Nine new compounds from the root bark of Lycium chinense and their α-glucosidase inhibitory activity

Abstract

Lycium chinense Mill. is a deciduous shrub in the Solanaceae family that is known for its fruits (Lycii fructus) and root bark (Lycii cortex). In our ongoing search for α-glucosidase inhibitors from the root bark of L. chinense, lyciumflavane A, one new flavane with an unusual benzofuran unit, one new amide possessing a naphthalene skeleton, one new sesquiterpene, three new lignan glucosides, and three new phenolic glucosides were isolated along with eight known compounds. Their structures were elucidated using NMR, HRESIMS, UV, ECD, and IR spectroscopic data. Their α-glucosidase inhibitory activity was screened using acarbose as a positive control (IC₅₀ = 385 μM). Compound 1 showed strong inhibitory activity against α-glucosidase (IC₅₀ = 20.89 μM).

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Introduction

Lycium chinense Mill. is cultivated in the mainland of China and is an important source for health foods and traditional Chinese medicine.^1–3 The root bark of L. chinense, named Digupi in China, is conventionally used in traditional Chinese medicine prescriptions to treat diabetes, coughs, hypertension, and fever. Modern pharmacological studies revealed that extracts from the root bark of L. chinense can lower serum glucose levels and improve insulin resistance.^4,5 Previously, phytochemical investigations of this plant have shown the presence of alkaloids, lignanamides, cyclopeptides, lignans, and sterols.^6–12

With the aim of discovering new bioactive natural products with hypoglycemic effects from the root bark of L. chinense, an oral sucrose tolerance test (OSTT) was performed. The results demonstrated that the water-soluble portion of the root bark of L. chinense obtained from an 80% EtOH extract could significantly decrease the postprandial blood glucose levels in normal ICR mice at a dose of 200 mg kg⁻¹ (Fig. S1, ESI†), which is similar to the hypoglycemic effect of acarbose (20 mg kg⁻¹). Through bioactivity-guided isolation, one new flavane with an unusual benzofuran unit, one new amide possessing a naphthalene skeleton, one new sesquiterpene, three new lignan glucosides, and three new phenolic glucosides were obtained from the root bark of L. chinense along with eight known compounds (Fig. 1). In this paper, we reported the isolation and structure elucidation of compounds 1–9 and evaluated these compounds as α-glucosidase inhibitors.

Fig. 1 Chemical structures of 1–17.

Results and discussion

The molecular formula of 1 was determined to be C₂₂H₂₂O₄ by the positive ion peak at m/z 351.1586 [M + H]⁺ in the HRESIMS, which indicated 12 degrees of unsaturation. The ¹H NMR spectrum of 1 (Table 1) revealed an ABX system aromatic ring at δ_H 6.88 (1H, d, J = 8.0 Hz), 6.29 (1H, dd, J = 2.0, 8.0 Hz), and 6.18 (1H, d, J = 2.0 Hz), a pentasubstituted aromatic ring at δ_H 7.17 (1H, s), three olefinic protons at δ_H 6.88 (1H, d, J = 2.0 Hz), 7.91 (1H, d, J = 2.0 Hz), and 5.07 (1H, m), an oxymethylene proton at δ_H 5.08 (1H, br d, J = 11.0 Hz), three methylene protons at δ_H 3.49 (1H, m), 3.42 (1H, m), 2.82 (1H, m), 2.66 (1H, m), 2.09 (1H, m), and 1.88 (1H, m), and two methyl groups at δ_H 1.67 (3H, s) and 1.61 (3H, s). The ¹³C NMR and HSQC spectra of 1 displayed 22 carbon resonances including an isopentene group, two olefinic carbons and a C6–C3–C6 unit, which could be attributed to a flavone skeleton.

Table 1 ¹H NMR data of 1–6 in DMSO-d₆ (500 MHz, δ in ppm, J in Hz)

