New Rare Sinapoyl Acylated Flavonoid Glycosides Obtained from the Seeds of Lepidium apetalum Willd.

Seven new rare sinapoyl acylated flavonoid glycosides, apetalumosides A1 (1), B8 (2), B9 (3), B10 (4), B11 (5), B12 (6), and C1 (7) were isolated from the seeds of Lepidium apetalum Willd. Their structures were elucidated by chemical and spectroscopic methods.

1. Introduction

In the course of our characterization studies on bioactive constituents from Lepidium apetalum Willd [1], we have reported the isolation and structure elucidation of nine new flavonoid glycosides, apetalumosides A, B1–B7, and C, together with one known isolate, quercetin 3-O-(2,6-di-O-β-d-glucopyranosyl)-β-d-glucopyranoside obtained from the seeds of it. As a continuing study on L. apetalum seeds, we have isolated seven new rare sinapoyl acylated flavonoid glycosides, named as apetalumosides A1 (1), B8 (2), B9 (3), B10 (4), B11 (5), B12 (6), and C1 (7) from the herbal medicine. In this paper, we describe the isolation and structure elucidation of these new ones.

2. Results and Discussion

The seeds of L. apetalum were refluxed with 50% ethanol/water. Evaporation of the solvent under reduced pressure provided a 50% ethanol/water extract. The extract was subjected to kinds of column chromatography (CC) and finally preparative HPLC (PHPLC) to yield seven new rare sinapoyl acylated flavonoid glycosides, apetalumosides A1 (1), B8 (2), B9 (3), B10 (4), B11 (5), B12 (6), and C1 (7) (Figure 1).

Figure 1

The structure of compounds 1–7. (A), the moiety structure which indicated in the table for compounds 1–3 and 5–7; (4), an isolated structure of B10 (4).

Apetalumoside A (1), –49.0° (MeOH), was isolated as yellow powder. The IR spectrum of 1 showed absorption bands ascribable to hydroxyl (3394 cm−1), α,β-unsaturated ester (1701, 1630 cm−1), aromatic ring (1648, 1604, 1514, 1458 cm−1), and O-glycosidic linkage (1075 cm−1). The molecular formula, C56H62O30, of 1 was eatablished by negative-ion HRESI-TOF-MS (m/z 1213.3238 [M − H]−, calcd for C56H61O30 1213.3253). Acid hydrolysis of it yielded D-glucose, which was identified by retention time and optical rotation using chiral detection by HPLC analysis [1,2]. The 1H and 13C-NMR (DMSO-d6, Table 1) spectra of 1 showed signals assignable to an isorhamnetin aglycon [δ 3.85 (3H, s, 3′-OCH3), 6.18 (1H, br. s, H-6), 6.32 (1H, br. s, H-8), 6.90 (1H, d, J = 8.5 Hz, H-5′), 7.60 (1H, dd, J = 1.5, 8.5 Hz, H-6′), 7.74 (1H, d, J = 1.5 Hz, H-2′), 12.74 (1H, br. s, 5-OH)], three β-d-glucopyranosyl groups [δ 4.33 (1H, d, J = 8.0 Hz, H-1′′′′), 4.67 (1H, d, J = 7.5 Hz, H-1′′′), 5.74 (1H, d, J = 7.0 Hz, H-1′′)], together with two sinapoyl {6′′′-sinapoyl: δH 3.77 (6H, s, 3′′′′′,5′′′′′-OCH3), 6.27 (1H, d, J = 15.5 Hz, H-8′′′′′), 6.81 (2H, s, H-2′′′′′,6′′′′′), 7.38 (1H, d, J = 15.5 Hz, H-7′′′′′), and δC 166.4 (C-9′′′′′); 2′′′′-sinapoyl: δH 3.91 (6H, s, 3′′′′′′,5′′′′′′-OCH3), 6.28 (1H, d, J = 16.0 Hz, H-8′′′′′′), 7.08 (2H, s, H-2′′′′′′,6′′′′′′), 7.51 (1H, d, J = 16.0 Hz, H-7′′′′′′), and δC 165.6 (C-9′′′′′′)]. The 1H 1H COSY experiment (Figure 2) on 1 indicated the presence of partial structure written in bold lines. To assign the badly overlapped protons in sugar chemical shift range, HSQC-TOCSY experiment was determined. In the HSQC-TOCSY spectrum, the correlations between the following proton and carbon pairs were observed: δC 98.2 (C-1′′) and δH 3.04 (H-4′′), 3.21 (H-5′′), 3.46 (H-3′′), 3.51 (H-2′′), 5.74 (H-1′′); δC 66.7 (C-6′′) and δH 3.04 (H-4′′), 3.21 (H-5′′), 3.50, 3.75 (H2-6′′); δC 103.8 (C-1′′′) and δH 3.15 (H-2′′′), 3.24 (H-4′′′), 3.25 (H-3′′′), 3.40 (H-5′′′), 4.67 (H-1′′′); δC 63.3 (C-6′′′) and δH 3.24 (H-4′′′), 3.40 (H-5′′′), 4.20, 4.27 (H2-6′′′); δC 100.2 (C-1′′′′) and δH 2.68 (H-5′′′′), 2.87 (H-3′′′′), 3.15 (H-4′′′′), 4.33 (H-1′′′′), 4.45 (H-2′′′′); δC 60.3 (C-6′′′′) and δH 2.68 (H-5′′′′), 2.87 (H-3′′′′), 3.15 (H-4′′′′), 3.41, 3.48 (H2-6′′′′). Finally, in the HMBC experiment (Figure 2), long-range correlations were observed between δH 5.74 (H-1′′) and δC 132.7 (C-3); δH 4.67 (H-1′′′) and δC 82.1 (C-2′′); δH 4.33 (H-1′′′′) and δC 66.7 (C-6′′); δH 4.20, 4.27 (H2-6′′′) and δC 166.4 (C-9′′′′′); δH 4.45 (H-2′′′′) and δC 165.6 (C-9′′′′′′), then the connectivities between oligoglycoside moieties and aglycon or sinapoyl groups were characterized. On the basis of above mentioned evidence, the structure of apetalumoside A1 (1) was elucidated to be isorhamnetin 3-O-[β-d-(2-O-sinapoyl)-glucopyranosyl(1→6)]-β-d-(6-O-sinapoyl)-glucopyranosyl(1→2)-β-d-glucopyranoside.

