Bioactive protopanaxatriol type saponins isolated from the roots of Panax notoginseng (Burk.) F. H. Chen.

Seven new protopanaxatriol type saponins, 20S-sanchirhinosides A1 (1), A2 (2), A3 (3), A4 (4), A5 (5), and A6 (6), and sanchirhinoside B (7) were obtained as minor constituents from the root extract of Panax notoginseng (Burkill, F. H. Chen), which showed protection effects against antimycin A induced mitochondrial oxidative stress. Their structures were elucidated by chemical and spectroscopic methods (IR, HRESI-TOF-MS, 1D and 2D NMR). Among them, compounds 4, 6 and 7 showed significant protective effects against antimycin A-induced L6 cell injury.

1. Introdution

Reactive oxygen species (ROS) cause protein and DNA injuries and further induce pathological changes, such as heart failure [1], neuronal injury [2] and ischemia reperfusion [3]. A lot of natural products show potential ROS scavenging effects and are used as antioxidant agents.

Panax notoginseng (Burkill, F. H. Chen), have been cultivated in China for more than 400 years. As a traditional Chinese medicine, whose root components have several medicinal properties and are used for stenching the blood, dispersion of gore and reduction of the pain caused by blood diseases, etc. The main components in this plant were identified to be saponins, flavonoids, dencichine and polysaccharides [4]. During the course of our characterization studies on the bioactive constituents from the roots of P. notoginseng, the 70% EtOH extract showed significant protective effects against antimycin A-induced L6 cell injuries. Seven new protopanaxatriol type saponins: 20S-sanchirhinosides A1 (1), A2 (2), A3 (3), A4 (4), A5 (5), and A6 (6) and sanchirhinoside B (7) were obtained as minor constituents from it. In this paper, we report the protect effects of P. notoginseng 70% EtOH extract and new compounds 1–7 against antimycin A-induced mitochondrial oxidative stress.

2. Results and Discussion

The dried roots of P. notoginseng were refluxed with 70% ethanol-water. Evaporation of the solvent under reduced pressure provided a 70% ethanol-water extract. The extract were subjected to column chromatography (CC) and finally HPLC to give seven new protopanaxatriol type saponins: 20S-sanchirhinosides A1–A6 (1–6), and sanchirhinoside B (7) (Figure 1).

Figure 1

The structures of compounds 1–7.

20(S)-Sanchirhinoside A (1) was isolated as a white powder, [α]25D + 12.6° (MeOH). The IR spectrum showed absorption bands at 3,365, 1,717, and 1,654 cm−1 ascribable to hydroxyl, α,β-unsaturated ester, and olefin functions, respectively. The molecular formula, C40H66O10 of 1 was determined by positive-ion HRESI-TOF-MS (m/z 729.4543 [M + Na]+, calcd. for C40H66O10Na 729.4548). The 1H-NMR spectrum of 1 (Table 1, in C5D5N) showed signals assignable to nine methyls [δ 0.84, 1.07, 1.26, 1.43, 1.56, 1.64, 1.68, 2.08 (3H each, all s, H3-30, 19, 18, 21, 29, 27, 26, 28), 1.77 (3H, br. d, ca. J = 7 Hz, H3-4'')], three methines bearing oxygen functions [δ 3.51 (1H, dd, J = 5.0, 12.0 Hz, H-3), 3.93 (1H, m, H-12), 4.40 (1H, ddd, J = 3.5, 10.5, 10.5 Hz, H-6)], one trisubstituted olefin [δ 5.33 (1H, t, J = 7.0 Hz, H-24)], one α,β-unsaturated ester moiety [δ 6.06 (1H, br. d, ca. J = 16 Hz, H-2''), 7.12 (1H, dq, J = 7.0, 15.5 Hz, H-3'')], together with an anomeric proton signal at δ 5.06 (1H, d, J = 7.5 Hz, H-1'). The 13C-NMR spectrum displayed 40 carbons, including 30 carbons for the aglycon, six carbons for the sugar unit and four for a butenoyl group. Taken together the 1H- and 13C-NMR spectra suggested that 1 was a dammarane-type triterpene saponin derivative. The chemical shift of δC 61.5 (C-5) indicated that 1 was a protopanaxatriol type saponin [δC~56 and ~61 (C-5) for protopanaxadiol and protopanaxatriol type saponins, respectively]. In conjunction with analysis of the HSQC spectrum, the 1H- and 13C-NMR data for 1 were assigned as shown in Table 1 (in C5D5N) and Table 2 (determined in CD3OD). The 1H 1H COSY experiment on 1 indicated the presence of the partial structure written in bold lines. In HMBC experiment, long-range correlations were observed between the following protons and carbons: H3-18 and C-7−9, 14; H3-19 and C-1, 5, 9, 10; H3-21 and C-17, 20, 22; H3-26 and C-24, 25, 27; H3-27 and C-24−26; H3-28 and C-3−5, 29; H3-29 and C-3−5, 28; H3-30 and C-8, 13−15; H-1' and C-6; H-6' and C-1''; H-2'', 3'' and C-1''; H3-4'' and C-2'', 3'' (Figure 2). The stereochemistry of C-20 in 1 was clarified by comparing the chemical shifts of 13-, 16-, 17-, and 21−24-carbons of it [δ 23.1 (C-23), 27.0 (C-21), 27.1 (C-16), 35.9 (C-22), 48.3 (C-13), 54.8 (C-17), 126.3 (C-24)] with those of similar 20-epimers of the dammarane type compounds, 20R-gensenoside Rh1 [δ 22.6 (C-21), 22.6 (C-23), 26.6 (C-16), 43.1 (C-22), 48.7 (C-13), 50.5 (C-17), 125.9 (C-24)] [5], and 20(S)-gensenoside Rh1 [δ 23.0 (C-23), 26.9 (C-21), 27.1 (C-16), 35.9 (C-22), 48.3 (C-13), 54.8 (C-17), 126.4 (C-24)] [6], which was measured in the same solvent (C5D5N) as 1, the stereostructure of the 20-position in 1 was confirmed to be S orientation.