No.	1	2	3	4	5	6
2	5.08, br d (11.0)	7.16, s	5.65, s	7.07, d (2.0)	7.07, d (2.0)	6.97, d (2.0)
3	2.09, m
	1.88, m
4	2.82, m		8.05, d (16.5)
	2.66, m
5	6.88, d (8.0)		6.02, d (16.5)	6.67, d (8.0)	6.67, d (8.0)	6.77, d (8.0)
6	6.29, dd (2.0, 8.0)		1.81, s	6.80, dd (2.0, 8.0)	6.80, dd (2.0, 8.0)	7.00, d (8.0)
7		7.75, s		4.96, d (4.5)	4.95, d (4.5)	4.94, d (4.0)
8	6.18, d (2.0)			4.38, m	4.39, m	4.50, m
9				3.65, m	3.62, m	3.66, m
				3.21, m	3.23, m	3.21, m
2′				7.01, d (2.0)	7.03, d (2.0)	7.05, d (1.5)
3′			1.96, m
			1.73, m
4′			3.95, m
5′			1.63, m	6.96, d (8.0)	6.97, d (8.0)	6.67, d (8.0)
			1.57, m
6′	7.17, s			6.87, dd (2.0, 8.0)	6.89, dd (2.0, 8.0)	6.87, dd (8.0, 2.0)
7′	6.88, d (2.0)	7.83, s	0.99, s	6.43, d (16.0)	6.55, d (16.0)	6.55, d (16.5)
8′	7.91, d (2.0)		3.48, m	6.23, dt (6.0, 16.0)	6.20, m	6.23, dt (16.0)
9′			0.67, s	4.08, m	4.15, m	4.39, d (6.0)
					4.37, m	4.16, m
1′′	3.42, m		4.25, d (8.0)	3.98, d (7.5)	3.97, d (7.0)	4.41, d (7.5)
	3.49, m
2′′	5.07, m	7.06, d (8.5)	2.94, m	3.61, m	3.00, m	2.99, m
3′′		6.69, d (8.5)	3.28, m	3.12, m	3.01, m	3.13, m
4′′	1.67, s		3.11, m	3.11, m	2.99, m	3.03, m
5′′	1.61, s	6.69, d (8.5)	3.17, m	3.13, m	3.27, m	3.06, m
6′′		7.06, d (8.5)	4.49, d (10.5)	3.83, d (10.0)	3.48, m	3.68, m,
			4.29, m	3.49, m	3.85, m	3.57, d (1.0)
7′′		2.72, m
8′′		3.36, m
1′′′				4.89, d (3.0)	4.88, d (3.0)	4.20, d (8.0)
2′′′		7.08, d (8.5)	7.47, d (1.5)	3.83, m	3.78, m	3.00, m
3′′′		6.71, d (8.5)				3.14, m
4′′′				3.87, d (9.5)	3.85, m	3.04, m
				3.60, d (9.5)	3.59, m
5′′′		6.71, d (8.5)	7.15, d (8.5)	3.36, m	3.35, m	3.08, m
6′′′		7.08, d (8.5)	7.54, dd (8.5, 1.5)			3.68, m
						3.45, m
1′′′′			4.99, d (6.5)		4.20, d (8.0)
2′′′′			3.18, m		2.99, m
3′′′′			3.28, m		3.13, m
4′′′′			3.17, m		3.06, m
5′′′′			3.28, m		3.13, m
6′′′′			3.64, d (10.5)		3.42, m
			3.47, m		3.84, m
1′′′′					4.89, d (3.0)
2′′′′					3.71, m
4′′′′					3.59, m
					3.87, m
5′′′′					3.36, m
3-OCH3		3.93, s		3.71, s	3.71, s	3.78, s
5-OCH3		3.87, s
3′-OCH3				3.77, s	3.77, s	3.71, s
3′′′-OCH3			3.80, s

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From the HMBC spectrum, the correlations of H-2 [δ_H 5.08 (1H, d, J = 10.0 Hz)] to C-1′ (δ_C 135.6) and C-6′ (δ_C 109.1) as well as the correlations of H-6′ [δ_H 7.17 (1H, s)] to C-2 (δ_C 74.6), C-1′ (δ_C 135.6), C-2′ (δ_C 122.3), C-4′ (δ_C 143.5), and C-5′ (δ_C 126.2) confirmed the basic skeleton of the flavane. The correlations of H-5 [δ_H 6.88 (1H, d, J = 8.0 Hz)] to C-9 (δ_C 156.4) and C-7 (δ_C 155.7) suggested the existence of 7-OH at ring A of the flavane. Moreover, the isopentene group was connected to the C-2′ (δ_C 122.3) of ring B because the H-1′′ [δ_H 3.42 (1H, m)] was correlated with C-1′ (δ_C 135.6), C-2′ (δ_C 122.3), and C-3′ (δ_C 139.3). By considering the remaining two degrees of unsaturation and the molecular formula, the benzofuran unit was established, which was supported by the HMBC correlations (Fig. 2) from H-7′ [δ_H 6.88 (1H, d, J = 2.0 Hz)] to C-4′ (δ_C 143.5), C-5′ (δ_C 126.2), and C-8′ (δ_C 145.3) and from H-8′ [δ_H 7.91 (1H, d, J = 2.0 Hz)] to C-4′ (δ_C 143.5) and C-5′ (δ_C 126.2). In addition, the 2S configuration was confirmed by the negative Cotton effect at 280 nm in the ECD spectrum.¹⁵ So, compound 1 was established as shown, and was accorded the trivial name lyciumflavane A.

Fig. 2 Key HMBC correlations of 1, 2, and 4.

