New Iridoid Glycosides with Antidepressant Activity Isolated from Cyperus rotundus

Abstract

Based on bioactive screening results, two new iridoid glycosides, named rotunduside G (1) and rotunduside H (2), were isolated from the rhizomes of Cyperus rotundus, together with four known ones, negundoside (3), nishindaside (4), isooleuropein (5) and neonuezhenide (6). Their structures were elucidated on the basis of spectroscopic methods and from literature values. In mice models of despair, 1 and 2 showed significant antidepressant activity.

Depression which is associated with substantial disability is a major public health issue world-wide, with a high lifetime prevalence ranging from 2 to 15%.¹⁾ Recently, more herbal medicine has been used as alternative therapy for depression.^2–4) Due to its natural constituent and availability, natural herbs which obtained from natural sources are believed to provide less untoward effect profiles and provide greater effectiveness as compared to synthetic drug available over the market.⁵⁾ The rhizome of Cyperus rotundus is a kind of traditional Chinese medicine named “Xiangfuzi,” which is widely used in folk medicine as an anti-inflammatory, antidepressant, analgesic, and antiemetic remedy for dysentery and women’s diseases.^6,7) In previous research, several phenolic glycosides with antidepressant-like effect from the rhizomes of Cyperus rotundus by our group.^7–10) In continuation of our search for new natural phenolic glycosides from this medicine plant, which has been proven to possess antidepressant activity, two new iridoid glycosides, rotunduside G (1) and rotunduside H (2), were isolated from the rhizomes of Cyperus rotundus, with four known iridoid glycosides (3–6). The latter were identified with negundoside (3), nishindaside (4), isooleuropein (5) and neonuezhenide (6)^11–14) (Fig. 1). In this paper, we evaluated the antidepressant-like effect of these iridoid glycosides using two classical behavioral models of antidepressants screening known as forced swimming test (FST) and tail suspension test (TST).

Fig. 1. Chemical Structures of Compounds 1–6 Isolated from the Rhizomes of Cyperus rotundus

Results and Discussion

The phytochemical study of 95% aqueous ethanol extract obtained from the rhizomes of Cyperus rotundus afforded six compounds. The structures of new compounds, rotunduside G (1) and rotunduside H (2) were determined by the one dimensional (1D)- and 2D-NMR elucidations, and mass spectral analysis.

Rotunduside G (1) was obtained as a white amorphous powder. Its molecular formula was assigned as C₄₃H₅₆O₂₅ on the basis of positive-ion high resolution electrospray ionization mass spectrometry (HR-ESI-MS) (m/z 995.3009 [M+Na]⁺) and ¹³C-NMR data. Infrared (IR) spectrum showed the absorption bands for hydroxyl (3200–3445 cm⁻¹), phenyl (1612, 1590 cm⁻¹) and α,β-unsaturated carboxyl (1699 cm⁻¹) groups. Positive result of Wieffering field test indicated that 1 could be an iridoid.¹⁵⁾ The following key proton signals were obviously observed in the ¹H-NMR spectra of 1: five olefinic protons at δ 7.55 (1H, d, J=16 Hz), 7.63 (1H, s), 6.29 (1H, d, J=16 Hz), 5.77 (1H, dq, J=2.1, 2.0, 2.0, 2.0 Hz), and 5.37 (1H, t, J=6.8 Hz), four oxygenated methylene groups at δ 4.24, 4.19 (each 1H, d, J=14 Hz), 4.17 (1H, dd, J=12.0, 2.0 Hz), 3.86 (1H, m), 4.53 (1H, dd, J=12.1, 2.0 Hz), 4.26 (1H, dd, J=12.1, 6.1 Hz), and 4.56 (2H, d, J=6.8 Hz), a methoxyl proton at δ 3.58 (3H, s), two aromatic protons at δ 6.69 (2H, s), and three anomeric protons at δ 4.79 (1H, d, J=7.8 Hz), 4.48 (1H, d, J=7.6 Hz) and 4.88 (1H, d, J=7.6 Hz). The ¹³C-NMR spectra of 1 also displayed a pair of signals due to the α,β-unsaturated ester groups at δ 168.6, 167.6, 150.3, 147.8, 115.3 and 111.6, and three anomeric carbons at δ 100.5, 103.9 and 102.8. Acid hydrolysis of 1 yielded D-glucose and D-glucuronic acid, which were identified by comparison with the respective authentic samples by gas chromatography (GC) analysis.

