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New triacetic acid lactone glycosides from the fruits of Forsythia suspensa and their nitric oxide production inhibitory activity
Carbohydrate Research 488 (2020) 107908
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
New triacetic acid lactone glycosides from the fruits of Forsythia suspensa and their nitric oxide production inhibitory activity
T
Siyuan Shao, Yanan Yang, Ziming Feng, Jianshuang Jiang, Peicheng Zhang∗ State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China
ARTICLE INFO
ABSTRACT
Keywords: Forsythia suspensa Triacetic acid lactone glycosides Forsyphensides A–C GC analysis NO production inhibitory activity
Three new triacetic acid lactone (TAL) glycosides, forsyphensides A–C (1–3) were isolated from the fruits of Forsythia suspensa. Their structures were elucidated by comprehensive spectroscopic techniques. The absolute configurations of their monosaccharides were determined by GC analysis. Notably, forsyphensides A–C were relatively rare TAL glycosides identified from plants. Compound 1 exhibited inhibitory activity against the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in microglia BV2 cells with the inhibition rate of 69.40%.
1. Introduction
2. Results and discussion
Natural products have continued to play an important role in drug discovery in human diseases for their novel structures and comprehensive bioactivities during the past years [1]. Forsythia suspensa, one kind of Chinese traditional medicine (TMC) coming from the Oleaceae family, contains various types of natural products for drug development [2]. Up to now, plenty of chemical constituents have been obtained from F. suspensa including phenylethanoid glycosides, lignans, terpenes, flavones and alkaloids [3–8]. Pharmacological studies demonstrated that F. suspensa possessed various bioactivities [8], among which antiinflammatory effect was the one attracted much attention [9–11]. During our research for seeking bioactive constituents from the n-butonal portion of the fruits of F. suspensa, three new compounds forsyphensides A–C (1–3) possessing a triacetic acid lactone (TAL) glycoside structure were obtained (Fig. 1, Experimental section in Supplementary material). To the best of our knowledge, they were relatively rare components isolated from higher plants [12,13]. Their structures were identified by comprehensive analysis of the spectroscopic data. The configurations of the monosaccharides were determined by GC analysis. The nitric oxide (NO) production inhibitory activities of compounds 1–3 were assayed on the lipopolysacchride (LPS)-induced NO production in microglia BV2 cells. Herein, we report the isolation, structural elucidation and bioactive evaluation of these isolates.
Compound 1 was isolated as a yellow amorphous powder with the molecular formula of C27H32O15 identified by the positive HRESIMS ion peak at m/z 619.1638 [M + Na]+ (calcd. for C27H32O15Na: 619.1633), suggesting 12 degrees of unsaturation (Fig. S4 in Supplementary material). The IR spectrum (Fig. S3 in Supplementary material) showed the presence of hydroxyl groups (3398 cm−1), carbonyl groups (1693 cm−1), olefinic bonds (1634 cm−1) and phenyl groups (1604, 1564 and 1514 cm−1). The 1H NMR data (Table 1) displayed four AA'BB’ system aromatic protons at δH 7.54 (d, 2H, J = 8.5 Hz, H-2′ and H-6′) and 6.77 (d, 2H, J = 8.5 Hz, H-3′ and H-5′); a pair of olefinic protons at δH 7.61 (d, 1H, J = 16.0 Hz, H-7′) and 6.37 (d, 1H, J = 16.0 Hz, H-8′); two single olefinic protons at δH 5.64 (d, 1H, J = 2.0 Hz, H-2) and 6.08 (d, 1H, J = 2.0 Hz, H-4); and one methyl group at δH 2.18 (s, 3H, H-6). In addition, two anomeric protons at δH 5.09 (d, 1H, J = 8.0 Hz, H-7), 4.38 (d, 1H, J = 8.0 Hz, H-13) and plenty of signals at δH 3.04–4.48 were observed, which suggested the existence of two β-sugars in compound 1. The 13C NMR data (Table 1) displayed 27 carbons, including six quaternary carbons, 18 methine carbons, two methylene carbons and one methyl carbon. Of them, 12 carbons were assigned as two hexoses combined with the HSQC spectrum (Fig. S7 in Supplementary material). They were determined to be D-glucose by GC analysis (Fig. S1 in Supplementary material) [14]. Furthermore, nine carbons were consisted of a 4-hydroxylcinnamoyl group through HMBC correlations of H-7′ to C-2′, H-8′ to C-1′ and C-9′
∗ Corresponding author. Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 2 Nan Wei Road, Xicheng District, Beijing, 100050, PR China. E-mail address: [email protected] (P. Zhang).
https://doi.org/10.1016/j.carres.2020.107908 Received 19 September 2019; Received in revised form 28 November 2019; Accepted 5 January 2020 Available online 07 January 2020 0008-6215/ © 2020 Published by Elsevier Ltd.
Carbohydrate Research 488 (2020) 107908
S. Shao, et al.
Fig. 1. Structures of compounds 1–3 from F. suspensa.