Table 1

1H and 13C-NMR data for 1 in DMSO-d6 (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
2	155.9	-	4′′′	69.6	3.24 (dd, 9.0, 9.0)
3	132.7	-	5′′′	73.9	3.40 (m)
4	177.3	-	6′′′	63.3	4.20 (br. d, ca. 12)
5	161.1	-			4.27 (dd, 5.5, 12.0)
6	98.9	6.18 (br. s)	1′′′′	100.2	4.33 (d, 8.0)
7	164.7	-	2′′′′	73.6	4.45 (dd, 8.0, 9.0)
8	94.0	6.32 (br. s)	3′′′′	74.2	2.87 (dd, 9.0, 9.0)
9	156.2	-	4′′′′	69.9	3.15 (dd, 8.0, 9.0)
10	103.8	-	5′′′′	76.4	2.68 (m)
1′	120.9	-	6′′′′	60.3	3.41 (dd, 5.0, 11.0)
2′	112.7	7.74 (d, 1.5)			3.48 (br. d, ca. 11)
3′	147.0	-	1′′′′′	124.2	-
4′	149.6	-	2′′′′′,6′′′′′	106.0	6.81 (s)
5′	115.3	6.90 (d, 8.5)	3′′′′′,5′′′′′	147.9	-
6′	122.8	7.60 (dd, 1.5, 8.5)	4′′′′′	138.2	-
5-OH	-	12.74 (br. s)	7′′′′′	145.2	7.38 (d, 15.5)
3′-OCH3	55.7	3.85 (s)	8′′′′′	114.3	6.27 (d, 15.5)
1′′	98.2	5.74 (d, 7.0)	9′′′′′	166.4	-
2′′	82.1	3.51 (dd, 7.0, 8.5)	3′′′′′,5′′′′′-OCH3	55.9	3.77 (s)
3′′	76.3	3.46 (dd, 8.5, 8.5)	1′′′′′′	124.6	-
4′′	69.4	3.04 (dd, 8.5, 9.0)	2′′′′′′,6′′′′′′	106.0	7.08 (s)
5′′	78.1	3.21 (m)	3′′′′′′,5′′′′′′	148.1	-
6′′	66.7	3.50 (dd, 4.5, 11.5)	4′′′′′′	138.3	-
		3.75 (br. d, ca. 12)	7′′′′′′	145.3	7.51 (d, 16.0)
1′′′	103.8	4.67 (d, 7.5)	8′′′′′′	114.8	6.28 (d, 16.0)
2′′′	74.3	3.15 (dd, 7.5, 8.0)	9′′′′′′	165.6	-
3′′′	76.3	3.25 (dd, 8.0, 9.0)	3′′′′′′,5′′′′′′-OCH3	56.2	3.91 (s)

Figure 2

The main 1H 1H COSY and HMBC correlations of 1–4.

Apetalumoside B (2), was obtained as yellow powder with negative rotation ( –41.6°, in MeOH). The molecular formula, C44H50O26, of 2 was determined by negative-ion HRESI-TOF-MS (m/z 993.2522 [M − H]−, calcd for C44H49O26 993.2518). Acid hydrolysis of it yielded d-glucose, which was identified by the same method as 1 [1,2]. The 1H and 13C (DMSO-d6, Table 2) and various 2D NMR experiments including 1H 1H COSY, HSQC, HMBC, and HSQC-TOCSY spectra of 2 indicated the presences of a quercetin aglycon [δ 6.16 (1H, d, J = 2.0 Hz, H-6), 6.26 (1H, d, J = 2.0 Hz, H-8), 6.89 (1H, d, J = 8.5 Hz, H-5′), 7.55 (1H, d, J = 2.0 Hz, H-2′), 7.59 (1H, dd, J = 2.0, 8.5 Hz, H-6′), 12.64 (1H, br. s, 5-OH)], three β-d-glucopyranosyls [δ 4.03 (1H, d, J = 7.5 Hz, H-1′′′′), 4.69 (1H, d, J = 7.5 Hz, H-1′′′), 5.65 (1H, d, J = 7.0 Hz, H-1′′)], and a sinapoyl [δH 3.77 (6H, s, 3′′′′′,5′′′′′-OCH3), 6.30 (1H, d, J = 16.0 Hz, H-8′′′′′), 6.83 (2H, s, H-2′′′′′,6′′′′′), 7.39 (1H, d, J = 16.0 Hz, H-7′′′′′); δC 166.4 (C-9′′′′′)]. According to the correlations from 1H 1H COSY, HSQC, and HSQC-TOCSY experiments, the 1H and 13C-NMR data for three β-D-glucopyranosyl groups were assigned in detail. Furthermore, in the HMBC experiments, long-range correlations between δH 5.65 (H-1′′) and δC 132.8 (C-3); δH 4.69 (H-1′′′) and δC 83.0 (C-2′′); δH 4.03 (H-1′′′′) and δC 67.8 (C-6′′); δH 4.21, 4.31 (H2-6′′′) and δC 166.4 (C-9′′′′′) were observed (Figure 2). Consequently, the structure of apetalumoside B8 (2) was determined as quercetin 3-O-[β-d-glucopyranosyl(1→6)]-β-d-(6-O-sinapoyl)-glucopyranosyl(1→2)-β-d-glucopyranoside.

