Triterpenoids with Promoting Effects on the Differentiation of PC12

Article pubs.acs.org/jnp

Triterpenoids with Promoting Effects on the Differentiation of PC12 Cells from the Steamed Roots of Panax notoginseng Cheng-Zhen Gu,†,‡ Jun-Jiang Lv,† Xiao-Xia Zhang,† Yi-Jun Qiao,† Hui Yan,† Yan Li,† Dong Wang,† Hong-Tao Zhu,† Huai-Rong Luo,† Chong-Ren Yang,† Min Xu,*,† and Ying-Jun Zhang*,† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: The roots of Panax notoginseng, an important Chinese medicinal plant, have been used traditionally in both the raw and processed forms, due to the different chemical constituents and bioactivities found. Thirty-eight dammarane-type triterpenoid saponins were isolated from the steam-processed roots of P. notoginseng, including 18 new substances, namely, notoginsenosides SP1−SP18 (1−18). The structures of 1−18 were determined on the basis of spectroscopic analysis and acidic hydrolysis. The absolute configuration of the hydroxy group at C-24 in 1−4, 19, and 20 was determined in each case by Mo2(AcO)4-induced circular dichroism. The new compounds were found to feature a diversity of highly oxygenated side chains, formed by hydrolysis of the C-20 sugar moiety followed by dehydration, dehydrogenation, epoxidation, hydroxylation, or methoxylation of the main saponins in the raw roots. The new saponins 1, 2, 6−8, 14, and 17 and the known compounds 20−27 showed promoting effects on the differentiation of PC12 cells, at a concentration of 10 μM.

M

notoginsenosides ST-1−ST-5.17 One of these substances, notoginsenoside ST-4, was found to be a promising agent for herpes simplex virus infection, having EC50 values of 16.5 and 19.4 μM for HSV-1 and HSV-2, respectively.18 This preliminary study also showed that the total saponins of the steamed roots of P. notoginseng could enhance the neurite outgrowth of NGFmediated PC12 cells at a concentration of 50 μg/mL. In order to determine the bioactive principles with neurite outgrowthpromoting activities, a large-scale and detailed phytochemical study on the processed roots of P. notoginseng was carried out, which led to the isolation of 15 protopanaxadiol- and 23 protopanaxatriol-type saponins, including 18 new metabolites (1−18). The structures of these new compounds were determined by spectroscopic analysis and acidic hydrolysis, using 13C−1H NMR spin-coupling constants (2,3JC,H) and Mo2(AcO)4-induced circular dichroism (CD). A possible transformation pathway of the new compounds in the processed roots of P. notoginseng is also proposed. Moreover, all the isolates were tested for their neurotrophic activities on PC12 cells and cytotoxicity against five human cancer cell lines (HL-60, SMMC-7712, A-549, MCF-7, and SW480), and the results obtained are discussed herein.

ethods for the processing of traditional Chinese medicines (TCM) have been developed over thousands of years. The processing methods have been used not only to produce new medicinal or curative effects but also to reduce or eliminate the toxicity and side effects of the raw medicinal materials. They involve steaming, baking, cooking, frying, and soaking with wine, vinegar, ginger juice, and/or other liquids. Panax notoginseng (Burkill) F. H. Chen ex C. Y. Wu & K. M. Feng (Araliaceae) is a significant medicinally used species, and its roots have been used traditionally in both the raw and processed forms. The raw form has been used to arrest various internal or external hemorrhages, eliminate bruises, and treat swelling and pain, as well as blood stasis and clots.1 In contrast, the steamed root has been reported to generate a tonic that can be used to nourish the blood and increase the production of various blood cells in anemic conditions.2 To date, over 80 triterpenoid saponins have been isolated and reported from P. notoginseng,3−5 among which notoginsenoside R1 and ginsenosides Rg1, Re, Rb1, and Rd are the main saponins of the raw roots. The pharmacological activities of these saponins are mainly associated with cardiovascular and cerebrovascular diseases.6−11 Eight saponins that are absent in the raw form, namely, ginsenosides 20(R/S)-Rh1, Rk3, Rh4, 20(S/R)-Rg3, Rk1, and Rg5, have been found to be the main constituents in the processed roots of P. notoginseng. Thus, the chemical components are different in the raw and processed roots of P. notoginseng.12−16 Our previous study on the steam-processed roots of P. notoginseng led to the isolation of five new saponins, © XXXX American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION The air-dried raw roots of P. notoginseng were crushed into small grains and steamed at 120 °C with a pressure of 0.12 MPa Received: January 11, 2015

A

DOI: 10.1021/acs.jnatprod.5b00027 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

for 12 h.16 The 80% aqueous MeOH extract was subjected to repeated column chromatography over D-101 resin, silica gel, Diaion HP20SS, RP-18, and MCI-gel CHP20P, followed by semipreparative HPLC, to afford 18 new dammarane-type triterpenoid saponins (1−18), together with 20 known compounds. The known substances were identified as notoginsenosides T4 (19)19 and ST-1 (20),17 ginsenosides 20(R)-Rh1 (21),20 20(R)-Rg3 (22),21 Rk2 (23),22 20(S)- (24) and 20(R)- (25) 6″-O-acetylginsenoside Rg3,23,24 20(R)-25hydroxyginsenoside Rh1 (26),20 25-hydroxyginsenoside Rk3 (27),25 ginsenosides 20(S)-Rh1,20 Rk3,22 Rh4,22 20(S)-Rg3,21 Rk1,22 Rg5,22 20(S)- and 20(R)-Rh2,26 and Rh3,22 20(R)dammarane-3β,6α,12β,20,25-pentol,27 and sanchinoside B1,28 respectively, using authentic samples and by comparison of their spectroscopic data with literature values. Among them, ginsenosides 20(S/R)-Rh1, 20(S/R)-Rg3, Rh4, Rk3, Rk1, and Rg5 are the main saponins found in the steamed processed roots of

P. notoginseng, and 25-hydroxyginsenoside Rk3 (27) was isolated from P. notoginseng for the first time. Compound 1 was obtained as a white, amorphous powder, and its molecular formula was determined to be C42H72O15 by the positive-mode HRESIMS (m/z 839.4766 [M + Na]+), corresponding to seven degrees of unsaturation. The IR spectrum indicated the presence of hydroxy groups (3423 cm−1) and an olefin moiety (1633 cm−1). The 13C NMR and DEPT spectra showed 42 carbon resonances. Twelve of these corresponded to two hexosyl units (δC 105.2, 83.4, 78.0, 71.6, 78.3, 62.7 and 106.1, 77.2, 78.2, 72.6, 78.3, 62.8) (Table 2), which were determined to be D-glucosyl moieties on the basis of acidic hydrolysis, followed by GC analysis of the corresponding trimethylsilylated L-cysteine adduct. The other 30 carbons (Table 2) were assigned to nine methines with three oxygen-bearing (δC 89.0, 71.0, and 80.1) and two olefinic (δC 136.1 and 130.3) methines, eight methyls, seven methylenes, and six quaternary carbons, of which two are B



Article

Table 1. 1H (600 MHz) NMR Data of Compounds 1−3, 11, and 17 in Pyridine-d5 (δ in ppm) no. 1 2 3 5 6 7 9 11 12 13 15 16 17 18 19 21 22 23 24 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ MeO

1

2

3

11

0.74 m 1.48 m 1.81 m 2.19 m 3.29 br d (9.6) 0.67 br d (11.4) 1.35 m 1.47 m 1.21 m 1.47 m 1.39 m 1.40 m 2.02 m 3.95 m 2.04 m 1.03 m 1.59 m 1.60 m 1.91 m 2.41 m 1.01 s 0.79 s 1.58 s 6.51 d (15.6) 6.44 dd (15.6, 6.0) 4.47 d (6.0) 1.60 s 1.61 s 1.29 s 1.10 s 0.97 s 4.95 d (7.8) 4.28 m 4.30 m 4.17 m 3.95 m 4.39 m 4.58 m 5.41 d (7.2) 4.17 m 4.28 m 4.36 m 3.95 m 4.51 m 4.51 m

0.73 m 1.46 m 1.80 m 2.18 m 3.28 dd (4.2, 11) 0.66 br d (12.0) 1.34 m 1.47 m 1.21 m 1.43 m 1.39 m 1.48 m 2.00 m 3.94 m 1.99 m 1.03 m 1.59 m 1.57 m 1.92 m 2.40 m 1.01 s 0.78 s 1.57 s 6.42 d (15.6) 6.55 dd (15.6, 6.6) 4.50 d (6.6) 1.62 s 1.63 s 1.29 s 1.10 s 0.97 s 4.94 d (7.8) 4.28 m 4.34 m 4.16 m 3.95 m 4.37 m 4.58 m 5.40 d (7.2) 4.16 m 4.27 m 4.37 m 3.95 m 4.50 m 4.50 m

0.75 m 1.50 m 1.84 m 2.22 m 3.31 dd (4.2, 12.0) 0.68 br d (12.0) 1.37 m 1.49 m 1.22 m 1.44 m 1.41 m 1.41 m 2.07 m 3.95 m 2.02 m 1.01 m 1.55 m 1.44 m 1.87 m 2.41 m 1.03 s 0.84 s 1.57 s 6.37 d (15.6) 6.47 dd (15.6, 6.6) 4.47 d (6.6) 1.57 s 1.58 s 1.31 s 1.13 s 0.93 s 4.96 d (7.8) 4.29 m 4.29 m 4.18 m 3.96 m 4.39 m 4.60 m 5.42 d (7.8) 4.17 m 4.36 m 4.38 m 3.96 m 4.40 m 4.52 m

