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New phenylpropanoid-substituted flavan-3-ols and flavonols from the leaves of Uncaria rhynchophylla
Fitoterapia 116 (2017) 17–23
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
New phenylpropanoid-substituted flavan-3-ols and flavonols from the leaves of Uncaria rhynchophylla Ruxin Li a, Jintang Cheng a, Mengjiao Jiao a, Li Li b, Cong Guo a, Sha Chen a, An Liu a,⁎ a b
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China Institute of Materia Medica, Chinese Academy of Medical Sciences, and Peking Union Medical College, Beijing 100050, China
a r t i c l e
i n f o
Article history: Received 12 October 2016 Received in revised form 9 November 2016 Accepted 10 November 2016 Available online 12 November 2016 Chemical compounds studied in this article: Cinchonain Ia (PubChem CID: 10456516) Cinchonain Ib (PubChem CID: 442675) Cinchonain Ic (PubChem CID: 21676383) Cinchonain Id (PubChem CID: 21676382) Quercetin (PubChem CID: 5280343) (−)-epicatechin (PubChem CID: 72276) Methyl caffeate (PubChem CID: 689075) Quercetin-3-O-robinobioside (PubChem CID: 10371536) Rutin (PubChem CID: 5280805)
a b s t r a c t Uncariols A (1) and B (2), two new phenylpropanoid-substituted flavan-3-ols, and (±)-uncariols C (3a/3b) and D (4a/4b), two pairs of new phenylpropanoid-substituted flavonol enantiomers, together with nine known compounds (5–13), were isolated from the leaves of Uncaria rhynchophylla. The structures of 1–4 were established primarily by NMR and HRESIMS experiments. The absolute configurations of the new ones were deduced via the circular dichroism (CD) and quantum chemical calculations of the electronic circular dichroic (ECD) spectra. In addition, all of the isolated compounds showed potent antioxidant activity in the DPPH radical scavenging test. © 2016 Published by Elsevier B.V.
Keywords: Flavan-3-ols Flavonols Enantiomers Uncaria rhynchophylla Antioxidant activity
1. Introduction The genus Uncaria (Rubiaceae) comprises 34 species worldwide, widely distributed in the tropical areas of Southeast Asia, Africa and South America [1]. Several species of this genus showed significant cytotoxic, antioxidant, anti-inflammatory, antiviral and vasodilating activities [2–6]. Previous chemical investigations on this genus have led to identification of a series of alkaloids, triterpenes and flavonoid derivatives [2–7]. In Chinese Pharmacopoeias, the dried hooks with stem of Uncaria rhynchophylla (Miq.) Jacks. have been used as a well-known traditional Chinese medicine “Gou-teng” to relieve hypertension, epilepsy, ⁎ Corresponding author. E-mail address: [email protected] (A. Liu).
http://dx.doi.org/10.1016/j.fitote.2016.11.005 0367-326X/© 2016 Published by Elsevier B.V.
preeclampsia and associated symptoms such as headaches and dizziness [8]. Compared with the intensive investigations of the hooks of U. rhynchophylla, few chemical studies have been conducted on the leaves of U. rhynchophylla. It is known that the growth of the hooks of U. rhynchophylla, which are widely used in clinical practice, often takes a couple of years. As a result, the clinical use of the hooks is restricted by the availability of U. rhynchophylla. On the contrary, the leaves of U. rhynchophylla could be collected every year without great damage to the plant. In order to promote the sustainable utilization of U. rhynchophylla, we performed extensive chemical and bioactive investigations on the ethanol extract of the leaves. Bioassay-directed fractionation of the extract led to the isolation of two new phenylpropanoidsubstituted flavan-3-ols, named uncariols A (1) and B (2), and two pairs of new phenylpropanoid-substituted flavonol enantiomers, named (±)-uncariols C (3a/3b) and D (4a/4b), together with nine
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R. Li et al. / Fitoterapia 116 (2017) 17–23
known compounds (5–13) (Fig. 1). Details of the isolation, structure elucidation, plausible biosynthetic pathway and antioxidant activities of these compounds are reported herein.
Metworks and Mass Frontier 7.0 software package were used for data collection and data analysis. 2.2. Plant material
2. Experimental The leaves of U. rhynchophylla were collected at Guiyang City (Guizhou Province, China) in March 2014 and were identified by Dr. Wei Sun (Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences). A voucher specimen (201,403 M) was deposited in the herbarium at the Department of Medicinal Plants, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (Beijing 100700, China).
2.1. General experimental procedures Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were recorded on Shimadzu Biospec-1601 spectrophotometer. CD spectra were recorded on a JASCO J-815 spectropolarimeter. A Bruker Vertex 70 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. 1H and 13C NMR data were acquired with a Bruker Avance-600 spectrometer using solvent signals (methanol-d4: δH 3.30/ δC 49.0) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HR-ESI-MS data were obtained using an LTQ Orbitrap Velos Pro mass spectrometer equipped with an electrospray ionization (ESI) source. The fragmentor and capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas. The temperature of the drying gas was set at 350 °C. The flow rate of the drying gas and the pressure of the nebulizer were 10 L/min and 10 psi, respectively. All MS experiments were performed in positive ion mode. Full-scan spectra were acquired over a scan range of m/z 100–1000 at 1.03 spectra/s. Xcalibur, 13
11
O
3'
10
HO
O
OH OH 4''
3''
8
1''
9
8a
7 5
O
3
4a
OH OH OH
O
O
HO
1'
9
OH
O
HO
O
OH
OH
OH
OH O
OH
OH O 4a 9S 4b 9R
3a 9S 3b 9R
1 9R 2 9S
OH
OH OH OH
O
OH OH
OH O
OH OH
O
OH
OH 4'
2
Air-dried leaves of U. rhynchophylla (7.5 kg) were extracted with 95% ethanol for three times(60 L × 2 h), followed by combination, concentration, and suspension in water. It was subsequently partitioned successively with CHCl3, EtOAc and n-BuOH. TLC analyses were used to guide the next isolation project. The EtOAc part (70 g) was chromatographed on a silica gel column eluted with petroleum ether/ EtOAc (from 1:0 to 0:1) and then EtOAc/MeOH (from 50:1 to 0:1), to yield six fractions (Fr. A–F). Fraction C (16 g) was chromatographed on a silica gel column eluted with CH2Cl2/MeOH (from 100:1 to 0:1),
OH
OH
12
O
2.3. Extraction and isolation
O
HO
HO
HO
O
HO
OH O
OH
OH
O
OH OH
OH O 9
O
OH
7 9R 8 9S
5 9S 6 9R
OH HO
O
OH HO
OH 10
OH O O
O
OH
O
O
OH
4''
HO
O
OH 11 Fig. 1. Structures of compounds 1–13.
OH
O
HO
O OH
OH
OH
OH 12 4''R 13 4''S
R. Li et al. / Fitoterapia 116 (2017) 17–23
to yield five subfractions (Fr. CA–CE). Fraction CC (3.7 g) was chromatographed over a RP-18 column (MeOH/H2O from 2:8 to 10:0) to yield five fractions (Fr. CC1–CC5). Fraction CC2 (0.8 g) was purified by a MCI gel column (MeOH/H2O 50:50, v/v) followed by semi-preparative HPLC eluted with MeOH/H2O (37:63, v/v, 2 mL/min) to yield compound 5 (2.1 mg), 8 (1.9 mg), 2 (1.2 mg) and 1 (1.8 mg). Fraction CC3 (0.6 g) was purified by a MCI gel column (MeOH/H2O 50:50, v/v) followed by semi-preparative HPLC eluted with MeOH/H2O (45:55, v/ v, 2 mL/min) to obtain 7 (2.2 mg) and 6 (2.8 mg). Fraction CC4 (0.6 g) was purified by preparative HPLC eluted with MeOH/H2O (45:55, v/v, 10 mL/min) to obtain compound 3 (5.0 mg) and 4 (4.0 mg). Compound 3 was separated over a chiral AD-H column (4.6 mm × 250 mm) with nhexane/isopropanol/CH3OH (76:18:6, v/v/v) as mobile phase, to obtain enantiomers (+)-3 (2.1 mg) and (−)-3 (1.9 mg), compound 4 was separated over a chiral AD-H column (4.6 mm × 250 mm) with n-hexane/ isopropanol/MeOH (80:15:5, v/v/v), to obtain enantiomers (+)-4 (1.6 mg) and (−)-4 (1.5 mg). Fraction CD (2.5 g) was separated over a RP-18 column (MeOH/H2O from 3:7 to 10:0) to give four fractions (Fr. CD1–CD4). Fraction CD3 (0.5 g) was purified by Sephadex LH-20 (MeOH) followed by preparative HPLC eluted with ACN/H2O (26:74, v/v, 10 mL/min) to yield compound 9 (3.0 mg). The n-BuOH part (90 g) was fractionated by a macroporous resin PRP-512B column eluted with EtOH/H2O (40–100%) to give four fractions F1–F4. Fraction2 (24 g) was chromatographed over a MCI gel column (EtOH/H2O from 1:1 to 9:1) to yield four fractions (Fr. 2A–2D). Fraction2B (5 g) was chromatographed over a RP-18 column (MeOH/ H2O from 3:7 to 10:0) to yield four fractions (Fr. 2BA–2BD). Fraction 2BB (0.7 g) was purified by Sephadex LH-20 (MeOH) followed by preparative HPLC eluted with MeOH/H2O (30:70, v/v, 10 mL/min) to yield compound 10 (5.6 mg). Fraction 2BC (0.8 g) was purified by Sephadex LH-20 (MeOH/H2O 1:1) followed by preparative HPLC eluted with CH3CN/H2O (15:85, v/v, 10 mL/min) to yield compound 12 (8.0 mg) and 13 (8.5 mg). Fraction2C (4 g) was separated by a RP-18 column (MeOH/H2O from 2:8 to 10:0) followed by preparative HPLC eluted with MeOH/H2O (35:65, v/v, 10 mL/min) to yield compound 11 (2.5 mg).