Table 2

1H and 13C-NMR data for 2 in DMSO-d6 (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
2	155.5	-	1′′′	104.3	4.69 (d, 7.5)
3	132.8	-	2′′′	74.3	3.17 (dd, 7.5, 8.5)
4	177.2	-	3′′′	76.2	3.26 (dd, 8.5, 9.0)
5	161.1	-	4′′′	69.4	3.29 (dd, 9.0, 9.0)
6	98.6	6.16 (d, 2.0)	5′′′	73.8	3.51 (m)
7	164.1	-	6′′′	63.0	4.21 (br. d, ca. 11)
8	93.4	6.26 (d, 2.0)			4.31 (dd, 5.0, 11.0)
9	156.1	-	1′′′′	103.2	4.03 (d, 7.5)
10	103.7	-	2′′′′	73.2	2.83 (dd, 7.5, 8.5)
1′	121.0	-	3′′′′	76.4	2.96 (dd, 8.5, 8.5)
2′	116.1	7.55 (d, 2.0)	4′′′′	69.6	3.02 (dd, 8.5, 8.5)
3′	144.8	-	5′′′′	76.4	2.87 (m)
4′	148.4	-	6′′′′	60.7	3.40 (dd, 5.0, 11.5)
5′	115.2	6.89 (d, 8.5)			3.55 (br. d, ca. 12)
6′	121.8	7.59 (dd, 2.0, 8.5)	1′′′′′	124.2	-
5-OH	-	12.64 (br. s)	2′′′′′,6′′′′′	105.8	6.83 (s)
1′′	98.0	5.65 (d, 7.0)	3′′′′′,5′′′′′	147.8	-
2′′	83.0	3.53 (dd, 7.0, 8.5)	4′′′′′	138.1	-
3′′	76.1	3.52 (dd, 8.5, 9.0)	7′′′′′	145.2	7.39 (d, 16.0)
4′′	69.1	3.27 (dd, 9.0, 9.0)	8′′′′′	114.3	6.30 (d, 16.0)
5′′	76.2	3.32 (m)	9′′′′′	166.4	-
6′′	67.8	3.45 (dd, 5.0, 11.5)	3′′′′′,5′′′′′-OCH3	55.9	3.77 (s)
		3.78 (br. d, ca. 12)

Apetalumoside B (3), –61.6° (MeOH). Negative-ion HRESI-TOF-MS determination suggested the molecular formula of it was C55H60O30 (m/z 1199.3095 [M − H]−, calcd for C55H59O30 1199.3097). The proton and carbon signals in 1H and 13C-NMR spectra (DMSO-d6, Table 3) were very similar to those of 1, except for the signals due to the aglycon, quercetin [δ 6.15 (1H, br. s, H-6), 6.20 (1H, br. s, H-8), 6.86 (1H, d, J = 8.5 Hz, H-5′), 7.52 (1H, dd, J = 1.5, 8.5 Hz, H-6′), 7.57 (1H, d, J = 1.5 Hz, H-2′), 12.76 (1H, br. s, 5-OH)]. The linkage positions of sugar parts with aglycon and two sinapoyl groups were elucidated by the HMBC determination (Figure 2), which showed long-range correlations between δH 5.69 (1H, d, J = 7.5 Hz, H-1′′) and δC 132.6 (C-3); δH 4.63 (1H, d, J = 7.5 Hz, H-1′′′and δC 82.6 (C-2′′); δH 4.27 (1H, d, J = 8.0 Hz, H-1′′′′) and δC 66.6 (C-6′′); δH [4.15 (1H, br. d, ca. J = 12 Hz), 4.28 (1H, dd, J = 4.5, 11.5 Hz), H2-6′′′] and δC 166.4 (C-9′′′′′); δH 4.42 (1H, dd, J = 8.0, 9.0 Hz, H-2′′′′) and δC 165.5 (C-9′′′′′′). Meanwhile, the badly overlapped protons in sugar chemical shift range were assigned by HSQC-TOCSY experiment. Finally, the presence of d-glucose was proved by acid analysis [1,2]. Then the structure of apetalumoside B9 (3) was elucidated as quercetin 3-O-[β-d-(2-O-sinapoyl)-glucopyranosyl(1→6)]-β-d-(6-O-sinapoyl)-glucopyranosyl(1→2)-β-d-glucopyranoside.

Table 3

1H and 13C-NMR data for 3 in DMSO-d6 (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
2	156.0	-	4′′′	69.4	3.23 (dd, 8.0, 8.0)
3	132.6	-	5′′′	73.8	3.41 (m)
4	177.3	-	6′′′	63.1	4.15 (br. d, ca. 12)
5	161.0	-			4.28 (dd, 4.5, 11.5)
6	98.7	6.15 (br. s)	1′′′′	100.1	4.27 (d, 8.0)
7	163.9	-	2′′′′	73.7	4.42 (dd, 8.0, 9.0)
8	93.7	6.20 (br. s)	3′′′′	74.2	2.79 (dd, 9.0, 9.0)
9	156.1	-	4′′′′	69.6	3.15 (dd, 8.0, 9.0)
10	103.9	-	5′′′′	76.3	2.69 (m)
1′	121.0	-	6′′′′	60.1	3.44 (m)
2′	116.1	7.57 (d, 1.5)	1′′′′′	124.2	-
3′	144.7	-	2′′′′′,6′′′′′	105.9	6.79 (s)
4′	148.5	-	3′′′′′,5′′′′′	147.8	-
5′	115.2	6.86 (d, 8.5)	4′′′′′	138.1	-
6′	121.6	7.52 (dd, 1.5, 8.5)	7′′′′′	145.1	7.34 (d, 16.0)
5-OH	-	12.76 (br. s)	8′′′′′	114.3	6.25 (d, 16.0)
1′′	97.9	5.69 (d, 7.5)	9′′′′′	166.4	-
2′′	82.6	3.51 (dd, 7.5, 8.5)	3′′′′′,5′′′′′-OCH3	55.9	3.74 (s)
3′′	76.1	3.39 (dd, 8.0, 8.5)	1′′′′′′	124.5	-
4′′	69.2	3.04 (dd, 8.0, 9.0)	2′′′′′′,6′′′′′′	105.9	7.07 (s)
5′′	78.1	3.16 (m)	3′′′′′′,5′′′′′′	148.1	-
6′′	66.6	3.43 (dd, 5.5, 11.5)	4′′′′′′	138.2	-
		3.69 (br. d, ca. 12)	7′′′′′′	145.1	7.49 (d, 16.0)
1′′′	104.2	4.63 (d, 7.5)	8′′′′′′	115.0	6.32 (d, 16.0)
2′′′	74.3	3.14 (dd, 7.5, 8.0)	9′′′′′′	165.5	-
3′′′	76.2	3.24 (dd, 8.0, 8.0)	3′′′′′′,5′′′′′′-OCH3	56.2	3.88 (s)