Table 1

1H- and 13C-NMR data for compound 1 in C5D5N (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
1	39.5	1.05 (m), 1.74 (m)	22	35.9	1.71 (m), 2.08 (m)
2	27.9	1.85 (m), 1.90 (m)	23	23.1	2.32 (m), 2.62 (m)
3	78.7	3.51 (dd, 5.0, 12.0)	24	126.3	5.33 (t, 7.0)
4	40.3	—	25	130.8	—
5	61.5	1.43 (d, 11.5)	26	25.8	1.68 (s)
6	80.0	4.40 (ddd, 3.5, 10.5, 10.5)	27	17.7	1.64 (s)
7	45.7	1.97 (dd, 10.5, 10.5)2.35 (m)	28	31.6	2.08 (s)
		2.52 (dd, 5.0, 10.5)	29	16.5	1.56 (s)
8	41.3	—	30	17.0	0.84 (s)
9	50.3	1.59 (m)	1'	106.2	5.06 (d, 7.5)
10	39.8	—	2'	75.4	4.06 (dd, 7.5, 9.0)
11	32.1	1.59 (m), 2.15 (m)	3'	79.2	4.22 (dd, 9.0, 9.0)
12	71.1	3.93 (m)	4'	71.6	4.00 (dd, 9.0, 9.0)
13	48.3	2.10 (dd, 10.5, 10.5)	5'	75.2	4.07 (m)
14	51.7	—	6'	65.2	4.77 (dd, 6.5, 12.0)
15	32.2	1.59 (m), 2.15 (m)			5.11 (br. d, ca. 12)
16	27.1	1.43 (m), 1.87 (m)	1''	166.5	—
17	54.8	2.35 (m)	2''	123.4	6.06 (br. d, ca. 16)
18	17.5	1.26 (s)	3''	144.8	7.12 (dq, 7.0, 15.5)
19	17.7	1.07 (s)	4''	17.9	1.77 (br. d, ca. 7)
20	73.0	—
21	27.0	1.43 (s)

Table 2

1H- and 13C-NMR data for compound 1 in CD3OD (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δCb	δHb (J in Hz)
1	40.2	1.06 (m), 1.75 (m)	22	36.3	1.37 (m), 1.54 (m)
2	27.6	1.57 (m), 1.63 (m)	23	23.3	1.98 (m), 2.15 (m)
3	79.9	3.10 (dd, 5.0, 10.5)	24	126.2	5.14 (t, 7.0)
4	40.4	—	25	132.1	—
5	61.9	1.11 (d, 10.5)	26	26.0	1.68 (s)
6	80.7	4.07 (ddd, 3.0, 10.5, 10.5)	27	17.84	1.62 (s)
7	45.9	1.59 (m), 2.00 (m)	28	31.3	1.34 (s)
8	42.0	—	29	16.3	0.97 (s)
9	50.9	1.45 (m)	30	17.1	0.91 (s)
10	40.5	—	1'	105.7	4.43 (d, 7.5)
11	32.0	1.20 (m), 1.85 (m)	2'	75.5	3.21 (dd, 7.5, 9.0)
12	72.0	3.53 (m)	3'	78.7	3.35 (dd, 9.0, 9.0)
13	48.5	1.72 (dd, 11.0, 11.0)	4'	71.8	3.23 (dd, 9.0, 9.0)
14	52.5	—	5'	75.3	3.52 (m)
15	32.2	1.02 (m), 1.49 (m)	6'	65.3	4.16 (dd, 6.0, 11.5)
16	27.4	1.28 (m), 1.86 (m)			4.45 (br. d, ca. 12)
17	55.1	2.03 (m)	1''	168.0	—
18	17.7	1.06 (s)	2''	123.5	5.88 (dd, 2.0, 15.0)
19	17.78	0.99 (s)	3''	146.5	7.00 (dq, 7.0, 15.0)
20	74.4	—	4''	18.3	1.88 (dd, 2.0, 7.0)
21	26.5	1.15 (s)3.63 (1H, m, overlapped)

Figure 2

The main 1H 1H COSY and HMBC correlations of 1 and 2.

Acid hydrolysis yielded D-glucose, which was identified by HPLC analysis by its retention time and optical rotation using chiral detection [7,8]. On the basis of above mentioned evidence, the structure of 1 was characterized to be 20(S)-sanchirhinoside A1.