1.47 m 0.73 m 1.82 m 2.20 m 3.29 dd (4.2, 11.4) 0.66 br d (11.4) 1.34 m 1.47 m 1.20 m 1.43 m 1.37 m 1.42 m 1.92 m 3.90 m 2.08 m 1.06 m 1.65 m 1.55 m 1.97 m 2.77 m 0.97 s 0.78 s 2.01 s 6.20d (8.4) 5.19 dd (8.4, 3.6) 3.79 d(3.6) 1.63 s 1.63 s 1.29 s 1.11 s 0.93 s 4.95d (7.8) 4.28 m 4.35 m 4.18 m 3.96 m 4.38 m 4.61 m 5.41 d (7.8) 4.17 m 4.28 m 4.38 m 3.96 m 4.38 m 4.60 m

methylenes [δC 35.9 (C-22), and 23.0 (C-23)] in 20(S)ginsenoside Rg3, signals for a trans-disubstituted double bond [δH 6.51 (1H, d, J = 15.6 Hz) and 6.44 (1H, dd, J = 6.0, 15.6 Hz)] occurred for 1, in addition to an oxymethine (δC 80.1, δH 4.47) and an oxygen-bearing quaternary carbon (δC 72.7). This suggested that the side chain of compound 1 has two more hydroxy groups than 20(S)-ginsenoside Rg3. The transdisubstituted double bond in 1 was located between C-22 and C-23, on the basis of the 1H−1H COSY correlations of an olefinic proton at δH 6.44 (H-23) with another olefinic proton at δH 6.51 (H-22) and the oxymethine proton at δH 4.47 (d, J = 6.0 Hz, H-24), as well as the HMBC correlations of H-22 with C-20 (δC 74.1), C-21 (δC 28.8), C-23 (δC 130.3), and C-24 (δC 80.1), H-23 (δH 6.44) with C-22 (δC 136.1) and C-24 (δC 80.1), H-24 (δH 4.47) with C-22, C-23, C-25 (δC 72.7) and C26 (δC 27.0), and H-26 (δH 1.60) and H-27 (δH 1.61) with both C-24 and C-25. On the basis of the above HMBC correlations, the oxymethine at δC 80.1 and the oxygen-bearing quaternary carbon at δC 72.7 were assigned at C-24 and C-25, respectively. Furthermore, the sugar location and sequence could be confirmed by HMBC correlations from the glucosyl H-1′ (δH 4.95) to the aglycone C-3 (δC 89.0) and from the terminal glucosyl H-1″ (δH 5.41) to the inner glucosyl C-2′ (δC 83.4). The absolute configuration of the side chain in 1 was determined from the 1H NMR coupling constants, the 13C NMR chemical shifts, and analysis of the Mo2(AcO)4-induced circular dichroism data. The large coupling constant of J22,23 (15.6 Hz) suggested the E-geometry of Δ22,23. The deshielded chemical shifts of C-21 (δC 28.8) and C-17 (δC 53.7) indicated the 20S configuration in 1, compared to a 20R configuration [C-21 (δC 22.8) and C-17 (δC 50.7)].20 The absolute configuration of C-24 was determined using Mo2(AcO)4induced circular dichroism. The aglycone 1a was mixed with Mo2(AcO)4 in DMSO to give a metal complex, which displayed a positive Cotton effect at 296 nm (Figure 1), suggesting the 24S configuration for 1a, according to the Snatzke rule.29,30 From the above evidence, the structure of notoginsenoside SP1 (1) was determined as (3β,12β,20S,22E,24S)-3,12,20,24,25pentahydroxydammar-22-ene-3-O-β-D-glucopyranosyl-(1→2)β-D-glucopyranoside. The molecular formula of compound 2 was determined to be C42H72O15, on the basis of the HRESIMS (m/z 839.4763 [M + Na]+), which was the same value as that of 1. The NMR data of 2 (Tables 1 and 2) were identical to those of 1, except for a difference for the upfield shift of H-22 to δH 6.42 ppm (vs δH 6.51 ppm in 1) and the downfield shift of H-23 to δH 6.55 ppm (vs δH 6.44 ppm in 1). The chemical shifts of C-21 (δC 29.3) and C-17 (δC 53.7) indicated the 20S configuration of 2,20 and the large coupling constant of J22,23 (15.6 Hz) suggested the Egeometry of Δ22,23, the same as those of 1. However, in a coHPLC analysis of 1 and 2, their retention times were different [33.5 min (1) and 33.1 min (2)], revealing compound 2 to be a stereoisomer of 1 at C-24, with the absolute configuration of C24 in 2 being R, opposite that of 1. Therefore, notoginsenoside SP2 (2) was determined to be (3β,12β,20S,22E,24R)3,12,20,24,25-pentahydroxydammar-22-ene-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside. Notoginsenoside SP3 (3) gave the same molecular formula as 1, namely, C42H72O15, on the basis of the HRESIMS [m/z 855.4494 [M + K]+]. The NMR data of 3 (Tables 1 and 2) were similar to those of 1. These similarities, together with the fact that 1 and 3 gave identical molecular formulas, indicated 3

17 1.48 0.72 2.20 1.82 3.29

m m m m m

0.67 br d (12.0) 1.37 m 1.49 m 1.23 m 1.47 m 1.38 m 1.44 m 1.91 m 3.89 m 2.02 m 1.11 m 1.69 m 1.54 m 2.01 m 2.88 m 1.03 s 0.81 s 1.92 s 5.58 d (9.6) 4.11 dd (9.6, 7.8) 3.13 d (7.8) 1.24 s 1.47 s 1.31 s 1.13 s 0.97 s 4.96d (7.8) 4.28 m 4.35 m 4.18 m 3.96 m 4.39 m 4.60 m 5.41 d(7.2) 4.16 m 4.28 m 4.38 m 3.96 m 4.39 m 4.60 m 3.45 s

oxygen-bearing (δC 74.1 and 72.7). In the 1H NMR spectrum (Table 1), two trans-coupled olefinic protons [δH 6.51 (1H, d, J = 15.6 Hz) and 6.44 (1H, dd, J = 6.0, 15.6 Hz)] and eight singlet methyls (δH 1.01, 0.79,1.58, 1.60, 1.61, 1.29, 1.10, and 0.97) were observed, in addition to two anomeric protons at δH 4.95 (1H, d, J = 7.8 Hz, H-1′) and δH 5.41 (1H, d, J = 7.2 Hz, H-1″), suggesting the two glucosyl moieties as being β. The above spectroscopic data were similar to those of 20(S)ginsenoside Rg3,21 a protopanaxadiol-type disaccharide saponin, except for the C-20 side chain. Instead of a trisubstituted double bond between C-24 and C-25 and two aliphatic C



Article

Table 2. 13C (150 MHz) NMR Data of Compounds 1−6, 11, 17, and 18 in Pyridine-d5 (δ in ppm) no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ MeO

1 39.5 26.7 89.0 39.7 56.4 18.4 35.2 39.9 50.4 36.9 32.1 71.0 50.1 51.9 31.5 27.0 53.7 15.9 16.4 74.1 28.8 136.1 130.3 80.1 72.7 27.0 25.6 28.1 16.6 17.3 105.2 83.4 78.0 71.6 78.3 62.7 106.1 77.2 78.2 72.6 78.3 62.8

2 t t d s d t t s d s t d d s t t d q q s q d d d s q q q q q d d d d d t d d d d d t

39.5 26.7 89.0 39.7 56.4 18.4 35.2 39.9 50.4 36.9 32.0 71.0 50.1 51.9 31.5 27.0 53.7 15.9 16.4 74.0 29.3 136.3 130.4 80.0 72.8 26.5 25.7 28.1 16.6 17.3 105.1 83.3 78.3 71.6 78.1 62.7 106.0 77.2 78.0 71.6 78.1 62.8


39.6 27.2 89.4 40.2 56.9 18.9 35.6 40.5 50.8 37.4 32.5 71.2 50.2 52.3 31.8 27.8 52.5 16.3 16.9 74.0 21.9 141.5 128.8 80.5 73.2 27.2 26.3 28.6 17.1 17.7 105.7 83.9 78.5 72.1 78.7 63.3 106.5 77.7 78.8 72.1 78.7 63.1


39.7 28.0 78.6 40.4 61.4 80.2 45.6 41.1 50.1 39.4 32.1 70.8 49.4 51.7 31.4 27.3 52.2 17.4 17.7 73.5 21.5 140.4 128.3 79.7 72.8 26.5 25.9 31.8 16.4 17.0 106.1 75.5 79.7 71.8 78.2 63.1

5 t t d s d d t s d s t d d s t t d q q s q d d d s q q q q q d d d d d t

39.9 t 28.4 t 78.9 d 40.8 s 61.8 d 80.5d 45.6 t 41.7 s 51.0 d 40.1 s 33.1 t 72.8 d 51.7 d 51.3 s 32.8 t 29.8 t 50.4 d 17.8 q 18.2 q 142.0 s 15.4 q 127.7 d 69.0 d 80.8 d 73.6 s 28.6 q 27.1 q 32.2 q 16.8 q 17.2 q 106.5 d 75.9 d 80.1 d 72.2 d 78.6 d 63.4 t

6 39.7 27.9 78.5 40.3 61.4 80.0 45.3 41.2 50.4 39.4 32.5 72.0 50.7 50.7 32.3 27.9 50.9 17.3 17.7 140.1 13.1 127.9 69.2 80.7 72.9 28.9 25.5 31.7 16.3 16.6 106.0 75.4 79.6 71.7 78.1 63.0


39.2 26.8 88.9 39.7 56.4 18.4 35.3 40.2 50.8 37.0 32.6 72.2 51.6 51.0 32.5 29.1 50.0 15.8 16.5 144.7 15.0 127.3 68.3 80.2 73.2 28.1 26.7 28.1 16.6 17.0 105.2 83.4 78.3 71.6 78.3 62.8 106.0 77.1 78.2 71.6 78.3 62.7


39.2 26.7 89.0 39.7 56.5 18.4 35.4 40.2 50.8 37.0 32.7 72.1 51.1 51.0 32.8 29.3 50.8 15.8 16.5 146.5 14.0 120.4 78.1 66.8 57.3 25.0 20.0 28.2 16.6 16.9 105.3 83.5 78.4 71.6 78.4 62.7 106.2 77.2 78.0 71.6 78.4 62.8 55.6

