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Iridoids and sesquiterpenoids from the roots of Valeriana jatamansi Jones
Fitoterapia 102 (2015) 27–34
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Iridoids and sesquiterpenoids from the roots of Valeriana jatamansi Jones Fa-Wu Dong a,b, Liu Yang a, Zhi-Kun Wu c, Wei-Gao a, Chen-Ting Zi a, Dan Yang a, Huai-Rong Luo a, Jun Zhou a, Jiang-Miao Hu a,⁎ a 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 b University of the Chinese Academy of Sciences, Beijing 100039, People's Republic of China c Lijiang Forest Ecosystem Research Station, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People's Republic of China
a r t i c l e
i n f o
Article history: Received 15 December 2014 Accepted in revised form 28 January 2015 Available online 7 February 2015 Keywords: Valeriana jatamansi Iridoid Sesquiterpenoid AChE
a b s t r a c t Three new iridoids, jatamanvaltrates R–S (1–2) and jatamanin Q (3), as well as three new sesquiterpenoids, valeriananoids D–E (4, 5) and clovane-2β-isovaleroxy-9α-ol (6), together with nine known compounds were isolated from the roots of Valeriana jatamansi Jones. Compound 2 was the first reported iridoid with fatty acid esters in the Valerianaceae family. The structures of new compounds were established on the basis of extensive spectroscopic analysis. Moreover, all the isolates were evaluated for inhibitory activity on acetylcholinesterase (AChE). © 2015 Elsevier B.V. All rights reserved.
1. Introduction Valeriana jatamansi Jones (Valerianaceae family), also named as Valeriana wallichii DC., an annual herb of the 200 species in genus Valeriana, is mainly distributed in China and mainland India [1]. The roots and rhizomes of the plant were used as a traditional Chinese medicine (TCM) for the treatment of various diseases, including insomnia, epilepsy, insanity, and nervous disorders [2–5]. Previous phytochemical investigations on this specie resulted in the isolation of sesquiterpenoids [5,6], essentials oils [5,7,8], flavone glycosides [9], valepotriates and acylated iridoids [10–19]. In the course of our continual search for bioactive natural products on nervous systems from genus Valeriana [20–23], the roots of V. jatamansi were investigated and got six new compounds, including three iridoids jatamanvaltrates R–S (1–2) and jatamanin Q (3), three sesquiterpenoids, valeriananoids D–E (4, 5) and clovane-2βisovaleroxy-9α-ol (6), together with nine known compounds.
⁎ Corresponding author. Tel.: +86 871 6522 3264; fax: +86 871 6522 3261. E-mail address: [email protected] (J.-M. Hu).
http://dx.doi.org/10.1016/j.fitote.2015.01.021 0367-326X/© 2015 Elsevier B.V. All rights reserved.
Compound 2 was the first reported iridoid with fatty acid esters in the Valerianaceae family. In view of traditional usage of V. jatamansi at nervous disorders, and it is reported that the chloroform and ethylacetate fractions, the essential oils of V. jatamansi and the related species of genus Valeriana exhibited activity against acetylcholinesterase (AChE) [22–27], which involves in learning, memory, Alzheimer's, and Parkinson's disease [28–31]. Thus, all the isolates were evaluated to know whether these compounds were the effective constituents on acetylcholinesterase (AChE) inhibitory activity of this plant (Fig. 1). Herein, we describe the isolation, structural elucidation of these new compounds and the AChE inhibitory activities of the 15 isolates. 2. Experimental 2.1. General experimental procedures Optical rotations were recorded on a JASCO model 1020 polarimeter (Horiba, Tokyo, Japan). ESIMS and HRESIMS were
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F.-W. Dong et al. / Fitoterapia 102 (2015) 27–34
Fig. 1. Structures of compounds 1–6.
run on an API QSTAR time-of-flight (AB-MDS Sciex, Concord, ON, Canada) or a Shimadzu LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan) or an Agilent G6230 TOF MS (Agilent Technologies, Palo Alto, USA), while EIMS and HREIMS were carried out on an Waters AutoSpec Premier p776 spectrometer (Waters, Millford, MA, USA). IR (KBr) spectra were obtained using a Tenor 27 spectrophotometer (Bruker Optics GmbH, Ettlingen, Germany). UV spectra were measured on a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Kyoto, Japan). 1D and 2D NMR spectra were performed on a Bruker AM-400, DRX-500, AV 600 and an Avance III 600 spectrometer (Bruker, Bremerhaven, Germany) with TMS as the internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. MPLC was performed on a Dr-Flash-S MPLC system (Lisui, Suzhou, China). Silica gel (200–300 mesh) for column chromatography (CC) and TLC was obtained from Qindao Marine Chemical Factory, Qingdao, China. Sephadex LH-20 was purchased from Amersham Biosciences, Sweden; RP-C18 gel (40–63 μm, Merck, Darmstadt, Germany), MCI gel (75–150 μm, Mitsubishi Chemical Corporation, Japan). Fractions were monitored by TLC, and spots were visualized by UV light and sprayed with 5% sulfuric acid in EtOH, followed by heating. 2.2. Plant material The roots of V. jatamansi were purchased in July 2012 from Yunnan Hongxiang Yixintang Pharmaceutical Co., Ltd. and identified by Dr. Zhi-Kun Wu. A voucher specimen (KUN No. 0864803) has been deposited at Herbarium of Kunming Institute of Botany, the Chinese Academy of Sciences, Kunming, China. 2.3. Extraction and isolation The roots of V. jatamansi (32.5 kg) were powdered and extracted with 95% EtOH (60 L) at room temperature (28 h) for 3 times. The combined extracts were concentrated under reduced pressure to afford a crude residue (3.65 kg) and then suspended in water (4.0 L) and partitioned with EtOAc (4.0 L × 5).
The EtOAc layer (1.72 kg) was subjected to silica gel column chromatography (CC, 200–300 mesh), then eluted with a gradient of petroleum ether/acetone (1:0–0:1) to afford 7 fractions (Fr.1–Fr.7). Fraction 1 (192.4 g) was passed through silica gel CC (petroleum ether/acetone gradient, 1:0–10:1) to give 7 subfractions (Fr.1.1–Fr.1.7). Fr.1.2 (49.5 g) was separated by repeated silica gel CC (petroleum ether/acetone, 1:0–50:1), then passed through Sephadex LH-20 column (CHCl3/MeOH, 1:1) to yield compound 5 (17.0 mg). Fr.1.5 (4.2 g) was chromatographed on Sephadex LH-20 column and eluted with MeOH to obtain 7 subfractions (Fr.1.5.1 Fr.1.5.7). Fr.1.5.4 (312 mg) was subjected to Sephadex LH-20 column (MeOH), then followed by RP-18 (MeOH/H2O gradient, 50:50–90:10) to give compounds 6 (14.0 mg), 7 (14.0 mg) and 13 (94.0 mg). Fraction 3 (55.5 g) was subjected to MCI gel (MeOH/H2O, 70:30–95:5) to give 5 subfractions. Fr.3.2 (5.45 g) was further separated by MPLC (MeOH/H2O gradient, 40:60–60:40), followed by a silica gel (petroleum ether/acetone, 20:1–15:1) and then passed through Sephadex LH-20 column (CHCl3/MeOH, 1:1) to yield compound 1 (34.0 mg). Fraction 4 (100.3 g) was chromatographed on silica gel CC (200–300 mesh) (petroleum ether/acetone, 50:1–1:1) to yield 4 fractions (Fr.4.1–Fr.4.4). Fraction 4.2 (28.3 g) was applied to a MCI gel (MeOH/H2O gradient, 60:40–85:15) to afford 3 subfractions (Fr.4.2.1– Fr.4.2.3). Fr.4.2.2 (9.0 g) by repeated silica gel CC (petroleum ether/acetone, 20:1–5:1), then followed by RP-18 (MeOH/H2O gradient, 50:50–85:15) and Sephadex LH-20 (MeOH) to yield compounds 2 (5 mg) and 8 (53 mg). Fraction 6 (193.7 g) was separated over silica gel CC, eluted with petroleum ether/ acetone (20:1–0:1), to afford 8 fractions ((Fr.6.1–Fr.6.8). Fr.6.4 (16.3 g) was subjected to MCI gel (MeOH/H2O gradient, 30:70–100:0) to obtain 7 subfractions (Fr.6.4.1– Fr.6.4.7). Fr.6.4.2 (4.16 g) was purified by Sephadex LH20 (MeOH) to give 12 (96 mg). Fr.6.5 (70.4 g) was chromatographed on repeated silica gel CC (petroleum ether/acetone, 10:1–0:1) to afford 8 subfractions (Fr.6.5.1– Fr.6.5.8). Fr.6.5.4 (33.6 g) was successively separated by MPLC (MeOH/H2O gradient, 20:80–85:15) and repeated silica gel CC (petroleum ether/EtOAc, 5:1–1:1), followed by RP-18 (MeOH/H2O gradient, 30:70–75:25) and purified by