19
2.3.1. Uncariol A (1) Pale red oil; [α]25 D − 86.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 282 (2.78) nm; CD (c 2.0 × 10−4 M, MeOH) λmax (Δε) 233 (−7.50), 272 (− 1.94), 296 (+ 1.19); IR (KBr) νmax 3416, 1688, 1605, 1440, 1268, 813 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 499.1602 [M + H]+ (calcd for C26H27O10, 499.1599).
2.3.2. Uncariol B (2) Pale red oil; [α]25 D − 47.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 282 (2.89) nm; CD (c 3.3 × 10−4 M, MeOH) λmax (Δε) 222 (− 11.34), 271 (− 0.66), 288 (− 1.67); IR (KBr) νmax 3424, 1688, 1607, 1440, 1270, 814 cm− 1; 1H and 13C NMR data see Table 1; HRESIMS m/z 499.1603 [M + H]+ (calcd for C26H27O10, 499.1599).
2.3.3. Uncariol C (3) Yellow powder; UV (MeOH) λmax (log ε): 259 (2.60), 274 (2.56) nm; IR (KBr) νmax 3415, 1682, 1665, 1625, 1599, 1519, 1371, 1324, 1006, 822 cm−1; 1H and 13C NMR data see Table 2; HRESIMS m/z 497.1083 [M + H]+ (calcd for C25H21O11, 497.1078). (−)-3: yellow powder; [α]25 D − 37.8 (c 0.1, MeOH); CD (c 2.0 × 10−4 M, MeOH) λmax (Δε) 263 (−3.95), 326 (−1.79). (+)-3: yellow powder; [α]25 D + 39.2 (c 0.1, MeOH); CD (c 1.4 × 10−4 M, MeOH) λmax (Δε) 261 (+4.19), 329 (+2.12).
2.3.4. Uncariol D (4) Yellow powder; UV (MeOH) λmax (log ε): 260 (2.63), 274 (2.61) nm; IR (KBr) νmax 3322, 1683, 1669, 1623, 1599, 1518, 1372, 1324, 1006, 822, 791 cm−1; 1H and 13C NMR data see Table 2; HRESIMS m/z 511.1254 [M + H]+ (calcd for C26H23O11, 511.1242). (−)-4: yellow powder; [α]25 D − 68.5 (c 0.1, MeOH); CD (c 2.0 × 10−4 M, MeOH) λmax (Δε) 265 (−1.80), 345 (−0.61). (+)-4: yellow powder; [α]25 D + 66.2 (c 0.1, MeOH); CD (c 1.4 × 10−4 M, MeOH) λmax (Δε) 259 (+3.39), 329 (+1.59).
Table 1 1 H (600 MHz) and 13C (150 MHz) NMR data of 1 and 2 in methanol-d4 (δ in ppm).
Pos 2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″
1 δH (J in Hz) 4.72, brs 4.12, m 2.86, dd (4.8, 16.8); 2.72, dd (2.4, 16.8)
5.97, s
4.97, t (7.8) 3.20, dd (7.8, 15.0); 3.24, dd (7.8, 15.0) 3.97, q (7.2) 1.09, t (7.2) 6.99, d (1.8)
6.76, d (8.4) 6.80, dd (8.4, 1.8) 6.81, overlap
6.51, d (7.8) 6.66, dd (7.8, 1.8)
δC
HMBC
80.0, CH 67.3, CH 29.7, CH2
3, 4, 1′, 2′, 6′
100.1, qC 155.8, qC 96.7, CH 155.6, qC 110.8, qC 155.2, qC 37.0, CH 39.5, CH2 175.9, qC 61.3, CH2 14.4, CH3 132.3, qC 115.4, CH 145.9, qC 145.7, qC 115.9, CH 119.6, CH 137.8, qC 116.5, CH 145.3, qC 143.8, qC 115.5, CH 120.1, CH
2, 3, 4a, 8a
2 δH (J in Hz) 4.83, brs 4.13, m 2.87, dd (4.8, 16.8); 2.75, dd (1.8, 16.8)
4a, 5, 8
5.97, s
7, 8, 10, 11, 1″, 2″, 6″ 8, 9, 11, 1″
5.00, t (7.8) 3.12, dd (7.8, 14.4); 3.18, dd (8.4, 14.4)
11, 13 12
3.98, q (7.2) 1.10, t (7.2)
2, 4′, 6′
6.93, brs
1′, 3′ 2, 2′, 4′
6.74, brs 6.74, brs
9, 4″, 6″
6.85, d (1.8)
1″, 3″ 9, 2″, 4″
6.60, d (7.8) 6.69, dd (7.8, 1.8)
δC 79.8, CH 67.5, CH 29.6, CH2 100.1, qC 155.8, qC 96.6, CH 155.6, qC 110.4, qC 155.2, qC 37.1, CH 39.5, CH2 175.8, qC 61.2, CH2 14.4, CH3 132.4, qC 115.2, CH 145.9, qC 145.6, qC 115.9, CH 119.5, CH 137.8, qC 116.5, CH 145.4, qC 143.9, qC 115.7, CH 120.2, CH
20
R. Li et al. / Fitoterapia 116 (2017) 17–23
Table 2 1 H (600 MHz) and 13C (150 MHz) NMR data of 3 and 4 in methanol-d4 (δ in ppm).
Pos 2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″
3 δH (J in Hz)
δC
4 δH (J in Hz)
HMBC
δC
4a, 5, 8
6.22, s
5.23, t (7.8) 3.30, overlap
148.5, qC 137.1, qC 177.6, qC 104.7, qC 160.9, qC 99.3, CH 163.6, qC 109.7, qC 155.6, qC 36.6, CH 38.7, CH2
7, 8, 8a, 10, 11, 1″, 2″, 6″ 8, 9, 11, 1″
5.23, t (7.2) 3.24, dd (7.2, 15.0); 3.33, overlap
3.53, s
175.4, qC 52.0, CH3
11
3.95, q (7.2) 1.02, t (7.2)
4′, 6′
7.78, s
1′, 3′
6.86, d (8.4) 7.46, brs
9, 4″, 6″
6.79, s
1″, 3″ 9, 2″, 4″
6.64, d (8.2) 6.68, d (8.2)
6.22, s
7.77, s
6.86, d (8.4) 7.45, brs 6.78, d (1.7)
6.64, d (8.2) 6.68, dd (8.2, 1.7)
124.4, qC 116.6, CH 146.2, qC 148.8, qC 116.3, CH 121.9, CH 135.9, qC 115.9, CH 146.0, qC 144.6, qC 116.1, CH 119.7, CH
2.4. DPPH radical scavenging assay The DPPH (α,α-Diphenyl-β-picrylhydrazyl) radical scavenging activity was measured according to the reported method [9]. DPPH and EtOH were used as stable free radical reagent and blank, respectively. The sample was dissolved in EtOH and then diluted to achieve concentrations of 200, 20 and 2 μg/mL. Each test sample (20 μL) was immediately mixed with freshly prepared DPPH solution (180 μL). The mixture was shaken and allowed to stand at room temperature in the dark for 20 min. The absorbance was measured at 517 nm. The scavenging activity was estimated based on the percentage of DPPH radical scavenged using the following equation: DPPH radical scavenging effect (%) = [(control absorbance − sample absorbance) / control absorbance] × 100. The IC50 value (μM) is the concentration of inhibition at which the DPPH radicals were scavenged by 50%. α-Tocopherol was used as positive control. Each sample was assayed in triplicate.