20(S)-Sanchirhinoside A (2) was obtained as white powder with positive rotation ([α]D25 + 7.4°). The molecular formula, C43H72O14, of 2 was determined by positive-ion HRESI-TOF-MS (m/z 835.4832 [M + Na]+, calcd for C43H72O14Na 835.4814). Acid hydrolysis of 2 yielded D-glucose and D-xylose, which was identified by the same method as 1 [7,8]. The 1H and 13C (C5D5N, Table 3) and various 2D NMR experiments including 1H 1H COSY, HSQC, and HMBC spectra of 2 indicated the presence of a 20S-protopanaxatriol type aglycon [9] [δH 0.92, 1.00, 1.23, 1.40, 1.44, 1.64, 1.66, 2.06 (3H each, all s, H3-30, 19, 18, 29, 21, 27, 26, 28), 1.40 (1H, d, J = 11.0 Hz, H-5), 3.49 (1H, dd, J = 5.0, 11.5 Hz, H-3), 3.94 (1H, m, H-12), 4.34 (1H, m, H-6); δC 23.0 (C-23), 26.8 (C-16), 27.1 (C-21), 35.9 (C-22), 48.4 (C-13), 54.8 (C-17), 126.3 (C-24); a β-D-glucopyranosyl [δ 5.00 (1H, d, J = 7.5 Hz, H-1')]; a β-D-xylopyranosyl [δ 5.76 (1H, d, J = 7.0 Hz, H-1'')]; together with an acetyl group [δH 2.08 (3H, s, H3-2'''); δC 21.0 (C-2'''), 170.9 (C-1''')]. Furthermore, in the HMBC experiments, long-range correlations between the following protons and carbons were observed: H-1' and C-6; H-1'' and C-2'; H-6' and C-1'' (Figure 2). Consequently, the structure of 2 was determined and named as 20(S)-sanchirhinoside A2.

Table 3

1H- and 13C-NMR data for compound 2 in C5D5N (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
1	39.5	1.00 (m), 1.70 (m)	23	23.0	2.30 (m), 2.62 (m)
2	27.8	1.85 (m)	24	126.3	5.33 (t, 7.0)
3	78.7	3.49 (dd, 5.0, 11.5)	25	130.8	—
4	40.1	—	26	25.8	1.66 (s)
5	61.2	1.40 (d, 11.0)	27	17.7	1.64 (s)
6	78.8	4.34 (m)	28	31.8	2.06 (s)
7	45.4	1.95 (dd, 10.5, 10.5) 2.35 (m)	29	17.0	1.40 (s)
		2.34 (dd, 5.0, 10.5)	30	17.0	0.92 (s)
8	41.2	—	1'	103.4	5.00 (d, 7.5)
9	50.1	1.56 (m)	2'	80.1	4.36 (dd, 7.5, 8.0)
10	39.7	—	3'	79.3	4.32 (m)
11	32.1	1.56 (m), 2.15 (m)	4'	71.3	3.99 (dd, 9.0, 9.0)
12	71.0	3.94 (m)	5'	75.0	3.94 (m)
13	48.4	2.09 (dd, 10.5, 10.5)	6'	65.0	4.61 (dd, 6.0, 11.5)
14	51.7	—			5.05 (br. d, ca. 12)
15	31.4	1.21 (dd, 10.0, 10.0)	1''	105.0	5.76 (d, 7.0)
		1.76 (dd, 10.0, 10.0)	2''	75.8	4.18 (m)
16	26.8	1.44 (m), 1.87 (m)	3''	78.8	4.16 (m)
17	54.8	2.34 (m)	4''	71.3	4.25 (m)
18	17.3	1.23 (s)	5''	67.3	3.66 (dd, 11.0, 11.0)
19	17.7	1.00 (s)			4.34 (m)
20	73.1	—	1'''	170.9
21	27.1	1.44 (s) 3.63 (1H, m, overlapped)	2'''	21.0	2.08 (s)
22	35.9	1.74 (m), 2.08 (m)

20(S)-Sanchirhinosides A (3) and A (4) were both obtained as white powders with positive rotation ([α]D25 + 19.7° for 3, and +23.2° for 4, respectively, both in MeOH). The same molecular formula, C41H70O13, of 3 and 4 were determined by positive-ion HRESI-TOF-MS (m/z 793.4720 [M + Na]+ for 3, 793.4715 [M + Na]+ for 4, respectively, calcd for C41H70O13Na 793.4709). With acid hydrolysis with 1 M HCl, both of them gave D-glucose and L-arabinose [7,8]. Compared with 20S-gensenoside Rh1 [6] showed it to be similar except for the signals of an α-L-arabinopyranosyl moiety in the 1H and 13C (C5D5N, Table 4) data of 3 [δH 4.96 (1H, d, J = 8.0 Hz, H-1''); δC 66.9 (C-5''), 69.6 (C-4''), 72.6 (C-2''), 75.3 (C-3''), 98.7 (C-1'')]. On the other hand, the 13C-NMR chemical shift of the carbon in the 20-position was shifted from 73.0 [6] to 83.0, which indicated that C-20 was linked with a sugar. Furthermore, in the HMBC experiments, long-range correlations between H-1' and C-6, H-1'' and C-20 were observed (Figure 3). Meanwhile, the 1H- and 13C-NMR (C5D5N, Table 5) and various 2D NMR experiments including 1H 1H COSY, HSQC, and HMBC spectra of 4 showed the same fragments as 3, including a 20S-protopanaxatriol type aglycon [δH 0.94, 1.03, 1.16, 1.48, 1.60, 1.60, 1.62, 1.98 (3H each, all s, H3-30, 19, 18, 29, 27, 26, 21, 28), 1.40 (1H, d, J = 10.5 Hz, H-5), 3.48 (1H, dd, J = 5.5, 10.5 Hz, H-3), 4.18 (1H, m, H-12), 4.37 (1H, m, H-6)], a β-D-glucopyranoside [δ 5.20 (1H, d, J = 7.5 Hz, H-1'')], and an α-L-arabipyranoside [δ 4.98 (1H, d, J = 8.0 Hz, H-1')]. In the HMBC experiments, long-range correlations between H-1' and C-6, H-1'' and C-20 were observed (Figure 3). On the basis of above mentioned evidence, the structures of 3 and 4 were elucidated as 20(S)-sanchirhinosides A3 and A4, respectively, as shown in Figure 3.