F.-W. Dong et al. / Fitoterapia 102 (2015) 27–34
Sephadex LH-20 (MeOH) to yield compounds 4 (60.4 mg), 3 (12.0 mg), 9 (19.0 mg), 10 (33.0 mg), 11 (7.0 mg) and 14 (20.0 mg) and 15 (322.0 mg). 2.4. Spectroscopic data Jatamanvaltrate R (1): colorless oil, [а] D23 = + 5.58 (c 0.42, MeOH). UV (MeOH) λmax (log εmax): 201 (1.40) nm; IR (KBr) νmax (cm− 1): 3448, 2960, 1740, 1626, 1450, 1371, 1294, 1243, 1187, 1119, 1040, and 961 cm− 1; 1H NMR and 13C NMR data, see Table 1; positive ESIMS m/z 407 [M + Na]+; and HRESIMS m/z 407.1680 [M + Na]+ (calcd for C19 H28 O8 Na, 407.1682). Jatamanvaltrate S (2): colorless oil, [а] D23 = + 12.67 (c 0.25, MeOH). UV (MeOH) λmax (log εmax): 201 (1.58) nm; IR (KBr) νmax (cm−1): 3432, 2925, 1743, 1628, 1465, 1372, 1293, 1242, 1165, 1120, 1038, and 935 cm−1; 1H NMR and 13C NMR data, see Table 1; positive ESIMS m/z 617 [M+Na]+; and HRESIMS m/z 617.3660 [M+Na]+ (calcd for C33 H54 O9 Na, 617.3666). Jatamanin Q (3): colorless oil, [а] D23 = + 8.37 (c 0.14, MeOH). UV (MeOH) λmax (log εmax): 201 (1.43) nm; IR (KBr) νmax (cm−1): 3432, 2927, 2876, 1631, 1454, 1383, 1201, 1182, 1142, 1119, 1091, 1072, 1043, 997, 972, and 951 cm−1; 1H NMR (CD3OD, 600 MHz): δ 4.72 (1H, d, J = 2.4 Hz, H-1), 4.10 (1H, dd, J = 3.0, 1.2 Hz, H-3a), 3.85 (1H, dd, J = 3.0, 1.2 Hz, H-3b), 1.74 (1H, br s, H-4), 2.62 (1H, ddd, J = 10.8, 10.2, 1.8 Hz, H-5), 1.88 (1H, m, H-6a), 1.90 (1H, m, H-6b), 4.10 (1H, m, H-7), 1.94 (1H, m, H-8), 2.07 (1H, ddd, J = 12.0, 10.2, 2.4 Hz, H-9), 1.03 (3H, d, J = 7.2 Hz, H-10), 4.11 (1H, dd, J = 6.0, 3.0 Hz, H-11a),
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3.86 (1H, dd, J = 6.0, 3.0 Hz, H-11b); 13C NMR (CD3OD, 150 MHz): δ 93.2(CH, C-1), 70.2 (CH2, C-3), 31.5 (CH, C-4), 37.5 (CH, C-5), 37.2 (CH2, C-6), 77.5 (CH, C-7), 42.6 (CH, C-8), 51.0 (CH, C-9), 13.5(CH3, C-10), 64.2 (CH2, C-11); positive ESIMS m/z 203 [M+H]+; and HREIMS m/z 202.1210 [M]+ (calcd for C10 H18 O4, 202.1205). Valeriananoid D (4): colorless oil, ½a 23 D ¼ −61:20 (c 0.31, MeOH). UV (MeOH) λmax (log εmax): 204 (2.20), 217 (2.39), 234 (2.10) and 325 (3.51) nm; IR (KBr) νmax (cm−1): 3440, 2950, 2928, 1691, 1631, 1605, 1515, 1464, 1429, 1382, 1270, 1179, 1162, 1124, 1032, and 982 cm−1; 1H NMR and 13C NMR data, see Table 2; positive ESIMS m/z 437 [M+Na]+; and HRESIMS m/z 437.2294 [M+Na]+ (calcd for C25 H34O5 Na, 437.2304). Valeriananoid E (5): colorless oil, ½a 23 D ¼ −39:40 (c 0.87, MeOH). UV (MeOH) λmax (log εmax): 201 (1.34), 219 (1.10) nm; IR (KBr) νmax (cm−1): 3443, 2926, 2855, 1731, 1631, 1463, 1375, 1257, 1182, 1153, 1111, 1090, 1013, and 999 cm−1; 1 H NMR and 13C NMR data, see Table 2; positive ESIMS m/z 525 [M+Na]+; and HRESIMS m/z 525.4265 [M+Na]+ (calcd for C33 H58 O3 Na, 525.4278). Clovane-2β-isovaleroxy-9α-ol (6): colorless crystal (Me2CO); mp 203–205 °C; ½a 23 D ¼ −10:18 (c 0.24, MeOH). UV (MeOH) λmax (log εmax): 201 (1.53) nm; IR (KBr) νmax (cm−1): 3439, 2955, 2928, 2867, 1731, 1630, 1464, 1383, 1369, 1293, 1237, 1191, 1168, 1120, 1098, 1056, and 1032 cm−1; 1H NMR and 13C NMR data, see Table 2; positive ESIMS m/z 345 [M+Na]+; and HRESIMS m/z 345.2403 [M+Na]+ (calcd for C20 H34 O3 Na, 345.2406).
Table 1 1 H NMR (400 MHz) and 13C NMR (100 MHz) data of compounds 1–2 (δ in ppm, J in Hz). Position
1 3 4 5 6 7 8 9 10 11 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴
1a,b
2a
δH
δC
δH
δC
5.28, d (3.1) 5.24, s
96.9, d 95.2, d 154.3, s 78.2, s 48.1, t
6.41, d (3.2) 5.29, s
91.3, d 95.8, d 152,7, s 77.9, s 48.6, t
2.41, dd (14.3, 7.3) 1.95, dd (14.4, 2.8) 4.84, dd (7.2, 2.7) 2.56, d (2.9) 4.41, d (11.7) 4.22, d (11.7) 5.26, s 5.08, s 3.76, 3.49, m 1.13, t (7.0)
2.17, m 2.04, m 0.93, d (6.6) 0.93, d (6.6) 2.01, s
75.6, d 83.5, s 46.9, d 65.3, t 107.8, t 64.5, t 15.4, q
173.9, s 44.0, t 26.7, d 22.8, q 23.2, q 173.5, s 21.0, q
The 1H NMR data of the palmitoyl at C-10 of 2: δH 1.28 (20H, m, H-6″–15″), 0.89 (3H, t, J = 6.0 Hz, H-16″). The 13C NMR data of the palmitoyl at C-10 of 2: δC 30.1–30.7 (t, C-6″–13″), 33.0 (t, C-14″), 23.7 (t, C-15″). 14.4 (q, C-16″). a Recorded in methanol-d4. b Recorded in pyridine-d5.
2.47, dd (14.8, 7.2) 1.99, dd (14.8, 2.8) 4.81, dd (7.1, 2.8) 2.69, d (2.0) 4.46, d (11.6) 4.24, d (11.6) 5.32, s 5.15, s 2.22, m 2.06, m 0.93, d (6.4) 0.93, d (6.4) 2.32, t (3.2) 1.59, m 1.28, m 1.28, m 2.03, s
75.5, d 84.0, s 46.2, d 65.1, t 108.8, t 173.3, s 43.9, t 26.6, d 22.6, q 22.7, q 174.7, s 35.0, t 25.9, t 30.1, t 30.1, t 171.3, s 21.3, q
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Table 2 1 H NMR (400 MHz) and 13C NMR (100 MHz) data of compounds 4–6 (δ in ppm, J in Hz). 4a Position
δH
1
2.08, dd (14.0, 7.6) 1.61, dd (14.0, 7.6) 4.83, d (8.4) 1.46, t (8.0)
2 3 4 5 6 7 8 9 10 11
1.72, dd (11.2, 4.4) 1.60, dd (11.2, 4.4) 1.48, m 1.35, m 1.94, m 1.40, m
5b
6a
δC
δH
δC 35.9, t
76.1, d 44.8, d
1.91, dd (14.0, 7.6) 1.63, dd (14.0, 7.6) 4.77, dd (9.6, 7.6) 1.44, t (6.8)
40.0, s 76.3, s 32.9, t 29.5, t 28.9, d 44.0, d 41.4, s 24.6, t
12
1.56, m 1.39, m 0.80 d (6.7)
13 14 15
1.18, s 1.09, s 0.89, s
1.72, dd (14.0, 6.4) 1.48, dd (14.0, 6.4) 1.52, m 1.34, m 1.94, m 1.41, m
74.2, d 43.3, d 39.0, s 75.0, s 32.6, t 28.1, t 27.6, d 42.3, Cd 40.0, s 23.4, t
19.2, q
1.54, m 1.37, m 0.79 d (6.8)
25.5, q 29.1, q 20.9, q
1.14, s 1.09, s 0.89, s
25.0, q 27.8, q 19.9, q
18.7, q
δH
δC 45.8, s
4.80, dd (8.3, 5.8) 1.76, dd (12.4, 5.6) 1.53, dd (12.4, 5.6) 1.52, m 1.47, m 1.37, m 1.36, m 1.13, m 3.24, br s 1.99, m 1.58, m 1.65, m 1.16, m 1.48, m 0.97, m 0.91, s 1.06, s 0.87, s
83.5, d 45.4, t 39.1, s 51.6, d 21.8, t 34.3, t 35.6, s 75.4, d 27.3, t 28.6, t 36.5, t 25.8, q 31.9, q 29.0, q
The signals of the substituent at C-2 in 1H NMR. For 4 (the feruloyl group): δ 6.28 (1H, d, J = 15.9 Hz, H-2′), 7.54 (1H, d, J = 15.9 Hz, H-3′), 7.15 (1H, d, J = 1.7 Hz, H-5′), 6.79 (1H, d, J = 8.1 Hz, H-6′), 7.02 (1H, dd, J = 8.1, 1.7 Hz, H-9′), 3.86 (3H, s, H-OMe-6′). For 5 (the oleoyl group): δ 2.23 (2H, t, J = 7.6 Hz, H-2′), 1.58, 1.06 (2H, m, H-3′), 1.24–1.29 (20H, m, H-4′–7′, and 12′–17′), 1.97 (4H, m, H-8′, 11′), 5.29 (2H, m, H-9′, 10′), 0.87 (3H, t, J = 6.8 Hz, H-18′). For 6 (the isovaleroxy group): δ 2.18 (2H, d, J = 7.2 Hz, H-2′), 2.01 (1H, m, H-3′), 0.92 (3H, d, J = 7.2 Hz, H-4′), 0.95 (3H, d, J = 6.4 Hz, H-5′). In 13C NMR. For 4 (the feruloyl group): δ 168.9 (C, C-1′), 116.2 (CH, C-2′), 146.4 (CH, C-3′), 127.7 (C, C-4′), 111.5 (CH, C-5′), 149.3 (C, C-6′), 150 (C, C-7′), 116.4 (CH, C-8′), 124.1 (CH, C-9′), 56.4 (CH3, C-OMe-6′). For 5 (the oleoyl group): δ 173.5 (C, C-1′), 34.9 (CH2, C-2′), 24.9 (CH2, C-3′), 28.4–29.6 (CH2, C-4′–7′, and 12′–16′), 31.9 (CH2, C-8′, 11′), 129.7–129.9 (CH, C-9′, 10′), 22.6 (CH2, C-17′), 14.1 (CH3, C-18′). For 6 (the isovaleroxy group): δ 174.7 (C, C-1′), 44.6 (CH2, C-2′), 26.9 (CH, C-3′), 22.7 (CH3, C-4′), 22.8 (CH3, C-5′). a Recorded in methanol-d4. b Recorded in CDCl3.
2.5. Acetylcholinesterase inhibitory activity Acetylcholinesterase (AChE) inhibitory activity of the isolated compounds was assayed by the spectrophotometric method developed by Ellman et al. [32]. AchE (Sigma Chemical) was used as substrate in the assay. Compounds were dissolved in DMSO. The reaction mixture contained 200 μL of phosphate buffer (pH 8.0), 50 μM of test compound soln, and 40 μL of AchE soln (0.02 U/mL). The mixture was incubated for 20 min (37 °C). Then, the reaction was initiated by the addition of 40 μL of DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM), respectively. The hydrolysis of acetylthiocholine was monitored at 405 nm every 30 s for 1 h. Tacrine was used as positive control with final concentration of 0.333 μM. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E − S) / E × 100 (E is the activity of the enzyme without test compound and S is the activity of enzyme with test compound).