3. Results and discussion Compound 1 had the molecular formula C26H26O10 as assigned by the HR-ESI-MS (m/z 499.1602 [M + H]+, calcd as 499.1599) with fourteen degrees of unsaturation. The IR absorptions indicate the presence of hydroxyl (3416 cm−1) and carbonyl (1688 cm−1) groups. The 13C NMR (Table 1), DEPT spectra, along with HSQC experiments, showed resonances for one methyl groups, three methylenes (including an oxymethylene at δC 61.3), ten methines, and twelve quaternary carbons (including one carboxylic carbon at δC 175.9). The 1H NMR spectrum (Table 1) revealed the occurence of a one-proton aromatic singlet at δH 5.97 and the aromatic ABX-type resonances at δH 6.76–6.99. Correspondingly, the 13C NMR (Table 1) spectrum exhibited the aromatic carbon signals displayed in the region of δC 96.7–155.6. The above evidence, together with the characteristic protons at δH 4.72 (1H, brs) and δH 4.12 (1H, m), implied that compound 1 had a epicatechin skeleton [10,11]. In addition, the signals at δH 3.20 (1H, dd, J = 7.8, 15.0 Hz), δH 3.24 (1H, dd, J = 7.8, 15.0 Hz), δH 4.97 (1H, t, J = 7.8 Hz), and δC 175.9 (C = O), as well as an extra set of ABX-type aromatic protons at δH 6.51– 6.81 indicated the presence of a phenylpropanoid (C6–C3) unit [12,13].
148.4, qC 137.0, qC 177.6, qC 104.6, qC 160.8, qC 99.3, CH 163.6, qC 109.5, qC 155.5, qC 36.6, CH 38.9, CH2 174.8, qC 61.4, CH2 14.3, CH3 124.3, qC 116.5, CH 146.1, qC 148.7, qC 116.2, CH 121.8, CH 135.8, qC 115.8, CH 145.9, qC 144.5, qC 116.0, CH 119.6, CH
The HMBC peaks of H-10/C-8 and H-9/C-8 (Fig. 2) indicated that the C6–C3 substituent was located at C-8. The aforementioned data suggested that compound 1 was very similar to 8-[(1R)-1-(3,4Dihydroxyphenyl)-3-methoxy-3-oxopropyl]-3-epicatechin [13], with the only difference being the methoxy group was replaced by an ethoxy group. The planar structure of 1 was further confirmed by the interpretation of COSY and HMBC NMR spectra as shown in Fig. 2. The stereochemistry of compound 1 was deduced as follows. A C-2/ C-3 cis relationship in the C ring was supported by negligible coupling between H-2 and H-3 [10,12]. The absolute configuration at the chiral center C-9 in 1 was deduced by application of the circular dichroism (CD) exciton chirality method. The CD spectrum of 1 showed positive and negative Cotton effects at 296 and 272 nm, respectively, similar to that observed in the model compounds catiguanin A [10], allowing assignment of the 9R absolute configuration. Furthermore, comparison of the experimental CD spectrum of 1 with the calculated ECD spectra for the 1a and 1b enantiomers supported the above assignment (Fig. 3). Therefore, the absolute configuration of 1 was deduced to be 2R, 3R, 9R.
OH O
OH OH
O
OH HO
O OH OH 1H-1H
COSY HMBC
Fig. 2. Key 1H\ \1H COSY and HMBC correlations for 1.
R. Li et al. / Fitoterapia 116 (2017) 17–23
Fig. 3. Experimental CD spectrum of 1 in MeOH and the calculated ECD spectra of (2S,3S,9S)-1a and (2R,3R,9R)-1b.
Compound 2 was assigned the same molecular formula C26H26O10 (fourteen degrees of unsaturation) as 1 by HR-ESI-MS (m/z 499.1603 [M + H]+, calcd as 499.1599). The 1H and 13C NMR spectroscopic data (Table 1) of 2 was very similar to that of 1, suggesting that 2 had the same gross structure as 1. Although the typical ABX spin system is not observed in the B ring of compound 2, the chemical shifts of C-3' and C-4' in 2 were at δ 144–153, demonstrating that the B ring of 2 is a 3,4-dioxygenated aromatic ring rather than 3,5-dioxygenated aromatic ring [14]. The same coupling between H-2 and H-3 in 2 and 1 revealed a cis relationship for the two protons [10,12]. The absolute configuration at the chiral center C-9 in 2 was deduced by application of the circular dichroism (CD) exciton chirality method. The CD spectrum of 2 showed negative and positive Cotton effects at 288 and 271 nm, respectively, similar to that observed in the model compounds catiguanin B [10], allowing assignment of the 9S absolute configuration. Comparison of the experimental CD spectrum of 2 with the calculated ECD spectra for the 2a and 2b enantiomers supported the above assignment (Fig. 4). Therefore, the absolute configuration of 2 was deduced to be 2R, 3R, 9S. The elemental composition of compound 3 was established as C25H20O11 (sixteen degrees of unsaturation) by HR-ESI-MS (m/z 497.1083 [M + H]+, calcd as 497.1078). The 1H and 13C NMR spectroscopy data (Table 1), along with HSQC experiments, showed resonances for one methyl, one methylene, eight methines, twenty aromatic carbons (seven of which were protonated), and two carboxylic carbons. Analysis of the NMR data of 3 (Table 2) revealed the presence of the same 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one moiety as that typically found in japonicin A [15]. In addition to the above-mentioned fragment, the signals at δH 3.30 (2H, m), δH 5.23 (1H, t, J = 7.8 Hz), and δC 177.6 (COO), as well as an ABX-type aromatic resonances at δ 6.64 (d, J = 8.2 Hz), 6.68 (dd, J = 8.2, 1.7 Hz), and 6.78 (d, J = 1.7 Hz) indicated the presence of a phenylpropanoid (C6–C3) unit [12]. Also, the signals at δ 177.6 and 3.53 (3H, s) showed the presence of a methyl ester group. The HMBC cross-peaks of H-9/C-7, C-8 and C-8a
21
Fig. 4. Experimental CD spectrum of 2 in MeOH and the calculated ECD spectra of (2R,3R,9S)-2a and (2S,3S,9R)-2b.
established the quercetin and phenylpropanoate units were linked via C-8 and C-9, completing the gross structure of 3 as shown (Fig. 1). It could be presumed that uncariol C (3) is a pair of enantiomers since the CD spectrum is a line as well as the small optical activity ([α]25 D − 4.26). Luckily, with the help of CHIRALPAK AD-H column, (+)-uncariol C (3a) and (−)-uncariol C (3b) with the opposite Cotton
Fig. 5. Experimental CD spectrum of 3a and 3b in MeOH and the calculated ECD spectra of (9S)-3a and (9R)-3b.