Through detailed analysis of its 1H and 13C NMR data (Table 1) indicated that the structure of 3 was highly similar with that of 2. The main difference between them was that three ABX system protons at δH 7.14 (br s, 1H, H-2″), 7.08 (d, 1H, J = 8.0 Hz, H-5″) and 7.01 (br d, 1H, J = 8.0 Hz, H-6″) in 2 was replaced by four AA'BB’ system protons at δH 7.62 (d, 2H, J = 8.0, H-2″ and H-6″) and 7.04 (d, 2H, J = 8.0, H-3″ and H-5″) in 3. The difference indicated that the caffeoyl group of 2 was instead of by a 4-hydroxylcinnamyl group of 3. Therefore, the structure of 3 was built by the HMBC correlations of H-7 to C-3, H-13 to C-8, H18 to C-9′, H-12 to C-9″ and H-10″ to C-4′′ (Fig. 2) and named as forsyphenside C. Forsyphensides A–C (1–3) were three new triacetic acid lactone glycosides from the fruits of F. suspensa. Notably, they were relatively rare TAL glycoside derivatives isolated from higher plants. In the biosynthetic view, the key intermediate TAL unit may be derived from acetyl-CoA and malonyl-CoA via ester condensation and cyclization reactions catalyzed by ketoacyl synthases (Scheme 1) [16]. After the biosynthesis of TAL unit, the UDP (uridine diphosphate)-glucoses were first coupled with it by glycosyltransferase and then compound 1 was established by the combination of hydroxylcinnamoyl-CoA and the TAL di-glycosides via hydroxycinnamoyl transferase [17,18]. Compounds 2 and 3 were finally built by further reactions with caffeoyl-CoA/hydroxylcinnamoyl-CoA and UDP-glucoses. All the compounds were evaluated on the LPS induced NO production in microglia BV2 cells for their inhibitory activities [19]. As a result, compound 1 displayed inhibitory effect against the NO production with the inhibition rate of 69.40% at the concentration of 10 μM, which was close with the 82.50% inhibition of curcumin (10 μM) as the positive control (Table 2). However, compounds 2 and 3 just exhibited weak inhibitory effects. Analysis of the structural-activity relationship of the three compounds indicated that the extra caffeoyl/4-hydroxylcinnamoyl group and glucose may decrease their inhibitory activities against NO production.
(Fig. 2). The remaining six carbons were not assigned easily for their uncommon characteristic carbon chemical shifts. Fortunately, the HMBC correlations of H-6 to C-4 and C-5, H-4 to C-2 and H-2 to C-1 were observed in the HMBC spectrum (Fig. S8 in Supplementary material) and offered the key hints. Combination of the chemical shifts and their 2D NMR correlations suggested that the six carbons established a triacetic acid lactone moiety [15]. Then, a glucose was linked to C-3 of the TAL unit via the HMBC correlation of H-7 to C-3. Another glucose was connected to the former one through the correlation of H-13 to C-8. The 4-hydroxylcinnamoyl group was connected to C-18 of the latter glucose through H-18 to C-9′. Thus, the structure of 1 was established and named as forsyphenside A. Compound 2, a brown amorphous powder, gave the molecular formula of C42H48O23 by the HRESIMS signal at m/z 943.2480 [M + Na]+ (calcd. for C42H48O23Na: 943.2479) (Fig. S12 in Supplementary material). Comparison of its 1H and 13C NMR data (Table 1) with those of 1 showed three more ABX system aromatic protons at δH 7.14 (br s, 1H, H-2″), 7.08 (d, 1H, J = 8.0 Hz, H-5″) and 7.01 (br d, 1H, J = 8.0 Hz, H-6″); two more olefinic protons at δH 7.49 (d, 1H, J = 16.0 Hz, H-7″) and 6.32 (d, 1H, J = 16.0 Hz, H-8″), a more anomeric protons at δH 4.87 (d, 1H, J = 7.0 Hz, H-10″) and five more proton signals at δH 3.04–3.70, which indicated the presence of an extra caffeoyl group and a glucose and in 2. The HMBC correlations of H-7 to C-3, H-13 to C-7 and H-18 to C-9′ suggested the four identical units of 1 and 2 had the same connection type (Fig. 2). Subsequently, the caffeoyl group was linked to C-12 by the correlation of H-12 to C-9′′. The glucose was connected to C-4″ by the correlation of H-10″ to C-4′′. Consequently, the structure of 2 was established and named as forsyphenside B. Compound 3 was isolated as a brown amorphous powder. Its molecular formula of C42H48O22 was identified by the HRESIMS adduct peak at m/z 927.2514 [M + Na]+ (calcd. for C42H48O22Na: 927.2529), which was 16 mass less than 2 (Fig. S20 in Supplementary material). 2