Compound 2 was obtained as a yellow amorphous powder. The molecular formula, C₃₀H₃₀N₂O₇, was established by the protonated molecular ion peak at m/z 531.2131 [M + H]⁺ (calcd for C₃₀H₃₁N₂O_7, 531.2131) in the HRESIMS, which corresponded to 17 degrees of unsaturation. In the ¹H NMR spectrum of 2 (Table 1), the presence of two p-tyramine moieties was deduced from two AA′BB′ spin system aromatic rings [δ_H 7.06 (2H, d, J = 8.5 Hz), 6.69 (2H, d, J = 8.5 Hz), 7.08 (2H, d, J = 8.5 Hz) and 6.71 (2H, d, J = 8.5 Hz)], two pairs of methylene proton signals [δ_H 2.72 (4H, m) and 3.36 (4H, m)], and two NH signals [δ_H 8.40 (1H, t, J = 5.5 Hz) and 8.34 (1H, t, J = 5.5 Hz)]. Simultaneously, another three aromatic protons at δ_H 7.16 (1H, s), 7.75 (1H, s), and 7.83 (1H, s), and two methoxy groups at δ_H 3.93 (3H, s) and 3.87 (3H, s) were also observed. The ¹³C NMR spectrum (Table 2) of 2 revealed 30 carbon signals, 20 of which were assigned to two p-tyramine moieties, two carbonyl groups and two methoxy groups, and the remaining 10 carbons were assigned to a naphthalene unit combined with the degrees of unsaturation. The HMBC correlations (Fig. 2) of H-8′′/C-9, H-8′′′/C-9′, H-7/C-9, and H-7′/C-9′ suggested that 2 was an amide possessing a naphthalene skeleton. The linkage points of the two methoxy groups were confirmed to be at C-3 and C-5 based on the correlations between the methoxy protons (δ_H 3.93) with C-3 and the methoxy protons (δ_H 3.87) with C-5 in the HMBC spectrum. Thus, compound 2 was elucidated as shown and was named lyciumamide A.

Table 2 ¹³C NMR data of 1–6 in DMSO-d₆ (125 MHz, δ in ppm)

No.	1	2	3	4	5	6
1		123.2	177.5	128.6	128.4	129.7
2	74.6	102.4	126.8	111.6	111.7	111.8
3	29.6	151.0	141.4	147.1	147.2	149.6
4	24.6	139.8	131.0	145.8	145.8	145.7
5	129.9	140.5	129.9	114.7	114.7	115.1
6	108.0	126.7	20.5	120.1	120.1	119.7
7	155.7	126.3		76.1	76.3	77.6
8	102.8	126.8		82.9	83.0	81.8
9	156.4	168.4		60.5	60.6	59.8
10	112.1
1′	135.6		81.0	130.2	129.7	129.7
2′	122.3		85.3	110.0	110.1	109.7
3′	139.3		42.0	149.6	149.6	147.6
4′	143.5		72.8	147.7	148.1	147.6
5′	126.2		41.8	115.7	115.7	114.6
6′	109.1		47.5	119.1	119.4	119.4
7′	107.7	120.0	19.5	128.5	131.7	131.4
8′	145.3	131.7	75.0	128.4	123.8	124.1
9′		168.6	16.0	61.6	68.6	68.7
1′′	23.9	129.6	102.2	100.3	100.2	102.1
2′′	123.8	129.5	73.3	73.3	73.4	74.2
3′′	129.8	115.0	77.1	76.6	76.6	77.1
4′′	25.4	155.6	70.6	70.0	70.0	69.9
5′′	17.7	115.0	73.6	75.3	75.3	76.8
6′′		129.5	64.3	67.3	67.4	61.0
7′′		34.1
8′′		41.1
1′′′		129.6	123.0	109.3	109.2	102.2
2′′′		129.7	112.7	75.9	75.9	73.5
3′′′		115.1	148.6	78.8	78.8	76.9
4′′′		155.6	150.7	73.4	73.3	70.1
5′′′		115.1	114.5	63.3	63.1	76.5
6′′′		129.7	122.8			61.1
7′′′		34.3	165.1
8′′′		41.1
1′′′′			99.6		101.8
2′′′′			73.1		73.4
3′′′′			76.8		76.6
4′′′′			69.4		70.3
5′′′′			76.5		75.6
6′′′′			60.4		67.7
1′′′′					109.3
2′′′′					75.9
3′′′′					78.8
4′′′′					73.2
5′′′′					63.3
3-OCH3		55.8		55.4	55.7	55.5
5-OCH3		60.4
3′-OCH3				55.7	55.4	55.4
3′′′-OCH3			55.7

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Compound 3 was isolated as a yellow powder. The molecular formula was established to be C₃₅H₄₈O₁₈ by the HRESIMS ion peak at m/z [M + Na]⁺ 779.2723 (C₃₅H₄₈O₁₈Na, calcd for 779.2733). The UV, IR, and NMR spectra of 3 were similar to dihydrophaseic acid 4′-O-(6′′-O-galloyl)-β-D-glucopyranoside;¹⁶ the differences between the compounds were that 3 contained an additional glucose unit and a vanilloyl group in place of the galloyl in dihydrophaseic acid 4′-O-(6′′-O-galloyl)-β-D-glucopyranoside. In the HMBC spectrum of 3, the correlations from H-1′′′′ (δ_H 4.99) to C-4′′′ (δ_C 150.7) established the linkage position of the additional glucose. Compound 3 was assigned as lyciumoside A.