3. Results and discussion Compound 1 was obtained as colorless oil with the molecular formula C19H28O8 as assigned by an m/z 407.1680 [M+Na]+ (calcd for C19H28O8 Na, 407.1682) from the positive HRESIMS experiment The IR (KBr, v) absorption bands at
3448 cm−1, 1740 cm−1 and 1626 cm−1, suggesting the presence of hydroxy, ester C_O and olefinic groups. The 1H NMR spectroscopic data (Table 1) displayed four methyls [at δH 2.01 (3H, s, H-2‴), 1.13 (3H, t, J = 7.0 Hz, H-2′) and 0.93 (6H, d, J = 6.6 Hz, H-4″, 5″)], three oxygenated methines [at δH 5.28 (1H, d, J = 3.1 Hz, H-1), 5.24 (1H, s, H-3) and 4.84 (1H, dd, J = 7.2, 2.7 Hz, H-7)], two characteristic isolated methylene groups [at δH 4.41, 4.22 (each 1H, d, J = 11.7 Hz, H-10) and 3.76, 3.49 (each 1H, m, H-1′)], and a terminal olefinic bond [at δH 5.26 and 5.08 (each 1H, s, H-11)]. The 13C NMR in combination with DEPT data (Table 1) exhibited nineteen carbon signals, including four methyls, five methylenes (two O-bearing), and five methines (three oxygenated), as well as five quaternary (two ester C_O groups, two O-bearing and one sp2) carbons. A detailed comparison of the NMR spectroscopic data (Table 1) with valeriandoid C [15] indicated that the two compounds have a similar structure, with an oxo-bridge between C-3 and C-8. The only difference between 1 and valeriandoid C was the hydroxy group at C-1 in valeriandoid C was replaced by an ethoxy group in 1. According to the HMBC spectrum, the correlations (Fig. 2) from H-1 (δH 5.28, d, J = 3.1 Hz) and H-2′ (δH 1.13, t, J = 7.0 Hz) to C-1′ (δC 64.5), from H-10a (δH 4.41, d, J = 11.7 Hz), H-10b (δH 4.22, d, J = 11.7 Hz), H-2″ (δH 2.17, m) and H-3″ (δH 2.04, m) to C-1″ (δC 173.9), and from H-7 (δH 4.84, dd, J = 7.2, 2.7 Hz) and H-2‴ (δH 2.01, s) to C-1‴ (δC 171.2), demonstrated that the ethoxyl, acetoxyl and
F.-W. Dong et al. / Fitoterapia 102 (2015) 27–34
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Fig. 2. Selected COSY, HMBC and ROESY correlations of compound 1.
isovaleroxyl groups were assigned to and located at C-1, C-7 and C-10, respectively. The relative configuration of 1 was established by a combination of the ROESY experiment (Fig. 2) and a comparison with the spectroscopic data of those reported ones [14–16,18,19], as well as on the basis of the biogenetic ground and the molecular modeling with a rigid epoxy-bridge skeleton. Generally, naturally occurring iridoids display an a-orientation for H-1 and β-orientation for H-9 [10–19]. According to the molecular model, the oxo-bridge from C-3 to C-8 could only be α-oriented, and the OH-5 and H-9 could only be β-oriented [14–16,18,19]. Moreover, the ROESY cross peaks of H-7/H-6α, H-9/H-6β and H-9/H-10, but not of H-7/H9 and H-10 indicated α-orientation of H-7 which was the same as valeriandoid C and 1, 5-dihydroxy-3, 8-epoxyvalechlorine [15,19]. Therefore, the structure of 1 was elucidated and named as jatamanvaltrate R. Compound 2 possessed the molecular formula C33 H54 O9, as determined by positive-ion HRESIMS at m/z 617.3660 [M+Na]+ (calcd 617.3666). The 1H, 13C NMR and DEPT data of 2 (Table 1) were similar to those of 1, suggesting that they were structural
analogues, except for the presence of a palmitoyl group [δC 174.7 (s, C-1″), 35.0 (t, C-2″), 25.9 (t, C-3″), 30.1–30.7 (t, C-4″–13″), 33.0 (t, C-14″), 23.7 (t, C-15″), 14.4 (q, C-16″)] in 2 rather than an ethoxy group [δC 64.5 (t, C-1′), 15.4 (q, C-2′)] in 1. The HMBC correlations of H-1 (δH 6.41, d, J = 3.2 Hz), H-2′ (δH 2.22, m) and H-3′ (δH 2.06, m) with C-1′ (δC 173.3), and of H-10a (δH 4.46, d, J = 11.6 Hz), H-10b (δH 4.26, d, J = 11.6 Hz), H-2″ (δH 2.32, t, J = 3.2 Hz) and H-3″ (δH 1.59, m) with C-1″ (δC 174.7) indicated the isovaleroxy group and palmitoyl were located at C-1and C-10, respectively. Comparison of the spectroscopic data and key ROESY correlations with those of reported iridoids and 1 [14–16,18,19] the relative configurations at C-1, C-3, C-5, C-7, C-8 and C-9 in compound 2 were demonstrated to be identical to 1. Consequently, 2 was defined and named as jatamanvaltrate S. The molecular formula of 3, C10H18O4, was established by positive-ion HREIMS at m/z 202.1210 [M]+, corresponding to two indices of hydrogen deficiency. Its 1H and 13C NMR (DEPT) spectrum displayed one methyl [δH 1.03 (3H, d, J = 7.2 Hz, H-10); δC 13.5 (C-10)], three methylenes [two oxygenated at δH 4.10 3.85 (each 1H, dd, J = 3.0, 1.2 Hz, H-3) and 4.11, 3.86
Fig. 3. Selected COSY, HMBC and ROESY correlations of compound 3.
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Fig. 4. Selected COSY, HMBC and ROESY correlations of compound 4.
(each 1H, dd, J = 6.0, 3.0 Hz, H-11); δC 70.2 (C-3) and 62.2 (C-11)], and six methines [two oxymethines at δH 4.72 (1H, d, J = 2.4 Hz, H-1) and 4.10 (1H, m, H-7); δC 93.2 (C-1) and 77.5 (C-7)], which were closely related to those of (1R, 4R, 5R, 7S, 8R, 9R)-1,7-dihydroxy-10,11-dimethylperhydrocyclopenta[c]pyran [33]. Except for the methyl at C-4 in (1R, 4R, 5R, 7S, 8R, 9R)1,7-dihydroxy-10,11-dimethylperhydrocyclopenta[c]pyran was replaced by a hydroxymethyl in 3, which was confirmed by the HBMC correlations from H-11 to C-3, C-4, and C-5. The HMBC correlations (Fig. 4) of δH 1.03 (H-10) with δC 42.6 (C-8), 51.0 (C-9) and 77.5 (C-7); of δH 4.11, 3.86 (H-11) with 31.5 (C-4), 37.5 (C-5) and 70.2 (C-3); as well as the 1H-1H COSY cross-peaks from 1.03 (H-10) to 1.94 (1H, m, H-8), and from 4.11, 3.86 (H-11) to 1.74 (1H, br s, H-4) readily confirmed the assignments. The relative configuration of 3 was determined by a combination of the ROESY spectrum (Fig. 3) and a careful comparison of spectroscopic data of 3 with those reported iridoids [10–19,33]. For those known naturally occurring iridoids, H-9 and H-5 possessed β-orientations, while H-1 was α-oriented. The ROESY cross peaks of H-4/H-6α, H-6α/ H-1, H-5/H-11, H-9 and H-6β, H-9/H-10 and H-5, and H-7/ H-6α and H-8, but not of H-7/H-10 and H-9 indicated that H-10 and H-11 were β-oriented, while H-4, H-7 and H-8 were α-oriented. Hence, the structure of 3 was established as (1R, 4S, 5R, 7S, 8R, 9R)-1,7-dihydroxy-11-hydroxymethyl-10methylperhydrocyclopenta[c]pyran and named as jatamanin Q. Compound 4 exhibited a deprotonated molecular ion [M + Na]+ at m/z 437.2294 (calcd for 437.2304) from HRESIMS, requiring 9° of unsaturation.. The IR spectrum of 4 displayed the presence of hydroxy (3440 cm− 1), ester C_O group (1691 cm− 1) and carbon–carbon double bond (1631 cm− 1). The 1H NMR spectroscopic data (Table 2) of 4 displayed a double bond [δH 6.28 (1H, d, J = 15.9 Hz, H-2′)
and 7.54 (1H, d, J = 15.9 Hz, H-3′)], three aromatic protons [δH 7.15 (1H, d, J = 1.7 Hz, H-5′), 6.79 (1H, d, J = 8.1 Hz, H-8′) and 7.02 (1H, dd, J = 8.1, 1.7 Hz, H-9′)], an oxygenated methine [δH 4.80 (1H, dd, J = 7.4, 6.1 Hz, H-2)], four methyl groups [δH 0.80 (3H, d, J = 6.7 Hz, H-12), 1.18 (3H, s, H-13), 1.09 (3H, s, H-14) and 0.89 (3H, s, H-15)], and a methoxyl [δH 3.86 (3H, s, H-OMe-6′)]. The 13C NMR and DEPT spectroscopic data (Table 2), in combination with HSQC spectra, exhibited twentyfive carbon signals, including an α, β-unsaturated ester carbonyl carbon [δC 168.9 (s, C-1′)], a phenyl ring (including three protonated carbons, two oxygenated carbons and a quaternary carbon), two olefinic methines [δC 116.2 (d, C-2′) and 146.4 (d, C-3′)], a methoxy group, and a sesquiterpenoid skeleton [four methyls, four methylenes, four methines (an oxymethine), as well as three quaternary carbons (one oxygenated)]. A careful comparison of the NMR data (Table 2) of 4 with those of the known compound valeriananoid C [5] indicated that they were structural analogues. The only difference was the AcO group at C-2 in valeriananoid C which was replaced by a feruloyl group in 4. A detailed analysis of HSQC, HMBC and 1H–1H COSY spectra supported the assignment of all protons and carbon signals. The position of the feruloyl group and methoxy group at C-2 and C-6′, respectively, was confirmed based on the HMBC correlations (Fig. 5) from H-2 (δH 4.80, dd, J = 7.4, 6.1 Hz), H-2′ (δH 6.28, d, J = 15.9 Hz) and H-3′ (δH 7.54, d, J = 15.9 Hz) to C-1′ (δC 168.9), and from OMe (δH 3.86, s) to C-6′ (δC 149.3). The relative configuration of 4 was established by the ROESY experiment (Fig. 5) and a detailed comparison of spectroscopic data of 4 with valeriananoids B and C, for which the absolute configurations were confirmed by X-ray crystallographic analysis [5]. The α-orientations of H-2, H-3, H-8, H-14 and H15 in 4 were established by the correlations of H-15/H-1α and H-8, H-2/H-1α and H-3, and H-3/H-14. The β-orientations of H-11, H-12 and H-13 were demonstrated by the cross-peaks
13
7 15
H HO
6
5
8 12 9
1 10
O
14 4
2
11
O Fig. 5. Selected COSY, HMBC and ROESY correlations of compound 6.