18 t t d s d t t s d s t d d s t t d q q s q d d d s q q q q q d d d d d t d d d d d t q

40.1 28.4 78.8 40.8 61.7 80.5 45.8 41.5 51.1 39.8 33.6 72.0 52.8 51.7 33.4 30.0 52.3 17.8 18.2 167.9 17.7 120.6 196.4 67.0 61.1 25.2 19.1 32.2 16.8 17.2 106.5 75.8 80.1 72.2 78.6 63.4

t t d s d d t s d s t d d s t t d q q s q d s d s q q q q q d d d d d t

693.4188 [M + Na]+). In the same manner as for 3, the molecular formula and the NMR data (Tables 2 and 3) of 4 were closely comparable to those of notoginsenoside T4 (19), a protopanaxatriol-type monodesmoside with an unknown absolute configuration at C-24.19 In fact, the major differences in the 13C NMR spectrum of 4 were the upfield shifts of C-17, C-21, and C-23 and the downfield shift of C-22, which revealed that 4 might be a geometrical isomer of 19 at C-20 and C-24. The chemical shifts of C-17 [δC 52.2 (4) and 53.7 (19)] and C21 [δC 21.5 (4) and 29.2 (19)] indicated the 20R and 20S configurations in 4 and 19, respectively.20 Moreover, the absolute configurations of C-24 in 4 and 19 were determined, respectively, to be S and R, according to the positive (4a) and negative (19a) Cotton effects at 310 nm (Figures 1 and 2) in the Mo2(AcO)4-induced CD experiments of 4a and 19a. The absolute configuration of C-24 in notoginsenoside T4 (19) was

to be an epimer of 1. The large coupling constant of J22,23 (15.6 Hz) indicated the E-geometry of Δ22,23, which was identical to that of 1. However, the upfield shifts of C-17, C-21, and C-23 by 1.2, 6.9, and 1.5 ppm, respectively, and downfield shift of C22 by 5.4 ppm in 3 indicated that the configurations of C-20 and C-24 of 3 are different from those of 1. The 20R configuration in 3 was confirmed by the chemical shift of C-21 (δC 21.9) and C-17 (δC 52.5).20 Moreover, the negative Cotton effect at 310 nm (Figure 2) in the Mo2(AcO)4-induced CD experiment of aglycone 3a confirmed C-24 in 3 as having the R configuration. Thus, the structure of notoginsenoside SP3 (3) was established as (3β,12β,20R,22E,24R)-3,12,20,24,25-pentahydroxydammar-22-ene-3-O-β-D-glucopyranosyl-(1→2)-β-Dglucopyranoside. The molecular formula of notoginsenoside SP4 (4) was elucidated as C36H62O11, on the basis of the HRESIMS (m/z D



Article

The relative configurations at C-23 and C-24 of compounds 5 and 6 were established using Murata’s method.31 In the hetero half-filtered TOCSY (HETLOC) spectrum of 5, the small coupling constant of 3JH‑23,H‑24 (3.0 Hz) indicated that H23 and H-24 are gauche, suggesting the presence of multiple rotamers of A1, A2, the alternating A1/A2, B1, B2, and the alternating B1/B2. The small coupling constants of 2JC‑23,H‑24 (0 Hz), 2JC‑24,H‑23 (−0.8 Hz), and 3JC‑22,H‑24 (0.8 Hz) indicated that 5 is mainly in the A1 configuration. In compound 6, the medium coupling constant of 3JH‑23,H‑24 (4.8 Hz) suggested the presence of two or more alternating rotamers (A2/A3, A1/A3, B2/B3, or B1/B3). The small 3JC‑22,H‑24 (0.8 Hz) and medium 2 JC‑24,H‑23 values (−2.4 Hz) indicated that the alternating rotamer A1/A3 is predominant (Figure 3). Thus, Murata’s method suggested that the relative configurations of C-23, C-24 in 5 and 6 are both threo. In the same manner as for 5 and 6, the relative configurations of 7 (J23,24 = 4.8 Hz, threo) and 8 (J23,24 = 1.8 Hz, threo) were proposed. The absolute configurations of C-23 in compounds 5−8 were determined on the basis of the chemical shift of the C-21 methyl group.32 It has been reported that the absolute configuration of C-23 can affect the chemical shift of the C21 methyl group. The deshielded chemical shift of C-21 showed an S configuration at C-23, and the comparatively shielded chemical shift of C-21 indicated a 23R configuration.32 Therefore, the chemical shifts of C-21 [δC 15.4 (5), 13.1 (6), 20.3 (7), and 19.5 (8)] suggested the absolute configurations of 5 and 7 are 23S and 24R, while 6 and 8 are 23R and 24S, respectively. Accordingly, the structures of notoginsenosides SP5−SP8 were assigned as (3β,6α,12β,20E,23S,24R)-(5), (3β,6α,12β,20E,23R,24S)-(6), (3β,6α,12β,20Z,23S,24R)-(7), and (3β,6α,12β,20Z,23R,24S)-3,6,12,23,24,25- hexahydroxydammar-20(22)-ene-6-O-β-D-glucopyranoside (8), respectively. Both notoginsenosides SP9 (9) and SP10 (10) gave the same molecular formula of C37H64O11, as deduced from the HRESIMS. Their NMR data (Tables 3 and 5) were closely related to those of 5 and 6, except that both 9 and 10 were shown to possess one more methoxy group [δH 3.40 (9), 3.34 (10), δC 55.3 (9), 55.1 (10)] in the side chain. The methoxy group was determined to be located at C-23, based on the HMBC correlation of the methoxy protons with C-23. Compounds 9 and 10 were also found to be C-23, C-24 diastereomers. In the same manner as for 5−7, the relative configurations of 9 (J23,24 = 1.8 Hz, threo) were constructed. However, in compound 10, the 1H NMR coupling constant of 3 JH‑23,H‑24 (6.6 Hz) was more than 5.0 Hz. The HETLOC spectrum of 10 was therefore measured, from which the small 3 JC‑22,H‑24 (0.8 Hz) and large 2JC‑24,H‑23 (−4.0 Hz) values obtained suggested that the alternating rotamer B1/B3 is predominant (Figure 3), indicating the erythro configuration of C-23, C-24 in 10. Furthermore, the chemical shifts of C-21 [δC 15.2 (9) and 13.3 (10)] supported 9 as 23S, 24R and 10 as 23R, 24R.32 On the basis of the above evidence, notoginsenosides SP9 and SP10 were determined as (3β,6α,12β,20E,23S,24R)- (9) and (3β,6α,12β,20E,23R,24R)23-methoxy-3,6,12,24,25-pentadroxydammar-20(22)-ene-6-Oβ-D-glucopyranoside (10), respectively. Compound 11 gave a molecular formula of C42H72O15, as established by the HRESIMS (m/z 839.4767 [M + Na]+). The NMR data (Tables 1 and 2) of 11 were closely related to those of notoginsenoside ST-2,17 a protopanaxadiol-type saponin with a glucopyranosyl (1→2) glucopyranosyl unit linked to C-

Figure 1. Mo2(OAc)4-induced CD (ICD) spectra of 1a and 4a.

Figure 2. Mo2(OAc)4-induced CD (ICD) spectra of 3a and 19a.

therefore determined as R for the first time, and the structure of notoginsenoside SP4 (4) was identified as (3β,6α,12β,20R,22E,24S)-3,6,12,20,24,25-hexahydroxydammar22-ene-6-O-β-D-glucopyranoside. Compounds 5−8 were analyzed and found to have the same molecular formula of C36H62O11, as established from their HRESIMS data. The NMR data (Tables 2, 3, and 5) indicated them all to have a protopanaxatriol skeleton and a glucopyranosyl unit in their molecule, which were quite similar to notoginsenoside ST-1 (20).17 Nevertheless, on comparing with the side chain of 20 with two hydroxy groups at C-24 and C-25, compounds 5−8 were found to have one more oxymethine [δH 5.20 (5), 5.08 (6), 5.36 (7), and 5.42 (8), δC 69.0 (5), 69.2 (6), 68.3 (7), and 67.2 (8)] in the side chain, instead of a C-23 methylene group at 20. The additional oxymethine was assigned as C-23, on the basis of the 1H−1H COSY and HMBC spectra. The aforementioned data suggested that compounds 5−8 are all 23-hydroxylated analogues of notoginsenoside ST-1 (20), with different absolute configurations of the side chain. In the ROESY experiments, compounds 7 and 8 showed correlations between H-21 and H-22, indicating that the double bonds between C-20 and C-22 are in the Z configuration in both cases. However, the chemical shifts of C-21 in 5 and 6 were shifted upfield, respectively, by 4.9 and 7.2 ppm, relative to 7, suggesting the double bonds between C-20 and C-22 to have E-geometry in both 5 and 6. The 1H and 13C NMR data revealed that compounds 5 and 6, as well as 7 and 8, are C-23, C-24 diastereomers. E



Article

Table 3. 1H (600 MHz) NMR Data of Compounds 4−10 in Pyridine-d5 (δ in ppm) no. 1 2 3 5 6 7

9 11 12 13 15 16 17 18 19 21 22 23 24 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′

4 1.02 m 1.69 m 1.88 m 1.95 m 3.56 dd (4.2, 11.4) 1.45 d (10.8) 4.46 m 1.94 m 2.55 dd (3, 12.6) 1.58 m 1.58 m 2.19 m 3.94 m 2.03 m 1.08 m 1.58 m 1.36 m 1.78 m 2.36 m 1.25 s 1.06 s 1.57 s 6.40 d (15.6) 6.47 dd (15.6, 5.4) 4.49 d (5.4) 1.57 s 1.59 s 2.10 s 1.64 s 0.79 s 5.06 d (7.8) 4.13 t (7.8) 4.31 t (9.0) 4.26 t (9.0) 3.99 m 4.40 dd (11.4, 5.4) 4.57 dd (11.4, 2.4)