Carbohydrate Research 488 (2020) 107908
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Table 1 1 H NMR (500 MHz) and13C NMR (125 MHz) data of compounds 1–3 in DMSO‑d6. Position
1
2
δH 1 2
5.64 (d, 2.0)
3 4
6.08 (d, 2.0)
5 6 7
2.18 5.09 8.0) 3.46 3.48 3.27 3.62
8 9 10 11 12 13 14 15 16 17 18 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 11″ 12″ 13″ 14″ 15″
7.54 (d, 8.5)
δC
δH
δC
δH
δC
163.6 89.9
5.63 (s)
163.6 89.9
5.64 (s)
163.6 89.9
168.9 99.7
6.06 (s)
168.9 99.7
6.06 (s)
168.9 99.7
(s) (d,
163.1 19.4 98.5
(m) (m) (m) (m)
79.1 74.1 72.7 75.1
3.70 (m) 3.64, m 4.38 (d, 8.0) 3.04 (m) 3.18 (m) 3.16 (m) 3.48 (m) 4.48 (m) 4.04, m
2.16 5.08 8.0) 3.45 3.46 3.17 3.70
59.6
6.77 (d, 8.5) 6.77 7.54 7.61 6.37
(d, (d, (d, (d,
8.5) 8.5) 16.0) 16.0)
(s) (d,
163.0 19.4 98.5
(m) (m) (m) (m)
79.1 74.1 69.9 73.9
4.48 (m) 4.10, m 4.39 (d, 8.0) 3.04 (m) 3.19 (m) 3.25 (m) 3.45 (m) 4.40 (m) 4.21, m
102.7 73.2 76.2 69.9 73.9 63.5 125.0 130.4
3
115.8 160.0 115.8 130.4 145.2 113.7 166.5
6.79 (d, 8.0) 6.79 7.54 7.52 6.38
(d, (d, (d, (d,
7.54 (d, 8.0)
8.0) 8.0) 16.0) 16.0)
7.14 (br s) 7.08 7.01 7.49 6.32
(d, 8.0) (br d, 8.0) (d, 16.0) (d, 16.0)
4.87 (d, 7.0) 3.04 (m) 3.33 (m) 3.26 (m) 3.63 (m) 3.70 (m) 3.62, m
3. Experimental section
63.7 102.7 73.2 76.2 69.9 73.8 63.2
115.9 160.1 115.9 130.4 145.0 113.8 166.5 128.6 115.2 146.8 147.2 115.6 120.7 144.8 114.9 166.3 101.1 73.2 75.6 72.6 75.1 59.6
124.9 130.4
2.17 5.09 8.0) 3.46 3.44 3.17 3.71
(s) (d,
163.0 19.4 98.6
(m) (m) (m) (m)
79.2 74.1 70.0 73.8
4.51 (m) 4.08, m 4.40 (d, 8.5) 3.05 (m) 3.19 (m) 3.25 (m) 3.53 (m) 4.40 (m) 4.17, m 7.52 (d, 8.5)
63.7 102.7 73.1 76.2 69.8 73.8 63.3
6.78 (d, 8.5) 6.78 7.52 7.51 6.36
(d, (d, (d, (d,
8.5) 8.5) 16.0) 16.0)
7.62 (d, 8.0) 7.04 (d, 8.0) 7.04 7.62 7.61 6.40
(d, (d, (d, (d,
8.0) 8.0) 16.0) 16.0)
5.01 (d, 7.5) 3.04 (m) 3.30 (m) 3.25 (m) 3.63 (m) 3.71 (m) 3.61, m
124.8 130.4
115.9 160.2 115.9 130.4 145.0 113.8 166.5 127.8 130.0 116.4 158.9 116.4 130.0 144.4 116.2 166.2 99.6 73.2 75.1 72.6 76.2 59.6
experiments were performed using an Agilent 7890A series system with a capillary column, HP-5 (60 m × 0.25 mm, with a 0.25 μm film; Dikma Technologies Inc., Beijing, People's Republic of China).
3.1. General experimental procedures UV spectra were collected by a JASCO V650 spectrometer (Thermo Scientific, Waltham, MA, USA). Optical rotations were recorded by a JASCO P-2000 polarimeter (JASCO, Easton, MD, USA). IR spectra were performed on a Nicolet 5700 spectrometer (Thermo Scientific, Waltham, MA, USA). The HRESIMS trials were obtained by an Agilent 6520 LC-QTOF mass spectrometer (Agilent Technologies, Waldbronn, Germany). The 1D and 2D NMR spectra were recorded by Bruker 500 MHz spectrometer and the values were given in ppm (Bruker-Biospin, Billerica, MA, USA). Column chromatography was performed on macroporous resin (Diaion HP-20, Mitsubishi Chemical Corp., Tokyo, Japan) and Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden). Preparative HPLC was a Shimadzu LC-10AT equipped with an SPD-10A detector (Shimadzu Corp., Tokyo, Japan) using a YMC-Pack ODS-A column (250 mm × 20 mm, 5 μm; YMC Corp., Kyoto, Japan). GC
3.2. Plant material The fruits of Forsythia suspensa were collected in December 2011 from Yuncheng City of Shanxi Province, People's Republic of China. It was identified by Lin Ma (Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing). A voucher specimen (ID-S-2597) has been deposited at the Herbarium of Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing. 3.3. Extraction and isolation The dried fruits of Forsythia suspensa (90 kg) were shattered and extracted with 75% C2H5OH for three times under reflux and filtered to 3
Carbohydrate Research 488 (2020) 107908
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Fig. 2. Key HMBC (H→C) and TOCSY (–) correlations of compounds 1–3.