Comparison of ¹H- and ¹³C-NMR spectra of 1 with those of rotunduside B⁷⁾ indicated that 1 had one more glucuronic acid [δ_H 4.88 (1H, d, J=7.6 Hz); δ_C 102.8, 72.9, 75.3, 71.6, 74.8, 171.7]¹⁶⁾ attached at C-4‴ (δ_C 141.3). The suggestion was in accord with the observation of the downfield shift of C-4‴ signal from δ 140.1 in rotunduside B to δ 141.3 in 1. This was further established by the heteronuclear multiple bond correlation (HMBC) correlation from H-1″″ [δ_H 4.88 (1H, d, J=7.6 Hz)] to C-4‴ (δ_C 141.3). Meantime, the occurrence of a prenyl group in the molecule could be easily deduced from the ¹H- and ¹³C-NMR spectra [δ_H 4.56 (2H, d, J=6.8 Hz), 5.37 (1H, t, J=6.8 Hz), 1.68 (3H, s), 1.77 (3H, s); δ_C 60.8, 121.7, 138.4, 25.9, and 18.1].¹⁷⁾ The detailed 2D-NMR analysis of ¹H–¹H correlated spectroscopy (¹H–¹H-COSY) and HMBC correlations also implied that 1 had a prenyl group (Fig. 2). Moreover, the prenyl group was attached to the C-6″″ (δ_C 171.7) position of the glucuronic acid, which was supported by the HMBC correlation between H-1″‴ [δ_H 4.56 (2H, d, J=6.8 Hz)] to C-6″″ (δ_C 171.7) (Fig. 2). Therefore, the structure of 1, which was established as shown in 1, is a new natural compound, which we named rotunduside G.

Fig. 2. Key HMBC and ¹H–¹H-COSY Correlations of Compounds 1 and 2

Rotunduside H (2) was isolated as a white amorphous powder. Its HR-ESI-MS exhibited a quasi-molecular ion peak at m/z 1011.2954 (calculated value 1011.2958) [M+Na]⁺, which indicated a molecular formula of C₄₃H₅₆O₂₆. The ¹H-NMR spectrum of 2 exhibited signals due to four olefinic protons at δ 7.63 (1H, s), 7.54 (1H, d, J=16 Hz), 6.29 (1H, d, J=16 Hz), 5.79 (1H, dq, J=2.1, 2.0, 2.0, 2.0 Hz), along with four oxygenated methylene groups at δ 4.23, 4.17 (each 1H, d, J=13.8 Hz), 4.52 (1H, dd, J=12.0, 2.0 Hz), 4.27 (1H, dd, J=12.0, 6.1 Hz), 4.15 (1H, dd, J=12.4, 2.2 Hz), 3.87 (1H, dd, J=12.4, 6.6 Hz), 5.66 (2H, s). The signals of three anomeric protons at δ 4.81 (1H, d, J=7.5 Hz), 4.49 (1H, d, J=7.8 Hz), 4.89 (1H, d, J=7.4 Hz) were also found. Acid hydrolysis of 2 was performed in the same manner as that of 1 and D-glucose and D-glucuronic acid as sugar residue were identified by GC analysis from 2. Analysis of the ¹³C-NMR spectrum revealed 43 carbon signals, including one carbonyl at δ_C 205.8 and three anomeric carbon signals at δ_C 100.7, 104.1, 102.9. A pair of α,β-unsaturated ester signals were found at δ 168.8, 167.8, 150.3, 147.9, 115.5, 111.4. The ¹H- and ¹³C-NMR spectroscopic data of 2 were similar to those of 1, with the exception of a 3″‴-methyl-2″‴-oxobutyl group [δ_H 5.66 (2H, s), 2.87 (1H, m), 1.23 (6H, d, J=8.0 Hz); δ_C 68.4, 205.8, 36.7, 17.4, 17.4] at C-6″″ in 2, instead of a prenyl group in 1. This was revealed by the ¹H–¹H correlations of the spin system H-3″‴/H-4″‴/H-5″‴ as well as the HMBC correlations from H-1″‴ to C-6″″, C-2″‴, and C-3″‴, from H-3″‴ to C-1″‴, C-2″‴, C-4″‴, and C-5″‴ (Fig. 2). Thus, the structure of 2, which was established as shown in 2, is a new natural compound, which we named rotunduside H.