1.02 1.68 1.88 1.96 3.56

5

6

m m m m m

1.02 m 1.69 m 1.87 m 1.94 m 3.55 dd (4.8, 11.4) 1.44 d (10.8) 4.47 m 1.95 m 2.54 dd (3.0, 12.6) 1.53 m 1.47 m 2.04 m 3.86 m 1.99 m 1.13 m 1.68 m 1.46 m 1.78 m 2.79 m 1.24 s 1.04 s 1.92 s 6.11 d (9.0) 5.08 dd (9.0, 4.8) 3.88 d(4.8) 1.67 s 1.59 s 2.10 s 1.64 s 0.78 s 5.06 d (7.8) 4.13 t (8.4) 4.30 t (8.4) 4.26 t (9.0) 3.98 m 4.41dd (11.4, 5.4) 4.56 dd (2.4, 11.4)

1.45 d (10.2) 4.46 m 1.96 m 2.55 dd (3.0, 12.6) 1.58 m 1.56 m 2.06 m 3.91 m 2.10 m 1.14 m 1.72 m 1.50 m 1.72 m 2.72 m 1.20 s 1.03 s 1.99 s 6.18 d (8.4) 5.20 dd (8.4, 3.0) 3.78 d (3.0) 1.63 s 1.64 s 2.11 s 1.63 s 0.79 s 5.06 d (7.8) 4.14 t (7.8) 4.31 m 4.28 m 3.99 m 4.41 m 4.57 br d (10.8)

7 1.03 1.70 1.87 1.95 3.55

m m m m m

1.45 d (10.2) 4.45 m 1.95 m 2.55 dd (3.0, 12.6) 1.56 m 1.49 m 2.06 m 3.90 m 2.08 m 1.12 m 1.69 m 1.43 m 1.83 m 3.55 m 1.25 s 1.06 s 1.92 s 5.87 d(9.0) 5.36 dd (9.0, 4.8) 4.00 d (4.8) 1.69 s 1.68 s 2.11 s 1.64 s 0.82 s 5.07 d (7.8) 4.13 t (7.8) 4.30 t (8.4) 4.20 t (9.0) 3.90 m 4.41dd (5.4, 11.4) 4.60 dd (2.4, 11.4)

MeO

8 1.07 1.71 1.82 1.95 3.55

m m m m m

9 0.97 1.63 1.83 1.90 3.52

10

m m m m m

1.03 1.71 1.87 1.94 3.55

m m m m m

1.46 d (10.2) 4.47 m 1.95 m 2.56 dd (3.0, 12.6) 1.61 m 1.50 m 2.09 m 3.96 m 2.07 m 1.18 m 1.70 m 1.52 m 1.70 m 3.61 m 1.25 s 1.06 s 1.85 s 6.00 d (7.8) 5.42 dd (1.8, 7.8) 3.72 d(1.8) 1.61 s 1.66 s 2.10 s 1.65 s 0.80 s 5.07 d (7.8) 4.14 m 4.31 m 4.27 m 3.99 m 4.41, m

1.41 d (12.6) 4.42 m 1.92 m 2.51 dd (3.0, 15.0) 1.52 m 1.43 m 2.02 m 3.86 m 2.09 m 1.08 m 1.73 m 1.89 m 1.44 m 2.69 m 1.21 s 1.00 s 1.98 s 5.78 d (10.0) 4.49 dd (10.0, 4.8) 3.75 d (4.8) 1.55 s 1.52 s 2.07 s 1.60 s 0.80 s 5.02 d (7.8) 4.10 t (9.0) 4.26 m 4.23 m 3.94 m 4.37 m

1.44 d (10.8) 4.46 m 1.97 m 2.55 br d (12.6) 1.55 m 1.48 m 2.05 m 3.86 m 2.03 m 1.17 m 1.74 m 1.52 m 1.85 m 2.82 m 1.26 s 1.05 s 1.95 s 5.72 d(9.6) 4.35 dd (9.6, 6.6) 3.90 d (6.6) 1.65 s 1.52 s 2.10 s 1.64 s 0.80 s 5.07 d (7.2) 4.13 t (7.8) 4.31 m 4.26 m 3.99 m 4.41 m

4.57 br d (11.4)

4.53 br d (13.8) 3.40

4.57 br d (10.8) 3.34

Compounds 12 and 13 were determined also to be C-23, C-24 diastereomers. The coupling constants of J23,24 found for 12 and 13 were 8.4 and 7.8 Hz, respectively. Compared with those of compounds 5−10, the relative configurations of C-23 and C-24 in 12 and 13 were proposed as erythro, due to their relatively large coupling constants of J23,24 (>5.0 Hz). In order to confirm this proposition, the carbon−proton spin-coupling constants of 2,3 JC,H, as well as 3JC‑22,H‑24 and 3JC‑25,H‑23 of 13 were measured from the HETLOC and phase-sensitive (ps)-HMBC NMR spectra. The medium 3JH‑23,H‑24 (7.8 Hz), small 3JC‑22,H‑24 (0.8 Hz), and medium 3JC‑25,H‑23 (5.4 Hz) values suggested that the alternating rotamer B1/B3 is predominant (Figure 3) in 13, corresponding to the erythro isomer. Due to compound 12 sharing a similar coupling constant of J23,24 with that of 13, the C-23, C-24 configurations of 12 were also established as erythro. Furthermore, the chemical shifts of C-21 [δC 13.7 (12) and

3, and a methoxy group at C-23 of the side chain. However, C23 in notoginsenoside ST-2 was substituted by a hydroxy group in 11. The side chain of 11 was identical to that of 5 with Δ20,22 and three hydroxy groups at C-23, C-24, and C-25. The relative configurations of 11 (J23,24 = 3.6 Hz, threo) were established due to similar 1H NMR coupling constants to those of 5. The chemical shift of C-21 [δC 15.0 (11)] was also consistent with that of 5 (δC 15.4), indicating the 23S and 24R absolute configuration of 11. Therefore, the structure of notoginsenoside SP11 (11) was elucidated as (3β,12β,20E,23S,24R)3,12,23,24,25-pentahydroxydmmar-20(22)-ene-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside. The NMR data (Tables 4 and 5) of both 12 and 13 were closely comparable to those of notoginsenoside T1,19 a protopanaxatriol-type saponin with unknown configurations of the C-23 hydroxy and the C-24, C-25 epoxy groups. F



Article

Table 4. 1H (600 MHz) NMR Data of Compounds 12−16 and 18 in Pyridine-d5 (δ in ppm) no. 1 2 3 5 6 7

9 11 12 13 15 16 17 18 19 21 22 23 24 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′

MeO

12

13

0.99 m 1.67 m 1.86 m 1.94 m 3.55 dd (4.2, 11.4) 1.43 d (10.2) 4.45 m 1.95 m 2.54 dd (3, 12.6) 1.55 m 1.46 m 2.03 m 3.90 m 2.01 m 1.16 m 1.69 m 1.44 m 1.69 m 2.83 m 1.23 s 1.03 s 1.87 s 5.93 d (9.6) 4.72 dd (9.6, 8.4) 3.27 d (8.4) 1.30 s 1.51 s 2.10 s 1.63 s 0.82 s 5.05 d (7.8) 4.13 t (8.4) 4.31 t (8.4) 4.26 t (7.8) 3.99 m 4.41 dd (5.4, 11.4) 4.57 dd (2.4, 11.4)

1.03 m 1.69 m 1.89 m 1.95 m 3.56 dd (4.8, 12.0) 1.46 d (10.8) 4.46 m 1.97 m 2.56 dd (2.4, 12.6) 1.58 m 1.50 m 2.07 m 3.97 m 2.09 m 1.17 m 1.73 m 1.45 m 1.73 m 2.79 m 1.25 s 1.05 s 1.95 s 5.88 d (9.0) 4.68 dd (9.0, 7.8) 3.27 d (7.8) 1.31 s 1.38 s 2.11 s 1.65 s 0.84 s 5.00 d (7.8) 4.14 t (8.4) 4.31 t (8.4) 4.26 t (9.0) 3.99 m 4.40 dd (5.4, 12.0) 4.57 dd (2.4, 11.4)

14 0.99 1.66 1.84 1.92 3.54

15

m m m m m

1.04 1.69 1.87 1.94 3.54

m m m m m

1.07 1.69 1.88 1.96 3.57

16

18

m m m m m

1.02 m 1.68 m 1.86 m 1.94 m 3.55 dd (4.8, 11.4) 1.45 d (10.8) 4.46 m 1.97 m 2.57 dd (3, 12.6) 1.57 m 1.46 m 2.06 m 3.88 m 2.16 m 1.21 m 1.76 m 1.68 m 1.88 m 2.82 m 1.25 s 1.04 s 2.54 s 6.78 s

1.43 d (10.2) 4.45 m 1.95 m 2.55 dd (3, 12.6) 1.55 m 1.55 m 2.02 m 3.88 m 2.00 m 1.26 m 1.72 m 1.47 m 1.87 m 2.81 m 1.25 s 1.05 s 1.91 s 5.55 d (9.6) 4.09 dd (9.6, 7.8) 3.11 d(7.8) 1.24 s 1.47 s 2.11 s 1.65 s 0.84 s 5.06 d (7.8) 4.13 t (7.8) 4.30 m 4.26 m 3.99 m 4.40 m

1.46 d (10.2) 4.47 m 1.94 m 2.57 dd (2.4, 12.6) 1.61 m 1.50 m 2.10 m 3.99 m 2.07 m 1.21 m 1.72 m 1.54 m 1.79 m 3.47 m 1.26 s 1.07 s 1.89 s 5.61 d (8.4) 4.81 dd (8.4, 7.8) 3.23 d (7.8) 1.32 s 1.35 s 2.11 s 1.65 s 0.84 s 5.07 d (7.8) 4.14 t (7.8) 4.31 t (8.4) 4.26 t (9.0) 4.00 m 4.41 m