Table 4

1H- and 13C-NMR data for compound 3 in C5D5N (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
1	39.5	1.02 (m), 1.74 (m)	23	23.2	2.22 (m), 2.50 (m)
2	28.0	1.85 (m), 1.93 (m)	24	125.9	5.28 (t, 7.0)
3	78.7	3.50 (dd, 5.0, 11.5)	25	131.1	—
4	40.4	—	26	25.8	1.62 (s)
5	61.4	1.41 (d, 10.5)	27	17.8	1.63 (s)
6	80.2	4.42 (ddd, 3.0, 10.5, 10.5)	28	31.8	2.08 (s)
7	45.2	1.94 (m), 2.50 (m)	29	16.4	1.61 (s)
8	41.1	—	30	17.2	0.81 (s)
9	50.0	1.51 (m)	1'	106.0	5.02 (d, 8.0)
10	39.7	—	2'	75.5	4.09 (dd, 8.0, 8.0)
11	31.0	1.51 (m), 2.05 (m)	3'	79.7	4.25 (m)
12	70.1	4.11 (m)	4'	71.9	4.21 (dd, 8.0, 9.0)
13	49.2	1.98 (dd, 10.5, 10.5)	5'	78.2	3.95 (m)
14	51.3	—	6'	63.1	4.37 (dd, 5.0, 12.0)
15	30.6	1.06 (m), 1.65 (m)			4.52 (dd, 1.5, 12.0)
16	26.6	1.30 (m), 1.75 (m)	1''	98.7	4.96 (d, 8.0)
17	51.5	2.48 (m)	2''	72.6	4.38 (dd, 8.0, 8.5)
18	17.60	1.17 (s)	3''	75.3	4.15 (dd, 3.0, 8.5)
19	17.55	1.03 (s)	4''	69.6	4.27 (m)
20	83.0	—	5''	66.9	3.75 (dd, 3.0, 11.0)
21	22.2	1.56 (s) 3.63 (1H, m, overlapped)			4.26 (m)
22	36.1	1.79 (m), 2.38 (m)

Figure 3

The main 1H 1H COSY and HMBC correlations of 3 and 4.

Table 5

1H- and 13C-NMR data for compound 4 in C5D5N (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
1	39.5	1.01 (m), 1.73 (m)	23	23.2	2.23 (m), 2.50 (m)
2	27.9	1.84 (m), 1.91 (m)	24	126.0	5.26 (t, 7.0)
3	78.6	3.48 (dd, 5.5, 10.5)	25	131.0	—
4	40.2	—	26	25.8	1.60 (s)
5	61.4	1.40 (d, 10.5)	27	17.8	1.60 (s)
6	79.8	4.37 (m)	28	31.7	1.98 (s)
7	45.4	1.97 (m), 2.39 (m)	29	16.6	1.48 (s)
8	41.2	—	30	17.3	0.94 (s)
9	50.0	1.55 (m)	1'	106.4	4.98 (d, 8.0)
10	39.7	—	2'	72.6	4.51 (dd, 8.0, 8.5)
11	31.0	1.55 (m), 2.09 (m)	3'	75.0	4.24 (dd, 3.0, 8.5)
12	70.2	4.18 (m)	4'	69.1	4.39 (m)
13	49.3	2.00 (dd, 10.5, 10.5)	5'	66.1	3.86 (dd, 3.0, 13.0)
14	51.4	—			4.38 (m)
15	30.8	0.99 (m), 1.61 (m)	1''	98.3	5.20 (d, 7.5)
16	26.6	1.36 (m), 1.82 (m)	2''	75.2	4.01 (dd, 7.5, 8.5)
17	51.6	2.55 (m)	3''	79.4	4.25 (dd, 8.5, 8.5)
18	17.6	1.16 (s)	4''	71.7	4.18 (dd, 8.5, 9.0)
19	17.5	1.03 (s)	5''	78.3	3.94 (m)
20	83.3	—	6''	62.9	4.34 (dd, 5.0, 11.5)
21	22.4	1.62 (s)3.63 (1H, m, overlapped)			4.50 (dd, 1.5, 11.5)
22	36.2	1.84 (m), 2.41 (m)

20(S)-Sanchirhinosides A (5) and A (6) were both isolated as white powders with positive optical rotations ([α]D25 + 105.3° for 5, and +3.1° for 6, respectively, both in MeOH). The molecular formula, C47H80O18, of 5 was determined from positive-ion HRESI-TOF-MS (m/z 955.5248 [M + Na]+, calcd. for C47H80O18Na 955.5237). On the other hand, the molecular formula, C53H90O23, of 6 (m/z 1117.5725 [M + Na]+, calcd for C53H90O23Na 1117.5765), was determined from HRESI-TOF-MS, too. Acid hydrolysis of 5 and 6 with 1 M HCl liberated D-glucose (from 5 and 6), D-xylose (from 6), and L-arabinose (from 5) [7,8]. Both the 1H- and 13C-NMR spectra of 5 and 6 (C5D5N, Table 6 for 5, and Table 7 for 6) indicated the presence of a 20S-protopanaxatriol type aglycon [9]. In conjunction with analysis of HSQC and HSQC-TOCSY spectra, the 1H- and 13C-NMR data for 5 and 6 were assigned. Meanwhile, in the HMBC experiment for compound 5, the long-range correlations were observed between the following proton and carbon pairs: δH 5.10 (1H, d, J = 7.5 Hz, H-1') and δC 78.8 (C-6); δH 6.60 (1H, d, J = 2.5 Hz, H-1'') and δC 79.2 (C-2'); δH 5.16 (1H, d, J = 7.5 Hz, H-1''') and δC 83.3 (C-20) (Figure 4). On the other hand, the correlations between δH 4.93 (1H, d, J = 7.5 Hz, H-1') and δC 79.5 (C-6); δH 5.76 (1H, d, J = 7.0 Hz, H-1'') and δC 80.2 (C-2'); δH 5.11 (1H, d, J = 7.0 Hz, H-1''') and δC 83.5 (C-20); δH 5.09 (1H, d, J = 7.5 Hz, H-1'''') and δC 70.3 (C-6''') were observed in HMBC experiment on compound 6. Consequently, compounds 5 and 6 were determined as 20(S)-sanchirhinosides A and A, respectively.