22
R. Li et al. / Fitoterapia 116 (2017) 17–23 Table 3 DPPH radical scavenging activities of compounds 1–13. Compounds
IC50 (μM)
1 2 3a 3b 4a 4b 5 6 7 8 9 10 11 12 13 α-Tocopherola
22.26 16.12 10.28 11.32 12.67 14.34 15.72 8.27 3.22 5.84 7.52 8.21 5.35 8.14 2.13 9.53
a
Positive control.
effects in CD spectra and opposite optical rotations were successfully obtained, which was further confirmed by the same 1D NMR data of 3a and 3b. The absolute configurations of compounds 3a and 3b were determined by comparing the experimental with the computational electronic circular dichroism (ECD) spectra (Fig. 5). Thus, the absolute configurations at C-9 in 3a and 3b are S and R, respectively. Compound 4 has the molecular formula C26H22O11, as deduced from the HR-ESI-MS (m/z 511.1254 [M + H]+, calcd as 511.1242), indicating sixteen degrees of unsaturation. The 1H and 13C NMR spectra were closely related to those of 3, except that the methyl ester group was replaced by the ethyl ester group in 4, which was confirmed by relevant 1 H\\1H COSY and HMBC data, the 1H\\1H COSY NMR data of 4 showed
one isolated spin-system of C-12–C-13. Furthermore, the multiple HMBC correlation networks of H3-13/C-12; and H2-12/C-11 implying that the ethoxy group is bonded to C-11, completing the planar structure of 4 as shown (Fig. 1). Uncariol D (4) is also a pair of enantiomers since the CD spectrum is a line. With the help of CHIRALPAK AD-H column, (+)-uncariol D (4a) and (−)-uncariol D (4b) with the opposite Cotton effects in CD spectra and opposite optical rotations were successfully obtained, which was further confirmed by the same 1D NMR data of 4a and 4b. The absolute configuration at the chiral center C-9 in 4a and 4b was established by comparing their circular dichroism (CD) spectra with those of 3a and 3b. The signs of the Cotton effects of 4a and 4b (SI, Figs. S29 and S30) are in accordance with those of 3a and 3b. Thus, the absolute configurations at C-9 in 4a and 4b are S and R, respectively, corresponding to those in 3a and 3b. The structures of the known compounds, cinchonain Ia (5) [12], cinchonain Ib (6) [12], cinchonain Ic (7) [12], cinchonain Id (8) [12], quercetin (9) [16], (−)-epicatechin (10) [17], methyl caffeate (11) [18], quercetin-3-O-robinobioside (12) [19] and rutin (13) [20] were identified on the basis of their spectroscopic data and by comparison with those in the literature. Antioxidant activities of compounds 1–13 were evaluated in the DPPH radical scavenging assay. All the isolates showed comparable DPPH radical scavenging effects with IC50 values of 2.13–22.26 μM, when compared to the positive control (α-Tocopherol) with IC50 value of 9.53 μM (Table 3). In summary, two new phenylpropanoid-substituted flavan-3-ols, and two pairs of new phenylpropanoid-substituted flavonol enantiomers, together with nine known compounds were isolated and identified from the leaves of U. rhynchophylla. Although flavonoids are widely distributed in numerous plants, natural products with
OH 12
robinobiose -H2O
13
rutinose -H2O
OH HO
O
11 H+
OH
3a/3b 4a/4b
OH O 9 OH
OH
OH HO
H2C COCoA COOH
O OH OH O
COCoA
OH OH 5-8
11
HO
O
-H2O
11 OH
OH
10
Scheme 1. Plausible biosynthetic pathway of compounds 1–13.
H+
1-2
R. Li et al. / Fitoterapia 116 (2017) 17–23
phenylpropanoid-substituted flavan-3-ols and flavonols are relatively rare, especially in the genus Uncaria. Up to date, only cinchonain Ia and Ib are isolated from this genus [3]. Furthermore, the absolute configurations of these new phenylpropanoid-substituted flavan-3-ols and flavonols were established for the first time via the quantum chemical calculations of ECD spectra. In addition, the plausible biosynthetic pathway of compounds 1–13 (Scheme 1) was proposed and all of the isolated compounds showed significant antioxidant activity in the DPPH radical scavenging test.
[5]
[6]
[7]
[8]
Conflict of interest The authors declare no conflicts of interest. Acknowledgments We gratefully acknowledge financial support from the National Scientific and Technological Major Project for “Significant New Drugs Creation” (Grant no. 2014ZX09304307-001) and the independent topics supported by operational expenses for basic research of China Academy of Chinese Medical Sciences (L2015032).
[9] [10]
[11]
[12]
[13]
[14]
Appendix A. Supplementary data [15]
Supplementary data to this article can be found online at doi:10. 1016/j.fitote.2016.11.005.
[16]
References
[17]
[1] M.E. Heitzman, C.C. Neto, E. Winiarz, A.J. Vaisberg, G.B. Hammond, Ethnobotany, phytochemistry and pharmacology of Uncaria (Rubiaceae), Phytochemistry 66 (2005) 5–29. [2] J.S. Lee, J. Kim, B.Y. Kim, H.S. Lee, J.S. Ahn, Y.S. Chang, Inhibition of phospholipase Cγ1 and cancer cell proliferation by triterpene esters from Uncaria rhynchophylla, J. Nat. Prod. 63 (2000) 753–756. [3] C. Wirth, H. Wagner, Pharmacologically active procyanidines from the bark of Uncaria tomentosa, Phytomedicine 4 (1997) 265–266. [4] M. Sandoval, N.N. Okuhama, X.J. Zhang, L.A. Condezo, J. Lao, F.M. Angeles, R.A. Musah, P. Bobrowski, M.J.S. Miller, Anti-inflammatory and antioxidant activities of
[18]
[19] [20]
23
cat's claw (Uncaria tomentosa and Uncaria guianensis) are independent of their alkaloid content, Phytomedicine 9 (2002) 325–337. D. Yuan, B. Ma, C.F. Wu, J.Y. Yang, L.J. Zhang, S.K. Liu, L.J. Wu, Y. Kano, Alkaloids from the leaves of Uncaria rhynchophylla and their inhibitory activity on NO production in lipopolysaccharide-activated microglia, J. Nat. Prod. 71 (2008) 1271–1274. K. Wang, X.Y. Zhou, Y.Y. Wang, M.M. Li, Y.S. Li, L.Y. Peng, X. Cheng, Y. Li, Y.P. Wang, Q.S. Zhao, Macrophyllionium and macrophyllines A and B, oxindole alkaloids from Uncaria macrophylla, J. Nat. Prod. 74 (2011) 12–15. N. Aimi, T. Shito, K. Fukushima, Y. Itai, C. Aoyama, K. Kunisawa, S. Sakai, J. Haginiwa, K. Yamasaki, Studies on plants containing indole alkaloids. VIII indole alkaloid glycosides and other constituents of the leaves of Uncaria rhynchophylla MIQ, Chem. Pharm. Bull. 30 (1982) 4046–4051. The State Pharmacopoeia Committee of China, Pharmacopoeia of the People's Republic of China, I, Chemical Industry Press, Beijing, China, 2005 P180. M.S. Blois, Antioxidant determinations by the use of a stable free radical, Nature 181 (1958) 1199–1200. W.X. Tang, H. Hioki, K. Harada, M. Kubo, Y. Fukuyama, Antioxidant phenylpropanoid-substituted epicatechins from Trichilia catigua, J. Nat. Prod. 70 (2007) 2010–2013. H.F. Chen, T. Tanaka, G.I. Nonaka, T. Fujioka, K. Mihashi, Phenylpropanoid-substituted catechins from Castanopsis hystrix and structure revision of cinchonains, Phytochemistry 33 (1993) 183–187. G. Nonaka, I. Nishioka, Tannins and related compounds. VII. phenylpropanoidsubstituted epicatechins, cinchonains from Cinchona succirubra. (1), Chem. Pharm. Bull. 30 (1982) 4268–4276. H.L. Huang, Z.Q. Lu, G.T. Chen, J.Q. Zhang, W. Wang, M. Yang, D.A. Guo, Phenylpropanoid-substituted catechins and epicatechins from Smilax china, Helv. Chim. Acta. 90 (2007) 1751–1757. Y.N. Yang, H. Zhu, Z. Chen, F. Liu, Y.W. An, Z.M. Feng, J.S. Jiang, P.C. Zhang, NMR spectroscopic method for the assignment of 3,5-dioxygenated aromatic rings in natural products, J. Nat. Prod. 78 (2015) 705–711. F. Xu, G.A. Zou, Y.Q. Liu, H.A. Aisa, Chemical constituents from seeds of Lactuca sativa, Chem. Nat. Compd. 48 (2012) 574–576. N.H. Abdullah, F. Salim, R. Ahmad, Chemical constituents of malaysian U. cordata var. ferruginea and their in vitro α-glucosidase inhibitory activities, Molecules 21 (2016) 525. S. Xu, M.Y. Shang, G.X. Liu, F. Xu, X. Wang, C.C. Shou, S.Q. Cai, Chemical constituents from the rhizomes of Smilax glabra and their antimicrobial activity, Molecules 18 (2013) 5265–5287. S.V. Dyck, G. Lemière, T. Jonckers, R. Dommisse, Synthesis of 4-O-methylcedrusin. Selective protection of catechols with diphenyl carbonate, Molecules 5 (2000) 153–161. T.G. Nam, S.M. Lee, J.H. Park, D.O. Kim, N.I. Baek, S.H. Eom, Flavonoid analysis of buckwheat sprouts, Food Chem. 170 (2015) 97–101. K. Kazuma, N. Noda, M. Suzuki, Malonylated flavonol glycosides from the petals of Clitoria ternatea, Phytochemistry 62 (2003) 229–237.