F.-W. Dong et al. / Fitoterapia 102 (2015) 27–34
of H-13/H-1β and H-12/H-11. According to a careful comparison of spectroscopic data, the configurations of 4 were consistent with those in valeriananoid B–C [5]. Therefore, the structure of 4 was elucidated and named as valeriananoid D. Compound 5 was assigned to be C33 H58 O3 on the basis of positive-ion HRESIMS data (m/z 525.4265 [M+Na]+, calcd for 525.4278). Analysis of the 1H, 13C NMR and DEPT data of 5 (Table 2) revealed that the structure of 5 was similar to those of 4, except for the presence of an oleoyl group in 5 [δC 173.5 (C-1′), 34.9 (C-2′), 24.9 (C-3′), 28.–29.6 (C-4′–7′, and 12′–16′), 31.9 (C-8′, 11′), 129.9 (C-9′), 129.7 (C-10′), 22.6 (C-17′) and 14.1 (C-18′)] rather than a feruloyl moiety in 4. The HMBC correlations (Fig. 2) of H-2 (δH 4.77, dd, J = 9.6, 7.6 Hz), H-2′ (δH 2.23, t, J = 7.6 Hz) and H-3′ (δH 1.58, m) with C-1′ (δC 173.5) indicated that the oleoyl group was located at C-2. The relative configuration of 5 was the same as that of 4 and valeriananoid C as was deduced by the ROESY spectrum and detailed comparison of the 1H NMR coupling constants of 4 with valeriananoid C [5]. Consequently, 5 was defined and named as valeriananoid E. Compound 6 had the molecular formula C20 H34 O10, which was determined by [M+Na]+ ion at m/z 345.2403 in the HRESIMS. Its IR spectrum exhibited the absorptions at 3439 and 1731 cm−1, suggesting the presence of OH group and ester carbonyl. The 1H NMR data of 7 (Table 2) disclosed two oxygenated methines [δH 4.80 (1H, dd, J = 8.3, 5.8 Hz), 3.24 (1H, br s)], a methylene [δH 2.18 (2H, d, J = 7.2 Hz)], and five methyls [δH 1.06 (3H, br s), 0.95 (3H, d, J = 6.4 Hz), 0.92 (3H, d, J = 7.2 Hz), 0.91 (3H, br s) and 0.87 (3H, br s)] groups. Its 13C NMR and DEPT spectra (Table 2) displayed signals corresponding to five methyls, seven methylenes, four methines (two oxygenated ones), and four quaternary carbons (an ester carbonyl group). The aforementioned information indicated that 6 shared the same skeleton as clovanediol [34], except that the hydroxy group at C-2 in clovanediol was replaced by an isovaleroxy moiety in compound 6. The substituent patterns of 6 were further confirmed by a series of the 2D-NMR (1H–1H COSY, HSQC and HMBC) spectra. In the HMBC spectrum (Fig. 5), the correlations from H-2 (δH 4.80, dd, J = 8.3, 5.8 Hz), H-2′ (δ 2.18, d, J = 7.2 Hz) and H-3′ (δ 2.01, m) to C-1′ (δC 175.4) revealed that the isovaleroxy group was located at C-2. The relative configuration of 6 was determined by a combination of the ROESY spectrum (Fig. 5) and a careful comparison of spectroscopic data of 6 with those reported clovanediol [34,35]. The α-orientations of H-2, H-12 and H-13 in 6 were established by the correlations of H-2/H-12 and H-13, H-12/H-13. The β-orientations of H-5, H-9, H-14 and H-15 were assigned by the cross-peaks of H-5 with H-14, of H-5 and H-15 with H-7β, of H-7β with H-9, of H-9 with H-10, H-11β and H-15, and of H-10β and H-11β with H-5. On the basis of these data, the configuration of 6 was identical with that of clovanediol [32,33]. Thus, compound 6 was assigned as clovane-2β-isovaleroxy-9α-ol. The known compounds were identified as valeriananoids A–C (7–9) [5], volvaltrate B (10) [19], valeriotetrate A (11) [19], valeriotetrate B (12) [13], 8, 11-desoidodidrovaltrate (13) [36], rupesin E (14) [14], and (3S, 4R, 5S, 7S, 8S, 9S)-3, 8-ethoxy-7hydroxy-4, 8-dimethylperhydrocyclopenta [c] pyran (15) [14]. Compounds 1–15 were evaluated for their inhibitory activity of AChE by the Ellman method [32]. At the concentration of 50 μM, all of the compounds exhibited acetylcholine
33
esterase activity inhibition ratios of less than 10%. The positive control, tacrine, showed an inhibition rate of 47.6% at 0.33 μM. Previous bioassay has reported that the chloroform and ethylacetate fractions of V. jatamansi exhibited a significant activity against acetylcholinesterase (AChE) [24], which indicated that some compounds of these layers may possess the inhibitory effects on AChE. The essential oils of V. jatamansi, which were characterized by the presence of monoterpenoids and sesquiterpenoids as a major composition [7], were evaluated and showed inhibitory activities [26]. Moreover, some sesquiterpenoids from other related species of genus Valeriana were tested on AChE in the course of our continual search for neuroprotective compounds; a few of them displayed activities against AChE. Unfortunately, all the isolates including 9 iridoids and 6 sesquiterpenoids from the EtOAc part displayed no activity. The results together with the above argument led one to assume that the iridoids may not be the active constituents on the inhibitory effects of AChE as large numbers of iridoids have been reported before. But the sesquiterpenoids potentially possessed the activity, especially the ones from the essential oils even though the isolated sesquiterpenoids showed no effects. Acknowledgments The authors are grateful to the staffs of the analytical group at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, for measuring the spectral data. This work was financially supported by Yunnan Province (No. 2013IB021). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.01.021. References [1] Mathela CS, Chanotiya CS, Sammal SS, Pant AK, Pandey S. Compositional diversity of terpenoids in the Himalayan Valeriana genera. Chem Biodivers 2005;2:1174–82. [2] Piccinelli AL, Arana S, Caceres A, Bianca RE, Sorrentino R, Rastrelli L. New lignans from the roots of Valeriana prionophylla with antioxidative and vasorelaxant activities. J Nat Prod 2004;67:1135–40. [3] Gilani AH, Khan AU, Jabeen Q, Subhan F, Ghafar R. Antispasmodic and blood pressure lowering effects of Valeriana wallichii are mediated through K+ channel activation. J Ethnopharmacol 2005;100:347–52. [4] Fernández S, Wasowski C, Paladini AC, Marder M. Sedative and sleepenhancing properties of linarin, a flavonoid-isolated from Valeriana officinalis. Pharmacol Biochem Behav 2004;77:399–404. [5] Ming DS, Yu DQ, Yang YY, He CH. The structures of three novel sesquiterpenoids from Valeriana jatamansi Jones. Tetrahedron Lett 1997; 38:5205–8. [6] Xu J, Yang B, Guo YQ, Jin DQ, Guo P, Liu CZ, et al. Neuroprotective bakkenolides from the roots of Valeriana jatamansi. Fitoterapia 2011;82: 849–53. [7] Verma RS, Verma RK, Padalia RC, Chauhan A, Singh A, Singh HP. Chemical diversity in the essential oil of Indian Valerian (Valeriana jatamansi Jones). Chem Biodivers 2011;8:1921–9. [8] Stahi E, Schütz E. The supercritical extraction on valepotriates from Valeriana officinalis L. Planta Med 1980;40:262–70. [9] Tang YP, Liu X, Yu B. Two new flavone glycosides from Valeriana jatamansi. J Asian Nat Prod Res 2003;5:257–61. [10] Yu LL, Huang R, Han CR, Lv YP, Zhao Y, Chen YG. New iridoid triesters from Valeriana jatamansi. Helv Chim Acta 2005;88:1059–62. [11] Becker H, Chavadej S. Tissue cultures of Valerianaceae. Part VII. Valepotriate production of normal and colchicine-treated cell suspension cultures of Valeriana wallichii. J Nat Prod 1985;48:17–21.
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[12] Tang YP, Liu X, Yu B. Iridoids from the rhizomes and roots of Valeriana jatamansi. J Nat Prod 2002;65:1949–52. [13] Lin S, Shen YH, Li HL, Yang XW, Chen T. Acylated iridoids with cytotoxicity from Valeriana jatamansi. J Nat Prod 2009;72:650–5. [14] Lin S, Chen T, Liu XH, Shen YH, Li HL, Shan L, et al. Iridoids and lignans from Valeriana jatamansi. J Nat Prod 2010;73:632–8. [15] Xu J, Zhao P, Guo YQ, Xie CF, Jin DQ, Ma YG, et al. Iridoids from the roots of Valeriana jatamansi and their neuroprotective effects. Fitoterapia 2011;82: 1133–6. [16] Li YD, Wu ZY, Li HM, Li HZ, Li RT. Iridoids from the roots of Valeriana jatamansi. Helv Chim Acta 2013;96:424–30. [17] Wang SJ, Qiu XQ, Zhua JY, Ma XQ, Lin B, Zheng CJ, et al. Two new iridoids from the root and rhizome of Valeriana jatamansi Jones. Helv Chim Acta 2014;97:722–6. [18] Lin S, Zhang ZX, Chen T, Ye J, Dai WX, Shan L, et al. Characterization of chlorinated valepotriates from Valeriana jatamansi. Phytochemistry 2013; 85:185–93. [19] Lin S, Shen YH, Zhang ZX, Li HL, Shan L, Liu RH, et al. Revision of the structures of 1,5-dihydroxy-3,8-epoxyvalechlorine, volvaltrate B, and valeriotetrate C from Valeriana jatamansi and V. officinalis. J Nat Prod 2010;73:1723–6. [20] Wang PC, Hu JM, Ran XH, Chen ZQ, Jiang HZ, Liu YQ, et al. Iridoids and sesquiterpenoids from the roots of Valeriana officinalis. J Nat Prod 2009;72: 1682–5. [21] Wang PC, Ran XH, Chen R, Luo HR, Liu YQ, Zhou J, et al. Germacrane-type sesquiterpenoids from the roots of Valeriana officinalis var. latifolia. J Nat Prod 2010;73:1563–7. [22] Wang PC, Ran XH, Chen R, Luo HR, Ma QY, Liu YQ, et al. Sesquiterpenoids and lignans from the roots of Valeriana officinalis L. Chem Biodivers 2011; 8:1908–13. [23] Wang PC, Ran XH, Luo HR, Ma QY, Liu YQ, Zhou J, et al. Phenolic compounds from the roots of Valeriana officinalis var. latifolia. J Braz Chem Soc 2013;24:1544–8. [24] Khuda F, Iqbal Z, Zakiullah KA, Shah Y, Khan A. Report: screening of selected medicinal plants for their enzyme inhibitory potential—a validation of their ethnopharmacological uses. Pak J Pharm Sci 2014;27: 593–6.