1.48 d (10.8) 4.48 m 2.02 m 2.58 dd (3, 13.2) 1.58 m 1.51 m 2.07 m 3.84 m 2.14 m 1.23 m 1.76 m 1.41 m 1.95 m 3.30 m 1.27 s 1.06 s 2.04 s 5.33 d (9.0) 4.26 dd (9.0, 7.8) 3.16 d (7.8) 1.25 s 1.63 s 2.13 s 1.66 s 0.90 s 5.09 d (7.8) 4.16 t (7.8) 4.32 m 4.26 m 4.01 m 4.41 m

4.56 br d (11.4) 3.43 s

4.58 br d (11.4)

4.59 br d (10.8) 3.43 s

3.58 s 1.29 s 1.34 s 2.12 s 1.64 s 0.84 s 5.08 d (7.8) 4.14 t (7.8) 4.30 t (7.8) 4.27 t (9.0) 3.99 m 4.41 dd (5.4, 11.4) 4.57 dd (2.4, 11.4)

Accordingly, notoginsenoside SP14 (14) was identified as (3β,6α,12β,20E,23S,24S)-24,25-epoxy-23-methoxy-3,6,12-trihydroxydammar-20(22)-ene-6-O-β-D-glucopyranoside. Compound 15 was shown to have the same planar structure as 12 and 13, by analysis of its NMR data (Tables 4 and 5). The ROESY correlation between H-21 and H-22 revealed the Z-geometry of Δ20,22 in 15. The large coupling constant of J23,24 [7.8 Hz (15)] suggested that the relative configuration of C-23, C-24 in 15 is erythro, being the same as compounds 12−14. Moreover, the same chemical shift of C-21 (δC 19.5) as that in 8 indicated the 23R and 24R configurations in 15. Notoginsenoside SP15 (15) was therefore assigned as (3β,6α,12β,20Z,23R,24R)-24,25-epoxy- 3,6,12,23-tetrahydroxydammar-20(22)-ene-6-O-β-D-glucopyranoside. The molecular formula of 16 was determined to be C37H62O10, on the basis of the HRESIMS (m/z 689.4241 [M

14.1 (13)] indicated the absolute configurations of 12 (23R,24R) and 13 (23S,24S), respectively. Therefore, compounds 12 and 13 (notoginsenosides SP12 and SP13) were determined as (3β,6α,12β,20E,23R,24R)- and (3β,6α,12β,20E,23S,24S)-24,25-epoxy-3,6,12,23-tetrahydroxydammar-20(22)-ene-6-O-β-D-glucopyranoside, respectively. On the basis of detailed analysis of the NMR data (Tables 4 and 5), compound 14 was determined to have the same planar structure as notoginsenoside T2,19 a protopanaxatriol-type saponin with unknown configurations of the C-23 methoxy and the C-24/C-25 epoxy groups. The relative configuration of C-23, C-24 of 14 was established as erythro due to the same coupling constant of J23,24 [7.8 Hz (14)] as those of 12 (8.4 Hz) and 13 (7.8 Hz). Moreover, the chemical shift of C-21 (δC 14.4) was similar to that of 13 [C-21 (δC 14.1)] and indicated that the absolute configurations of 14 are also 23S and 24S. G



Article

Table 5. 13C (150 MHz) NMR Data of Compounds 7−10 and 12−16 in Pyridine-d5 (δ in ppm) no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′ MeO

7 39.9 28.4 79.0 40.8 61.9 80.6 45.8 41.1 51.1 40.1 33.0 72.4 51.1 51.5 33.1 28.8 41.7 17.8 18.2 140.6 20.3 128.8 68.3 81.0 73.6 29.6 26.4 32.1 16.8 17.1 106.6 75.6 80.1 72.3 78.7 63.4


39.9 28.0 78.9 40.8 61.8 80.5 45.7 41.7 51.2 40.1 32.7 73.1 51.7 51.7 33.6 28.4 41.5 17.6 18.2 144.2 19.5 127.7 67.2 80.3 73.5 28.4 27.5 32.2 16.8 17.1 106.5 75.9 80.2 72.2 78.7 63.4


39.5 27.9 78.5 40.3 61.4 80.0 45.1 41.3 50.7 39.7 33.1 72.1 51.5 50.9 32.3 29.5 50.3 17.3 17.7 145.1 15.2 123.1 78.2 80.5 72.4 28.3 26.0 31.7 16.3 16.8 106.0 75.4 79.6 71.8 78.1 63.1 55.3

10 t t d s d d t s d s t d d s t t d q q s q d d d s q q q q q d d d d d t q

12

39.5 t 27.9 t 78.5 d 40.3 s 61.4 d 80.1 d 45.4 t 41.3 s 50.5 d 39.7 s 32.2 t 72.2 d 50.7 d 50.7 s 32.5 t 28.4 t 51.2 d 17.4 q 17.7 q 143.4 s 13.3 q 123.0 d 79.4 d 80.6 d 72.5 s 29.2 q 24.8 q 31.7 q 16.4 q 16.7 q 106.0 d 75.4 d 79.6 d 71.8 d 78.1d 63.1 t 55.1 q

39.5 27.9 78.6 40.4 61.4 80.1 45.2 41.7 50.7 39.7 32.5 72.3 50.9 50.7 32.7 28.6 50.6 17.3 17.7 143.0 13.7 124.7 68.6 68.7 58.4 25.2 20.1 31.7 16.3 16.7 106.0 75.5 79.7 71.9 78.2 63.1


39.5 27.9 78.6 40.4 61.4 80.1 45.3 41.3 50.6 39.7 32.5 72.3 50.6 51.0 32.4 29.2 50.4 17.4 17.7 143.6 14.1 124.3 68.4 68.7 58.7 25.1 19.9 31.7 16.3 16.8 106.0 75.5 79.7 71.7 78.2 63.1


39.8 28.4 78.9 40.8 61.8 80.5 45.7 41.7 51.0 40.1 33.1 72.5 51.1 51.3 33.2 29.6 51.1 17.7 18.2 146.9 14.4 120.7 78.4 67.1 57.8 25.4 20.4 32.2 16.8 17.1 106.5 75.9 80.1 72.2 78.7 63.4 56.1

15 t t d s d d t s d s t d d s t t d q q s q d d d s q q q q q d d d d d t q

39.9 28.4 78.7 41.6 61.8 80.5 45.8 41.8 51.1 40.1 32.6 73.1 51.7 51.8 33.6 27.9 41.6 17.6 18.2 146.0 19.5 123.6 67.8 68.7 58.2 25.6 19.8 32.1 16.8 17.1 106.5 75.9 80.1 72.2 78.9 63.5


39.9 t 28.4 t 79.0 d 40.9 s 61.8 d 80.4 d 45.7 t 41.8 s 51.0 d 40.1 s 33.5 t 72.3 d 50.4 d 51.6 s 32.9 t 30.0 t 42.1 d 17.8 q 18.2 q 147.4 s 21.3 q 121.1 d 77.7 d 67.1 d 58.0 s 25.5 q 20.4 q 32.1 q 16.8 q 17.2 q 106.4 d 75.9 d 80.2 d 72.2 d 78.7 d 63.5t 56.3 q

similar to that in 7 [C-21 (δC 20.3)] indicated the absolute configuration of 16 to be 23S and 24S. Thus, notoginsenoside SP16 (16) was elucidated as (3β,6α,12β,20Z,23S,24S)-24,25epoxy-23-methoxy-3,6,12-trihydroxydammar-20(22)-ene-6-Oβ-D-glucopyranoside. Compound 17 gave a molecular formula of C43H72O14, on the basis of the HRESIMS (m/z 835.4820 [M + Na]+). The 1H and 13CNMR data (Tables 1 and 2) of 17 were similar to those of notoginsenoside ST-2,17 a protopanaxatriol-type saponin, except for the signals due to the side chain. Moreover, compound 17 had one more degree of unsaturation than notoginsenoside ST-2. Since the number of double bonds in 17 was the same as that in notoginsenoside ST-2, it was concluded there must be one more ring on the side chain of 17. The oxygen bridge between C-24 and C-25 and a methoxy group at C-23 were determined according to the 1H−1H COSY and HMBC correlations. The J23,24 value of 17 (7.8 Hz) indicated that C-23, C-24 in 17 are erythro configured, since the chemical shift of C-21 (δC 14.0) was identical to that of C-21 of 13 (δC 14.1). This suggested that the absolute configuration of 17 is 23S, 24S. Therefore, the structure of notoginsenoside SP17 (17) was elucidated as (3β,12β,20E,23S,24S)-24,25-epoxy-23-

Figure 3. Newman projections of C-22−C-25 in compounds 5, 6, 10, and 13.