Table 6

1H- and 13C-NMR data for compound 5 in C5D5N (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
1	39.5	0.96 (m), 1.69 (m)	26	25.8	1.61 (s)
2	27.8	1.75 (m), 1.85 (m)	27	17.8	1.61 (s)
3	78.7	3.48 (dd, 5.0, 11.5)	28	32.0	2.13 (s)
4	40.2	—	29	17.15	1.49 (s)
5	61.2	1.37 (d, 10.5)	30	17.21	0.83 (s)
6	78.8	4.41 (m)	1'	103.9	5.10 (d, 7.5)
7	45.5	1.92 (m), 2.41 (m)	2'	79.2	4.30 (dd, 7.5, 8.5)
8	41.2	—	3'	78.3	4.17 (m)
9	49.9	1.49 (m)	4'	72.0	4.17 (m)
10	39.7	—	5'	77.9	3.88 (m)
11	30.9	1.48 (m), 2.05 (m)	6'	62.8	4.32 (m)
12	70.3	4.10 (m)			4.48 (br. d, 11)
13	49.0	1.97 (dd, 10.5, 10.5)	1''	108.6	6.60 (d, 2.5)
14	51.4	—	2''	82.2	5.12 (br. s)
15	30.7	1.03 (m), 1.64 (m)	3''	77.6	4.93 (br. s)
16	26.6	1.28 (m), 1.75 (m)	4''	86.0	4.93 (br. s)
17	51.7	2.47 (m)	5''	62.4	4.18 (m)
18	17.4	1.17 (s)			4.30 (br. d, ca. 12)
19	17.5	0.96 (s)	1'''	98.3	5.16 (d, 7.5)
20	83.3	—	2'''	75.2	4.00 (dd, 7.5, 8.5)
21	22.4	1.60 (s)3.63 (1H, m, overlapped)	3'''	79.2	4.24 (dd, 8.5, 8.5)
22	36.0	1.81 (m), 2.39 (m)	4'''	71.6	4.19 (dd, 8.5, 9.0)
23	23.3	2.25 (m), 2.50 (m)	5'''	78.3	3.92 (m)
24	126.0	5.27 (t, 7.0)	6'''	62.9	4.32 (m)
25	131.0	—			4.48 (br. d, ca. 11)

Table 7

1H- and 13C-NMR data for compound 6 in C5D5N (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
1	39.5	0.94 (m), 1.71 (m)	1'	103.6	4.93 (d, 7.5)
2	27.8	1.81 (m)	2'	80.2	4.39 (dd, 7.5, 8.5)
3	78.9	3.48 (dd, 5.0, 11.0)	3'	79.9	4.35 (dd, 8.5, 8.5)
4	40.2	—	4'	71.8	4.18 (m)
5	61.3	1.37 (d, 10.0)	5'	78.0	3.83 (m)
6	79.5	4.32 (m)	6'	62.9	4.31 (m)
7	45.0	1.93 (m), 2.35 (m)			4.57 (br. d, ca. 11)
8	41.2	—	1''	104.9	5.76 (d, 7.0)
9	50.0	1.48 (dd, 11.0, 11.0)	2''	75.9	4.16 (dd, 7.0, 8.5)
10	39.7	—	3''	78.8	4.25 (m)
11	30.9	1.50 (m), 2.04 (m)	4''	71.3	4.25 (m)
12	70.2	4.16 (m)	5''	67.3	3.66 (dd, 10.5, 10.5)
13	49.2	1.98 (dd, 10.5, 10.5)			4.33 (m)
14	51.4	—	1'''	98.1	5.11 (d, 7.0)
15	30.7	1.07 (m), 1.61 (m)	2'''	74.9	3.90 (dd, 7.0, 9.0)
16	26.6	1.28 (m), 1.72 (m)	3"'	79.3	4.17 (m)
17	51.6	2.51 (m)	4'''	71.6	4.05 (m)
18	17.59	1.15 (s)	5'''	77.1	4.06 (m)
19	17.55	0.97 (s)	6'''	70.3	4.31 (m)
20	83.5	—			4.72 (br. d, ca. 11)
21	22.3	1.63 (s)3.63 (1H, m, overlapped)	1''''	105.4	5.09 (d, 7.5)
22	36.2	1.80 (m), 2.40 (m)	2''''	75.3	4.04 (m)
23	23.2	2.39 (m), 2.60 (m)	3"''	78.36	4.21 (m)
24	126.0	5.32 (t, 7.0)	4''''	71.7	4.21 (m)
25	131.1	—	5''''	78.41	3.92 (m)
26	25.8	1.61 (s)	6''''	62.8	4.36 (m)
27	18.0	1.67 (s)			4.51 (br. d, ca. 12)
28	31.7	2.06 (s)
29	16.7	1.46 (s)
30	17.2	0.80 (s)

Figure 4

The main 1H 1H COSY and HMBC correlations of 5 and 6.