Apetalumoside B (4) was obtained as yellow powder with negative rotation ( –66.4°, in MeOH). Acid hydrolysis with 1 M HCl, it gave d-glucose [1,2]. The molecular formula of 4, C44H50O26 (m/z 993.2537 [M − H]−, calcd for C44H49O26 993.2518), was the same as that of 2. And the 1H and 13C (DMSO-d6, Table 4) together with various 2D NMR experiments of 4 showed the same fragments {quercetin aglycon [δ 6.39 (1H, d, J = 1.5 Hz, H-6), 6.58 (1H, d, J = 1.5 Hz, H-8), 6.90 (1H, d, J = 8.5 Hz, H-5′), 7.57 (1H, d, J = 1.5 Hz, H-2′), 7.61 (1H, dd, J = 1.5, 8.5 Hz, H-6′), 12.70 (1H, br. s, 5-OH)], three β-d-glucopyranosyl groups [δ 4.68 (1H, d, J = 7.5 Hz, H-1′′′), 5.03 (1H, d, J = 7.5 Hz, H-1′′′′), 5.68 (1H, d, J = 6.5 Hz, H-1′′)], and a sinapoyl [δH 3.75 (6H, s, 3′′′′′,5′′′′′-OCH3), 6.25 (1H, d, J = 15.5 Hz, H-8′′′′′), 6.78 (2H, s, H-2′′′′′,6′′′′′), 7.36 (1H, d, J = 15.5 Hz, H-7′′′′′); δC 166.4 (C-9′′′′′)]} as 2. But comparison the 1H and 13C-NMR data of 6–8 positions in 4 [δH 6.39 (H-6), 6.58 (H-8); δC 94.0 (C-8), 99.1 (C-6), 162.5 (C-7)] with those in 2 [δH 6.16 (H-6), 6.26 (H-8); δC 93.4 (C-8), 98.6 (C-6), 164.1 (C-7)] revealed a glycoside substitution shift around the 7-position. Meanwhile, the 13C-NMR data of C-6′′ of 4 (δC 60.4) shifted to high field compared with that of 2 (δC 67.8), which meant there was no substitution at C-6′′ position for compound 4. Furthermore, in the HMBC experiment, long-range correlations were observed between δH 5.68 (H-1′′) and δC 133.1 (C-3); δH 4.68 (H-1′′′) and δC 83.6 (C-2′′); δH 5.03 (H-1′′′′) and δC 162.5 (C-7); δH [4.20 (1H, br. d, ca. J = 12 Hz), 4.31 (1H, dd, J = 5.0, 11.5 Hz), H2-6′′′] and δC 166.4 (C-9′′′′′). Consequently, the structure of apetalumoside B10 (4) was determined as quercetin 3-O-β-d-(6-O-sinapoyl)-glucopyranosyl(1→2)-β-d-glucopyranoside-7-O-β-d-glucopyranoside.

Table 4

1H and 13C-NMR data for 4 in DMSO-d6 (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
2	156.1	-	1′′′	104.5	4.68 (d, 7.5)
3	133.1	-	2′′′	74.4	3.17 (dd, 7.5, 9.0)
4	177.4	-	3′′′	76.1	3.26 (dd, 8.0, 9.0)
5	160.7	-	4′′′	69.5	3.25 (dd, 8.0, 8.0)
6	99.1	6.39 (d, 1,5)	5′′′	73.8	3.53 (m)
7	162.5	-	6′′′	63.1	4.20 (br. d, ca. 12)
8	94.0	6.58 (d, 1.5)			4.31 (dd, 5.0, 11.5)
9	155.7	-	1′′′′	99.6	5.03 (d, 7.5)
10	105.4	-	2′′′′	73.0	3.28 (dd, 7.5, 8.0)
1′	120.8	-	3′′′′	76.2	3.34 (dd, 8.0, 9.0)
2′	116.2	7.57 (d, 1.5)	4′′′′	69.5	3.19 (dd, 9.0, 9.0)
3′	144.8	-	5′′′′	77.0	3.44 (m)
4′	148.7	-	6′′′′	60.5	3.49 (m, overlapped)
5′	115.2	6.90 (d, 8.5)			3.72 (br. d, ca. 11)
6′	121.9	7.61 (dd, 1.5, 8.5)	1′′′′′	124.1	-
5-OH	-	12.70 (br. s)	2′′′′′,6′′′′′	105.8	6.78 (s)
1′′	97.7	5.68 (d, 6.5)	3′′′′′,5′′′′′	147.8	-
2′′	83.6	3.49 (m, overlapped)	4′′′′′	138.1	-
3′′	76.3	3.49 (m, overlapped)	7′′′′′	145.1	7.36 (d, 15.5)
4′′	69.4	3.13 (dd, 8.0, 8.0)	8′′′′′	114.3	6.25 (d, 15.5)
5′′	77.4	3.10 (m)	9′′′′′	166.4	-
6′′	60.4	3.27 (m, overlapped)	3′′′′′,5′′′′′-OCH3	55.9	3.75 (s)
		3.49 (m, overlapped)