Compound 4 was obtained as a white powder. The molecular formula, C₃₁H₄₂O₁₆, was established by a sodiated molecular ion peak observed at m/z 693.2377 [M + Na]⁺ (calcd for C₃₁H₄₂O₁₆Na, 693.2365) in the HRESIMS. The ¹H NMR spectrum of 4 (Table 1) exhibited two ABX spin system aromatic protons at δ_H 7.07 (1H, d, J = 2.0 Hz), 6.67 (1H, d, J = 8.0 Hz), 6.80 (1H, dd, J = 2.0, 8.0 Hz), 7.01 (1H, d, J = 2.0 Hz), 6.96 (1H, d, J = 8.0 Hz), and 6.87 (1H, dd, J = 2.0, 8.0 Hz), and two olefinic protons at δ_H 6.43 (1H, d, J = 16.0 Hz) and 6.23 (1H, dt, J = 6.0, 16.0 Hz). Two oxygenated methine protons at δ_H 4.96 (1H, d, J = 4.5 Hz) and 4.38 (1H, m), four oxygenated methylene protons at δ_H 3.65 (1H, m), 3.21 (1H, m), and 4.08 (2H, m), two methoxy groups at δ_H 3.71 (3H, s) and 3.77 (3H, s), and two anomeric protons at δ_H 3.98 (1H, d, J = 7.5 Hz) and 4.89 (1H, d, J = 3.0 Hz) were observed in the upfield region. The ¹³C NMR and HSQC spectra displayed a total of 31 carbon signals. Apart from one glucopyranose moiety, one apiofuranose moiety, and two methoxy groups, the remaining 18 carbon signals were assigned to two C6–C3 units. The key HMBC correlations (Fig. 2) of H-7 at δ_H 4.96 with C-1, 2, 6, 8, and 9, of H-7′ at δ_H 6.43 with C-1′, 2′, 6′, 8′, and 9′, and of H-8 at δ_H 4.38 with C-4′ at δ_C 147.7 suggested that 4 was an 8-O-4′ system neolignan.¹⁷ The methoxy groups were confirmed to be at C-3 and C-3′ based on the HMBC correlations of the methoxy groups at δ_H 3.71 and 3.77 with C-3 and C-3′, respectively. The HMBC correlation of H-1′′ (δ_H 3.98) with C-7 (δ_C 76.1) revealed that the glucopyranose unit was attached to C-7, while the correlation of H-1′′′ (δ_H 4.89) with C-6′′ (δ_C 67.3) suggested that the apiofuranose unit was attached to C-6′′. On the basis of the above analysis, the planner structure of 4 was similar to that of ligusinenoside D,¹⁸ except for the position of sugar moiety. Acid hydrolysis of 4 yielded 4a, D-glucopyranose and D-apiofuranose, which were identified by GC analysis of their trimethylsilyl L-cysteine derivatives. Furthermore, the β-linkage of D-glucopyranose and D-apiofuranose was determined by their anomeric protons at δ_H 3.98 (1H, d, J = 7.5 Hz) and δ_H 4.89 (1H, d, J = 3.0 Hz). The ¹H NMR experiment of 4a was performed in CDCl₃. A small coupling constant (J_7,8 = 4.5 Hz) between H-7 and H-8 indicated the erythro configuration of C-7 and C-8 unambiguously.¹⁹ This result combined with the negative Cotton effect at 231 nm in the CD spectrum of 4 established the 7S,8R configurations for 4.¹⁹ From the above data, the structure of 4 was assigned as shown (Fig. 1), and this compound was named lyciumlignan A.

The molecular formula of 5 was determined to be C₄₂H₆₀O₂₅ on the basis of the sodiated molecular ion peak observed at m/z 987.3316 [M + Na]⁺ in the HRESIMS. A comparison of the UV and NMR data of 5 with those of 4 revealed that the aglycone of 5 was also an 8-O-4′ system neolignan. Its ¹H NMR spectrum exhibited four anomeric protons at δ_H 3.97 (1H, d, J = 7.0 Hz), 4.88 (1H, d, J = 3.0 Hz), 4.20 (1H, d, J = 8.0 Hz), and 4.89 (1H, d, J = 3.0 Hz), which indicated the existence of two glucopyranose units and two apiofuranose units with the β-linkage. The key HMBC correlations of H-1′′ (δ_H 3.97) to C-7 (δ_C 76.3), H-1′′′′ (δ_H 4.20) to C-9′ (δ_C 68.6), H-1′′′ (δ_H 4.88) to C-6′′ (δ_C 67.4), and H-1′′′′ (δ_H 4.89) to C-6′′′′ (δ_C 67.7) clearly presented the planner structure for compound 5 as 4,9-dihydroxy-3,3′-dimethoxy-7-en-8,4′-oxyneolignan-7-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranosyl-9′-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside. Acid hydrolysis of 5 resulted in 5a, D-glucopyranose, and D-apiofuranose. The D-configurations of these two sugars were determined by GC analysis. In the ¹H NMR spectrum of 5a (CDCl₃), the characteristic coupling constant of H-7 (J_7,8 = 7.5 Hz) allowed for the determination of the thero configuration of C-7 and C-8.¹⁸ By considering the negative Cotton effects at 231 nm (Δε −1.82) in 5 that were exhibited in the CD spectrum, the 7R,8R configurations were confirmed.¹⁸ Thus, 5 was characterized as shown and named lyciumlignan B.