Sanchirhinoside B (7), [α]25D + 14.7° (MeOH), was isolated as a white powder. The molecular formula, C42H70O13, of 7 was determined by positive-ion HRESI-TOF-MS (m/z 805.4700 [M + Na]+, calcd. for C42H70O13Na 805.4709). The 1H-, 13C-NMR (C5D5N, Table 8) and various 2D NMR experiments, including 1H 1H COSY, HSQC, and HMBC of 7 suggested the presence of eight methyls, two olefinic protons, three methines bearing oxygen functions, together with two anomeric proton signals, which indicated that 7 was a dammarane-type triterpene saponin derivative with two double bonds. Comparison of the 1H- and 13C-NMR spectra of 7 with those of ginsenoside Rh4 [10] indicated that the two compounds had the same C-17 side chain. The stereochemistry of the double bond at C-20(22) was determined by a NOESY experiment. In the NOESY spectrum for 7, the correlation signal between δH 1.77 (3H, s, H3-21) and δH 1.74, 2.81 (1H each, both m, H2-23) was observed (Figure 5). Consequently, the configuration of double bond at C-20(22) was supposed to be E. Furthermore, in HMBC experiment, long-range correlations were observed between δH 5.01 (H-1') and δC 80.0 (C-6); δH 4.98 (H-1'') and δC 77.1 (C-12). Finally, acid hydrolysis of 7 only liberated D-glucose [7,8]. Therefore, the structure of 7 was concluded to be sanchirhinoside B as shown in Figure 5.

Table 8

1H- and 13C-NMR data for compound 7 in C5D5N (500 MHz for 1H and 125 MHz for 13C).

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
1	39.2	0.88 (m), 1.51 (m)	23	27.8	1.74 (m), 2.81 (m)
2	28.0	1.84 (m)	24	124.8	5.41 (t, 7.0)
3	78.6	3.52 (dd, 5.0, 11.0)	25	130.4	—
4	40.4	—	26	25.9	1.71 (s)
5	61.5	1.37 (d, 10.5)	27	17.9	1.62 (s)
6	80.0	4.38 (m)	28	31.8	2.06 (s)
7	45.1	1.88 (m), 2.47 (m)	29	16.3	1.59 (s)
8	41.2	—	30	16.9	0.73 (s)
9	50.4	1.43 (m)	1'	105.9	5.01 (d, 7.5)
10	39.8	—	2'	75.5	4.09 (dd, 7.5, 8.0)
11	28.1	1.22 (m), 2.15 (m)	3'	79.7	4.24 (dd, 8.0, 9.0)
12	77.1	4.12 (m)	4'	72.5	4.11 (dd, 8.0, 9.0)
13	48.9	1.98 (dd, 10.5, 10.5)	5'	77.9	3.98 (m)
14	51.1	—	6'	63.1	4.37 (dd, 5.0, 12.0)
15	32.7	1.10 (m), 1.66 (m)			4.51 (dd, 2.0, 12.0)
16	29.4	1.44 (m), 1.78 (m)	1''	101.2	4.98 (d, 7.5)
17	49.6	2.77 (m)	2''	75.3	3.90 (dd, 7.5, 8.0)
18	17.2	1.09 (s)	3''	78.6	4.27 (dd, 8.0, 9.0)
19	17.7	0.91 (s)	4''	71.8	4.22 (dd, 9.0, 9.0)
20	138.4	—	5''	78.2	3.94 (m)
21	13.6	1.77 (s)3.63 (1H, m, overlapped)	6''	63.7	4.36 (dd, 5.0, 12.0)
22	123.4	5.55 (t, 7.0)			4.59 (dd, 2.0, 12.0)

Figure 5

The main 1H 1H COSY, HMBC and NOE correlations of 7.

Furthermore, the protective effects of P. notoginseng 70% EtOH extract and new compounds 1–7 against antimycin A-induced mitochondrial oxidative stress were determined. The 70% ethanolic extract and compounds 4, 6 and 7 showed significant protective effects against antimycin A-induced L6 cell injury (Table 9).

Table 9

Cell survival rate of P. notoginseng extract and compounds 1–7 on L6 cells treated with antimycin A.

Sample	Cell survival rate (%)
Normal	100.0 ± 0.0 **
Control	45.9 ± 0.1
Probucol	56.1 ± 1.1 **
P. notoginseng ext.	55.3 ± 1.2 *
1	50.8 ± 1.9
2	56.8 ± 2.5
3	54.2 ± 1.5
4	59.3 ± 2.1 *
5	57.2 ± 3.1
6	59.0 ± 2.1 *
7	57.4 ± 1.6 *

Values represent the mean ± SD of determinations (n = 8). * p < 0.05; ** p < 0.01 vs. control group. Administrated concentration of probucol and 1–7 were 10 μmol/L, P. notoginseng ext. was 10 μg/mL. N = 8.

3. Experimental

3.1. General

Optical rotations were measured on a Rudolph Autopol® IV automatic polarimeter. IR spectra were recorded on a Varian 640-IR FT-IR spectrophotometer. UV spectra were obtained on a Varian Cary 50 UV-Vis spectrophotometer. NMR spectra were determined on a Bruker 500 MHz NMR spectrometer at 500 MHz for 1H- and 125 MHz for 13C-NMR, with TMS as an internal standard. Positive- and Negative-ion HRESI-TOF-MS were recorded on an Agilent Technologies 6520 Accurate-Mass Q-Tof LC/MS spectrometer. 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), Sephadex LH-20 (Ge Healthcare Bio-Sciences, Uppsala, Sweden), and ODS (40–63 μm, YMC Co., Ltd., Tokyo, Japan). A Cosmosil 5C18-MS-II (20 mm i.d. × 250 mm, Nakalai Tesque, Inc., Tokyo, Japan) preparative HPLC (PHPLC) column was used to purify the constituents. TLC plates pre-coated with silica gel GF254 (Tianjin Silida Technology Co., Ltd., Tianjin, China) were used to detect the purity of isolates by spraying with 10% aqueous H2SO4-EtOH, followed by heating.