Apetalumoside B (5) was isolated as yellow powder, too. It had the molecular formula, C50H60O31, deduced from the negative-ion HRESI-TOF-MS (m/z 1155.3063 [M − H]−, calcd for C50H59O31 1155.3046). On acid hydrolysis and identification with HPLC analysis, the presence of D-glucose was determined [1,2]. The 1H and 13C (DMSO-d6, Table 5) together with 1H 1H COSY, HSQC, HMBC, and HSQC-TOCSY spectra revealed it had the same aglycon, quercetin as 2–4 [δ 6.38 (1H, d, J = 2.0 Hz, H-6), 6.55 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, d, J = 8.5 Hz, H-5′), 7.57 (1H, d, J = 2.0 Hz, H-2′), 7.58 (1H, dd, J = 2.0, 8.5 Hz, H-6′), 12.63 (1H, br. s, 5-OH)]. On the other hand, there were four β-d-glucopyranosyl groups [δ 3.99 (1H, d, J = 7.5 Hz, H-1′′′′), 4.66 (1H, d, J = 7.5 Hz, H-1′′′), 5.01 (1H, d, J = 7.5 Hz, H-1′′′′′), 5.62 (1H, d, J = 6.5 Hz, H-1′′)], and a sinapoyl [δH 3.75 (6H, s, 3′′′′′′,5′′′′′′-OCH3), 6.26 (1H, d, J = 16.0 Hz, H-8′′′′′′), 6.80 (2H, s, H-2′′′′′′,6′′′′′′), 7.36 (1H, d, J = 16.0 Hz, H-7′′′′′′); δC 166.4 (C-9′′′′′′)]} in 5. There was one more β-d-glucopyranosyl in 5 than in 4. Moreover, the 13C-NMR data of C-6′′ for 5 (δC 67.8) shifted to low field compared with that of 4 (δC 60.4), which indicated C-6′′ position might be substituted with β-d-glucopyranosyl in 5. Meanwhile, in the HMBC experiment (Figure 3), long-range correlation was observed between δH 3.99 (1H, d, J = 7.5 Hz, H-1′′′′) and δC 67.8 (C-6′′). Finally, in the HSQC-TOCSY spectra, the correlations between δC 97.9 (C-1′′) and δH 3.25 (H-4′′), 3.32 (H-5′′), 3.50 (H-3′′), 3.52 (H-2′′), 5.62 (H-1′′); δH 3.78 (H-6b′′) and δC 67.8 (C-6′′), 69.2 (C-4′′), 76.3 (C-5′′); δC 104.5 (C-1′′′) and δH 3.16 (H-2′′′), 3.25 (H-4′′′), 3.26 (H-3′′′), 3.51 (H-5′′′), 4.66 (H-1′′′); δH 4.19, 4.30 (H2-6′′′) and δC 63.1 (C-6′′′), 69.4 (C-4′′′), 73.8 (C-5′′′); δC 103.2 (C-1′′′′) and δH 2.80 (H-2′′′′), 2.85 (H-5′′′′), 2.90 (H-3′′′′), 2.98 (H-4′′′′), 3.99 (H-1′′′′); δC 60.7 (C-6′′′′) and δH 2.85 (H-5′′′′), 2.90 (H-3′′′′), 2.98 (H-4′′′′), 3.38, 3.54 (H2-6′′′′); δC 99.8 (C-1′′′′′) and δH 3.19 (H-4′′′′′), 3.32 (H-3′′′′′), 3.27 (H-2′′′′′), 3.43 (H-5′′′′′), 5.01 (H-1′′′′′); δC 60.6 (C-6′′′′′) and δH 3.19 (H-4′′′′′), 3.32 (H-3′′′′′), 3.43 (H-5′′′′′), 3.49, 3.72 (H2-6′′′′′) were observed, then the badly overlapped protons in sugar chemical shift range were assigned clearly. On the basis of above mentioned evidence, the structure of 5 was determined to be quercetin 3-O-[β-d-glucopyranosyl(1→6)]-β-d-(6-O-sinapoyl)-glucopyranosyl(1→2)-β-d-glucopyranoside-7-O-β-d-glucopyranoside.

Table 5

1H and 13C-NMR data for 5 in DMSO-d6 (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
2	156.2	-	4′′′	69.4	3.25 (m, overlapped)
3	133.1	-	5′′′	73.8	3.51 (m)
4	177.3	-	6′′′	63.1	4.19 (br. d, ca. 12)
5	160.7	-			4.30 (dd, 4.5, 12.0)
6	99.2	6.38 (d, 2.0)	1′′′′	103.2	3.99 (d, 7.5)
7	162.6	-	2′′′′	73.1	2.80 (dd, 7.5, 8.0)
8	94.3	6.55 (d, 2.0)	3′′′′	76.2	2.90 (dd, 8.0, 9.0)
9	155.7	-	4′′′′	69.6	2.98 (dd, 9.0, 9.0)
10	105.5	-	5′′′′	76.4	2.85 (m)
1′	120.8	-	6′′′′	60.7	3.38 (dd, 5.5, 11.5)
2′	116.3	7.57 (d. 2.0)			3.54 (br. d, ca. 12)
3′	144.8	-	1′′′′′	99.8	5.01 (d, 7.5)
4′	148.7	-	2′′′′′	73.1	3.27 (dd, 7.5, 8.0)
5′	115.2	6.88 (d, 8.5)	3′′′′′	76.2	3.32 (dd, 8.0, 9.0)
6′	121.9	7.58 (dd, 2.0, 8.5)	4′′′′′	69.5	3.19 (dd, 9.0, 9.0)
5-OH	-	12.63 (br. s)	5′′′′′	77.0	3.43 (m)
1′′	97.9	5.62 (d, 6.5)	6′′′′′	60.6	3.49 (dd, 4.0, 11.5)
2′′	83.2	3.52 (dd, 6.5, 8.0)			3.72 (br. d, ca. 12)
3′′	76.1	3.50 (dd, 8.0, 8.0)	1′′′′′′	124.2	-
4′′	69.2	3.25 (m, overlapped)	2′′′′′′,6′′′′′′	105.9	6.80 (s)
5′′	76.3	3.32 (m)	3′′′′′′,5′′′′′′	147.8	-
6′′	67.8	3.43 (dd, 5.5, 11.5)	4′′′′′′	138.1	-
		3.78 (dd, 3.5, 11.5)	7′′′′′′	145.1	7.36 (d, 16.0)
1′′′	104.5	4.66 (d, 7.5)	8′′′′′′	114.3	6.26 (d, 16.0)
2′′′	74.4	3.16 (dd, 7.5, 8.0)	9′′′′′′	166.4	-
3′′′	76.2	3.26 (m, overlapped)	3′′′′′′,5′′′′′′-OCH3	55.9	3.75 (s)

Figure 3

The main 1H 1H COSY and HMBC correlations of 5–7.