Detailed analysis of the 1D NMR spectra (Tables 1 and 2) suggested that the structure of 6 was similar to that of 5. The difference between these two compounds was the absence of two apiofuranose units at C-6′′ and C-6′′′ in 6. Using the same method that was described for 5, the absolute configurations of C-7 and C-8 in 6 were established as 7R,8S. Therefore, the structure of 6 (lyciumlignan C) was determined to be (7R,8S)-4,9-dihydroxy-3,3′-dimethoxy-7-en-8,4′-oxyneolignan-7-O-β-D-glucopyranosyl-9′-O-β-D-glucopyranoside.

Compound 7 exhibited a molecular formula of C₂₀H₃₀O₁₂ according to the positive HRESIMS ion observed at m/z 485.1633 [M + Na]⁺. The ¹H NMR spectrum (Table 3) of 7 revealed a set of ABX system aromatic protons at δ_H 7.00 (1H, d, J = 2.0 Hz), 6.88 (1H, overlap), and 6.88 (1H, overlap), two oxymethylene protons at δ_H 4.70 (1H, d, J = 12.0 Hz) and 4.50 (1H, d, J = 12.0 Hz), and two methoxyl protons at δ_H 3.73 (3H, s) and 3.74 (3H, s). Moreover, two anomeric protons at δ_H 4.16 (1H, d, J = 8.0 Hz) and 4.24 (1H, d, J = 7.5 Hz) observed in the upfield region confirmed the presence of two sugar moieties with a β-linkage. The ¹³C NMR spectrum showed 20 carbon signals that could be assigned to a glucopyranose unit, a xylopyranose unit, two methoxy groups, and a benzylalcohol moiety. The HMBC correlation peaks of the two methoxy groups at δ_H 3.73 and 3.74 with C-3 and C-4 confirmed the linkage positions of these methoxy groups. The sequence of two sugar units was determined to be β-xylopyranosyl-(1→6)-O-β-glucopyranosyl based on correlations from H-1′′ to C-6′ in the HMBC spectrum. The unusual downfield shift of the H-7 (δ_H 4.70, 4.50) and C-7 (δ_C 69.3) was an important indicator that the sugar moiety occurred at C-7. This was supported by the HMBC correlation that was observed from H-1′ (δ_H 4.16) to C-7. Finally, the structure of 7 was elucidated as 3′,4′-dimethoxy-benzylalcohol-1-O-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranoside.

Table 3 ¹H and ¹³C NMR data of 7–9 in DMSO-d₆ (500 MHz for ¹H NMR, 125 MHz for ¹³C NMR)

No.	7		No.	8		No.	9
	δH (J in Hz)	δC		δH (J in Hz)	δC		δH (J in Hz)	δC
1		130.3	1		133.6	1		132.0
2	7.00, br s	111.4	2	6.71, s	104.8	2	7.25, d (9.0)	127.6
3		148.1	3		152.7	3	6.71, d (9.0)	115.4
4		148.5	4		136.5	4		157.2
5	6.88, overlap	111.9	5		152.7	5	6.71, d (9.0)	115.4
6	6.88, overlap	120.2	6	6.71, s	104.8	6	7.25, d (9.0)	127.6
7	4.70, d (12.0)	69.3	7	4.70, d (12.5)	69.3	7	6.53, d (16.0)	132.0
	4.50, d (12.0)			4.54, d (12.5)
8			8			8	6.10, dt (16.0)	122.4
9			9			9	4.44, m	68.8
							4.13, m
1′	4.16, d (8.0)	101.5	1′	4.17, d (7.5)	101.7	1′	4.19, d (7.5)	101.7
2′	3.01, m	73.3	2′	3.00, m	73.3	2′	2.98, m	73.4
3′	3.08, m	76.6	3′	3.08, m	76.5	3′	3.13, m	76.6
4′	3.55, m	70.0	4′	3.55, m	70.0	4′	2.99, m	70.3
5′	3.27, m	75.8	5′	3.24, m	75.8	5′	3.25, m	75.6
6′	3.95, m	68.4	6′	3.95, d (10.5)	68.4	6′	3.41, d (3.0)	67.7
	3.54, m			3.55, m			3.85, brd
1′′	4.24, d (7.5)	104.0	1′′	4.24, d (8.0)	104.1	1′′	4.88, d (3.0)	109.2
2′′	2.97, m	73.3	2′′	2.98, m	73.4	2′′	3.75, m	75.9
3′′	3.05, m	76.6	3′′	3.14, m	76.6	3′′		78.8
4′′	3.27, m	69.6	4′′	3.27, m	69.6	4′′	3.82, d (9.5)	73.2
							3.59, d (9.5)
5′′	3.02, m	65.7	5′′	3.68, m	65.7	5′′	3.34, m	63.1
	3.68, m			3.02, m
3-OCH3	3.73, s	55.4	3,5-OCH3	3.73, s	59.9
4-OCH3	3.74, s	55.5	4-OCH3	3.63, s	55.8

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A comparison of the IR, UV, and NMR data (Table 3) of 8 with those of 7 revealed that the difference between these two compounds was the presence of an additional methoxy group at C-5 in 8. This result was supported by the HMBC correlation from OCH₃-3, 5 to C-3, 5, and the singlet proton signal at δ_H 6.71 (2H, s) in the ¹H NMR spectrum. So, compound 8 was elucidated as 3′,4′,5′-trimethoxy-benzylalcohol-1-O-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranoside.