3.2. Plant Material

The dried roots of P. notoginseng (Burkill, F. H. Chen) were collected from Wenshan, Guangxi province, China and identified by Dr. Li Tianxiang. The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM (No. 20120505).

3.3. Extraction and Isolation

The dried roots of P. notoginseng (5.0 kg) were refluxed twice with 70% ethanol-water (volume) for 2 times. Evaporation of the solvent under reduced pressure provided a 70% ethanol-water extract (480.2 g). The residue was dissolved in H2O, then subjected to D101 CC [EtOH-H2O (0:100 → 50:50 → 100:0, v/v) to afford three fractions (Fr. 1−3). Fraction 3 (120.0 g) was subjected to silica gel CC [CHCl3 → CHCl3-MeOH (100:3 → 100:7, v/v) → CHCl3-MeOH-H2O (10:3:1 → 7:3:1 → 6:4:1, v/v/v, lower layer)] to give 12 fractions (Fr. 1–12). Fraction 7 (8.0 g) was subjected to normal phase silica gel CC [CHCl3 → CHCl3-MeOH-H2O (40:3:1 → 30:3:1 → 20:3:1 → 10:3:1, v/v/v, lower layer) → MeOH] to yield fourteen fractions (Fr. 7-1-1–7-1-14). Fraction 7-6 (97.9 mg) was purified by prepared HPLC (PHPLC) [MeOH-H2O (70:30, v/v)], and sanchirhinoside A1 (1, 2.9 mg) was obtained. Fraction 8 (4.0 g) was isolated by ODS CC [MeOH-H2O (40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 100:0, v/v] to give 11 fractions (Fr. 8-1–8-11). Fractions 8-5 (46.6 mg), 8-6 (80.5 mg), and 8-8 (40.2 mg) were purified by PHPLC [MeOH-H2O (60:40, v/v)] to yield sanchirhinosides A4 (4, 1.6 mg), B (7, 3.3 mg), and A2 (2, 7.7mg), respectively. Fraction 9 (16.0 g) was subjected to ODS CC [MeOH-H2O (30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 100:0, v/v)] to afford nine fractions (Fr. 9-1–9-9). Fraction 9-7 (113.8 mg) was purified by PHPLC [MeOH-H2O (60:40, v/v)], and sanchirhinoside A3 (3, 7.6 mg) was obtained. Fraction 10 (3.6 g) was separated by ODS CC [MeOH-H2O (10:90 → 20:80 → 30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 100:0, v/v)] to afford 15 fractions (Fr. 10-1–10-15). Fraction 10-7 (393.8 mg) was purified by PHPLC [MeOH-H2O (50:50, v/v)] to give sanchirhinoside A5 (5, 8.2 mg). Fraction 12 (10.0 g) was subjected to ODS CC [MeOH-H2O (10:90 → 20:80 → 30:70 → 40:60 → 50:50 → 60:40 → 100:0, v/v)] to give 13 fractions (Fr.12-1–12-13). Fraction 12-9 (107.8 mg) was further purified by silica gel CC [CHCl3-MeOH-H2O (7:3:1, v/v/v, lower layer) to yield sanchirhinoside A6 (6, 12.7 mg).

20S-Sanchirhinoside A (1): White powder. [α]D25 + 12.6° (c = 0.12, MeOH); IR νmax (KBr) cm−1: 3,365, 2,928, 2,872, 1,717, 1,654, 1,457, 1,375, 1,316, 1,188, 1,085, 1,045. 1H-NMR (500 MHz, C5D5N) and 13C-NMR (125 MHz, C5D5N) spectroscopic data, see Table 1; 1H-NMR (500 MHz, CD3OD) and 13C- NMR (125 MHz, CD3OD) spectroscopic data, see Table 2. HRESI-TOF-MS: Positive-ion mode m/z 729.4543 [M + Na]+ (calcd’ for C40H66O10Na 729.4548); Negative-ion mode m/z 741.4364 [M + Cl]− (calcd for C40H66O10Cl 741.4350).

20S-Sanchirhinoside A (2): White powder. [α]D25 +7.4° (c = 0.33, MeOH); IR νmax (KBr) cm−1: 3,367, 2,931, 2,876, 1,733, 1,642, 1,456, 1,373, 1,242, 1,160, 1,075, 1,043. 1H NMR (500 MHz, C5D5N) and 13C-NMR (125 MHz, C5D5N) spectroscopic data, see Table 3. Positive-ion mode m/z 835.4832 [M + Na]+ (calcd. for C43H72O14Na 835.4814); Negative-ion mode m/z 847.4568 [M + Cl]− (calcd. for C43H72O14Cl 847.4616).

20S-Sanchirhinoside A (3): White powder. [α]D25 + 19.7° (c = 0.36, MeOH); IR νmax (KBr) cm−1: 3,367, 2,927, 2,875, 1,647, 1,457, 1,386, 1,253, 1,074, 1,027. 1H-NMR (500 MHz, C5D5N) and 13C-NMR (125 MHz, C5D5N) spectroscopic data, see Table 4. Positive-ion mode m/z 793.4720 [M + Na]+ (calcd. for C41H70O13Na 793.4709); Negative-ion mode m/z 815.4779 [M + COOH]− (calcd. for C42H71O15 815.4798).