[25] Ozdemir A, Turkoglu V, Demir H. In vitro effect of some plant extracts on acetylcholinesterase enzyme in human erythrocytes and serum. Fresenius Environ Bull 2013;22:2510–5. [26] Park IK. Fumigant toxicity of Oriental sweetgum (Liquidambar orientalis) and valerian (Valeriana wallichii) essential oils and their components, including their acetylcholinesterase inhibitory activity, against Japanese termites (Reticulitermes speratus). Molecules 2014;19:12547–58. [27] Wang Q, Wang C, Shu Z, Chan K, Huang S, Li Y, et al. Valeriana amurensis improves amyloid-beta 1–42 induced cognitive deficit by enhancing cerebral cholinergic function and protecting the brain neurons from apoptosis in mice. J Ethnopharmacol 2014;153:318–25. [28] Goodman CB, Soliman KF. Altered brain cholinergic enzymes activity in the genetically obese rat. Experientia 1991;47:833–5. [29] Léna C, Changeux JP. Allosteric nicotinic receptors, human pathologies. J Physiol Paris 1998;92:63–74. [30] Levey AI, Edmunds SM, Koliatsos V, Wiley RG, Heilman CJ. Expression of m1–m4 muscarinic acetylcholine receptor proteins in rat hippocampus and regulation by cholinergic innervation. J Neurosci 1995;15:4077–92. [31] Heese K, Low JW, Inoue N. Nerve growth factor, neural stem cells and Alzheimer's disease. Neurosignals 2006;15:1–12. [32] Ellman GL, Courtney KD, Andres VJ, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88–95. [33] Xie S, Uesato S, Inouye H, Fujita T, Murai F, Tagawa M, et al. Absolute structure of nepetaside, a new iridoid glucoside from Nepeta cataria. Phytochemistry 1988;27:469–72. [34] Henry H, Yasuhiro T, Tohru K, Sutardjo S. Constituents of Sindora sumatrana Miq. I. isolation and NMR spectral analysis of sesquiterpenes from the dried pods. Chem Pharm Bull 1994;42:138–46. [35] Gupta AS, Sukh D. Sesquiterpenes from the oleoresin of Dipterocarpus pilosus: humulene epoxide-III, caryophyllenol-I and caryophyllenol-II. Tetrahedron 1971;27:635–44. [36] Thies PW. Composition of valepotriates. Active components of valerian. Tetrahedron 1968;24:313–47.
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Iridoids and sesquiterpenoids from the roots of Valeriana jatamansi Jones Fa-Wu Dong a,b, Liu Yang a, Zhi-Kun Wu c, Wei-Gao a, Chen-Ting Zi a, Dan Yang a, Huai-Rong Luo a, Jun Zhou a, Jiang-Miao Hu a,⁎ a 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 b University of the Chinese Academy of Sciences, Beijing 100039, People's Republic of China c Lijiang Forest Ecosystem Research Station, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People's Republic of China
a r t i c l e
i n f o
Article history: Received 15 December 2014 Accepted in revised form 28 January 2015 Available online 7 February 2015 Keywords: Valeriana jatamansi Iridoid Sesquiterpenoid AChE
a b s t r a c t Three new iridoids, jatamanvaltrates R–S (1–2) and jatamanin Q (3), as well as three new sesquiterpenoids, valeriananoids D–E (4, 5) and clovane-2β-isovaleroxy-9α-ol (6), together with nine known compounds were isolated from the roots of Valeriana jatamansi Jones. Compound 2 was the first reported iridoid with fatty acid esters in the Valerianaceae family. The structures of new compounds were established on the basis of extensive spectroscopic analysis. Moreover, all the isolates were evaluated for inhibitory activity on acetylcholinesterase (AChE). © 2015 Elsevier B.V. All rights reserved.
1. Introduction Valeriana jatamansi Jones (Valerianaceae family), also named as Valeriana wallichii DC., an annual herb of the 200 species in genus Valeriana, is mainly distributed in China and mainland India [1]. The roots and rhizomes of the plant were used as a traditional Chinese medicine (TCM) for the treatment of various diseases, including insomnia, epilepsy, insanity, and nervous disorders [2–5]. Previous phytochemical investigations on this specie resulted in the isolation of sesquiterpenoids [5,6], essentials oils [5,7,8], flavone glycosides [9], valepotriates and acylated iridoids [10–19]. In the course of our continual search for bioactive natural products on nervous systems from genus Valeriana [20–23], the roots of V. jatamansi were investigated and got six new compounds, including three iridoids jatamanvaltrates R–S (1–2) and jatamanin Q (3), three sesquiterpenoids, valeriananoids D–E (4, 5) and clovane-2βisovaleroxy-9α-ol (6), together with nine known compounds.
⁎ Corresponding author. Tel.: +86 871 6522 3264; fax: +86 871 6522 3261. E-mail address: [email protected] (J.-M. Hu).
http://dx.doi.org/10.1016/j.fitote.2015.01.021 0367-326X/© 2015 Elsevier B.V. All rights reserved.
Compound 2 was the first reported iridoid with fatty acid esters in the Valerianaceae family. In view of traditional usage of V. jatamansi at nervous disorders, and it is reported that the chloroform and ethylacetate fractions, the essential oils of V. jatamansi and the related species of genus Valeriana exhibited activity against acetylcholinesterase (AChE) [22–27], which involves in learning, memory, Alzheimer's, and Parkinson's disease [28–31]. Thus, all the isolates were evaluated to know whether these compounds were the effective constituents on acetylcholinesterase (AChE) inhibitory activity of this plant (Fig. 1). Herein, we describe the isolation, structural elucidation of these new compounds and the AChE inhibitory activities of the 15 isolates. 2. Experimental 2.1. General experimental procedures Optical rotations were recorded on a JASCO model 1020 polarimeter (Horiba, Tokyo, Japan). ESIMS and HRESIMS were
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Fig. 1. Structures of compounds 1–6.
run on an API QSTAR time-of-flight (AB-MDS Sciex, Concord, ON, Canada) or a Shimadzu LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan) or an Agilent G6230 TOF MS (Agilent Technologies, Palo Alto, USA), while EIMS and HREIMS were carried out on an Waters AutoSpec Premier p776 spectrometer (Waters, Millford, MA, USA). IR (KBr) spectra were obtained using a Tenor 27 spectrophotometer (Bruker Optics GmbH, Ettlingen, Germany). UV spectra were measured on a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Kyoto, Japan). 1D and 2D NMR spectra were performed on a Bruker AM-400, DRX-500, AV 600 and an Avance III 600 spectrometer (Bruker, Bremerhaven, Germany) with TMS as the internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. MPLC was performed on a Dr-Flash-S MPLC system (Lisui, Suzhou, China). Silica gel (200–300 mesh) for column chromatography (CC) and TLC was obtained from Qindao Marine Chemical Factory, Qingdao, China. Sephadex LH-20 was purchased from Amersham Biosciences, Sweden; RP-C18 gel (40–63 μm, Merck, Darmstadt, Germany), MCI gel (75–150 μm, Mitsubishi Chemical Corporation, Japan). Fractions were monitored by TLC, and spots were visualized by UV light and sprayed with 5% sulfuric acid in EtOH, followed by heating. 2.2. Plant material The roots of V. jatamansi were purchased in July 2012 from Yunnan Hongxiang Yixintang Pharmaceutical Co., Ltd. and identified by Dr. Zhi-Kun Wu. A voucher specimen (KUN No. 0864803) has been deposited at Herbarium of Kunming Institute of Botany, the Chinese Academy of Sciences, Kunming, China. 2.3. Extraction and isolation The roots of V. jatamansi (32.5 kg) were powdered and extracted with 95% EtOH (60 L) at room temperature (28 h) for 3 times. The combined extracts were concentrated under reduced pressure to afford a crude residue (3.65 kg) and then suspended in water (4.0 L) and partitioned with EtOAc (4.0 L × 5).