Compound 9 was obtained as a white powder. The molecular formula, C₂₀H₂₈O_11, was established by the positive molecular ion peak at m/z 467.1531 [M + Na]⁺ (calcd for C₂₀H₂₈O₁₁Na, 467.1524) in the HRESIMS. The ¹H NMR spectrum of 9 revealed an AA′BB′ spin system [δ_H 7.25 (2H, d, J = 9.0 Hz) and 6.71 (2H, d, J = 9.0 Hz)], two trans olefince proton signals [δ_H 6.53 (1H, d, J = 16.0 Hz) and 6.10 (1H, d, 16.0 Hz)], two oxymethylene proton signals [δ_H 4.44 (1H, m) and 4.13 (1H, m)], and two anomeric proton signals [δ_H 4.19 (1H, d, J = 7.5 Hz) and 4.88 (1H, d, J = 3.0 Hz)]. In the ¹³C NMR spectrum (Table 3), aside from the 11 carbon signals of a glucopyranose unit and an apiofuranose unit, the remaining nine carbon signals could be attributed to a p-coumaryl alcohol moiety. Subsequently, the two sugar units were determined to be β-apiofuranosyl-(1→6)-O-β-glucopyranosyl by the characteristic correlation of H-1′′ with C-6′ in the HMBC spectrum. In combination with the HMBC correlation of H-1′ with C-9, compound 9 was defined as 4′-hydroxystyrone-1-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside.

Based on the spectroscopic data and a comparison with those found in the literature, the eight known compounds (Fig. 1) were established as kazinol A (10),^20,21 7,4′-dihydroxy-isopentene flavane (11),²² acacetin (12),²³ quercitrin (13),²⁴ maackianin (14),²⁵ maackiain (15),²⁶ (7R,8S)-4,9,9′-trihydroxy-3,3′-dimethoxy-7′-en-8,4′-oxyneolignan-7-O-β-D-glucopyranoside (16),²⁷ and 2-hydroxy-4-methoxybenzaldehyde-2-O-β-D-glucopyraneosyl-1-6-O-β-D-xylopyranoside (17).²⁸

Experimental

General experimental procedures

The optical rotations, UV spectra, and ECD spectra were measured on JASCO P-2000 polarimeter, JASCO V650 spectrometer, and JASCO J-815 spectrometer (JASCO, Easton, MD, USA), respectively. Infrared (IR) spectra were recorded on a Nicolet 5700 spectrometer (Thermo Scientific, Waltham, MA, USA). 1D and 2D NMR spectra were recorded with a Bruker 500 MHz spectrometer (Bruker-Biospin, Billerica, MA, USA). High-resolution electrospray ionization mass spectrometry (HRESIMS) was performed on an Agilent 6520 HPLC-Q-TOF (Agilent Technologies, Waldbronn, Germany). Flash chromatography was performed using a Combiflash RF200 apparatus (Teledyne Isco Corp., Lincoln, NE, USA). Preparative and semi-preparative HPLC (pHPLC and semi-pHPLC) were performed using a Shimadzu preparative chromatography system (Shimadzu Corp., Tokyo, Japan) with YMC-Pack ODS-A columns (250 mm × 20 mm, 5 μm; 250 mm × 10 mm, 5 μm; YMC Corp., Kyoto, Japan). GC analysis was performed using on an Agilent 7890A instrument. HPLC-DAD analysis was conducted on an Agilent 1200 instrument with a YMC C₁₈ column (250 mm × 4.6 mm, 5 μm).

Plant material

The root bark of the L. chinense was collected from Ningan Town, Zhongning County, Ningxia Hui autonomous region, People's Republic of China, in March 2012. A voucher specimen (ID-S-2592) was deposited in the Herbarium of the Institute of Materia Medica, Chinese Academy of Medical Science, Beijing.

Extraction and isolation

The powdered plant material (100 kg) was extracted three times with 80% EtOH (600 L) under reflux. The solvent was evaporated and the crude residue (8.0 kg) was partitioned with EtOAc (3 × 45 L). The EtOAc and H₂O solvent were removed under reduced pressure, which yielded Fr. 1 and Fr. 2, respectively. Fr. 2 (1.375 kg) was chromatographed with a macroporous resin column (HP-20, 200 × 15 cm) and eluted successively with H₂O, 15%, 30%, 50%, 70%, and 95% EtOH. The 30% EtOH fraction was concentrated and was further chromatographed over a macroporous resin (SP-700, 200 × 15 cm) and eluted successively with 15%, 20%, 25%, 30%, 45%, 50%, and 95% EtOH, which yielded fractions A–G.