+ Na]+). Compared to 15, compound 16 had one more methoxy group (δC 56.3, δH 3.43) located in the side chain. The HMBC correlation of the methoxy proton with C-23 (δC 77.7) indicated its location at C-23. The ROESY correlation between H-21 and H-22 revealed the Z-geometry of Δ20,22. Moreover, the J23,24 value (7.8 Hz) supported the erythro C-23, C-24 configuration, and the chemical shift of C-21 (δC 21.3) in being H



Article

roots of P. notoginseng, were also tested, in order to compare their bioactivities. As shown in Table 6, the new compounds 1,

methoxy-3,12-dihydroxydammar-20(22)-ene-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside. Compound 18 gave a molecular formula of C36H58O10, as deduced from the HRESIMS (m/z 673.3927 [M + Na]+), representing eight degrees of unsaturation. The NMR data of 18 were similar to those of ginsenoside Rh4,22 except for signals arising from the side chain. The 13C NMR data (Tables 2 and 4) at δC 196.4 indicated the presence of a ketone group in 18. Besides the five degrees of unsaturation due to the glucosyl and the four rings from the aglycon skeleton, three degrees of unsaturation were represented by the side chain of 18. However, the NMR data suggested that only one double bond (δC 167.9, 120.6) and one carbonyl group (δC 196.4) are present in 18. Thus, compound 18 was found to have an additional ring in the side chain. On the basis of the HMBC correlations, a double bond between C-20 and C-22, a carbonyl group at C-23, and an oxygen bridge between C-24 and C-25 were determined. The configuration of the double bond was identified as having E-geometry due to the chemical shift of C21 (δC 17.7), being similar to this carbon (δC 16.7) of cordialia A,33 while the Z-geometry corresponded to the relative deshielded chemical shift of C-21 (δC 20.2).34 Therefore, n o t o g i n s e n o si d e S P 1 8 ( 1 8 ) w a s d e t e r m i n e d a s (3β,6α,12β,20E,24)-24,25-epoxy-23-one-3,6,12-trihydroxydammar-20(22)-ene-6-O-β-D-glucopyranoside. In the present study, 18 new dammarane-type saponins featuring a highly oxygenated side chain were obtained from the steam-processed roots of P. notoginseng. Most of these exhibit a di- or trioxygenated side chain occurring as stereoisomers (1 and 2, 5 and 6, 7 and 8, 9 and 10, along with 12 and 13). These were obtained in pure form, and their absolute configurations at the side chains were determined. The relative configurations at C-23 and C-24 were determined using Murata’s method. The results suggested that a small coupling constant J23,24 (5.0 Hz) corresponded to the erythro configuration. The chemical shifts of C-21 revealed the absolute configurations of C-23 and C-24.32 Moreover, the absolute configurations of C-24 in 1, 3, 4, 19, and 20 were determined according to the positive (S configuration) or negative (R configuration) Cotton effects in the Mo2(AcO)4-induced CD experiments of their corresponding aglycons.29,30 It is noted that the R and S configurations of C-20 affected significantly the chemical shifts of C-17 and C-21, while the R and S configurations of C-24 (1−4 and 19) have no such influence on the chemical shifts of C-24 and C-25. The protopanaxatriol saponins, ginsenosides Rg1, Re, and notoginsenoside R1, and the protopanaxadiol saponins, ginsenosides Rb1 and Rd, are the five main saponins of the raw roots of P. notoginseng. After a steaming process conducted for 12 h at 0.12 MPa, these major saponins with one or two sugars at C-20 decreased in abundance, while other new saponins were formed, including the major products ginsenosides 20(R)-(21) and 20(S)-Rh1, Rk3, Rh4, 20(S)- and 20(R)(22) Rg3, Rk1, and Rg5. A proposed transformation pathway of these metabolites is shown in the Supporting Information (Figure S177). Since the processed roots of P. notoginseng have been used as a tonic by local people in its growing area in southwest China, all the compounds were evaluated for their ability to stimulate nerve growth factor (NGF)-mediated neurite outgrowth on PC12 cells. The ginsenosides Rg1, Re, Rb1, and Rd and notoginsenoside R1, which are main components in the raw

Table 6. Differentiation Rates of Compounds from the Processed Roots of P. notoginseng on PC12 Cells compound

differentiation rate (%)

blanka NGFb NGFc 1 2 6 7 8 14 17 20 21 22 23 24 25 26 27

0 3.6 21.7 8.2 10.8 9.9 11.4 10.9 11.6 11.4 9.3 8.5 9.5 8.9 9.3 13.4 8.6 8.3

a c

Blank with no added NGF. bNegative control: 5 ng/mL of NGF. Positive control: 50 ng/mL of NGF.

2, 6−8, 14, and 17 and the known metabolites 20−27 were found to promote the differentiation of PC12 cells after 72 h at a concentration of 10 μM, while the major compounds in the raw roots showed no promoting properties. Although the total saponins of the raw roots of P. notoginseng, ginsenosides Rg1, Rb1, and Rd and notoginsenoside R1, were reported to have some cytotoxicity for cancer cells,35−38 all the compounds from the steam-processed roots of P. notoginseng showed no cytotoxicity against the HL-60, SMMC-7712, A-549, MCF-7, and SW480 cell lines (IC50 values >10 μM). The present results may provide a scientific basis for ethnomedical usage of the processed P. notoginseng roots.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were performed on a JASCO P-1020 polarimeter. UV spectra were recorded on a Shimadzu UV-2401A spectrophotometer. IR spectra were measured on a Bruker Tensor 27 spectrometer with KBr pellets. 1D and 2D NMR spectra were run on Bruker DRX-400, -500, and AVANCE III-600 NMR spectrometers operating at 400, 500, and 600 MHz for 1H and 100, 125, and 150 MHz for 13C, respectively. The carbon−proton spin-coupling constants (2,3JC,H) were run on a Bruker AVANCE 800 MHz NMR spectrometer for 1H and 200 MHz for 13C. Coupling constants are expressed in hertz, and chemical shifts are given on the ppm scale with solvents as internal standard. ESIMS and HRESIMS were measured on a Bruker HCT/Esquire and an Agilent G6230. ICD was detected with a Chirascan circular dichroism spectrograph (Applied Photophysis, England). Preparative HPLC was carried out on an Agilent 1260 apparatus with a DAD detector. Semipreparative HPLC was performed on an Agilent 1260 liquid chromatograph with a 5 μm Thermo BDS Hypersil-C18 column (10 × 250 mm). Column chromatography (CC) was performed with Diaion 101 resin (Shandong Lukang Pharmaceutical Co., Ltd., People’s Republic of China), Diaion HP20SS (Mitsubishi Chemical Co., Tokyo, Japan), silica gel (200−300 mesh) (Qingdao Marine Chemical and Industrial Factory, Qingdao People’s Republic of China), MCI-gel CHP20P (75−100 μm) (Mitsubishi Chemical Co., Ltd.), and RP-8 or I



Article

RP-18 gel (40−60 μm) (Merck, Darmstadt, Germany). Fractions were monitored by TLC, and spots were visualized by heating the silica gel plates sprayed with 10% sulfuric ethanol solution. Plant Material. Air-dried roots of P. notoginseng were collected from Wenshan County, Yunnan Province, People’s Republic of China, in April 2011 and identified by one of the authors (C.-R.Y.). A voucher specimen (KIB-Z-00336) has been deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Science. The raw plant sample was crushed into small pieces and then steamed at high temperature (120 °C) and pressure (0.12 MPa) for 12 h, yielding a steamed P. notoginseng preparation. Extraction and Isolation. The steamed P. notoginseng (15.0 kg) was extracted with 80% aqueous MeOH (3 × 20 L, 2 days each) at room temperature. After removal of the organic solvent, the extract (3.0 kg) was subjected to Diaion 101 column chromatography (250 × 30 cm), eluting with H2O to remove saccharides and then with MeOH to give a total saponin fraction (2.12 kg), which were further fractionated on a silica gel column (250 × 30 cm), eluting with CHCl3−MeOH−H2O (85:15:1−75:25:2), to afford eight fractions (Frs. A−H). Fr. A (10 g) was chromatographed over an RP-18 column, eluting with MeOH−H2O (7:3 to 9:1), to yield three subfractions (Fr. A1− A3). Fr. A1 (45 mg) was purified by semipreparative HPLC (CH3CN−H2O, 7:3) to give 23 (9 mg) and ginsenoside Rh3 (16 mg). Fr. B (200 g) was applied to RP-18 CC (MeOH−H2O, 1:1 to 9:1), to give five subfractions (Fr. B1−B5). Fr. B1 (120 g) was purified by RP-18 CC (MeOH−H2O, 7:3), followed with recrystallization in MeOH−H2O (75:25), to yield ginsenoside Rk1 (40 g) and ginsenoside Rg5 (43 g). Fr. B2 (8 g) was applied to MCI-gel CHP20P (MeOH−H2O, 6:4 to 9:1) and RP-18 (MeOH−H2O, 75:25) CC, followed by recrystallization, to give ginsenoside 20(S)Rh2 (800 mg) and 20(R)-Rh2 (1 g). Fr. B3 (200 mg) was purified by RP-18 CC (MeOH−H2O, 78:22) to give 20(R)-dammarane3β,6α,12β,20,25-pentol (30 mg). Fr. C (250 g) was subjected to a Diaion HP20SS CC (MeOH− H2O, 4:6 to 9:1) to afford five subfractions (Frs. C1−C5). Frs. C1 (90 g) and C2 (102 g) were applied separately to RP-18 CC (MeOH− H2O, 64:36), followed by recrystallization in MeOH−H2O (60:40, 55:45), to give ginsenoside Rk3 (30 g), Rh4 (34 g), and 20(S)-Rg3 (38 g) and 22 (36 g), respectively. Fr. C4 (63 mg) was purified by semipreparative HPLC (CH3CN−H2O, 1:1) to give 24 (6 mg) and 25 (13 mg). Fr. D (150 g), Fr. E (50 g), Fr. F (40 g), Fr. G (30 g), and Fr. H (10 g) were separately applied to an RP-18 column, eluting with MeOH− H2O (4:6 to 9:1), to yield subfractions D1−D5, E1−E5, F1−F5, G1− G4, and H1−H4, respectively. Fr. D2 (96 g) was further recrystallized in MeOH−H2O (40:60) to yield 21 (33 g) and 20(S)-Rh1 (30 g). Fr. E1 (173 mg) was purified by semipreparative HPLC (CH3CN−H2O, 23:77 to 30:70) to give 1 (13 mg), 2 (12 mg), and 3 (11 mg). Fr. E2 (27 g) was subjected to RP-18 CC (MeOH−H2O, 43:57), followed with recrystallization in MeOH−H2O (35:65), to yield sanchinoside B1 (9 g) and 26 (4 g). Fr. E3 (196 mg) was purified by semipreparative HPLC (CH3CN−H2O, 33:67 to 40:60) to yield 18 (3 mg), 11 (15 mg), 17 (16 mg), and 27 (6 mg). Fr. F2 (182 mg) was purified by semipreparative HPLC (CH3CN− H2O, 23:77 to 28:72) to afford 4 (8 mg), 15 (15 mg), and 20 (7 mg). Fr. F3 (121 mg) was purified by semipreparative HPLC (CH3CN− H2O, 25:75 to 30:70) to yield 14 (12 mg) and 16 (14 mg). Fr. G3 (172 mg) was purified by semipreparative HPLC (CH3CN−H2O, 21:79 to 24:76) to yield 9 (6 mg), 10 (13 mg), 12 (15 mg), and 13 (17 mg). Fr. H1 (121 mg) was purified by semipreparative HPLC (CH3CN−H2O, 15:85 to 18:82) to afford 6 (21 mg), 7 (7 mg), and 19 (35 mg). Fr. H2 (133 mg) was purified by semipreparative HPLC (CH3CN−H2O, 19:81 to 22:78) to yield 5 (13 mg) and 8 (9 mg). Notoginsenoside SP1 (1): amorphous powder; [α]21 D −6.6 (c 0.86, MeOH); IR (KBr) νmax 3423, 2964, 2935, 2876, 1633, 1463, 1368, 1154, 1076, 1043, 659, 571 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive) m/z 839.4766 [M + Na]+ (calcd for C42H72O15Na, 839.4763).