Apetalumoside B (6), –84.0° (MeOH), was isolated as yellow powder. The molecular formula, C61H70O35, of 6 was determined from negative-ion HRESI-TOF-MS (m/z 1361.3625 [M − H]−, calcd for C61H69O35 1361.3625). Acid hydrolysis of 6 with 1 M HCl liberated D-glucose [1,2]. Comparison the 1H and 13C (DMSO-d6, Table 6) spectra with those of 5, revealed there was another sinapoyl [δH 3.87 (6H, s, 3′′′′′′′,5′′′′′′′-OCH3), 6.32 (1H, d, J = 16.0 Hz, H-8′′′′′′′), 7.06 (2H, s, H-2′′′′′′′,6′′′′′′′), 7.50 (1H, d, J = 16.0 Hz, H-7′′′′′′′); δC 165.5 (C-9′′′′′′′)] appeared in 6, and the 1H-NMR data of 2′′′′-position [δH 4.39 (1H, dd, J = 8.0, 8.0 Hz, H-2′′′′)] shifted to low field relatived to that of 5 [δH 2.80 (1H, dd, J = 7.5, 8.0 Hz, H-2′′′′). The above mentioned evidence suggested the another sinapoyl group linked with 2′′′′-position, which was certified by the long-range correlation between δH 4.39 (H-2′′′′) and δC 165.5 (C-9′′′′′′′) observed in the HMBC experiment. In conjunction with analysis of HSQC and HSQC-TOCSY spectra, the 1H and 13C-NMR data for 6 were assigned. Finally, the structure of apetalumoside B12 (6) was clarified to be quercetin 3-O-[β-d-(2-O-sinapoyl)-glucopyranosyl(1→6)]-β-d-(6-O-sinapoyl)-glucopyranosyl(1→2)-β-d-glucopyranoside-7-O-β-d-glucopyranoside.

Table 6

1H and 13C-NMR data for 6 in DMSO-d6 (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
2	156.8	-	1′′′′	100.2	4.22 (d, 8.0)
3	132.8	-	2′′′′	73.4	4.39 (dd, 8.0, 8.0)
4	177.4	-	3′′′′	74.0	2.71 (dd, 8.0, 8.0)
5	160.8	-	4′′′′	69.6	3.11 (dd, 8.0, 9.5)
6	99.3	6.40 (d, 1.5)	5′′′′	76.3	2.68 (m)
7	162.7	-	6′′′′	60.1	3.44 (m, overlapped)
8	94.8	6.51 (d, 1.5)			3.48 (m, overlapped)
9	155.5	-	1′′′′′	100.0	5.00 (d, 7.5)
10	105.5	-	2′′′′′	73.1	3.26 (dd, 7.5, 7.5)
1′	120.7	-	3′′′′′	76.2	3.30 (dd, 7.5, 8.5)
2′	116.3	7.58 (d, 1.5)	4′′′′′	69.6	3.16 (dd, 7.5, 8.5)
3′	144.8	-	5′′′′′	77.0	3.40 (m)
4′	148.8	-	6′′′′′	60.6	3.48 (m, overlapped)
5′	115.2	6.86 (d, 8.5)			3.75 (br. d, ca. 12)
6′	121.7	7.51 (dd, 1.5, 8.5)	1′′′′′′	124.1	-
5-OH	-	12.76 (br. s)	2′′′′′′,6′′′′′′	105.8	6.76 (s)
1′′	97.8	5.67 (d, 7.5)	3′′′′′′,5′′′′′′	147.8	-
2′′	82.8	3.48 (m, overlapped)	4′′′′′′	138.1	-
3′′	76.0	3.38 (dd, 7.5, 8.0)	7′′′′′′	145.2	7.33 (d, 16.0)
4′′	69.1	3.01 (dd, 8.0, 9.0)	8′′′′′′	114.3	6.22 (d, 16.0)
5′′	78.1	3.14 (m)	9′′′′′′	166.3	-
6′′	66.7	3.45 (m, overlapped)	3′′′′′′,5′′′′′′-OCH3	55.9	3.73 (s)
		3.67 (br. d, ca. 13)	1′′′′′′′	124.5	-
1′′′	104.4	4.61 (d, 7.5)	2′′′′′′′,6′′′′′′′	105.9	7.06 (s)
2′′′	74.4	3.13 (dd, 7.5, 8.0)	3′′′′′′′,5′′′′′′′	148.1	-
3′′′	76.2	3.24 (dd, 8.0, 8.0)	4′′′′′′′	138.2	-
4′′′	69.5	3.23 (dd, 8.0, 8.0)	7′′′′′′′	145.1	7.50 (d, 16.0)
5′′′	73.8	3.41 (m)	8′′′′′′′	114.9	6.32 (d, 16.0)
6′′′	63.2	4.15 (br. d, ca. 11)	9′′′′′′′	165.5	-
		4.28 (dd, 6.5, 11.0)	3′′′′′′′,5′′′′′′′-OCH3	56.1	3.87 (s)

Apetalumoside C (7), –47.2° (MeOH). Negative-ion HRESI-TOF-MS determination suggested the molecular formula of it was C55H60O29 (m/z 1183.3123 [M − H]−, calcd for C55H59O29 1183.3147). Treated 7 with 1 M HCl to yield D-glucose [1,2]. The proton and carbon signals in 1H and 13C-NMR spectra (DMSO-d6, Table 7) were very similar to those of 1, except for the signals due to the aglycon, kaempferol [δ 6.14 (1H, br. s, H-6), 6.27 (1H, br. s, H-8), 6.85 (2H, d, J = 8.5 Hz, H-3′,5′), 7.93 (2H, d, J = 8.5 Hz, H-2′,6′), 12.72 (1H, br. s, 5-OH)]. The linkage positions of sugar parts with aglycon and two sinapoyl groups were determined by the HMBC experiment, which showed long-range correlations between δH 5.58 (1H, d, J = 6.5 Hz, H-1′′) and δC 132.5 (C-3); δH 4.62 (1H, d, J = 7.5 Hz, H-1′′′) and δC 82.0 (C-2′′); δH 4.26 (1H, d, J = 8.0 Hz, H-1′′′′) and δC 66.7 (C-6′′); δH [4.17 (1H, br. d, ca. J = 12 Hz), 4.28 (1H, dd, J = 5.5, 11.5 Hz), H2-6′′′] and δC 166.4 (C-9′′′′′); δH 4.43 (1H, dd, J = 8.0, 9.0 Hz, H-2′′′′) and δC 165.4 (C-9′′′′′′). Moreover, the 1H-NMR data for four β-d-glucopyranosyl groups were assigned by HSQC and HSQC-TOCSY determination. On the basis of above mentioned evidence, the structure of apetalumoside C1 (7) was elucidated as kaempferol 3-O-[β-d-(2-O-sinapoyl)-glucopyranosyl(1→6)]-β-d-(6-O-sinapoyl)-glucopyranosyl(1→2)-β-d-glucopyranoside.