yield a crude extract (12.6 kg). The extract was suspended in water and successively separated by petroleum ether, ethyl acetate and n-butanol, respectively. The n-butanol section (4 kg) was then suspended in water to obtain an aqueous layer (1.5 kg). The layer was concentrated to yield a water-soluble portion and then chromatographed on a macroporous adsorption resin column, eluting with a mixture of C2H5OH–H2O to afford five fractions A–E. The fraction D (372 g) was subjected to column chromatography on HP-20 to yield six fractions (Fr. D1–D7). Fr. D5 (182 g) was performed on Sephadex LH-20, eluting with a mixture gradient of CH3OH–H2O (from 0:100 to 50:50) to afford 224 fractions (Fr. D5.1–D5.224). Among them, Fr. D5.41–D5.46 were purified by the reversed-phase preparative HPLC with CH3CN–H2O (23:77) to yield 1 (48 mg, 20.4 min). Fr. D5.62–D5.68 were combined and performed on the reversed-phase preparative HPLC with CH3CN–H2O (22:78) to yield 2 (7 mg, 27.9 min). Fr. D5.89–D5.98 were performed on the reversed-phase preparative HPLC with CH3CN–H2O (21:79) to yield 3 (12 mg, 28.5 min).
Table 2 Inhibition effects of compounds 1–3 (10 μM) on LPS-induced NO production. Group
Inhibition rate of NO (%)
Inhibition rate of Cell proliferation (%)
1 2 3 Curcumin
69.40 15.62 13.57 82.50
5.4 −3.2 1.7 8.4
3.3.1. Forsyphenside A (1)
20 -47.7 (c 0.10, CH3OH); UV D (CH3OH) λmax (log ε) 227 (4.16), 311 (4.41) nm; IR (KBr) νmax 3398, 2900, 1693, 1634, 1604, 1564, 1514, 1451, 1248, 1170, 1078 cm−1; 1 H NMR and 13C NMR data, see Table 1; HRESIMS m/z 619.1638 [M + Na]+ (calcd for C27H32O15Na, 619.1633). Yellow amorphous powder, [α]
3.3.2. Forsyphenside B (2)
Brown amorphous powder, [α]
Scheme 1. The possible biosynthetic pathway of triacetic acid lactone glycosides. 4
20 -52.9 (c 0.10, CH3OH); UV D
Carbohydrate Research 488 (2020) 107908
S. Shao, et al.
(CH3OH) λmax (log ε) 292 (4.55) nm; IR (KBr) νmax 3402, 2918, 1694, 1634, 1605, 1564, 1512, 1449, 1267, 1171, 1076 cm−1; 1H NMR and 13 C NMR data, see Table 1; HRESIMS m/z 943.2480 [M + Na]+ (calcd for C42H48O23Na, 943.2479).
Declaration of competing interest None. Acknowledgments
3.3.3. Forsyphenside C (3)
20 -81.1 (c 0.10, CH3OH); UV D (CH3OH) λmax (log ε) 222 (4.12), 292 (4.25) nm; IR (KBr) νmax 3404, 2903, 1692, 1636, 1605, 1565, 1512, 1450, 1245, 1175, 1075 cm−1; 1 H NMR and 13C NMR data, see Table 1; HRESIMS m/z 927.2514 [M + Na]+ (calcd for C42H48O22Na, 927.2529).
This work was financially supported by the National Natural Science Foundation of China (81673313) and the Innovation Fund for Medical Sciences (2016-I2M-1-010). The authors thank Yinghong Wang and Xiameng Jin for conducting the NMR experiments.
Brown amorphous powder, [α]
Appendix A. Supplementary data
3.4. Determination of the absolute configurations of sugars
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.carres.2020.107908.
Compound 1 (4 mg) was dissolved in 1 M HCl (2 mL) and incubated at 60 °C for 12 h. The mixture was concentrated under vacuum to produce a residue. The residue was suspended in water and extracted with EtOAc (3 × 2 mL). The aqueous layer was evaporated under vacuum to yield a residue. The residue was dissolved in 1 mL pyridine. LCysteine methyl ester hydrochloride (2 mg) was added, and the reaction was incubated at 60 °C for 2 h. Then, N-trimethylsilylimidazole (0.5 mL) was added after the mixture was evaporated. The mixture reaction was incubated at 60 °C for 2 h and partitioned between nhexane and water (3 × 2 mL). Compounds 2 and 3 were conducted as same as 1. Finally, the n-hexane extracts were subjected to GC analysis under the following conditions: capillary column, HP-5 (60 m × 0.25 mm, 0.25 μm, Dikma); detection temperature (FID), 300 °C; injection temperature, 300 °C; initial temperature, 200 °C; raised to 260 °C at a rate of 10 °C/min, and the final temperature was maintained for 30 min, then declined to 200 °C at a rate of 40 °C/min and the temperature was maintained for 1 min; carrier, N2. The D-glucose was confirmed by comparing the retention time of its derivative with the original sugar derivative in the same manner, which displayed retention time of 29.5 min.
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3.5. Anti-inflammatory activity assay of compounds 1–3 The BV2 cells were cultivated in DMEM with 10% fetal calf serum at 37 °C under 5% CO2 and 100% relative humidity for 24 h. After that, the cells in 96-well plates were treated with different samples (10 μM), following by adding in LPS and maintaining for another 24 h. Then, 100 μL aliquots of the supernatants were added to 100 μL of Griess reagent (0.1% naphthylethylenediamine and 1% sulfanilamide in a 5% H3PO4 solution) at room temperature for 20 min. NO production was measured by the concentration of nitrite in the supernatant. The absorbance was measured at 540 nm (curcumin as the positive control).