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
New phenylpropanoid-substituted flavan-3-ols and flavonols from the leaves of Uncaria rhynchophylla Ruxin Li a, Jintang Cheng a, Mengjiao Jiao a, Li Li b, Cong Guo a, Sha Chen a, An Liu a,⁎ a b
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China Institute of Materia Medica, Chinese Academy of Medical Sciences, and Peking Union Medical College, Beijing 100050, China
a r t i c l e
i n f o
Article history: Received 12 October 2016 Received in revised form 9 November 2016 Accepted 10 November 2016 Available online 12 November 2016 Chemical compounds studied in this article: Cinchonain Ia (PubChem CID: 10456516) Cinchonain Ib (PubChem CID: 442675) Cinchonain Ic (PubChem CID: 21676383) Cinchonain Id (PubChem CID: 21676382) Quercetin (PubChem CID: 5280343) (−)-epicatechin (PubChem CID: 72276) Methyl caffeate (PubChem CID: 689075) Quercetin-3-O-robinobioside (PubChem CID: 10371536) Rutin (PubChem CID: 5280805)
a b s t r a c t Uncariols A (1) and B (2), two new phenylpropanoid-substituted flavan-3-ols, and (±)-uncariols C (3a/3b) and D (4a/4b), two pairs of new phenylpropanoid-substituted flavonol enantiomers, together with nine known compounds (5–13), were isolated from the leaves of Uncaria rhynchophylla. The structures of 1–4 were established primarily by NMR and HRESIMS experiments. The absolute configurations of the new ones were deduced via the circular dichroism (CD) and quantum chemical calculations of the electronic circular dichroic (ECD) spectra. In addition, all of the isolated compounds showed potent antioxidant activity in the DPPH radical scavenging test. © 2016 Published by Elsevier B.V.
Keywords: Flavan-3-ols Flavonols Enantiomers Uncaria rhynchophylla Antioxidant activity
1. Introduction The genus Uncaria (Rubiaceae) comprises 34 species worldwide, widely distributed in the tropical areas of Southeast Asia, Africa and South America [1]. Several species of this genus showed significant cytotoxic, antioxidant, anti-inflammatory, antiviral and vasodilating activities [2–6]. Previous chemical investigations on this genus have led to identification of a series of alkaloids, triterpenes and flavonoid derivatives [2–7]. In Chinese Pharmacopoeias, the dried hooks with stem of Uncaria rhynchophylla (Miq.) Jacks. have been used as a well-known traditional Chinese medicine “Gou-teng” to relieve hypertension, epilepsy, ⁎ Corresponding author. E-mail address: [email protected] (A. Liu).
http://dx.doi.org/10.1016/j.fitote.2016.11.005 0367-326X/© 2016 Published by Elsevier B.V.
preeclampsia and associated symptoms such as headaches and dizziness [8]. Compared with the intensive investigations of the hooks of U. rhynchophylla, few chemical studies have been conducted on the leaves of U. rhynchophylla. It is known that the growth of the hooks of U. rhynchophylla, which are widely used in clinical practice, often takes a couple of years. As a result, the clinical use of the hooks is restricted by the availability of U. rhynchophylla. On the contrary, the leaves of U. rhynchophylla could be collected every year without great damage to the plant. In order to promote the sustainable utilization of U. rhynchophylla, we performed extensive chemical and bioactive investigations on the ethanol extract of the leaves. Bioassay-directed fractionation of the extract led to the isolation of two new phenylpropanoidsubstituted flavan-3-ols, named uncariols A (1) and B (2), and two pairs of new phenylpropanoid-substituted flavonol enantiomers, named (±)-uncariols C (3a/3b) and D (4a/4b), together with nine
18
R. Li et al. / Fitoterapia 116 (2017) 17–23
known compounds (5–13) (Fig. 1). Details of the isolation, structure elucidation, plausible biosynthetic pathway and antioxidant activities of these compounds are reported herein.
Metworks and Mass Frontier 7.0 software package were used for data collection and data analysis. 2.2. Plant material
2. Experimental The leaves of U. rhynchophylla were collected at Guiyang City (Guizhou Province, China) in March 2014 and were identified by Dr. Wei Sun (Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences). A voucher specimen (201,403 M) was deposited in the herbarium at the Department of Medicinal Plants, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (Beijing 100700, China).
2.1. General experimental procedures Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were recorded on Shimadzu Biospec-1601 spectrophotometer. CD spectra were recorded on a JASCO J-815 spectropolarimeter. A Bruker Vertex 70 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. 1H and 13C NMR data were acquired with a Bruker Avance-600 spectrometer using solvent signals (methanol-d4: δH 3.30/ δC 49.0) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HR-ESI-MS data were obtained using an LTQ Orbitrap Velos Pro mass spectrometer equipped with an electrospray ionization (ESI) source. The fragmentor and capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas. The temperature of the drying gas was set at 350 °C. The flow rate of the drying gas and the pressure of the nebulizer were 10 L/min and 10 psi, respectively. All MS experiments were performed in positive ion mode. Full-scan spectra were acquired over a scan range of m/z 100–1000 at 1.03 spectra/s. Xcalibur, 13
11
O
3'
10
HO
O
OH OH 4''
3''
8
1''
9
8a
7 5
O
3
4a
OH OH OH
O
O
HO
1'
9
OH
O
HO
O
OH
OH
OH
OH O
OH
OH O 4a 9S 4b 9R
3a 9S 3b 9R
1 9R 2 9S
OH
OH OH OH
O
OH OH
OH O
OH OH
O
OH
OH 4'
2
Air-dried leaves of U. rhynchophylla (7.5 kg) were extracted with 95% ethanol for three times(60 L × 2 h), followed by combination, concentration, and suspension in water. It was subsequently partitioned successively with CHCl3, EtOAc and n-BuOH. TLC analyses were used to guide the next isolation project. The EtOAc part (70 g) was chromatographed on a silica gel column eluted with petroleum ether/ EtOAc (from 1:0 to 0:1) and then EtOAc/MeOH (from 50:1 to 0:1), to yield six fractions (Fr. A–F). Fraction C (16 g) was chromatographed on a silica gel column eluted with CH2Cl2/MeOH (from 100:1 to 0:1),
OH
OH
12
O
2.3. Extraction and isolation
O
HO
HO
HO
O
HO
OH O
OH
OH
O
OH OH
OH O 9
O
OH
7 9R 8 9S
5 9S 6 9R
OH HO
O
OH HO
OH 10
OH O O
O
OH
O
O
OH
4''
HO
O
OH 11 Fig. 1. Structures of compounds 1–13.
OH
O
HO
O OH
OH
OH
OH 12 4''R 13 4''S
R. Li et al. / Fitoterapia 116 (2017) 17–23
to yield five subfractions (Fr. CA–CE). Fraction CC (3.7 g) was chromatographed over a RP-18 column (MeOH/H2O from 2:8 to 10:0) to yield five fractions (Fr. CC1–CC5). Fraction CC2 (0.8 g) was purified by a MCI gel column (MeOH/H2O 50:50, v/v) followed by semi-preparative HPLC eluted with MeOH/H2O (37:63, v/v, 2 mL/min) to yield compound 5 (2.1 mg), 8 (1.9 mg), 2 (1.2 mg) and 1 (1.8 mg). Fraction CC3 (0.6 g) was purified by a MCI gel column (MeOH/H2O 50:50, v/v) followed by semi-preparative HPLC eluted with MeOH/H2O (45:55, v/ v, 2 mL/min) to obtain 7 (2.2 mg) and 6 (2.8 mg). Fraction CC4 (0.6 g) was purified by preparative HPLC eluted with MeOH/H2O (45:55, v/v, 10 mL/min) to obtain compound 3 (5.0 mg) and 4 (4.0 mg). Compound 3 was separated over a chiral AD-H column (4.6 mm × 250 mm) with nhexane/isopropanol/CH3OH (76:18:6, v/v/v) as mobile phase, to obtain enantiomers (+)-3 (2.1 mg) and (−)-3 (1.9 mg), compound 4 was separated over a chiral AD-H column (4.6 mm × 250 mm) with n-hexane/ isopropanol/MeOH (80:15:5, v/v/v), to obtain enantiomers (+)-4 (1.6 mg) and (−)-4 (1.5 mg). Fraction CD (2.5 g) was separated over a RP-18 column (MeOH/H2O from 3:7 to 10:0) to give four fractions (Fr. CD1–CD4). Fraction CD3 (0.5 g) was purified by Sephadex LH-20 (MeOH) followed by preparative HPLC eluted with ACN/H2O (26:74, v/v, 10 mL/min) to yield compound 9 (3.0 mg). The n-BuOH part (90 g) was fractionated by a macroporous resin PRP-512B column eluted with EtOH/H2O (40–100%) to give four fractions F1–F4. Fraction2 (24 g) was chromatographed over a MCI gel column (EtOH/H2O from 1:1 to 9:1) to yield four fractions (Fr. 2A–2D). Fraction2B (5 g) was chromatographed over a RP-18 column (MeOH/ H2O from 3:7 to 10:0) to yield four fractions (Fr. 2BA–2BD). Fraction 2BB (0.7 g) was purified by Sephadex LH-20 (MeOH) followed by preparative HPLC eluted with MeOH/H2O (30:70, v/v, 10 mL/min) to yield compound 10 (5.6 mg). Fraction 2BC (0.8 g) was purified by Sephadex LH-20 (MeOH/H2O 1:1) followed by preparative HPLC eluted with CH3CN/H2O (15:85, v/v, 10 mL/min) to yield compound 12 (8.0 mg) and 13 (8.5 mg). Fraction2C (4 g) was separated by a RP-18 column (MeOH/H2O from 2:8 to 10:0) followed by preparative HPLC eluted with MeOH/H2O (35:65, v/v, 10 mL/min) to yield compound 11 (2.5 mg).