Fr. C (72 g) was subjected to Combiflash RF200 apparatus with a C₁₈ column (55 × 8 cm, 50 μm) and eluted with MeOH–H₂O (from 5 : 95 to 100 : 0) to provide Fr. C1–Fr. C25. Fr. C5 (3 g) was chromatographed over Sephadex LH-20 and eluted with gradient mixtures of MeOH–H₂O (from 10 : 90 to 95 : 5) to yield Fr. C5-1–C5-15. Fr. C5-4 was purified with pHPLC (MeOH–H₂O, 45 : 55) to yield 5 (20 mg). Fraction C5-11 was chromatographed over Sephadex LH-20 using MeOH–H₂O (from 0 : 100 to 60 : 40) as the gradient mobile phase system and was further purified with pHPLC to yield 3 (26 mg), 6 (16 mg), and 17 (35 mg). Fraction C7 (5 g) was subjected to the Rp-C₁₈ (50 μm) and eluted with MeOH–H₂O (from 10 : 90 to 100 : 0) to yield Fr. C7-1–C7-8. 4 (17 mg) was obtained from Fr. C7-3 by pHPLC (MeOH–H₂O, 30 : 70). Fr. C7-5 was purified using pHPLC with MeOH–H₂O (40 : 60) as the mobile phase to yield 16 (12 mg) and 9 (24 mg). Fr. C10 (1.8 g) was subjected to the Rp-C₁₈ (50 μm) and eluted with MeOH–H₂O (15 : 85) to give six fractions (Fr. C10-1–C10-6). Purification of Fr. C10-2 (1.8 g) with MeOH–H₂O (20 : 80) on pHPLC yielded 7 (32 mg) and 8 (19 mg).

Fraction G (4 g) was chromatographed over Sephadex LH-20 with a gradient of H₂O–MeOH (from 60 : 40 to 0 : 100) to yield Fr. G1–G11. Fr. G2 was purified by pHPLC (MeOH–H₂O, 50 : 50) to yield 13 (25 mg) and 2 (20 mg). 11 (33 mg) and 12 (27 mg) were obtained from Fr. G5 by pHPLC (MeOH–H₂O, 65 : 35) as the mobile phase. Fr. G7 was purified by pHPLC (MeOH–H₂O, 70 : 30) to yield 14 (29 mg) and 15 (22 mg). Fr. G9 and Fr. G10 were purified by pHPLC with MeOH–H₂O (70 : 30) to yield 1 (28 mg) and 10 (15 mg), respectively.

Structure characterization

Lyciumflavane A, 1. [α]²⁰_D −8.7 (c 0.10, MeOH); CD (MeOH) λ_max (Δε): 219 (−1.03) and 276 (−0.08) nm; UV (MeOH) λ_max (log ε): 207 (4.24), 255 (2.95), and 285 (2.53) nm; IR (KBr) ν_max: 3364, 2926, 1598, 1509, 1460, 1378, 1261, 1155, 1114, 1041, 956, and 801 cm⁻¹; and (+)-HRESIMS: m/z 351.1586 [M + H]⁺ (calcd for C₂₂H₂₃O_4, 351.1596). For the NMR data, see Tables 1 and 2.

Lyciumamide A, 2. UV (MeOH) λ_max (log ε): 202 (4.15), 237 (3.79), and 258 (4.05) nm; IR (KBr) ν_max: 3325, 2940, 1635, 1515, 1452 1241, 1122, 1087, 910, 827, and 560 cm⁻¹; and (+)-HRESIMS: m/z 531.2131 [M + H]⁺ (calcd for C₃₀H₃₁N₂O₇, 531.2131). For the NMR data, see Tables 1 and 2.

Lyciumoside A, 3. [α]²⁰_D −5.31 (c 0.10, MeOH); CD (MeOH) λ_max (Δε): 217 (−2.67), 251 (−0.77), and 276 (−0.31) nm; UV (MeOH) λ_max (log ε): 207 (4.25), 215 (4.28), 256 (4.26), and 297 (3.49) nR (KBr) ν_max: 3379, 2930, 1712, 1601, 1417, 1274, 1074, 1026, 883, and 764 cm⁻¹; and (+)-HRESIMS: m/z 779.2723 [M + Na]⁺ (calcd for C₃₅H₄₈O₁₈Na, 779.2738). For the NMR data, see Tables 1 and 2.

Lyciumlignan A, 4. [α]²⁰_D −45.5 (c 0.10 MeOH); CD (MeOH) λ_max (Δε): 231 (−1.10), 255 (+0.84), and 284 (+1.99); UV (MeOH) λ_max (log ε): 206 (4.57), 268 (3.89), and 301 (3.49); IR (KBr) ν_max: 3371, 2936, 2881, 1698, 1604, 1513, 1268, 1034, 823, and 621 cm⁻¹; and (+)-HRESIMS: m/z 693.2377 [M + Na]⁺ (calcd for C₃₁H₄₂O₁₆Na, 693.2371). For the NMR data, see Tables 1 and 2.

Lyciumlignan B, 5. [α]²⁰_D −66.7 (c 0.10 MeOH); CD (MeOH) λ_max (Δε): 231 (−1.82), 253 (+0.79), and 285 (+2.02); UV (MeOH) λ_max (log ε): 205 (4.54), 268 (3.95), and 304 (3.32); IR (KBr) ν_max: 3368, 2974, 2931, 2885, 1513, 1419, 1268, 1049, 825, and 617 cm⁻¹; and (+)-HRESIMS: m/z 987.3316 [M + Na]⁺ (calcd for C₄₂H₆₀O₂₅Na, 987.3321). For the NMR data, see Tables 1 and 2.