The EtOAc layer (1.72 kg) was subjected to silica gel column chromatography (CC, 200–300 mesh), then eluted with a gradient of petroleum ether/acetone (1:0–0:1) to afford 7 fractions (Fr.1–Fr.7). Fraction 1 (192.4 g) was passed through silica gel CC (petroleum ether/acetone gradient, 1:0–10:1) to give 7 subfractions (Fr.1.1–Fr.1.7). Fr.1.2 (49.5 g) was separated by repeated silica gel CC (petroleum ether/acetone, 1:0–50:1), then passed through Sephadex LH-20 column (CHCl3/MeOH, 1:1) to yield compound 5 (17.0 mg). Fr.1.5 (4.2 g) was chromatographed on Sephadex LH-20 column and eluted with MeOH to obtain 7 subfractions (Fr.1.5.1 Fr.1.5.7). Fr.1.5.4 (312 mg) was subjected to Sephadex LH-20 column (MeOH), then followed by RP-18 (MeOH/H2O gradient, 50:50–90:10) to give compounds 6 (14.0 mg), 7 (14.0 mg) and 13 (94.0 mg). Fraction 3 (55.5 g) was subjected to MCI gel (MeOH/H2O, 70:30–95:5) to give 5 subfractions. Fr.3.2 (5.45 g) was further separated by MPLC (MeOH/H2O gradient, 40:60–60:40), followed by a silica gel (petroleum ether/acetone, 20:1–15:1) and then passed through Sephadex LH-20 column (CHCl3/MeOH, 1:1) to yield compound 1 (34.0 mg). Fraction 4 (100.3 g) was chromatographed on silica gel CC (200–300 mesh) (petroleum ether/acetone, 50:1–1:1) to yield 4 fractions (Fr.4.1–Fr.4.4). Fraction 4.2 (28.3 g) was applied to a MCI gel (MeOH/H2O gradient, 60:40–85:15) to afford 3 subfractions (Fr.4.2.1– Fr.4.2.3). Fr.4.2.2 (9.0 g) by repeated silica gel CC (petroleum ether/acetone, 20:1–5:1), then followed by RP-18 (MeOH/H2O gradient, 50:50–85:15) and Sephadex LH-20 (MeOH) to yield compounds 2 (5 mg) and 8 (53 mg). Fraction 6 (193.7 g) was separated over silica gel CC, eluted with petroleum ether/ acetone (20:1–0:1), to afford 8 fractions ((Fr.6.1–Fr.6.8). Fr.6.4 (16.3 g) was subjected to MCI gel (MeOH/H2O gradient, 30:70–100:0) to obtain 7 subfractions (Fr.6.4.1– Fr.6.4.7). Fr.6.4.2 (4.16 g) was purified by Sephadex LH20 (MeOH) to give 12 (96 mg). Fr.6.5 (70.4 g) was chromatographed on repeated silica gel CC (petroleum ether/acetone, 10:1–0:1) to afford 8 subfractions (Fr.6.5.1– Fr.6.5.8). Fr.6.5.4 (33.6 g) was successively separated by MPLC (MeOH/H2O gradient, 20:80–85:15) and repeated silica gel CC (petroleum ether/EtOAc, 5:1–1:1), followed by RP-18 (MeOH/H2O gradient, 30:70–75:25) and purified by
F.-W. Dong et al. / Fitoterapia 102 (2015) 27–34
Sephadex LH-20 (MeOH) to yield compounds 4 (60.4 mg), 3 (12.0 mg), 9 (19.0 mg), 10 (33.0 mg), 11 (7.0 mg) and 14 (20.0 mg) and 15 (322.0 mg). 2.4. Spectroscopic data Jatamanvaltrate R (1): colorless oil, [а] D23 = + 5.58 (c 0.42, MeOH). UV (MeOH) λmax (log εmax): 201 (1.40) nm; IR (KBr) νmax (cm− 1): 3448, 2960, 1740, 1626, 1450, 1371, 1294, 1243, 1187, 1119, 1040, and 961 cm− 1; 1H NMR and 13C NMR data, see Table 1; positive ESIMS m/z 407 [M + Na]+; and HRESIMS m/z 407.1680 [M + Na]+ (calcd for C19 H28 O8 Na, 407.1682). Jatamanvaltrate S (2): colorless oil, [а] D23 = + 12.67 (c 0.25, MeOH). UV (MeOH) λmax (log εmax): 201 (1.58) nm; IR (KBr) νmax (cm−1): 3432, 2925, 1743, 1628, 1465, 1372, 1293, 1242, 1165, 1120, 1038, and 935 cm−1; 1H NMR and 13C NMR data, see Table 1; positive ESIMS m/z 617 [M+Na]+; and HRESIMS m/z 617.3660 [M+Na]+ (calcd for C33 H54 O9 Na, 617.3666). Jatamanin Q (3): colorless oil, [а] D23 = + 8.37 (c 0.14, MeOH). UV (MeOH) λmax (log εmax): 201 (1.43) nm; IR (KBr) νmax (cm−1): 3432, 2927, 2876, 1631, 1454, 1383, 1201, 1182, 1142, 1119, 1091, 1072, 1043, 997, 972, and 951 cm−1; 1H NMR (CD3OD, 600 MHz): δ 4.72 (1H, d, J = 2.4 Hz, H-1), 4.10 (1H, dd, J = 3.0, 1.2 Hz, H-3a), 3.85 (1H, dd, J = 3.0, 1.2 Hz, H-3b), 1.74 (1H, br s, H-4), 2.62 (1H, ddd, J = 10.8, 10.2, 1.8 Hz, H-5), 1.88 (1H, m, H-6a), 1.90 (1H, m, H-6b), 4.10 (1H, m, H-7), 1.94 (1H, m, H-8), 2.07 (1H, ddd, J = 12.0, 10.2, 2.4 Hz, H-9), 1.03 (3H, d, J = 7.2 Hz, H-10), 4.11 (1H, dd, J = 6.0, 3.0 Hz, H-11a),
29
3.86 (1H, dd, J = 6.0, 3.0 Hz, H-11b); 13C NMR (CD3OD, 150 MHz): δ 93.2(CH, C-1), 70.2 (CH2, C-3), 31.5 (CH, C-4), 37.5 (CH, C-5), 37.2 (CH2, C-6), 77.5 (CH, C-7), 42.6 (CH, C-8), 51.0 (CH, C-9), 13.5(CH3, C-10), 64.2 (CH2, C-11); positive ESIMS m/z 203 [M+H]+; and HREIMS m/z 202.1210 [M]+ (calcd for C10 H18 O4, 202.1205). Valeriananoid D (4): colorless oil, ½a 23 D ¼ −61:20 (c 0.31, MeOH). UV (MeOH) λmax (log εmax): 204 (2.20), 217 (2.39), 234 (2.10) and 325 (3.51) nm; IR (KBr) νmax (cm−1): 3440, 2950, 2928, 1691, 1631, 1605, 1515, 1464, 1429, 1382, 1270, 1179, 1162, 1124, 1032, and 982 cm−1; 1H NMR and 13C NMR data, see Table 2; positive ESIMS m/z 437 [M+Na]+; and HRESIMS m/z 437.2294 [M+Na]+ (calcd for C25 H34O5 Na, 437.2304). Valeriananoid E (5): colorless oil, ½a 23 D ¼ −39:40 (c 0.87, MeOH). UV (MeOH) λmax (log εmax): 201 (1.34), 219 (1.10) nm; IR (KBr) νmax (cm−1): 3443, 2926, 2855, 1731, 1631, 1463, 1375, 1257, 1182, 1153, 1111, 1090, 1013, and 999 cm−1; 1 H NMR and 13C NMR data, see Table 2; positive ESIMS m/z 525 [M+Na]+; and HRESIMS m/z 525.4265 [M+Na]+ (calcd for C33 H58 O3 Na, 525.4278). Clovane-2β-isovaleroxy-9α-ol (6): colorless crystal (Me2CO); mp 203–205 °C; ½a 23 D ¼ −10:18 (c 0.24, MeOH). UV (MeOH) λmax (log εmax): 201 (1.53) nm; IR (KBr) νmax (cm−1): 3439, 2955, 2928, 2867, 1731, 1630, 1464, 1383, 1369, 1293, 1237, 1191, 1168, 1120, 1098, 1056, and 1032 cm−1; 1H NMR and 13C NMR data, see Table 2; positive ESIMS m/z 345 [M+Na]+; and HRESIMS m/z 345.2403 [M+Na]+ (calcd for C20 H34 O3 Na, 345.2406).
Table 1 1 H NMR (400 MHz) and 13C NMR (100 MHz) data of compounds 1–2 (δ in ppm, J in Hz). Position
1 3 4 5 6 7 8 9 10 11 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴
1a,b
2a
δH
δC
δH
δC
5.28, d (3.1) 5.24, s
96.9, d 95.2, d 154.3, s 78.2, s 48.1, t
6.41, d (3.2) 5.29, s
91.3, d 95.8, d 152,7, s 77.9, s 48.6, t
2.41, dd (14.3, 7.3) 1.95, dd (14.4, 2.8) 4.84, dd (7.2, 2.7) 2.56, d (2.9) 4.41, d (11.7) 4.22, d (11.7) 5.26, s 5.08, s 3.76, 3.49, m 1.13, t (7.0)
2.17, m 2.04, m 0.93, d (6.6) 0.93, d (6.6) 2.01, s
75.6, d 83.5, s 46.9, d 65.3, t 107.8, t 64.5, t 15.4, q
173.9, s 44.0, t 26.7, d 22.8, q 23.2, q 173.5, s 21.0, q
The 1H NMR data of the palmitoyl at C-10 of 2: δH 1.28 (20H, m, H-6″–15″), 0.89 (3H, t, J = 6.0 Hz, H-16″). The 13C NMR data of the palmitoyl at C-10 of 2: δC 30.1–30.7 (t, C-6″–13″), 33.0 (t, C-14″), 23.7 (t, C-15″). 14.4 (q, C-16″). a Recorded in methanol-d4. b Recorded in pyridine-d5.
2.47, dd (14.8, 7.2) 1.99, dd (14.8, 2.8) 4.81, dd (7.1, 2.8) 2.69, d (2.0) 4.46, d (11.6) 4.24, d (11.6) 5.32, s 5.15, s 2.22, m 2.06, m 0.93, d (6.4) 0.93, d (6.4) 2.32, t (3.2) 1.59, m 1.28, m 1.28, m 2.03, s
75.5, d 84.0, s 46.2, d 65.1, t 108.8, t 173.3, s 43.9, t 26.6, d 22.6, q 22.7, q 174.7, s 35.0, t 25.9, t 30.1, t 30.1, t 171.3, s 21.3, q
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Table 2 1 H NMR (400 MHz) and 13C NMR (100 MHz) data of compounds 4–6 (δ in ppm, J in Hz). 4a Position
δH
1
2.08, dd (14.0, 7.6) 1.61, dd (14.0, 7.6) 4.83, d (8.4) 1.46, t (8.0)
2 3 4 5 6 7 8 9 10 11
1.72, dd (11.2, 4.4) 1.60, dd (11.2, 4.4) 1.48, m 1.35, m 1.94, m 1.40, m
5b
6a
δC
δH
δC 35.9, t
76.1, d 44.8, d
1.91, dd (14.0, 7.6) 1.63, dd (14.0, 7.6) 4.77, dd (9.6, 7.6) 1.44, t (6.8)
40.0, s 76.3, s 32.9, t 29.5, t 28.9, d 44.0, d 41.4, s 24.6, t
12
1.56, m 1.39, m 0.80 d (6.7)
13 14 15
1.18, s 1.09, s 0.89, s
1.72, dd (14.0, 6.4) 1.48, dd (14.0, 6.4) 1.52, m 1.34, m 1.94, m 1.41, m
74.2, d 43.3, d 39.0, s 75.0, s 32.6, t 28.1, t 27.6, d 42.3, Cd 40.0, s 23.4, t
19.2, q
1.54, m 1.37, m 0.79 d (6.8)
25.5, q 29.1, q 20.9, q
1.14, s 1.09, s 0.89, s
25.0, q 27.8, q 19.9, q
18.7, q
δH
δC 45.8, s
4.80, dd (8.3, 5.8) 1.76, dd (12.4, 5.6) 1.53, dd (12.4, 5.6) 1.52, m 1.47, m 1.37, m 1.36, m 1.13, m 3.24, br s 1.99, m 1.58, m 1.65, m 1.16, m 1.48, m 0.97, m 0.91, s 1.06, s 0.87, s
83.5, d 45.4, t 39.1, s 51.6, d 21.8, t 34.3, t 35.6, s 75.4, d 27.3, t 28.6, t 36.5, t 25.8, q 31.9, q 29.0, q
The signals of the substituent at C-2 in 1H NMR. For 4 (the feruloyl group): δ 6.28 (1H, d, J = 15.9 Hz, H-2′), 7.54 (1H, d, J = 15.9 Hz, H-3′), 7.15 (1H, d, J = 1.7 Hz, H-5′), 6.79 (1H, d, J = 8.1 Hz, H-6′), 7.02 (1H, dd, J = 8.1, 1.7 Hz, H-9′), 3.86 (3H, s, H-OMe-6′). For 5 (the oleoyl group): δ 2.23 (2H, t, J = 7.6 Hz, H-2′), 1.58, 1.06 (2H, m, H-3′), 1.24–1.29 (20H, m, H-4′–7′, and 12′–17′), 1.97 (4H, m, H-8′, 11′), 5.29 (2H, m, H-9′, 10′), 0.87 (3H, t, J = 6.8 Hz, H-18′). For 6 (the isovaleroxy group): δ 2.18 (2H, d, J = 7.2 Hz, H-2′), 2.01 (1H, m, H-3′), 0.92 (3H, d, J = 7.2 Hz, H-4′), 0.95 (3H, d, J = 6.4 Hz, H-5′). In 13C NMR. For 4 (the feruloyl group): δ 168.9 (C, C-1′), 116.2 (CH, C-2′), 146.4 (CH, C-3′), 127.7 (C, C-4′), 111.5 (CH, C-5′), 149.3 (C, C-6′), 150 (C, C-7′), 116.4 (CH, C-8′), 124.1 (CH, C-9′), 56.4 (CH3, C-OMe-6′). For 5 (the oleoyl group): δ 173.5 (C, C-1′), 34.9 (CH2, C-2′), 24.9 (CH2, C-3′), 28.4–29.6 (CH2, C-4′–7′, and 12′–16′), 31.9 (CH2, C-8′, 11′), 129.7–129.9 (CH, C-9′, 10′), 22.6 (CH2, C-17′), 14.1 (CH3, C-18′). For 6 (the isovaleroxy group): δ 174.7 (C, C-1′), 44.6 (CH2, C-2′), 26.9 (CH, C-3′), 22.7 (CH3, C-4′), 22.8 (CH3, C-5′). a Recorded in methanol-d4. b Recorded in CDCl3.