Table 7

1H and 13C-NMR data for 7 in DMSO-d6 (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
2	156.1	-	6′′′	63.2	4.17 (br. d, ca. 12)
3	132.5	-			4.28 (dd, 5.5, 11.5)
4	177.3	-	1′′′′	100.2	4.26 (d, 8.0)
5	161.1	-	2′′′′	73.6	4.43 (dd, 8.0, 9.0)
6	98.9	6.14 (br. s)	3′′′′	74.2	2.86 (dd, 9.0, 9.0)
7	164.5	-	4′′′′	69.9	3.11 (dd, 9.0, 9.0)
8	93.8	6.27 (br. s)	5′′′′	76.5	2.78 (m)
9	156.2	-	6′′′′	60.4	3.40 (m, overlapped)
10	103.7	-			3.52 (br. d, ca. 12)
1′	120.6	-	1′′′′′	124.2	-
2′,6′	130.7	7.93 (d, 8.5)	2′′′′′,6′′′′′	106.0	6.79 (s)
3′,5′	115.1	6.85 (d, 8.5)	3′′′′′,5′′′′′	147.8	-
4′	159.9	-	4′′′′′	138.2	-
5-OH	-	12.72 (br. s)	7′′′′′	145.1	7.37 (d, 15.5)
1′′	97.9	5.58 (d, 6.5)	8′′′′′	114.4	6.27 (d, 15.5)
2′′	82.0	3.41 (dd, 6.5, 8.0)	9′′′′′	166.4	-
3′′	76.2	3.40 (dd, 8.0, 9.0)	3′′′′′,5′′′′′-OCH3	55.9	3.73 (s)
4′′	69.2	3.01 (dd, 9.0, 9.0)	1′′′′′′	124.5	-
5′′	77.8	3.13 (m)	2′′′′′′,6′′′′′′	106.0	7.05 (s)
6′′	66.7	3.44 (dd, 6.5, 12.0)	3′′′′′′,5′′′′′′	148.1	-
		3.68 (br. d, ca. 12)	4′′′′′′	138.3	-
1′′′	103.9	4.62 (d, 7.5)	7′′′′′′	145.1	7.49 (d, 16.0)
2′′′	74.3	3.11 (dd, 7.5, 9.0)	8′′′′′′	115.0	6.29 (d, 16.0)
3′′′	76.3	3.21 (dd, 9.0, 9.0)	9′′′′′′	165.4	-
4′′′	69.5	3.23 (dd, 9.0, 9.0)	3′′′′′′,5′′′′′′-OCH3	56.1	3.87 (s)
5′′′	73.8	3.35 (m)

3. Experimental

3.1. General

UV spectra were recorded on a Varian Cary 50 UV-Vis spectrophotometer (Varian, Inc., Hubbardsdon, MA, USA). IR spectra were obtained on a Varian 640-IR FT-IR spectrophotometer (Varian Australia Pty Ltd, Mulgrave, Australia). Optical rotations were determined on a Rudolph Autopol® IV automatic polarimeter (Rudolph Research Analytical, Hackettstown NJ, USA). NMR spectra were measured on a Bruker AVANCE III 500 MHz NMR spectrometer (500 MHz for 1H and 125 MHz for 13C-NMR, Bruker BioSpin AG Industriestrasse 26 CH-8117, Fällanden, Switzerland) with TMS as an internal standard. Negative-ion HRESI-TOF-MS were determined on an Agilent 6520 Accurate-Mass Q-Tof MS spectrometer (drying gas, N2; flow rate, 8.0 L/min; temperature, 350 °C; nebulizer, 30 psig; capillary, −3500 V; fragmentor, 175 V; skimmer, 65 V; OCT RF V, 750 V. Mass range recorded m/z 100–1200, Agilent Technologies, Inc., Santa Clara, CA, USA).

Column chromatographies were performed on macroporous resin D101 (Haiguang Chemical Co., Ltd., Tianjin, China), Silica gel (48–75 μm, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), ODS (40–63 μm, YMC Co., Ltd., Tokyo, Japan), and Sephadex LH-20 (Ge Healthcare Bio-Sciences, Uppsala, Sweden), and Preparative HPLC (PHPLC) column, Cosmosil 5C18-MS-II (20 mm i.d. × 250 mm, 5 μM, Nakalai Tesque, Inc., Tokyo, Japan) were used to purify the constituents.

3.2. Plant Material

The seeds of L. apetalum were collected from Anguo city, China, and identified by Li Tianxiang. The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM (No. 20120501).

3.3. Extraction and Isolation

L. apetalum seeds (10 kg) were crushed and refluxed with 50% ethanol/water. Then, the 50% ethanol/water extract was partitioned in a CHCl3/H2O mixture (1:1, v/v), and CHCl3 and H2O layers were obtained. Then the H2O layer was subjected to D101 macroporous resin CC (H2O → 95% EtOH). As a result, H2O and 95% EtOH eluted fractions were given.

The EtOH fraction (80 g) was subjected to silica gel CC [CHCl3 → CHCl3/MeOH (100:3 → 100:5, v/v) → CHCl3/MeOH/H2O (10:3:1 → 6:4:1, v/v/v) → MeOH] to yield 16 fractions (Fr. 1–16). Fraction 12 was isolated by ODS CC [MeOH/H2O (10:90 → 20:80 → 30:70 → 40:60 → 50:50 → 70:30→ 100:0, v/v] to give 9 fractions (Fr. 12-1–12-9). Fraction 12-6 was separated by PHPLC [CH3CN/1% CH3COOH (18:82 → 100:0, v/v)] to obtain 14 fractions (Fr. 12-6-1–12-6-14). Fraction 12-6-11 was purified by PHPLC [CH3CN/1% CH3COOH (20:80, v/v)], and apetalumoside B9 (3, 16.1 mg) was obtained. Fraction 12-6-12 was isolated by PHPLC [CH3CN/1% CH3COOH (18:82, v/v)] to yield apetalumosides A1 (1, 23.7 mg) and C1 (7, 15.6 mg) respectively. Fraction 14 was separated by Sephadex LH-20 CC [MeOH/H2O (1:1, v/v)] to yield 7 fractions (Fr. 14-1–14-7). Fraction 14-5 was purified by PHPLC [CH3CN/1% CH3COOH (15:85, v/v)], as a result, 18 fractions (Fr. 14-5-1–14-5-18) were obtained. Fraction 14-5-5 was subjected to PHPLC [CH3CN/1% CH3COOH (14:86, v/v)] to give apetalumoside B12 (6, 11.4 mg). Fraction 14-5-7 was separated by Sephadex LH-20 CC [MeOH/H2O (1:1, v/v)] to afford three fractions (Fr. 14-5-7-1–14-5-7-3). Fraction 14-5-7-2 was subjected to PHPLC [CH3CN/1% CH3COOH (12:88, v/v)], and apetalumoside B10 (4, 19.8 mg) was obtained. Fraction 14-5-15 was further separated by PHPLC [MeOH/1% CH3COOH (34:66, v/v)] to give apetalumoside B8 (2, 50.2 mg). Fraction 16 was separated by PHPLC through gradient elution [MeOH/1% CH3COOH (20:80 → 25:75 → 30:70 → 35:65 → 40:60 → 50:50 → 60:40 → 100:0, v/v)] to obtain 26 fractions (Fr. 16-1–16-26). Fraction 16-10 was further isolated by PHPLC [CH3CN/1% CH3COOH (12:88, v/v)] to yield three fractions (Fr. 16-10-1–16-10-3). Fraction 16-10-2 was purified by Sephadex LH-20 CC [MeOH/H2O (1:1, v/v)], and apetalumoside B11 (5, 16.8 mg) was given.