5
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
New triacetic acid lactone glycosides from the fruits of Forsythia suspensa and their nitric oxide production inhibitory activity
T
Siyuan Shao, Yanan Yang, Ziming Feng, Jianshuang Jiang, Peicheng Zhang∗ State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China
ARTICLE INFO
ABSTRACT
Keywords: Forsythia suspensa Triacetic acid lactone glycosides Forsyphensides A–C GC analysis NO production inhibitory activity
Three new triacetic acid lactone (TAL) glycosides, forsyphensides A–C (1–3) were isolated from the fruits of Forsythia suspensa. Their structures were elucidated by comprehensive spectroscopic techniques. The absolute configurations of their monosaccharides were determined by GC analysis. Notably, forsyphensides A–C were relatively rare TAL glycosides identified from plants. Compound 1 exhibited inhibitory activity against the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in microglia BV2 cells with the inhibition rate of 69.40%.
1. Introduction
2. Results and discussion
Natural products have continued to play an important role in drug discovery in human diseases for their novel structures and comprehensive bioactivities during the past years [1]. Forsythia suspensa, one kind of Chinese traditional medicine (TMC) coming from the Oleaceae family, contains various types of natural products for drug development [2]. Up to now, plenty of chemical constituents have been obtained from F. suspensa including phenylethanoid glycosides, lignans, terpenes, flavones and alkaloids [3–8]. Pharmacological studies demonstrated that F. suspensa possessed various bioactivities [8], among which antiinflammatory effect was the one attracted much attention [9–11]. During our research for seeking bioactive constituents from the n-butonal portion of the fruits of F. suspensa, three new compounds forsyphensides A–C (1–3) possessing a triacetic acid lactone (TAL) glycoside structure were obtained (Fig. 1, Experimental section in Supplementary material). To the best of our knowledge, they were relatively rare components isolated from higher plants [12,13]. Their structures were identified by comprehensive analysis of the spectroscopic data. The configurations of the monosaccharides were determined by GC analysis. The nitric oxide (NO) production inhibitory activities of compounds 1–3 were assayed on the lipopolysacchride (LPS)-induced NO production in microglia BV2 cells. Herein, we report the isolation, structural elucidation and bioactive evaluation of these isolates.
Compound 1 was isolated as a yellow amorphous powder with the molecular formula of C27H32O15 identified by the positive HRESIMS ion peak at m/z 619.1638 [M + Na]+ (calcd. for C27H32O15Na: 619.1633), suggesting 12 degrees of unsaturation (Fig. S4 in Supplementary material). The IR spectrum (Fig. S3 in Supplementary material) showed the presence of hydroxyl groups (3398 cm−1), carbonyl groups (1693 cm−1), olefinic bonds (1634 cm−1) and phenyl groups (1604, 1564 and 1514 cm−1). The 1H NMR data (Table 1) displayed four AA'BB’ system aromatic protons at δH 7.54 (d, 2H, J = 8.5 Hz, H-2′ and H-6′) and 6.77 (d, 2H, J = 8.5 Hz, H-3′ and H-5′); a pair of olefinic protons at δH 7.61 (d, 1H, J = 16.0 Hz, H-7′) and 6.37 (d, 1H, J = 16.0 Hz, H-8′); two single olefinic protons at δH 5.64 (d, 1H, J = 2.0 Hz, H-2) and 6.08 (d, 1H, J = 2.0 Hz, H-4); and one methyl group at δH 2.18 (s, 3H, H-6). In addition, two anomeric protons at δH 5.09 (d, 1H, J = 8.0 Hz, H-7), 4.38 (d, 1H, J = 8.0 Hz, H-13) and plenty of signals at δH 3.04–4.48 were observed, which suggested the existence of two β-sugars in compound 1. The 13C NMR data (Table 1) displayed 27 carbons, including six quaternary carbons, 18 methine carbons, two methylene carbons and one methyl carbon. Of them, 12 carbons were assigned as two hexoses combined with the HSQC spectrum (Fig. S7 in Supplementary material). They were determined to be D-glucose by GC analysis (Fig. S1 in Supplementary material) [14]. Furthermore, nine carbons were consisted of a 4-hydroxylcinnamoyl group through HMBC correlations of H-7′ to C-2′, H-8′ to C-1′ and C-9′
∗ Corresponding author. Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 2 Nan Wei Road, Xicheng District, Beijing, 100050, PR China. E-mail address: [email protected] (P. Zhang).
https://doi.org/10.1016/j.carres.2020.107908 Received 19 September 2019; Received in revised form 28 November 2019; Accepted 5 January 2020 Available online 07 January 2020 0008-6215/ © 2020 Published by Elsevier Ltd.
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Fig. 1. Structures of compounds 1–3 from F. suspensa.