Notoginsenoside SP2 (2): amorphous powder; [α]21 D +5.6 (c 1.4, MeOH); IR (KBr) νmax 3418, 2965, 2944, 2877, 1632, 1452, 1371, 1165, 1077, 1028, 625, 576 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive) m/z 839.4763 [M + Na]+ (calcd for C42H72O15Na, 839.4763). Notoginsenoside SP3 (3): amorphous powder; [α]21 D −5.5 (c 1.7, MeOH); IR (KBr) νmax 3424, 2941, 2878, 1639, 1464, 1454, 1414, 1384, 1198, 1170, 1077, 1038, 979, 646, 602, 579 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive) m/z 855.4494 [M + K]+ (calcd for C42H72O15K, 855.4503). Notoginsenoside SP4 (4): amorphous powder; [α]21 D −12.9 (c 0.55, MeOH); IR (KBr) νmax 3428, 2962, 2930, 2876, 2856, 1636, 1464, 1414, 1384, 1154, 1077, 1033, 641, 534 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS (positive) m/z 693.4188 [M + Na]+ (calcd for C36H62O11Na, 693.4184). Notoginsenoside SP5 (5): amorphous powder; [α]21 D +10.2 (c 0.54, MeOH); IR (KBr) νmax 3424, 2961, 2933, 2876, 1631, 1462, 1384, 1156, 1075, 1029, 928, 574, 532 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS (positive) m/z 693.4188 [M + Na]+ (calcd for C36H62O11Na, 693.4184). NotoginsenosideSP6 (6): amorphous powder; [α]21 D +19.9 (c 1.4, MeOH); IR (KBr) νmax 3425, 2964, 2934, 2878, 1632, 1464, 1384, 1154, 1074, 1027, 928, 580, 530 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS (positive) m/z 693.4187 [M + Na]+ (calcd for C36H62O11Na, 693.4184). Notoginsenoside SP7 (7): amorphous powder; [α]21 D −5.7 (c 0.77, MeOH); IR (KBr) νmax 3421, 2964, 2935, 2878, 1629, 1462, 1384, 1155, 1074, 1031, 928, 891, 588, 533 cm−1; 1H and 13C NMR data, see Tables 3 and 5; HRESIMS (positive) m/z 693.4187 [M + Na]+ (calcd for C36H62O11Na, 693.4184). Notoginsenoside SP8 (8): amorphous powder; [α]21 D −4.7 (c 1.0, MeOH); IR (KBr) νmax 3423, 2962, 2933, 2876, 1631, 1462, 1383, 1155, 1076, 1031, 612, 552 cm−1; 1H and 13C NMR data, see Tables 3 and 5; HRESIMS (positive) m/z 693.4184 [M + Na]+ (calcd for C36H62O11Na, 693.4184). Notoginsenoside SP9 (9): amorphous powder; [α]24 D +22.2 (c 0.19, MeOH); IR (KBr) νmax 3426, 2960, 2933, 2877, 1631, 1457, 1384, 1155, 1074, 1030, 893, 573, 529 cm−1; 1H and 13C NMR data, see Tables 3 and 5; HRESIMS (positive) m/z 707.4326 [M + Na]+ (calcd for C37H64O11Na, 707.4341). Notoginsenoside SP10 (10): amorphous powder; [α]21 D −23.7 (c 0.43, MeOH); IR (KBr) νmax 3431, 2960, 2932, 2877, 1631, 1452, 1384, 1155, 1073, 1030, 928, 579, 530 cm−1; 1H and 13C NMR data, see Tables 3 and 5; HRESIMS (positive) m/z 707.4343 [M + Na]+ (calcd for C37H64O11Na, 707.4341). Notoginsenoside SP11 (11): amorphous powder; [α]21 D −9.4 (c 0.89, MeOH); IR (KBr) νmax 3424, 2944, 2877, 1632, 1552, 1463, 1453, 1385, 1161, 1077, 1030, 895, 593, 579 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive) m/z 839.4767 [M + Na]+ (calcd for C42H72O15Na, 839.4763). Notoginsenoside SP12 (12): amorphous powder; [α]21 D +19.0 (c 0.92, MeOH); IR (KBr) νmax 3425, 2961, 2929, 2875, 1631, 1583, 1383, 1153, 1075, 1026, 972, 578 cm−1; 1H and 13C NMR data, see Tables 4 and 5; HRESIMS (positive) m/z 675.4082 [M + Na]+ (calcd for C36H60O10Na, 675.4079). Notoginsenoside SP13 (13): amorphous powder; [α]21 D +17.0 (c 0.64, MeOH); IR (KBr) νmax 3421, 2959, 2931, 2875, 1631, 1460, 1382, 1154, 1073, 1029, 892, 574 cm−1; 1H and 13C NMR data, see Tables 4 and 5; HRESIMS (positive) m/z 675.4082 [M + Na]+ (calcd for C36H60O10Na, 675.4079). Notoginsenoside SP14 (14): amorphous powder; [α]21 D +3.9 (c 1.0, MeOH); IR (KBr) νmax 3431, 3426, 2960, 2932, 2878, 1633, 1452, 1380, 1152, 1071, 1027, 645, 561 cm−1; 1H and 13C NMR data, see Tables 4 and 5; HRESIMS (positive) m/z 689.4238 [M + Na]+ (calcd for C37H62O10Na, 689.4235). Notoginsenoside SP15 (15): amorphous powder; [α]21 D −24.2 (c 0.83, MeOH); IR (KBr) νmax 3424, 2960, 2935, 2876, 1631, 1453, 1383, 1074, 1034, 973, 596, 531 cm−1; 1H and 13C NMR data, see Tables 4 and 5; HRESIMS (positive) m/z 675.4084 [M + Na]+ (calcd for C36H60O10Na, 675.4079). J



Article

Notoginsenoside SP16 (16): amorphous powder; [α]21 D −13.7 (c 1.0, MeOH); IR (KBr) νmax 3433, 2961, 2934, 2877, 1631, 1553, 1460, 1384, 1073, 1033, 591, 538 cm−1; 1H and 13C NMR data, see Tables 4 and 5; HRESIMS (positive) m/z 689.4241 [M + Na]+ (calcd for C37H62O10Na, 689.4235). Notoginsenoside SP17 (17): amorphous powder; [α]21 D −9.0 (c 0.9, MeOH); IR (KBr) νmax 3423, 2944, 2876, 1632, 1564, 1453, 1414, 1384, 1160, 1076, 1038, 980, 627 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive) m/z 835.4820 [M + Na]+ (calcd for C43H72O14Na, 835.4814). Notoginsenoside SP18 (18): amorphous powder; [α]21 D +18.7 (c 0.57, MeOH); UV (MeOH) λmax (log ε) 257 (3.26) nm; IR (KBr) νmax 3432, 2960, 2932, 2875, 1630, 1614, 1384, 1155, 1075, 1033, 576 cm−1; 1H and 13C NMR data, see Tables 2 and 4; HRESIMS (positive) m/z 673.3927 [M + Na]+ (calcd for C36H58O10Na, 673.3922). Acid Hydrolysis of Compounds 1 and 6. Compounds 1 and 6 (each 5 mg) were hydrolyzed in 2 M HCl (5 mL) at 65 °C for 6 h, respectively. The reaction mixture was extracted with CHCl3 three times (3 × 5 mL). The aqueous layer was neutralized with 2 M NaOH and dried to produce a monosaccharide mixture. Next, a solution of the sugar mixture in pyridine (2 mL) was added to L-cysteine methyl ester hydrochloride (1.5 mg) and kept at 60 °C for 1 h. After this, trimethylsilylimidazole (1.5 mL) was added to the reaction mixture in an ice−water bath and kept at 60 °C for 30 min. The mixture was subjected to GC analysis, run on a Shimadzu GC-14C gas chromatograph, equipped with a 30 m × 0.32 mm i.d. 30QC2/AC-5 quartz capillary column and a H2 flame ionization detector with the following conditions: column temperature, 180−280 °C; programmed increase, 3 °C/min; carrier gas, N2 (1 mL/min); injector and detector temperature, 250 °C; injection volume, 4 μL; and split ratio 1/50. The configuration of the sugar moiety was determined by comparing the retention time with the derivatives of the authentic samples. The retention times of D-/L-glucose were 21.135/21.580 min, and the configuration of the respective sugar moiety from compounds 1 and 6 was determined as D-glucose. Enzymatic Hydrolysis of Compounds 1, 3, 4, 19, and 20. Compounds 1, 3, 4, 19, and 20 (each 6 mg) in MeOH−H2O (1:100, 20 mL) were hydrolyzed with cellulase (300 mg) at 37 °C for 4 days. The reaction mixture was subjected to an MCI-gel CHP20P column, eluted initially with 10% MeOH and then with 70% MeOH. The 70% MeOH fraction was evaporated to dryness and yielded the aglycones 1a, 3a, 4a, 19a, and 20a (1.5 mg, respectively) from 1, 3, 4, 19, and 20, respectively. Mo2(AcO)4-Induced Circular Dichroism. Mo2(AcO)4 (0.7 mg) dissolved in DMSO (1 mL) was prepared as the stock solution, to which aglycones 1a, 3a, 4a, 19a, and 20a (each 0.5 mg) were added separately. The CD spectrum was recorded immediately after mixing and scanned every 10 min for 30 min, to produce a stationary Mo2(AcO)4-induced circular dichroism spectrum for each of 1a, 3a, 4a, 19a, and 20a.29,30 Neurite Outgrowth-Promoting Activity. The neurotrophic activities of the test compounds were examined according to an assay using PC12 cells.39 Briefly, PC12 cells were maintained in F12 medium supplemented with 12.5% horse serum (Hyclone) and 2.5% fetal bovine serum (FBS) (Hyclone) and incubated at 5% CO2 and 37 °C. Test compounds were dissolved in DMSO. For the neurite outgrowth-promoting activity bioassay, PC12 cells were seeded at a density of 5 × 104 cells/mL in a 48-well plate coated with poly-Llysine. After 24 h, the medium was changed to one containing 10 μM of each test compound plus 5 ng/mL NGF (Sigma) or various concentrations of NGF (50 ng/mL for the positive control, 5 ng/mL for the negative control). The final concentration of DMSO was 0.05%, and the same concentration of DMSO was added into the negative control. After 72 h incubation, the neurite outgrowth was assessed under a phase-contrast microscope. Neurite processes with a length equal to or greater than the diameter of the neuron cell body were scored as neurite-bearing cells. The ratio of the neurite-bearing cells to total cells (with at least 100 cells examined/view area; 5 give viewing areas/well) was determined and expressed as a percentage.