Apetalumoside A (1): Yellow powder. –49.0° (c = 0.97, MeOH); IR νmax (KBr) cm−1: 3394, 2933, 1701, 1648, 1630, 1604, 1514, 1458, 1356, 1284, 1175, 1114, 1075, 826; UV λmax (MeOH) nm (log ε): 329 (4.56), 266 (4.26, sh), 236 (4.56). 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectroscopic data, see Table 1. HRESI-TOF-MS: Negative-ion mode m/z 1213.3238 [M − H]− (calcd for C56H61O30 1213.3253).

Apetalumoside B (2): Yellow powder. –41.6° (c = 0.99, MeOH); IR νmax (KBr) cm−1: 3367, 2936, 1700, 1650, 1628, 1606, 1514, 1456, 1361, 1287, 1196, 1077, 823, 597, 521; UV λmax (MeOH) nm (log ε): 334 (4.40), 266 (4.22, sh), 241 (4.38). 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectroscopic data, see Table 2. HRESI-TOF-MS: Negative-ion mode m/z 993.2522 [M − H]− (calcd for C44H49O26 993.2518).

Apetalumoside B (3): Yellow powder. –61.6° (c = 0.98, MeOH); IR νmax (KBr) cm−1: 3394, 2942, 1700, 1650, 1631, 1605, 1514, 1457, 1362, 1285, 1173, 1115, 1077, 827; UV λmax (MeOH) nm (log ε): 329 (4.60), 269 (4.26, sh), 238 (4.57). 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectroscopic data, see Table 3. HRESI-TOF-MS: Negative-ion mode m/z 1199.3095 [M − H]− (calcd for C55H59O30 1199.3097).

Apetalumoside B (4): Yellow powder. –66.4° (c = 0.98, MeOH); IR νmax (KBr) cm−1: 3366, 2925, 1698, 1652, 1628, 1601, 1515, 1456, 1342, 1283, 1200, 1075, 824; UV λmax (MeOH) nm (log ε): 333 (4.34), 269 (4.20, sh), 242 (4.36). 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectroscopic data, see Table 4. HRESI-TOF-MS: Negative-ion mode m/z 993.2537 [M − H]− (calcd for C44H49O26 993.2518).

Apetalumoside B (5): Yellow powder. –47.6° (c = 0.10, MeOH); IR νmax (KBr) cm−1: 3367, 2923, 1702, 1652, 1633, 1601, 1515, 1456, 1343, 1284, 1199, 1075, 824; UV λmax (MeOH) nm (log ε): 333 (4.44), 268 (4.32, sh), 241 (4.45). 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectroscopic data, see Table 5. HRESI-TOF-MS: Negative-ion mode m/z 1155.3063 [M − H]− (calcd for C50H59O31 1155.3046).

Apetalumoside B (6): Yellow powder. –84.0° (c = 0.99, MeOH); IR νmax (KBr) cm−1: 3367, 2924, 1700, 1654, 1631, 1600, 1515, 1457, 1342, 1281, 1176, 1073, 825; UV λmax (MeOH) nm (log ε): 330 (4.52), 270 (4.24, sh), 238 (4.50). 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectroscopic data, see Table 6. HRESI-TOF-MS: Negative-ion mode m/z 1361.3625 [M − H]− (calcd for C61H69O35 1361.3625).

Apetalumoside C (7): Yellow powder. –47.2° (c = 0.77, MeOH); IR νmax (KBr) cm−1: 3391, 2936, 1700, 1654, 1628, 1606, 1514, 1457, 1360, 1283, 1178, 1114, 1076, 831; UV λmax (MeOH) nm (log ε): 325 (4.47), 265 (4.20), 238 (4.44, sh). 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectroscopic data, see Table 7. HRESI-TOF-MS: Negative-ion mode m/z 1183.3123 [M − H]− (calcd for C55H59O29 1183.3147).

Acid Hydrolysis of 1–7: A solution of 1–7 (each 1.5 mg) in 1 M HCl (1 mL) was heated under reflux for 3 h, respectively. The reaction mixture was dealt, then analyzed by CH3CN/H2O (70:30, v/v; flow rate 1.0 mL/min) using the same condition as reference [1]. As result, d-glusose was detected from 1–7 by comparison of its retention time and optical rotation with that of authentic sample (tR 8.8 min, positive).

4. Conclusions

As results, seven new sinapoyl acylated flavonoid glycosides were obtained from L. apetalum seeds. Although various acylated flavonol glycosides distribute widely in the plant kingdom, sinapoylates such as the flavonoid glycosides reported in this paper are quite rare, which were found only in 21 species from eight family plants, including Cruciferae [3,4,5,6,7,8,9,10,11,12,13], Leguminosae [14,15], Apocynaceae [16], Solanaceae [17], Elaeagnaceae [18,19], Rubiaceae [20,21], Ranunculaceae [22], and Moraceae [23] until now. And almost of them were obtained from the Cruciferae family, which includes L. apetalum researched by our lab. This will have some guidance for plant taxonomy.