Lyciumlignan C, 6. [α]²⁰_D +16.93 (c 0.07 MeOH); CD (MeOH) λ_max (Δε): 230 (+0.80); UV (MeOH) λ_max (log ε): 205 (4.35), 271 (3.71), and 301 (3.61); IR (KBr) ν_max: 3384, 2930, 1565, 1513, 1420, 1267, 1078, 1031, 670, and 618 cm⁻¹; and (+)-HRESIMS: m/z 723.2485 [M + Na]⁺ (calcd for C₃₂H₄₄O₁₇Na, 723.2476). For the NMR data, see Tables 1 and 2.

3′,4′-Dimethoxy-benzylalcohol-1-O-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranoside, 7. [α]²⁰_D −35.9 (c 0.10 MeOH); UV (MeOH) λ_max (log ε): 203 (4.09), 221 (3.61), and 279 (2.93) nm; IR (KBr) ν_max: 3612, 3429, 2932, 2835, 1514, 1449, 1368, 1258, 1050, and 668 cm⁻¹; and (+)-HRESIMS: m/z 485.1633 [M + Na]⁺ (calcd for C₂₀H₃₀O₁₂Na, 485.1635). For the NMR data, see Table 3.

3′,4′,5′-Trimethoxy-benzylalcohol-1-O-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranoside, 8. [α]²⁰_D −15.9 (c 0.10 MeOH); UV (MeOH) λ_max (log ε): 207 (4.19), 233 (3.55), and 269 (3.17) nm; IR (KBr) ν_max: 3368, 2935, 1594, 1461, 1423, 1334, 1239, 1126, 1044, and 832 cm⁻¹; and (+)-HRESIMS: m/z 515.1741 [M + Na]⁺ (calcd for C₂₁H₃₂O₁₃Na, 515.1741). For the NMR data, see Table 3.

4′-Hydroxystyrone-1-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, 9. [α]²⁰_D −10.4 (c 0.10 MeOH); UV λ_max (MeOH) nm: 206 (4.25), 263 (4.05), and 297 (3.46); (+)-HRESIMS: m/z 467.1531 [M + Na]⁺ (calcd for C₂₀H₂₈O₁₁Na, 467.1529); and IR (KBr) ν_max: 3360, 2928, 1609, 1514, 1446, 1269, 1050, 853, 767, and 621 cm⁻¹. For the NMR data, see Table 3.

Determination of the absolute configuration of sugar

Compound 4 (8 mg) was dissolved in 0.5 M HCl (8 mL) and refluxed for 4 h. The reaction solution was then extracted with EtOAc (3 × 10 mL). The EtOAc layer was concentrated to create a residue that was further purified using pHPLC with MeOH–H₂O (45 : 55) to yield 4a (3.5 mg). The aqueous layer was evaporated under a vacuum to create a monosaccharide residue. The residue was mixed with L-cysteine methyl ester hydrochloride (2 mg), and was dissolved in fresh anhydrous pyridine (2.0 mL). The reaction mixture was maintained at 60 °C for 2 h. Then, the dried N-trimethylsilylimidazole (0.2 mL) was added to the mixture. The solution was incubated at 60 °C for 2 h and partitioned between n-hexane and H₂O. The n-hexane layer was subjected to GC analysis under the following conditions: capillary column, HP-5 (30 m × 0.32 mm, with a 0.25 μm film, Dikma); detector temperature, 300 °C; injection temperature, 300 °C; initial temperature, 200 °C that was raised to 280 °C at a rate of 10 °C min⁻¹; the final temperature was maintained for 30 min; detection, FID; and carrier gas, N₂ gas. D-glucose and D-apiose were confirmed by comparing the retention time of their derivatives to the standard sugar derivatized in a similar manner, which exhibited retention times of 20.5 and 14.5 min, respectively. The hydrolysis procedures for 5–9 were similar to that of 4. The D-xylose was confirmed by the retention time (14.9 min) of its derivative.

Inhibitory activity of α-glucosidase

The inhibitory activity of compounds 1–17 on α-glucosidase was determined by the same method as described in the literature.^13,14

Conclusions

In our ongoing effort to discover new bioactive natural products with hypoglycemic effects from the root bark of L. chinense, 17 compounds were isolated, including one new flavane with an unusual benzofuran unit, one new amide possessing a naphthalene skeleton, one new sesquiterpene, three new lignan glucosides, three new phenolic glucosides, and eight known compounds. A literature survey revealed that this is the first report of a flavane with an unusual benzofuran unit that showed strong inhibitory activity against α-glucosidase (IC₅₀ = 20.89 μM).

Acknowledgements

The research described in this publication was supported by the National Natural Science Foundation of China (No. 81303207) and the Beijing Natural Science Foundation (No. 7144227).

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Footnotes

† Electronic supplementary information (ESI) available: 1D NMR, 2D NMR HRMS, IR, and ECD spectra. See DOI: 10.1039/c6ra24751b

‡ These authors contributed equally.

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