2.5. Acetylcholinesterase inhibitory activity Acetylcholinesterase (AChE) inhibitory activity of the isolated compounds was assayed by the spectrophotometric method developed by Ellman et al. [32]. AchE (Sigma Chemical) was used as substrate in the assay. Compounds were dissolved in DMSO. The reaction mixture contained 200 μL of phosphate buffer (pH 8.0), 50 μM of test compound soln, and 40 μL of AchE soln (0.02 U/mL). The mixture was incubated for 20 min (37 °C). Then, the reaction was initiated by the addition of 40 μL of DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM), respectively. The hydrolysis of acetylthiocholine was monitored at 405 nm every 30 s for 1 h. Tacrine was used as positive control with final concentration of 0.333 μM. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E − S) / E × 100 (E is the activity of the enzyme without test compound and S is the activity of enzyme with test compound).
3. Results and discussion Compound 1 was obtained as colorless oil with the molecular formula C19H28O8 as assigned by an m/z 407.1680 [M+Na]+ (calcd for C19H28O8 Na, 407.1682) from the positive HRESIMS experiment The IR (KBr, v) absorption bands at
3448 cm−1, 1740 cm−1 and 1626 cm−1, suggesting the presence of hydroxy, ester C_O and olefinic groups. The 1H NMR spectroscopic data (Table 1) displayed four methyls [at δH 2.01 (3H, s, H-2‴), 1.13 (3H, t, J = 7.0 Hz, H-2′) and 0.93 (6H, d, J = 6.6 Hz, H-4″, 5″)], three oxygenated methines [at δH 5.28 (1H, d, J = 3.1 Hz, H-1), 5.24 (1H, s, H-3) and 4.84 (1H, dd, J = 7.2, 2.7 Hz, H-7)], two characteristic isolated methylene groups [at δH 4.41, 4.22 (each 1H, d, J = 11.7 Hz, H-10) and 3.76, 3.49 (each 1H, m, H-1′)], and a terminal olefinic bond [at δH 5.26 and 5.08 (each 1H, s, H-11)]. The 13C NMR in combination with DEPT data (Table 1) exhibited nineteen carbon signals, including four methyls, five methylenes (two O-bearing), and five methines (three oxygenated), as well as five quaternary (two ester C_O groups, two O-bearing and one sp2) carbons. A detailed comparison of the NMR spectroscopic data (Table 1) with valeriandoid C [15] indicated that the two compounds have a similar structure, with an oxo-bridge between C-3 and C-8. The only difference between 1 and valeriandoid C was the hydroxy group at C-1 in valeriandoid C was replaced by an ethoxy group in 1. According to the HMBC spectrum, the correlations (Fig. 2) from H-1 (δH 5.28, d, J = 3.1 Hz) and H-2′ (δH 1.13, t, J = 7.0 Hz) to C-1′ (δC 64.5), from H-10a (δH 4.41, d, J = 11.7 Hz), H-10b (δH 4.22, d, J = 11.7 Hz), H-2″ (δH 2.17, m) and H-3″ (δH 2.04, m) to C-1″ (δC 173.9), and from H-7 (δH 4.84, dd, J = 7.2, 2.7 Hz) and H-2‴ (δH 2.01, s) to C-1‴ (δC 171.2), demonstrated that the ethoxyl, acetoxyl and
F.-W. Dong et al. / Fitoterapia 102 (2015) 27–34
31
Fig. 2. Selected COSY, HMBC and ROESY correlations of compound 1.
isovaleroxyl groups were assigned to and located at C-1, C-7 and C-10, respectively. The relative configuration of 1 was established by a combination of the ROESY experiment (Fig. 2) and a comparison with the spectroscopic data of those reported ones [14–16,18,19], as well as on the basis of the biogenetic ground and the molecular modeling with a rigid epoxy-bridge skeleton. Generally, naturally occurring iridoids display an a-orientation for H-1 and β-orientation for H-9 [10–19]. According to the molecular model, the oxo-bridge from C-3 to C-8 could only be α-oriented, and the OH-5 and H-9 could only be β-oriented [14–16,18,19]. Moreover, the ROESY cross peaks of H-7/H-6α, H-9/H-6β and H-9/H-10, but not of H-7/H9 and H-10 indicated α-orientation of H-7 which was the same as valeriandoid C and 1, 5-dihydroxy-3, 8-epoxyvalechlorine [15,19]. Therefore, the structure of 1 was elucidated and named as jatamanvaltrate R. Compound 2 possessed the molecular formula C33 H54 O9, as determined by positive-ion HRESIMS at m/z 617.3660 [M+Na]+ (calcd 617.3666). The 1H, 13C NMR and DEPT data of 2 (Table 1) were similar to those of 1, suggesting that they were structural
analogues, except for the presence of a palmitoyl group [δC 174.7 (s, C-1″), 35.0 (t, C-2″), 25.9 (t, C-3″), 30.1–30.7 (t, C-4″–13″), 33.0 (t, C-14″), 23.7 (t, C-15″), 14.4 (q, C-16″)] in 2 rather than an ethoxy group [δC 64.5 (t, C-1′), 15.4 (q, C-2′)] in 1. The HMBC correlations of H-1 (δH 6.41, d, J = 3.2 Hz), H-2′ (δH 2.22, m) and H-3′ (δH 2.06, m) with C-1′ (δC 173.3), and of H-10a (δH 4.46, d, J = 11.6 Hz), H-10b (δH 4.26, d, J = 11.6 Hz), H-2″ (δH 2.32, t, J = 3.2 Hz) and H-3″ (δH 1.59, m) with C-1″ (δC 174.7) indicated the isovaleroxy group and palmitoyl were located at C-1and C-10, respectively. Comparison of the spectroscopic data and key ROESY correlations with those of reported iridoids and 1 [14–16,18,19] the relative configurations at C-1, C-3, C-5, C-7, C-8 and C-9 in compound 2 were demonstrated to be identical to 1. Consequently, 2 was defined and named as jatamanvaltrate S. The molecular formula of 3, C10H18O4, was established by positive-ion HREIMS at m/z 202.1210 [M]+, corresponding to two indices of hydrogen deficiency. Its 1H and 13C NMR (DEPT) spectrum displayed one methyl [δH 1.03 (3H, d, J = 7.2 Hz, H-10); δC 13.5 (C-10)], three methylenes [two oxygenated at δH 4.10 3.85 (each 1H, dd, J = 3.0, 1.2 Hz, H-3) and 4.11, 3.86
Fig. 3. Selected COSY, HMBC and ROESY correlations of compound 3.
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F.-W. Dong et al. / Fitoterapia 102 (2015) 27–34
Fig. 4. Selected COSY, HMBC and ROESY correlations of compound 4.
