m/z 797.2441 [M + ³⁵Cl]⁻ (calcd. for [C₃₇H₄₅O₁₇³⁵Cl]⁻:797.2429, Δ = + 1.9 ppm); m/z 798.2459 [M + ³⁵⁺³⁷Cl]⁻ (calcd. for [C₃₇H₄₅O₁₇³⁵⁺³⁷Cl]⁻: 798.2462, Δ = -0.4 ppm), m/z 799.2433 [M + ³⁷Cl]⁻ (calcd. for [C₃₇H₄₅O₁₇³⁷Cl]⁻: 799.2395, Δ = + 4.8 ppm); ¹H NMR (CD₃OD, 600 MHz) δ (ppm), ¹³C NMR (CD₃OD, 150 MHz) δ (ppm) data: see Table 2.

Acid Hydrolysis of Compound 1-3^11,12

Refer to Supplemental Material.

Nitric Oxide Assay

Refer to Supplemental Material.

Conclusions

Three new lignan glycosides, (+)-lyoniresinol 3α-O-β-D-allopyranoside (1), (-)-lyoniresinol 3α-O-β-D-allopyranoside (2), and 2α-[(3,5-dimethoxy-4-hydroxy)-benzoyl]-(-)-lyoniresinol-3α-O-β-D-glucopyranoside (3), and four known compounds, (+)-lyoniresinol 3α-O-β-D-glucopyranoside (4), (-)-lyoniresinol 3α-O-β-D-glucopyranoside (5), palicoside (6), and rutin (7) were isolated from the stems and leaves of S. vanprukii. Their chemical structures were elucidated by HR-ESI-MS, 1D and 2D NMR, and CD spectroscopic analysis. All the isolates exhibited inhibitory effects on NO production in LPS stimulated RAW 264.7 cells with IC₅₀ values ranging of 47.57 ± 4.01 to 83.62 ± 2.25 µM, compared to a value of 43.24 ± 2.41 µM for the positive control compound, L-NMMA.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221096173 - Supplemental material for Strychnovanosides A - C, Three New Lignan Glycosides from Strychnos vanprukii

Supplemental material, sj-docx-1-npx-10.1177_1934578X221096173 for Strychnovanosides A - C, Three New Lignan Glycosides from Strychnos vanprukii by Nguyen Chi Mai, Ninh Khac Ban, Truong Quang Trung, Dinh Thien Hoang, Bui Van Thanh, Do Thi Trang, Bui Huu Tai and Phan Van Kiem in Natural Product Communications