Through detailed analysis of its 1H and 13C NMR data (Table 1) indicated that the structure of 3 was highly similar with that of 2. The main difference between them was that three ABX system protons at δH 7.14 (br s, 1H, H-2″), 7.08 (d, 1H, J = 8.0 Hz, H-5″) and 7.01 (br d, 1H, J = 8.0 Hz, H-6″) in 2 was replaced by four AA'BB’ system protons at δH 7.62 (d, 2H, J = 8.0, H-2″ and H-6″) and 7.04 (d, 2H, J = 8.0, H-3″ and H-5″) in 3. The difference indicated that the caffeoyl group of 2 was instead of by a 4-hydroxylcinnamyl group of 3. Therefore, the structure of 3 was built by the HMBC correlations of H-7 to C-3, H-13 to C-8, H18 to C-9′, H-12 to C-9″ and H-10″ to C-4′′ (Fig. 2) and named as forsyphenside C. Forsyphensides A–C (1–3) were three new triacetic acid lactone glycosides from the fruits of F. suspensa. Notably, they were relatively rare TAL glycoside derivatives isolated from higher plants. In the biosynthetic view, the key intermediate TAL unit may be derived from acetyl-CoA and malonyl-CoA via ester condensation and cyclization reactions catalyzed by ketoacyl synthases (Scheme 1) [16]. After the biosynthesis of TAL unit, the UDP (uridine diphosphate)-glucoses were first coupled with it by glycosyltransferase and then compound 1 was established by the combination of hydroxylcinnamoyl-CoA and the TAL di-glycosides via hydroxycinnamoyl transferase [17,18]. Compounds 2 and 3 were finally built by further reactions with caffeoyl-CoA/hydroxylcinnamoyl-CoA and UDP-glucoses. All the compounds were evaluated on the LPS induced NO production in microglia BV2 cells for their inhibitory activities [19]. As a result, compound 1 displayed inhibitory effect against the NO production with the inhibition rate of 69.40% at the concentration of 10 μM, which was close with the 82.50% inhibition of curcumin (10 μM) as the positive control (Table 2). However, compounds 2 and 3 just exhibited weak inhibitory effects. Analysis of the structural-activity relationship of the three compounds indicated that the extra caffeoyl/4-hydroxylcinnamoyl group and glucose may decrease their inhibitory activities against NO production.
(Fig. 2). The remaining six carbons were not assigned easily for their uncommon characteristic carbon chemical shifts. Fortunately, the HMBC correlations of H-6 to C-4 and C-5, H-4 to C-2 and H-2 to C-1 were observed in the HMBC spectrum (Fig. S8 in Supplementary material) and offered the key hints. Combination of the chemical shifts and their 2D NMR correlations suggested that the six carbons established a triacetic acid lactone moiety [15]. Then, a glucose was linked to C-3 of the TAL unit via the HMBC correlation of H-7 to C-3. Another glucose was connected to the former one through the correlation of H-13 to C-8. The 4-hydroxylcinnamoyl group was connected to C-18 of the latter glucose through H-18 to C-9′. Thus, the structure of 1 was established and named as forsyphenside A. Compound 2, a brown amorphous powder, gave the molecular formula of C42H48O23 by the HRESIMS signal at m/z 943.2480 [M + Na]+ (calcd. for C42H48O23Na: 943.2479) (Fig. S12 in Supplementary material). Comparison of its 1H and 13C NMR data (Table 1) with those of 1 showed three more ABX system aromatic protons at δH 7.14 (br s, 1H, H-2″), 7.08 (d, 1H, J = 8.0 Hz, H-5″) and 7.01 (br d, 1H, J = 8.0 Hz, H-6″); two more olefinic protons at δH 7.49 (d, 1H, J = 16.0 Hz, H-7″) and 6.32 (d, 1H, J = 16.0 Hz, H-8″), a more anomeric protons at δH 4.87 (d, 1H, J = 7.0 Hz, H-10″) and five more proton signals at δH 3.04–3.70, which indicated the presence of an extra caffeoyl group and a glucose and in 2. The HMBC correlations of H-7 to C-3, H-13 to C-7 and H-18 to C-9′ suggested the four identical units of 1 and 2 had the same connection type (Fig. 2). Subsequently, the caffeoyl group was linked to C-12 by the correlation of H-12 to C-9′′. The glucose was connected to C-4″ by the correlation of H-10″ to C-4′′. Consequently, the structure of 2 was established and named as forsyphenside B. Compound 3 was isolated as a brown amorphous powder. Its molecular formula of C42H48O22 was identified by the HRESIMS adduct peak at m/z 927.2514 [M + Na]+ (calcd. for C42H48O22Na: 927.2529), which was 16 mass less than 2 (Fig. S20 in Supplementary material). 2