19
2.3.1. Uncariol A (1) Pale red oil; [α]25 D − 86.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 282 (2.78) nm; CD (c 2.0 × 10−4 M, MeOH) λmax (Δε) 233 (−7.50), 272 (− 1.94), 296 (+ 1.19); IR (KBr) νmax 3416, 1688, 1605, 1440, 1268, 813 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 499.1602 [M + H]+ (calcd for C26H27O10, 499.1599).
2.3.2. Uncariol B (2) Pale red oil; [α]25 D − 47.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 282 (2.89) nm; CD (c 3.3 × 10−4 M, MeOH) λmax (Δε) 222 (− 11.34), 271 (− 0.66), 288 (− 1.67); IR (KBr) νmax 3424, 1688, 1607, 1440, 1270, 814 cm− 1; 1H and 13C NMR data see Table 1; HRESIMS m/z 499.1603 [M + H]+ (calcd for C26H27O10, 499.1599).
2.3.3. Uncariol C (3) Yellow powder; UV (MeOH) λmax (log ε): 259 (2.60), 274 (2.56) nm; IR (KBr) νmax 3415, 1682, 1665, 1625, 1599, 1519, 1371, 1324, 1006, 822 cm−1; 1H and 13C NMR data see Table 2; HRESIMS m/z 497.1083 [M + H]+ (calcd for C25H21O11, 497.1078). (−)-3: yellow powder; [α]25 D − 37.8 (c 0.1, MeOH); CD (c 2.0 × 10−4 M, MeOH) λmax (Δε) 263 (−3.95), 326 (−1.79). (+)-3: yellow powder; [α]25 D + 39.2 (c 0.1, MeOH); CD (c 1.4 × 10−4 M, MeOH) λmax (Δε) 261 (+4.19), 329 (+2.12).
2.3.4. Uncariol D (4) Yellow powder; UV (MeOH) λmax (log ε): 260 (2.63), 274 (2.61) nm; IR (KBr) νmax 3322, 1683, 1669, 1623, 1599, 1518, 1372, 1324, 1006, 822, 791 cm−1; 1H and 13C NMR data see Table 2; HRESIMS m/z 511.1254 [M + H]+ (calcd for C26H23O11, 511.1242). (−)-4: yellow powder; [α]25 D − 68.5 (c 0.1, MeOH); CD (c 2.0 × 10−4 M, MeOH) λmax (Δε) 265 (−1.80), 345 (−0.61). (+)-4: yellow powder; [α]25 D + 66.2 (c 0.1, MeOH); CD (c 1.4 × 10−4 M, MeOH) λmax (Δε) 259 (+3.39), 329 (+1.59).
Table 1 1 H (600 MHz) and 13C (150 MHz) NMR data of 1 and 2 in methanol-d4 (δ in ppm).
Pos 2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″
1 δH (J in Hz) 4.72, brs 4.12, m 2.86, dd (4.8, 16.8); 2.72, dd (2.4, 16.8)
5.97, s
4.97, t (7.8) 3.20, dd (7.8, 15.0); 3.24, dd (7.8, 15.0) 3.97, q (7.2) 1.09, t (7.2) 6.99, d (1.8)
6.76, d (8.4) 6.80, dd (8.4, 1.8) 6.81, overlap
6.51, d (7.8) 6.66, dd (7.8, 1.8)
δC
HMBC
80.0, CH 67.3, CH 29.7, CH2
3, 4, 1′, 2′, 6′
100.1, qC 155.8, qC 96.7, CH 155.6, qC 110.8, qC 155.2, qC 37.0, CH 39.5, CH2 175.9, qC 61.3, CH2 14.4, CH3 132.3, qC 115.4, CH 145.9, qC 145.7, qC 115.9, CH 119.6, CH 137.8, qC 116.5, CH 145.3, qC 143.8, qC 115.5, CH 120.1, CH
2, 3, 4a, 8a
2 δH (J in Hz) 4.83, brs 4.13, m 2.87, dd (4.8, 16.8); 2.75, dd (1.8, 16.8)
4a, 5, 8
5.97, s
7, 8, 10, 11, 1″, 2″, 6″ 8, 9, 11, 1″
5.00, t (7.8) 3.12, dd (7.8, 14.4); 3.18, dd (8.4, 14.4)
11, 13 12
3.98, q (7.2) 1.10, t (7.2)
2, 4′, 6′
6.93, brs
1′, 3′ 2, 2′, 4′
6.74, brs 6.74, brs
9, 4″, 6″
6.85, d (1.8)
1″, 3″ 9, 2″, 4″
6.60, d (7.8) 6.69, dd (7.8, 1.8)
δC 79.8, CH 67.5, CH 29.6, CH2 100.1, qC 155.8, qC 96.6, CH 155.6, qC 110.4, qC 155.2, qC 37.1, CH 39.5, CH2 175.8, qC 61.2, CH2 14.4, CH3 132.4, qC 115.2, CH 145.9, qC 145.6, qC 115.9, CH 119.5, CH 137.8, qC 116.5, CH 145.4, qC 143.9, qC 115.7, CH 120.2, CH
20
R. Li et al. / Fitoterapia 116 (2017) 17–23
Table 2 1 H (600 MHz) and 13C (150 MHz) NMR data of 3 and 4 in methanol-d4 (δ in ppm).
Pos 2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″
3 δH (J in Hz)
δC
4 δH (J in Hz)
HMBC
δC
4a, 5, 8
6.22, s
5.23, t (7.8) 3.30, overlap
148.5, qC 137.1, qC 177.6, qC 104.7, qC 160.9, qC 99.3, CH 163.6, qC 109.7, qC 155.6, qC 36.6, CH 38.7, CH2
7, 8, 8a, 10, 11, 1″, 2″, 6″ 8, 9, 11, 1″
5.23, t (7.2) 3.24, dd (7.2, 15.0); 3.33, overlap
3.53, s
175.4, qC 52.0, CH3
11
3.95, q (7.2) 1.02, t (7.2)
4′, 6′
7.78, s
1′, 3′
6.86, d (8.4) 7.46, brs
9, 4″, 6″
6.79, s
1″, 3″ 9, 2″, 4″
6.64, d (8.2) 6.68, d (8.2)
6.22, s
7.77, s
6.86, d (8.4) 7.45, brs 6.78, d (1.7)
6.64, d (8.2) 6.68, dd (8.2, 1.7)
124.4, qC 116.6, CH 146.2, qC 148.8, qC 116.3, CH 121.9, CH 135.9, qC 115.9, CH 146.0, qC 144.6, qC 116.1, CH 119.7, CH
2.4. DPPH radical scavenging assay The DPPH (α,α-Diphenyl-β-picrylhydrazyl) radical scavenging activity was measured according to the reported method [9]. DPPH and EtOH were used as stable free radical reagent and blank, respectively. The sample was dissolved in EtOH and then diluted to achieve concentrations of 200, 20 and 2 μg/mL. Each test sample (20 μL) was immediately mixed with freshly prepared DPPH solution (180 μL). The mixture was shaken and allowed to stand at room temperature in the dark for 20 min. The absorbance was measured at 517 nm. The scavenging activity was estimated based on the percentage of DPPH radical scavenged using the following equation: DPPH radical scavenging effect (%) = [(control absorbance − sample absorbance) / control absorbance] × 100. The IC50 value (μM) is the concentration of inhibition at which the DPPH radicals were scavenged by 50%. α-Tocopherol was used as positive control. Each sample was assayed in triplicate.