Cytotoxicity Assay. As previously reported.40

■

ASSOCIATED CONTENT

S Supporting Information *

The 1H and 13C NMR, HSQC, HMBC, 1H−1H COSY, ROESY, and mass spectra for compounds 1−18, the 1H NMR and ESIMS data of the aglycon (1a, 3a, 4a, 19a, 20a), the HETLOC spectra of 5, 6, 10, and 13, and the ICD spectrum of the aglycon (20a). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jnatprod.5b00027.

■

AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +86-871-6522-3235. E-mail: [email protected] (Y.-J. Zhang). *E-mail: [email protected] (M. Xu). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors are grateful to the members of the analytical group at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Science, for measuring the spectroscopic data. This work was supported by the Science and Technology Planning Project of Yunnan Province, China (2013FC008), the 973 Program of Ministry of Science and Technology of China (2011CB915503), the National Natural Science Foundation of China (No. 81173477), and the 12th Five-Year National Science and Technology Supporting Program of China (2012BAI29B06).

■

REFERENCES

(1) The State Pharmacopoeia Commission of PR China.Pharmacopoeia of the People’s Republic of China; Chemistry Industry Press: Beijing, 2005; Vol. 1, pp 10−11. (2) State Administration of Traditional Chinese Medicine (People’s Republic of China). Zhong Hua Ben Cao Jin Xuan Ben; Shanghai Science and Technology Publishers: Shanghai, 1996; Vol. 1, pp 34−38. (3) Komakine, N.; Okasaka, M.; Takaishi, Y.; Kawazoe, K.; Murakami, K.; Yamada, Y. J. Nat. Med. 2006, 60, 135−137. (4) Wang, X. Y.; Wang, D.; Ma, X. X.; Zhang, Y. J.; Yang, C. R. Helv. Chim. Acta 2008, 91, 60−66. (5) Wang, C. Z.; McEntee, E.; Wicks, S.; Wu, J. A.; Yuan, C. S. J. Nat. Med. 2006, 60, 97−106. (6) Jiang, K. Y.; Qian, Z. N. Acta Pharmacol. Sin. 1995, 16, 399−402. (7) White, C. M.; Fan, C.; Chow, M. J. Clin. Pharmacol. 2000, 40, 1150−1153. (8) Yuan, J.; Guo, W.; Yang, B.; Liu, P.; Wang, Q.; Yuan, H. J. Tradit. Chin. Med. 1997, 17, 14−17. (9) Matsuura, H.; Kasai, R.; Tanaka, O.; Saruwatari, Y. I.; Fuwa, T.; Zhou, J. Chem. Pharm. Bull. 1983, 31, 2281−2287. (10) Li, S. H.; Chu, Y. Acta Pharmacol. Sin. 1999, 20, 551−554. (11) Sengupta, S.; Toh, S. A.; Sellers, L. A.; Skepper, J. N.; Koolwijk, P.; Leung, H. W.; Yeung, H. W.; Wong, R. N. S.; Sasisekharan, R.; Fan, T. P. D. Circulation 2004, 110, 1219−1225. (12) Toh, D. F.; New, L. S.; Koh, H. L.; Chan, E. C.Y. J. Pharm. Biomed. Anal. 2010, 52, 43−50. (13) Lau, A. J.; Toh, D. F.; Chua, T. K.; Pang, Y. K.; Woo, S. O.; Koh, H. L. J. Ethnopharmacol. 2009, 125, 380−386. (14) Lau, A. J.; Seo, B. H.; Woo, S. O.; Koh, H. L. J. Chromatogr. A 2004, 1057, 141−149. (15) Lau, A. J.; Woo, S. O.; Koh, H. L. J. Chromatogr. A 2003, 1011, 77−87. K



Article

(16) Wang, D.; Liao, P. Y.; Zhu, H. T.; Chen, K. K.; Xu, M.; Zhang, Y. J.; Yang, C. R. Food Chem. 2012, 132, 1808−1813. (17) Liao, P. Y.; Wang, D.; Zhang, Y. J.; Yang, C. R. J. Agric. Food Chem. 2008, 56, 1751−1756. (18) Pei, Y.; Du, Q.; Liao, P. Y.; Chen, Z. P.; Wang, D.; Yang, C. R.; Kitazato, K.; Wang, Y. F.; Zhang, Y. J. J. Asian Nat. Prod. Res. 2011, 13, 498−504. (19) Teng, R. W.; Li, H. Z.; Wang, D. Z.; Yang, C. R. Helv. Chim. Acta 2004, 87, 1270−1278. (20) Teng, R. W.; Li, H. Z.; Chen, J. T.; Wang, D. Z.; He, Y. N.; Yang, C. R. Magn. Reson. Chem. 2002, 40, 483−488. (21) Yang, T. R.; Kasai, R.; Zhou, J.; Tanaka, O. Phytochemistry 1983, 22, 1473−1478. (22) Park, I. H.; Kim, N. Y.; Han, S. B.; Kim, J. M.; Kwon, S. W.; Kim, H. J.; Park, M. K.; Park, J. H. Arch. Pharmacal Res. 2002, 25, 428−432. (23) Teng, R. W.; Ang, C. S.; McManus, D.; Armstrong, D.; Mau, S.; Bacic, A. Helv. Chim. Acta 2004, 87, 1860−1872. (24) Gebhardt, S.; Bihler, S.; Schubert-Zsilavecz, M.; Riva, S.; Monti, D.; Falcone, L.; Danieli, B. Helv. Chim. Acta 2002, 85, 1943−1959. (25) Wu, F. F.; Sun, H.; Wei, W. F.; Han, Y.; Wang, P.; Dong, T. W.; Yan, L. G.; Wang, X. J. J. Sep. Sci. 2011, 34, 3194−3199. (26) Li, H. Z.; Zhang, Y. J.; Yang, C. R. Nat. Prod. Res. Dev. 2006, 18, 549−554. (27) Cao, J. Q.; Fu, P.; Zhao, Y. Q. Chin. Tradit. Herbal Drugs 2013, 44, 137−140. (28) Wei, J. X.; Wang, L. A.; Du, H.; Li, R. Acta Pharmacol. Sin. 1985, 20, 288−293. (29) Di Bari, L. D.; Pescitelli, G.; Pratelli, C.; Pini, D.; Salvadori, P. J. Org. Chem. 2001, 66, 4819−4825. (30) Frelek, J.; Ikekawa, N.; Takatsuto, S.; Snatzke, G. Chirality 1997, 9, 578−582. (31) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana, K. J. Org. Chem. 1999, 64, 866−876. (32) Du, L.; Zhu, T. J.; Fang, Y. C.; Gu, Q. Q.; Zhu, W. M. J. Nat. Prod. 2008, 71, 1343−1351. (33) Kuroyanagi, M.; Kawahara, N.; Sekita, S.; Satake, M.; Hayashi, T.; Takase, Y.; Masuda, K. J. Nat. Prod. 2003, 66, 1307−1312. (34) Machida, K.; Kikuchi, M. Chem. Pharm. Bull. 1999, 47, 692− 694. (35) Feng, L. B.; Pan, X. F.; Sun, Z. L. China Pharmacist 2008, 11, 1185−1187. (36) Zhang, S.; Zhao, G. Chin. J. New Drugs 2011, 20, 953−958. (37) Yang, Z. G.; Sun, H. X.; Ye, Y. P. Chem. Biodiversity 2006, 3, 187−197. (38) Zheng, W. Q.; Zhu, M. J. Zhejiang Chin. Med. Univ. 2007, 31, 557−559. (39) Greene, L. A.; Tischler, A. S. Proc. Natl. Acad. Sci. U. S. A. 1976, 73, 2424−2428. (40) Lv, J. J.; Xu, M.; Wang, D.; Zhu, H. T.; Yang, C. R.; Wang, Y. F.; Li, Y.; Zhang, Y. J. J. Nat. Prod. 2013, 76, 926−932.

L


Triterpenoids with Promoting Effects on the Differentiation of PC12

Recommend Documents