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Table 1 1 H NMR (500 MHz) and13C NMR (125 MHz) data of compounds 1–3 in DMSO‑d6. Position
1
2
δH 1 2
5.64 (d, 2.0)
3 4
6.08 (d, 2.0)
5 6 7
2.18 5.09 8.0) 3.46 3.48 3.27 3.62
8 9 10 11 12 13 14 15 16 17 18 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 11″ 12″ 13″ 14″ 15″
7.54 (d, 8.5)
δC
δH
δC
δH
δC
163.6 89.9
5.63 (s)
163.6 89.9
5.64 (s)
163.6 89.9
168.9 99.7
6.06 (s)
168.9 99.7
6.06 (s)
168.9 99.7
(s) (d,
163.1 19.4 98.5
(m) (m) (m) (m)
79.1 74.1 72.7 75.1
3.70 (m) 3.64, m 4.38 (d, 8.0) 3.04 (m) 3.18 (m) 3.16 (m) 3.48 (m) 4.48 (m) 4.04, m
2.16 5.08 8.0) 3.45 3.46 3.17 3.70
59.6
6.77 (d, 8.5) 6.77 7.54 7.61 6.37
(d, (d, (d, (d,
8.5) 8.5) 16.0) 16.0)
(s) (d,
163.0 19.4 98.5
(m) (m) (m) (m)
79.1 74.1 69.9 73.9
4.48 (m) 4.10, m 4.39 (d, 8.0) 3.04 (m) 3.19 (m) 3.25 (m) 3.45 (m) 4.40 (m) 4.21, m
102.7 73.2 76.2 69.9 73.9 63.5 125.0 130.4
3
115.8 160.0 115.8 130.4 145.2 113.7 166.5
6.79 (d, 8.0) 6.79 7.54 7.52 6.38
(d, (d, (d, (d,
7.54 (d, 8.0)
8.0) 8.0) 16.0) 16.0)
7.14 (br s) 7.08 7.01 7.49 6.32
(d, 8.0) (br d, 8.0) (d, 16.0) (d, 16.0)
4.87 (d, 7.0) 3.04 (m) 3.33 (m) 3.26 (m) 3.63 (m) 3.70 (m) 3.62, m
3. Experimental section
63.7 102.7 73.2 76.2 69.9 73.8 63.2
115.9 160.1 115.9 130.4 145.0 113.8 166.5 128.6 115.2 146.8 147.2 115.6 120.7 144.8 114.9 166.3 101.1 73.2 75.6 72.6 75.1 59.6
124.9 130.4
2.17 5.09 8.0) 3.46 3.44 3.17 3.71
(s) (d,
163.0 19.4 98.6
(m) (m) (m) (m)
79.2 74.1 70.0 73.8
4.51 (m) 4.08, m 4.40 (d, 8.5) 3.05 (m) 3.19 (m) 3.25 (m) 3.53 (m) 4.40 (m) 4.17, m 7.52 (d, 8.5)
63.7 102.7 73.1 76.2 69.8 73.8 63.3
6.78 (d, 8.5) 6.78 7.52 7.51 6.36
(d, (d, (d, (d,
8.5) 8.5) 16.0) 16.0)
7.62 (d, 8.0) 7.04 (d, 8.0) 7.04 7.62 7.61 6.40
(d, (d, (d, (d,
8.0) 8.0) 16.0) 16.0)
5.01 (d, 7.5) 3.04 (m) 3.30 (m) 3.25 (m) 3.63 (m) 3.71 (m) 3.61, m
124.8 130.4
115.9 160.2 115.9 130.4 145.0 113.8 166.5 127.8 130.0 116.4 158.9 116.4 130.0 144.4 116.2 166.2 99.6 73.2 75.1 72.6 76.2 59.6
experiments were performed using an Agilent 7890A series system with a capillary column, HP-5 (60 m × 0.25 mm, with a 0.25 μm film; Dikma Technologies Inc., Beijing, People's Republic of China).
3.1. General experimental procedures UV spectra were collected by a JASCO V650 spectrometer (Thermo Scientific, Waltham, MA, USA). Optical rotations were recorded by a JASCO P-2000 polarimeter (JASCO, Easton, MD, USA). IR spectra were performed on a Nicolet 5700 spectrometer (Thermo Scientific, Waltham, MA, USA). The HRESIMS trials were obtained by an Agilent 6520 LC-QTOF mass spectrometer (Agilent Technologies, Waldbronn, Germany). The 1D and 2D NMR spectra were recorded by Bruker 500 MHz spectrometer and the values were given in ppm (Bruker-Biospin, Billerica, MA, USA). Column chromatography was performed on macroporous resin (Diaion HP-20, Mitsubishi Chemical Corp., Tokyo, Japan) and Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden). Preparative HPLC was a Shimadzu LC-10AT equipped with an SPD-10A detector (Shimadzu Corp., Tokyo, Japan) using a YMC-Pack ODS-A column (250 mm × 20 mm, 5 μm; YMC Corp., Kyoto, Japan). GC
3.2. Plant material The fruits of Forsythia suspensa were collected in December 2011 from Yuncheng City of Shanxi Province, People's Republic of China. It was identified by Lin Ma (Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing). A voucher specimen (ID-S-2597) has been deposited at the Herbarium of Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing. 3.3. Extraction and isolation The dried fruits of Forsythia suspensa (90 kg) were shattered and extracted with 75% C2H5OH for three times under reflux and filtered to 3
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Fig. 2. Key HMBC (H→C) and TOCSY (–) correlations of compounds 1–3.