3. Results and discussion Compound 1 had the molecular formula C26H26O10 as assigned by the HR-ESI-MS (m/z 499.1602 [M + H]+, calcd as 499.1599) with fourteen degrees of unsaturation. The IR absorptions indicate the presence of hydroxyl (3416 cm−1) and carbonyl (1688 cm−1) groups. The 13C NMR (Table 1), DEPT spectra, along with HSQC experiments, showed resonances for one methyl groups, three methylenes (including an oxymethylene at δC 61.3), ten methines, and twelve quaternary carbons (including one carboxylic carbon at δC 175.9). The 1H NMR spectrum (Table 1) revealed the occurence of a one-proton aromatic singlet at δH 5.97 and the aromatic ABX-type resonances at δH 6.76–6.99. Correspondingly, the 13C NMR (Table 1) spectrum exhibited the aromatic carbon signals displayed in the region of δC 96.7–155.6. The above evidence, together with the characteristic protons at δH 4.72 (1H, brs) and δH 4.12 (1H, m), implied that compound 1 had a epicatechin skeleton [10,11]. In addition, the signals at δH 3.20 (1H, dd, J = 7.8, 15.0 Hz), δH 3.24 (1H, dd, J = 7.8, 15.0 Hz), δH 4.97 (1H, t, J = 7.8 Hz), and δC 175.9 (C = O), as well as an extra set of ABX-type aromatic protons at δH 6.51– 6.81 indicated the presence of a phenylpropanoid (C6–C3) unit [12,13].
148.4, qC 137.0, qC 177.6, qC 104.6, qC 160.8, qC 99.3, CH 163.6, qC 109.5, qC 155.5, qC 36.6, CH 38.9, CH2 174.8, qC 61.4, CH2 14.3, CH3 124.3, qC 116.5, CH 146.1, qC 148.7, qC 116.2, CH 121.8, CH 135.8, qC 115.8, CH 145.9, qC 144.5, qC 116.0, CH 119.6, CH
The HMBC peaks of H-10/C-8 and H-9/C-8 (Fig. 2) indicated that the C6–C3 substituent was located at C-8. The aforementioned data suggested that compound 1 was very similar to 8-[(1R)-1-(3,4Dihydroxyphenyl)-3-methoxy-3-oxopropyl]-3-epicatechin [13], with the only difference being the methoxy group was replaced by an ethoxy group. The planar structure of 1 was further confirmed by the interpretation of COSY and HMBC NMR spectra as shown in Fig. 2. The stereochemistry of compound 1 was deduced as follows. A C-2/ C-3 cis relationship in the C ring was supported by negligible coupling between H-2 and H-3 [10,12]. The absolute configuration at the chiral center C-9 in 1 was deduced by application of the circular dichroism (CD) exciton chirality method. The CD spectrum of 1 showed positive and negative Cotton effects at 296 and 272 nm, respectively, similar to that observed in the model compounds catiguanin A [10], allowing assignment of the 9R absolute configuration. Furthermore, comparison of the experimental CD spectrum of 1 with the calculated ECD spectra for the 1a and 1b enantiomers supported the above assignment (Fig. 3). Therefore, the absolute configuration of 1 was deduced to be 2R, 3R, 9R.
OH O
OH OH
O
OH HO
O OH OH 1H-1H
COSY HMBC
Fig. 2. Key 1H\ \1H COSY and HMBC correlations for 1.
R. Li et al. / Fitoterapia 116 (2017) 17–23
Fig. 3. Experimental CD spectrum of 1 in MeOH and the calculated ECD spectra of (2S,3S,9S)-1a and (2R,3R,9R)-1b.
Compound 2 was assigned the same molecular formula C26H26O10 (fourteen degrees of unsaturation) as 1 by HR-ESI-MS (m/z 499.1603 [M + H]+, calcd as 499.1599). The 1H and 13C NMR spectroscopic data (Table 1) of 2 was very similar to that of 1, suggesting that 2 had the same gross structure as 1. Although the typical ABX spin system is not observed in the B ring of compound 2, the chemical shifts of C-3' and C-4' in 2 were at δ 144–153, demonstrating that the B ring of 2 is a 3,4-dioxygenated aromatic ring rather than 3,5-dioxygenated aromatic ring [14]. The same coupling between H-2 and H-3 in 2 and 1 revealed a cis relationship for the two protons [10,12]. The absolute configuration at the chiral center C-9 in 2 was deduced by application of the circular dichroism (CD) exciton chirality method. The CD spectrum of 2 showed negative and positive Cotton effects at 288 and 271 nm, respectively, similar to that observed in the model compounds catiguanin B [10], allowing assignment of the 9S absolute configuration. Comparison of the experimental CD spectrum of 2 with the calculated ECD spectra for the 2a and 2b enantiomers supported the above assignment (Fig. 4). Therefore, the absolute configuration of 2 was deduced to be 2R, 3R, 9S. The elemental composition of compound 3 was established as C25H20O11 (sixteen degrees of unsaturation) by HR-ESI-MS (m/z 497.1083 [M + H]+, calcd as 497.1078). The 1H and 13C NMR spectroscopy data (Table 1), along with HSQC experiments, showed resonances for one methyl, one methylene, eight methines, twenty aromatic carbons (seven of which were protonated), and two carboxylic carbons. Analysis of the NMR data of 3 (Table 2) revealed the presence of the same 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one moiety as that typically found in japonicin A [15]. In addition to the above-mentioned fragment, the signals at δH 3.30 (2H, m), δH 5.23 (1H, t, J = 7.8 Hz), and δC 177.6 (COO), as well as an ABX-type aromatic resonances at δ 6.64 (d, J = 8.2 Hz), 6.68 (dd, J = 8.2, 1.7 Hz), and 6.78 (d, J = 1.7 Hz) indicated the presence of a phenylpropanoid (C6–C3) unit [12]. Also, the signals at δ 177.6 and 3.53 (3H, s) showed the presence of a methyl ester group. The HMBC cross-peaks of H-9/C-7, C-8 and C-8a
21
Fig. 4. Experimental CD spectrum of 2 in MeOH and the calculated ECD spectra of (2R,3R,9S)-2a and (2S,3S,9R)-2b.
established the quercetin and phenylpropanoate units were linked via C-8 and C-9, completing the gross structure of 3 as shown (Fig. 1). It could be presumed that uncariol C (3) is a pair of enantiomers since the CD spectrum is a line as well as the small optical activity ([α]25 D − 4.26). Luckily, with the help of CHIRALPAK AD-H column, (+)-uncariol C (3a) and (−)-uncariol C (3b) with the opposite Cotton
Fig. 5. Experimental CD spectrum of 3a and 3b in MeOH and the calculated ECD spectra of (9S)-3a and (9R)-3b.
22
R. Li et al. / Fitoterapia 116 (2017) 17–23 Table 3 DPPH radical scavenging activities of compounds 1–13. Compounds
IC50 (μM)
1 2 3a 3b 4a 4b 5 6 7 8 9 10 11 12 13 α-Tocopherola
22.26 16.12 10.28 11.32 12.67 14.34 15.72 8.27 3.22 5.84 7.52 8.21 5.35 8.14 2.13 9.53
a
Positive control.