(each 1H, dd, J = 6.0, 3.0 Hz, H-11); δC 70.2 (C-3) and 62.2 (C-11)], and six methines [two oxymethines at δH 4.72 (1H, d, J = 2.4 Hz, H-1) and 4.10 (1H, m, H-7); δC 93.2 (C-1) and 77.5 (C-7)], which were closely related to those of (1R, 4R, 5R, 7S, 8R, 9R)-1,7-dihydroxy-10,11-dimethylperhydrocyclopenta[c]pyran [33]. Except for the methyl at C-4 in (1R, 4R, 5R, 7S, 8R, 9R)1,7-dihydroxy-10,11-dimethylperhydrocyclopenta[c]pyran was replaced by a hydroxymethyl in 3, which was confirmed by the HBMC correlations from H-11 to C-3, C-4, and C-5. The HMBC correlations (Fig. 4) of δH 1.03 (H-10) with δC 42.6 (C-8), 51.0 (C-9) and 77.5 (C-7); of δH 4.11, 3.86 (H-11) with 31.5 (C-4), 37.5 (C-5) and 70.2 (C-3); as well as the 1H-1H COSY cross-peaks from 1.03 (H-10) to 1.94 (1H, m, H-8), and from 4.11, 3.86 (H-11) to 1.74 (1H, br s, H-4) readily confirmed the assignments. The relative configuration of 3 was determined by a combination of the ROESY spectrum (Fig. 3) and a careful comparison of spectroscopic data of 3 with those reported iridoids [10–19,33]. For those known naturally occurring iridoids, H-9 and H-5 possessed β-orientations, while H-1 was α-oriented. The ROESY cross peaks of H-4/H-6α, H-6α/ H-1, H-5/H-11, H-9 and H-6β, H-9/H-10 and H-5, and H-7/ H-6α and H-8, but not of H-7/H-10 and H-9 indicated that H-10 and H-11 were β-oriented, while H-4, H-7 and H-8 were α-oriented. Hence, the structure of 3 was established as (1R, 4S, 5R, 7S, 8R, 9R)-1,7-dihydroxy-11-hydroxymethyl-10methylperhydrocyclopenta[c]pyran and named as jatamanin Q. Compound 4 exhibited a deprotonated molecular ion [M + Na]+ at m/z 437.2294 (calcd for 437.2304) from HRESIMS, requiring 9° of unsaturation.. The IR spectrum of 4 displayed the presence of hydroxy (3440 cm− 1), ester C_O group (1691 cm− 1) and carbon–carbon double bond (1631 cm− 1). The 1H NMR spectroscopic data (Table 2) of 4 displayed a double bond [δH 6.28 (1H, d, J = 15.9 Hz, H-2′)
and 7.54 (1H, d, J = 15.9 Hz, H-3′)], three aromatic protons [δH 7.15 (1H, d, J = 1.7 Hz, H-5′), 6.79 (1H, d, J = 8.1 Hz, H-8′) and 7.02 (1H, dd, J = 8.1, 1.7 Hz, H-9′)], an oxygenated methine [δH 4.80 (1H, dd, J = 7.4, 6.1 Hz, H-2)], four methyl groups [δH 0.80 (3H, d, J = 6.7 Hz, H-12), 1.18 (3H, s, H-13), 1.09 (3H, s, H-14) and 0.89 (3H, s, H-15)], and a methoxyl [δH 3.86 (3H, s, H-OMe-6′)]. The 13C NMR and DEPT spectroscopic data (Table 2), in combination with HSQC spectra, exhibited twentyfive carbon signals, including an α, β-unsaturated ester carbonyl carbon [δC 168.9 (s, C-1′)], a phenyl ring (including three protonated carbons, two oxygenated carbons and a quaternary carbon), two olefinic methines [δC 116.2 (d, C-2′) and 146.4 (d, C-3′)], a methoxy group, and a sesquiterpenoid skeleton [four methyls, four methylenes, four methines (an oxymethine), as well as three quaternary carbons (one oxygenated)]. A careful comparison of the NMR data (Table 2) of 4 with those of the known compound valeriananoid C [5] indicated that they were structural analogues. The only difference was the AcO group at C-2 in valeriananoid C which was replaced by a feruloyl group in 4. A detailed analysis of HSQC, HMBC and 1H–1H COSY spectra supported the assignment of all protons and carbon signals. The position of the feruloyl group and methoxy group at C-2 and C-6′, respectively, was confirmed based on the HMBC correlations (Fig. 5) from H-2 (δH 4.80, dd, J = 7.4, 6.1 Hz), H-2′ (δH 6.28, d, J = 15.9 Hz) and H-3′ (δH 7.54, d, J = 15.9 Hz) to C-1′ (δC 168.9), and from OMe (δH 3.86, s) to C-6′ (δC 149.3). The relative configuration of 4 was established by the ROESY experiment (Fig. 5) and a detailed comparison of spectroscopic data of 4 with valeriananoids B and C, for which the absolute configurations were confirmed by X-ray crystallographic analysis [5]. The α-orientations of H-2, H-3, H-8, H-14 and H15 in 4 were established by the correlations of H-15/H-1α and H-8, H-2/H-1α and H-3, and H-3/H-14. The β-orientations of H-11, H-12 and H-13 were demonstrated by the cross-peaks
13
7 15
H HO
6
5
8 12 9
1 10
O
14 4
2
11
O Fig. 5. Selected COSY, HMBC and ROESY correlations of compound 6.
F.-W. Dong et al. / Fitoterapia 102 (2015) 27–34
of H-13/H-1β and H-12/H-11. According to a careful comparison of spectroscopic data, the configurations of 4 were consistent with those in valeriananoid B–C [5]. Therefore, the structure of 4 was elucidated and named as valeriananoid D. Compound 5 was assigned to be C33 H58 O3 on the basis of positive-ion HRESIMS data (m/z 525.4265 [M+Na]+, calcd for 525.4278). Analysis of the 1H, 13C NMR and DEPT data of 5 (Table 2) revealed that the structure of 5 was similar to those of 4, except for the presence of an oleoyl group in 5 [δC 173.5 (C-1′), 34.9 (C-2′), 24.9 (C-3′), 28.–29.6 (C-4′–7′, and 12′–16′), 31.9 (C-8′, 11′), 129.9 (C-9′), 129.7 (C-10′), 22.6 (C-17′) and 14.1 (C-18′)] rather than a feruloyl moiety in 4. The HMBC correlations (Fig. 2) of H-2 (δH 4.77, dd, J = 9.6, 7.6 Hz), H-2′ (δH 2.23, t, J = 7.6 Hz) and H-3′ (δH 1.58, m) with C-1′ (δC 173.5) indicated that the oleoyl group was located at C-2. The relative configuration of 5 was the same as that of 4 and valeriananoid C as was deduced by the ROESY spectrum and detailed comparison of the 1H NMR coupling constants of 4 with valeriananoid C [5]. Consequently, 5 was defined and named as valeriananoid E. Compound 6 had the molecular formula C20 H34 O10, which was determined by [M+Na]+ ion at m/z 345.2403 in the HRESIMS. Its IR spectrum exhibited the absorptions at 3439 and 1731 cm−1, suggesting the presence of OH group and ester carbonyl. The 1H NMR data of 7 (Table 2) disclosed two oxygenated methines [δH 4.80 (1H, dd, J = 8.3, 5.8 Hz), 3.24 (1H, br s)], a methylene [δH 2.18 (2H, d, J = 7.2 Hz)], and five methyls [δH 1.06 (3H, br s), 0.95 (3H, d, J = 6.4 Hz), 0.92 (3H, d, J = 7.2 Hz), 0.91 (3H, br s) and 0.87 (3H, br s)] groups. Its 13C NMR and DEPT spectra (Table 2) displayed signals corresponding to five methyls, seven methylenes, four methines (two oxygenated ones), and four quaternary carbons (an ester carbonyl group). The aforementioned information indicated that 6 shared the same skeleton as clovanediol [34], except that the hydroxy group at C-2 in clovanediol was replaced by an isovaleroxy moiety in compound 6. The substituent patterns of 6 were further confirmed by a series of the 2D-NMR (1H–1H COSY, HSQC and HMBC) spectra. In the HMBC spectrum (Fig. 5), the correlations from H-2 (δH 4.80, dd, J = 8.3, 5.8 Hz), H-2′ (δ 2.18, d, J = 7.2 Hz) and H-3′ (δ 2.01, m) to C-1′ (δC 175.4) revealed that the isovaleroxy group was located at C-2. The relative configuration of 6 was determined by a combination of the ROESY spectrum (Fig. 5) and a careful comparison of spectroscopic data of 6 with those reported clovanediol [34,35]. The α-orientations of H-2, H-12 and H-13 in 6 were established by the correlations of H-2/H-12 and H-13, H-12/H-13. The β-orientations of H-5, H-9, H-14 and H-15 were assigned by the cross-peaks of H-5 with H-14, of H-5 and H-15 with H-7β, of H-7β with H-9, of H-9 with H-10, H-11β and H-15, and of H-10β and H-11β with H-5. On the basis of these data, the configuration of 6 was identical with that of clovanediol [32,33]. Thus, compound 6 was assigned as clovane-2β-isovaleroxy-9α-ol. The known compounds were identified as valeriananoids A–C (7–9) [5], volvaltrate B (10) [19], valeriotetrate A (11) [19], valeriotetrate B (12) [13], 8, 11-desoidodidrovaltrate (13) [36], rupesin E (14) [14], and (3S, 4R, 5S, 7S, 8S, 9S)-3, 8-ethoxy-7hydroxy-4, 8-dimethylperhydrocyclopenta [c] pyran (15) [14]. Compounds 1–15 were evaluated for their inhibitory activity of AChE by the Ellman method [32]. At the concentration of 50 μM, all of the compounds exhibited acetylcholine
33
esterase activity inhibition ratios of less than 10%. The positive control, tacrine, showed an inhibition rate of 47.6% at 0.33 μM. Previous bioassay has reported that the chloroform and ethylacetate fractions of V. jatamansi exhibited a significant activity against acetylcholinesterase (AChE) [24], which indicated that some compounds of these layers may possess the inhibitory effects on AChE. The essential oils of V. jatamansi, which were characterized by the presence of monoterpenoids and sesquiterpenoids as a major composition [7], were evaluated and showed inhibitory activities [26]. Moreover, some sesquiterpenoids from other related species of genus Valeriana were tested on AChE in the course of our continual search for neuroprotective compounds; a few of them displayed activities against AChE. Unfortunately, all the isolates including 9 iridoids and 6 sesquiterpenoids from the EtOAc part displayed no activity. The results together with the above argument led one to assume that the iridoids may not be the active constituents on the inhibitory effects of AChE as large numbers of iridoids have been reported before. But the sesquiterpenoids potentially possessed the activity, especially the ones from the essential oils even though the isolated sesquiterpenoids showed no effects. Acknowledgments The authors are grateful to the staffs of the analytical group at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, for measuring the spectral data. This work was financially supported by Yunnan Province (No. 2013IB021). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.01.021. References [1] Mathela CS, Chanotiya CS, Sammal SS, Pant AK, Pandey S. Compositional diversity of terpenoids in the Himalayan Valeriana genera. Chem Biodivers 2005;2:1174–82. [2] Piccinelli AL, Arana S, Caceres A, Bianca RE, Sorrentino R, Rastrelli L. New lignans from the roots of Valeriana prionophylla with antioxidative and vasorelaxant activities. J Nat Prod 2004;67:1135–40. [3] Gilani AH, Khan AU, Jabeen Q, Subhan F, Ghafar R. Antispasmodic and blood pressure lowering effects of Valeriana wallichii are mediated through K+ channel activation. J Ethnopharmacol 2005;100:347–52. [4] Fernández S, Wasowski C, Paladini AC, Marder M. Sedative and sleepenhancing properties of linarin, a flavonoid-isolated from Valeriana officinalis. Pharmacol Biochem Behav 2004;77:399–404. [5] Ming DS, Yu DQ, Yang YY, He CH. The structures of three novel sesquiterpenoids from Valeriana jatamansi Jones. Tetrahedron Lett 1997; 38:5205–8. [6] Xu J, Yang B, Guo YQ, Jin DQ, Guo P, Liu CZ, et al. Neuroprotective bakkenolides from the roots of Valeriana jatamansi. Fitoterapia 2011;82: 849–53. [7] Verma RS, Verma RK, Padalia RC, Chauhan A, Singh A, Singh HP. Chemical diversity in the essential oil of Indian Valerian (Valeriana jatamansi Jones). Chem Biodivers 2011;8:1921–9. [8] Stahi E, Schütz E. The supercritical extraction on valepotriates from Valeriana officinalis L. Planta Med 1980;40:262–70. [9] Tang YP, Liu X, Yu B. Two new flavone glycosides from Valeriana jatamansi. J Asian Nat Prod Res 2003;5:257–61. [10] Yu LL, Huang R, Han CR, Lv YP, Zhao Y, Chen YG. New iridoid triesters from Valeriana jatamansi. Helv Chim Acta 2005;88:1059–62. [11] Becker H, Chavadej S. Tissue cultures of Valerianaceae. Part VII. Valepotriate production of normal and colchicine-treated cell suspension cultures of Valeriana wallichii. J Nat Prod 1985;48:17–21.
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