yield a crude extract (12.6 kg). The extract was suspended in water and successively separated by petroleum ether, ethyl acetate and n-butanol, respectively. The n-butanol section (4 kg) was then suspended in water to obtain an aqueous layer (1.5 kg). The layer was concentrated to yield a water-soluble portion and then chromatographed on a macroporous adsorption resin column, eluting with a mixture of C2H5OH–H2O to afford five fractions A–E. The fraction D (372 g) was subjected to column chromatography on HP-20 to yield six fractions (Fr. D1–D7). Fr. D5 (182 g) was performed on Sephadex LH-20, eluting with a mixture gradient of CH3OH–H2O (from 0:100 to 50:50) to afford 224 fractions (Fr. D5.1–D5.224). Among them, Fr. D5.41–D5.46 were purified by the reversed-phase preparative HPLC with CH3CN–H2O (23:77) to yield 1 (48 mg, 20.4 min). Fr. D5.62–D5.68 were combined and performed on the reversed-phase preparative HPLC with CH3CN–H2O (22:78) to yield 2 (7 mg, 27.9 min). Fr. D5.89–D5.98 were performed on the reversed-phase preparative HPLC with CH3CN–H2O (21:79) to yield 3 (12 mg, 28.5 min).
Table 2 Inhibition effects of compounds 1–3 (10 μM) on LPS-induced NO production. Group
Inhibition rate of NO (%)
Inhibition rate of Cell proliferation (%)
1 2 3 Curcumin
69.40 15.62 13.57 82.50
5.4 −3.2 1.7 8.4
3.3.1. Forsyphenside A (1)
20 -47.7 (c 0.10, CH3OH); UV D (CH3OH) λmax (log ε) 227 (4.16), 311 (4.41) nm; IR (KBr) νmax 3398, 2900, 1693, 1634, 1604, 1564, 1514, 1451, 1248, 1170, 1078 cm−1; 1 H NMR and 13C NMR data, see Table 1; HRESIMS m/z 619.1638 [M + Na]+ (calcd for C27H32O15Na, 619.1633). Yellow amorphous powder, [α]
3.3.2. Forsyphenside B (2)
Brown amorphous powder, [α]
Scheme 1. The possible biosynthetic pathway of triacetic acid lactone glycosides. 4
20 -52.9 (c 0.10, CH3OH); UV D
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(CH3OH) λmax (log ε) 292 (4.55) nm; IR (KBr) νmax 3402, 2918, 1694, 1634, 1605, 1564, 1512, 1449, 1267, 1171, 1076 cm−1; 1H NMR and 13 C NMR data, see Table 1; HRESIMS m/z 943.2480 [M + Na]+ (calcd for C42H48O23Na, 943.2479).
Declaration of competing interest None. Acknowledgments
3.3.3. Forsyphenside C (3)
20 -81.1 (c 0.10, CH3OH); UV D (CH3OH) λmax (log ε) 222 (4.12), 292 (4.25) nm; IR (KBr) νmax 3404, 2903, 1692, 1636, 1605, 1565, 1512, 1450, 1245, 1175, 1075 cm−1; 1 H NMR and 13C NMR data, see Table 1; HRESIMS m/z 927.2514 [M + Na]+ (calcd for C42H48O22Na, 927.2529).
This work was financially supported by the National Natural Science Foundation of China (81673313) and the Innovation Fund for Medical Sciences (2016-I2M-1-010). The authors thank Yinghong Wang and Xiameng Jin for conducting the NMR experiments.
Brown amorphous powder, [α]
Appendix A. Supplementary data
3.4. Determination of the absolute configurations of sugars
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.carres.2020.107908.
Compound 1 (4 mg) was dissolved in 1 M HCl (2 mL) and incubated at 60 °C for 12 h. The mixture was concentrated under vacuum to produce a residue. The residue was suspended in water and extracted with EtOAc (3 × 2 mL). The aqueous layer was evaporated under vacuum to yield a residue. The residue was dissolved in 1 mL pyridine. LCysteine methyl ester hydrochloride (2 mg) was added, and the reaction was incubated at 60 °C for 2 h. Then, N-trimethylsilylimidazole (0.5 mL) was added after the mixture was evaporated. The mixture reaction was incubated at 60 °C for 2 h and partitioned between nhexane and water (3 × 2 mL). Compounds 2 and 3 were conducted as same as 1. Finally, the n-hexane extracts were subjected to GC analysis under the following conditions: capillary column, HP-5 (60 m × 0.25 mm, 0.25 μm, Dikma); detection temperature (FID), 300 °C; injection temperature, 300 °C; initial temperature, 200 °C; raised to 260 °C at a rate of 10 °C/min, and the final temperature was maintained for 30 min, then declined to 200 °C at a rate of 40 °C/min and the temperature was maintained for 1 min; carrier, N2. The D-glucose was confirmed by comparing the retention time of its derivative with the original sugar derivative in the same manner, which displayed retention time of 29.5 min.
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3.5. Anti-inflammatory activity assay of compounds 1–3 The BV2 cells were cultivated in DMEM with 10% fetal calf serum at 37 °C under 5% CO2 and 100% relative humidity for 24 h. After that, the cells in 96-well plates were treated with different samples (10 μM), following by adding in LPS and maintaining for another 24 h. Then, 100 μL aliquots of the supernatants were added to 100 μL of Griess reagent (0.1% naphthylethylenediamine and 1% sulfanilamide in a 5% H3PO4 solution) at room temperature for 20 min. NO production was measured by the concentration of nitrite in the supernatant. The absorbance was measured at 540 nm (curcumin as the positive control).
5