effects in CD spectra and opposite optical rotations were successfully obtained, which was further confirmed by the same 1D NMR data of 3a and 3b. The absolute configurations of compounds 3a and 3b were determined by comparing the experimental with the computational electronic circular dichroism (ECD) spectra (Fig. 5). Thus, the absolute configurations at C-9 in 3a and 3b are S and R, respectively. Compound 4 has the molecular formula C26H22O11, as deduced from the HR-ESI-MS (m/z 511.1254 [M + H]+, calcd as 511.1242), indicating sixteen degrees of unsaturation. The 1H and 13C NMR spectra were closely related to those of 3, except that the methyl ester group was replaced by the ethyl ester group in 4, which was confirmed by relevant 1 H\\1H COSY and HMBC data, the 1H\\1H COSY NMR data of 4 showed
one isolated spin-system of C-12–C-13. Furthermore, the multiple HMBC correlation networks of H3-13/C-12; and H2-12/C-11 implying that the ethoxy group is bonded to C-11, completing the planar structure of 4 as shown (Fig. 1). Uncariol D (4) is also a pair of enantiomers since the CD spectrum is a line. With the help of CHIRALPAK AD-H column, (+)-uncariol D (4a) and (−)-uncariol D (4b) with the opposite Cotton effects in CD spectra and opposite optical rotations were successfully obtained, which was further confirmed by the same 1D NMR data of 4a and 4b. The absolute configuration at the chiral center C-9 in 4a and 4b was established by comparing their circular dichroism (CD) spectra with those of 3a and 3b. The signs of the Cotton effects of 4a and 4b (SI, Figs. S29 and S30) are in accordance with those of 3a and 3b. Thus, the absolute configurations at C-9 in 4a and 4b are S and R, respectively, corresponding to those in 3a and 3b. The structures of the known compounds, cinchonain Ia (5) [12], cinchonain Ib (6) [12], cinchonain Ic (7) [12], cinchonain Id (8) [12], quercetin (9) [16], (−)-epicatechin (10) [17], methyl caffeate (11) [18], quercetin-3-O-robinobioside (12) [19] and rutin (13) [20] were identified on the basis of their spectroscopic data and by comparison with those in the literature. Antioxidant activities of compounds 1–13 were evaluated in the DPPH radical scavenging assay. All the isolates showed comparable DPPH radical scavenging effects with IC50 values of 2.13–22.26 μM, when compared to the positive control (α-Tocopherol) with IC50 value of 9.53 μM (Table 3). In summary, two new phenylpropanoid-substituted flavan-3-ols, and two pairs of new phenylpropanoid-substituted flavonol enantiomers, together with nine known compounds were isolated and identified from the leaves of U. rhynchophylla. Although flavonoids are widely distributed in numerous plants, natural products with
OH 12
robinobiose -H2O
13
rutinose -H2O
OH HO
O
11 H+
OH
3a/3b 4a/4b
OH O 9 OH
OH
OH HO
H2C COCoA COOH
O OH OH O
COCoA
OH OH 5-8
11
HO
O
-H2O
11 OH
OH
10
Scheme 1. Plausible biosynthetic pathway of compounds 1–13.
H+
1-2
R. Li et al. / Fitoterapia 116 (2017) 17–23
phenylpropanoid-substituted flavan-3-ols and flavonols are relatively rare, especially in the genus Uncaria. Up to date, only cinchonain Ia and Ib are isolated from this genus [3]. Furthermore, the absolute configurations of these new phenylpropanoid-substituted flavan-3-ols and flavonols were established for the first time via the quantum chemical calculations of ECD spectra. In addition, the plausible biosynthetic pathway of compounds 1–13 (Scheme 1) was proposed and all of the isolated compounds showed significant antioxidant activity in the DPPH radical scavenging test.
[5]
[6]
[7]
[8]
Conflict of interest The authors declare no conflicts of interest. Acknowledgments We gratefully acknowledge financial support from the National Scientific and Technological Major Project for “Significant New Drugs Creation” (Grant no. 2014ZX09304307-001) and the independent topics supported by operational expenses for basic research of China Academy of Chinese Medical Sciences (L2015032).
[9] [10]
[11]
[12]
[13]
[14]
Appendix A. Supplementary data [15]
Supplementary data to this article can be found online at doi:10. 1016/j.fitote.2016.11.005.
[16]
References
[17]
[1] M.E. Heitzman, C.C. Neto, E. Winiarz, A.J. Vaisberg, G.B. Hammond, Ethnobotany, phytochemistry and pharmacology of Uncaria (Rubiaceae), Phytochemistry 66 (2005) 5–29. [2] J.S. Lee, J. Kim, B.Y. Kim, H.S. Lee, J.S. Ahn, Y.S. Chang, Inhibition of phospholipase Cγ1 and cancer cell proliferation by triterpene esters from Uncaria rhynchophylla, J. Nat. Prod. 63 (2000) 753–756. [3] C. Wirth, H. Wagner, Pharmacologically active procyanidines from the bark of Uncaria tomentosa, Phytomedicine 4 (1997) 265–266. [4] M. Sandoval, N.N. Okuhama, X.J. Zhang, L.A. Condezo, J. Lao, F.M. Angeles, R.A. Musah, P. Bobrowski, M.J.S. Miller, Anti-inflammatory and antioxidant activities of
[18]
[19] [20]
23
cat's claw (Uncaria tomentosa and Uncaria guianensis) are independent of their alkaloid content, Phytomedicine 9 (2002) 325–337. D. Yuan, B. Ma, C.F. Wu, J.Y. Yang, L.J. Zhang, S.K. Liu, L.J. Wu, Y. Kano, Alkaloids from the leaves of Uncaria rhynchophylla and their inhibitory activity on NO production in lipopolysaccharide-activated microglia, J. Nat. Prod. 71 (2008) 1271–1274. K. Wang, X.Y. Zhou, Y.Y. Wang, M.M. Li, Y.S. Li, L.Y. Peng, X. Cheng, Y. Li, Y.P. Wang, Q.S. Zhao, Macrophyllionium and macrophyllines A and B, oxindole alkaloids from Uncaria macrophylla, J. Nat. Prod. 74 (2011) 12–15. N. Aimi, T. Shito, K. Fukushima, Y. Itai, C. Aoyama, K. Kunisawa, S. Sakai, J. Haginiwa, K. Yamasaki, Studies on plants containing indole alkaloids. VIII indole alkaloid glycosides and other constituents of the leaves of Uncaria rhynchophylla MIQ, Chem. Pharm. Bull. 30 (1982) 4046–4051. The State Pharmacopoeia Committee of China, Pharmacopoeia of the People's Republic of China, I, Chemical Industry Press, Beijing, China, 2005 P180. M.S. Blois, Antioxidant determinations by the use of a stable free radical, Nature 181 (1958) 1199–1200. W.X. Tang, H. Hioki, K. Harada, M. Kubo, Y. Fukuyama, Antioxidant phenylpropanoid-substituted epicatechins from Trichilia catigua, J. Nat. Prod. 70 (2007) 2010–2013. H.F. Chen, T. Tanaka, G.I. Nonaka, T. Fujioka, K. Mihashi, Phenylpropanoid-substituted catechins from Castanopsis hystrix and structure revision of cinchonains, Phytochemistry 33 (1993) 183–187. G. Nonaka, I. Nishioka, Tannins and related compounds. VII. phenylpropanoidsubstituted epicatechins, cinchonains from Cinchona succirubra. (1), Chem. Pharm. Bull. 30 (1982) 4268–4276. H.L. Huang, Z.Q. Lu, G.T. Chen, J.Q. Zhang, W. Wang, M. Yang, D.A. Guo, Phenylpropanoid-substituted catechins and epicatechins from Smilax china, Helv. Chim. Acta. 90 (2007) 1751–1757. Y.N. Yang, H. Zhu, Z. Chen, F. Liu, Y.W. An, Z.M. Feng, J.S. Jiang, P.C. Zhang, NMR spectroscopic method for the assignment of 3,5-dioxygenated aromatic rings in natural products, J. Nat. Prod. 78 (2015) 705–711. F. Xu, G.A. Zou, Y.Q. Liu, H.A. Aisa, Chemical constituents from seeds of Lactuca sativa, Chem. Nat. Compd. 48 (2012) 574–576. N.H. Abdullah, F. Salim, R. Ahmad, Chemical constituents of malaysian U. cordata var. ferruginea and their in vitro α-glucosidase inhibitory activities, Molecules 21 (2016) 525. S. Xu, M.Y. Shang, G.X. Liu, F. Xu, X. Wang, C.C. Shou, S.Q. Cai, Chemical constituents from the rhizomes of Smilax glabra and their antimicrobial activity, Molecules 18 (2013) 5265–5287. S.V. Dyck, G. Lemière, T. Jonckers, R. Dommisse, Synthesis of 4-O-methylcedrusin. Selective protection of catechols with diphenyl carbonate, Molecules 5 (2000) 153–161. T.G. Nam, S.M. Lee, J.H. Park, D.O. Kim, N.I. Baek, S.H. Eom, Flavonoid analysis of buckwheat sprouts, Food Chem. 170 (2015) 97–101. K. Kazuma, N. Noda, M. Suzuki, Malonylated flavonol glycosides from the petals of Clitoria ternatea, Phytochemistry 62 (2003) 229–237.