20S-Sanchirhinoside A (4): White powder. [α]D25 + 23.2° (c = 0.08, MeOH); IR νmax (KBr) cm−1: 3,366, 2,929, 2,872, 1,643, 1,457, 1,386, 1,255, 1,127, 1,073, 1,043. 1H-NMR (500 MHz, C5D5N) and 13C- NMR (125 MHz, C5D5N) spectroscopic data, see Table 5. Positive-ion mode m/z 793.4716 [M + Na]+ (calcd. for C41H70O13Na 793.4709); Negative-ion mode m/z 815.4468 [M + COOH]− (calcd. for C42H71O15 815.4798).

20S-Sanchirhinoside A (5): White powder. [α]D25 + 105.3° (c = 0.41, MeOH); IR νmax (KBr) cm−1: 3,367, 2,930, 2,875, 1,647, 1,457, 1,386, 1,310, 1,073, 1,042. 1H-NMR (500 MHz, C5D5N) and 13C- NMR (125 MHz, C5D5N) spectroscopic data, see Table 6. Positive-ion mode m/z 955.5248 [M + Na]+, calcd. for C47H80O18Na 955.5237); Negative-ion mode m/z 967.4950 [M + Cl]− (calcd. for C47H80O18Cl 967.5039).

20S-Sanchirhinoside A (6): White powder. [α]D25 + 3.1° (c = 0.55, MeOH); IR νmax (KBr) cm−1: 3,367, 2,929, 2,878, 1,645, 1,456, 1,386, 1,307, 1,074, 1,043. 1H-NMR (500 MHz, C5D5N) and 13C-NMR (125 MHz, C5D5N) spectroscopic data, see Table 7. Positive-ion mode m/z 1117.5725 [M + Na]+, calcd. for C53H90O23Na 1117.5765); Negative-ion mode m/z 1093.5731 [M - H]− (calcd. for C53H89O23 1093.5800).

Sanchirhinoside B (7): White powder. [α]D25 + 14.7° (c = 0.12, MeOH); IR νmax (KBr) cm−1: 3,367, 2,927, 2,874, 1,653, 1,457, 1,395, 1,151, 1,072, 1,024. 1H-NMR (500 MHz, C5D5N) and 13C-NMR (125 MHz, C5D5N) spectroscopic data, see Table 8. Positive-ion mode m/z 805.4700 [M + Na]+, calcd. for C42H70O13Na 805.4709); Negative-ion mode m/z 817.4518 [M + Cl]− (calcd. for C42H70O13Cl 817.4510).

3.4. Acid Hydrolysis of 1–7

A solution of new compounds 1–7 (each 1.5 mg) in 1 M HCl (1 mL) was heated under reflux for 3 h, respectively. The reaction mixture was neutralized with Amberlite IRA-400 (OH− form) and removed by filtration. The aqueous layer was subjected to the HPLC analysis under the following condition, respectively: HPLC column, Kaseisorb LC NH2-60-5, 4.6 mm i.d. × 250 mm (Tokyo Kasei Co. Ltd., Tokyo, Japan); detection, optical rotation [Chiralyser (IBZ Messtechnik GMBH, Hannover, Germany)]; mobile phase, CH3CN-H2O (75:25, v/v); flow rate 1.0 mL/min. As results, D-xylose (from 2, 6), L-arabinose (from 3–5), D-glucose (from 1–7) and were confirmed by comparison of the retention times with the authentic samples [tR: 8.8 min (D-xylose), 10.2 min (L-arabinose), and 13.1 min (D-glucose), all of them showed positive optical rotations].

3.5. Mitochondrial Oxidative Stress Protect Effects Assay

Antimycin A was used to induce mitochondrial oxidative stress [11]. Briefly, L6 cells (Cell Resource Center, IBMS, CAMS/PUMC, Beijing, China) were plated at a density of 5 × 104 cells/well in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Scientific, UT, USA) supplemented with 10% calf serum (Thermo Scientific) in a 96-well plate and were incubated at 37 °C for 24 h. Cells were treated with or without 10 μmol/L sample DMSO solution (final DMSO concentration was 0.5%). One hour later, medium was removed and 100 μg/mL antimycin A (Sigma Co. Ltd, MO, USA) in 200 μL DMEM was added to each well, The MTT assay was performed 24 h later to detect the cell survival rate. Probucol was used as positive control.

3.6. Statistical Analysis

Values are expressed as mean ± S.D. All the grouped data were statistically performed with SPSS 11.0. Significant differences between means were evaluated by one-way analysis of variance (ANOVA) and Tukey’s Studentized range test was used for post hoc evaluations. p < 0.05 was considered to indicate statistical significance.

4. Conclusions

Antimycin A is known to cause the leakage of superoxide radicals from cell mitochondria by inhibiting mitochondrial electron transport [12]. Compared with normal group, 100 µg/mL antimycin A induced significant L6 cell injury, while 10 µM probucol showed increased cell survival rate effects compared with the antimycin treated group. From the bioactive 70% EtOH extract of P. notoginseng roots, seven new protopanaxatriol type saponins, 20S-sanchirhinosides A1–A6 (1–6), and sanchirhinoside B (7) were obtained. Among the new compounds, 4, 6 and 7 showed significant protective effects against antimycin A-induced L6 cell injury. This research will benefit investigation of trace bioactive chemical constituents of P. notoginseng root. On the basis of the activity screening results, further studies of the antioxidant mechanisms of compounds 1–7 are necessary.