Compounds (1–6) were evaluated for their antidepressant activities on a forced swimming test (FST) and a tail suspension test (TST) in mice. In the despair mice models, rotunduside G (1) and rotunduside H (2) displayed significant antidepressant activity at the dosage of 50 mg/kg intragastrically (i.g.) (Table 1), which was close to the positive control fluoxetine (20 mg/kg). The other compounds showed weak antidepressant activity.

Table 1. Effect of Compounds 1–6 on the Forced Swimming Test (FST) and Tail Suspension Test (TST) in Mice (Mean±S.E.M.)

Sample	n	FST immobility duration (s)	Reduction (%)	TST immobility duration (s)	Reduction (%)
Control	8	90.3±36.1		114.6±19.9
Fluoxetine	8	33.5±12.9**	62.9	44.9±17.8**	60.8
1	8	34.1±14.3**	62.2	49.6±23.1**	56.7
2	8	36.7±11.8**	59.4	54.8±20.9**	52.2
3	8	66.4±21.4	26.5	99.1±25.3	13.5
4	8	89.1±16.6	1.3	100.4±27.5	12.4
5	8	71.3±18.5	21.0	97.3±24.4	15.1
6	8	83.8±14.7	7.2	89.6±29.1	21.8

** p<0.01, significant as compared to the control group.

Experimental

General

UV spectra were recorded on a Hewlett-Packard HP-845 UV-VIS spectrophotometer (Palo Alto, U.S.A.). Optical rotations were measured using a JASCO P-1010 digital polarimeter (Japan) and IR spectra were obtained from a PerkinElmer, Inc. Spectrum One FT-IR spectrometer (England). MS on a Finnigan LCQ Advantage Spectrometer (Thermo Scientific, U.S.A.) and a Shimadzu GC-MS model QP2010 Plus spectrophotometer (Japan), respectively. NMR spectra were recorded on 400 MHz FT-NMR spectrometer (Varian Inova AS 400, U.S.A.), and deuterium solvents for NMR were purchased from Merck Co., Ltd. Chemical shifts are given as δ values with reference to tetramethylsilane (TMS) as internal standard. Column chromatography separations were carried out on silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, P. R. China), ODS (50 mesh, AA12S50, YMC), MCI-gel CHP 20P (35–75 µm, Japan) and Diaion HP-20 (Pharmacia, Peapack, New Jersey, U.S.A.). The thin layer chromatography (TLC) analysis was carried out using a Kiesel gel 60 F₂₅₄ and RP-18 F_254S plates (Merck), and UV lamp (Spectroline Model ENF-240 C/F, Spectronics Corporation, U.S.A.) and 10% H₂SO₄ solution were used for detection. All other chemicals and reagents of analytical grade were obtained from Sigma-Aldrich, unless indicated otherwise.

Plant Materials

The rhizomes of Cyperus rotundus were collected in Zhanjiang, Guangdong Province of China in September 2009, and were identified by Professor Wenqing Yin (School of Chemistry & Chemical Engineering of Guangxi Normal University, Ministry of Education Key Laboratory of Chemistry and Molecular Engineering of Medicinal Resource, Guilin). A voucher specimen (No. 20090903) has been deposited in the authors’ laboratory.

Extraction and Isolation

The dry rhizomes of Cyperus rotundus (10 kg) were extracted three times under reflux with 95% aqueous EtOH (150 L×2 h). After removing the solvent under reduced pressure, the residue was suspended in water and then sequentially extracted with petroleum ether, CH₂Cl₂, EtOAc and n-BuOH. The n-BuOH extract (152 g) was submitted through a column chromatography (CC) of high porous absorption resin (Diaion HP-20), eluting with H₂O and CH₃OH. The methanol fraction (98 g) was repeatedly CC over normal and reverse phase silica gel to afford four fractions (Frs. 1–4). Fraction 1 was subjected to ODS CC eluting with CH₃OH–H₂O (0 : 1–1 : 0) and silica gel with CHCl₃–MeOH–H₂O (9 : 1 : 0.1–8 : 2 : 0.2) to give compound 3 (26 mg). Fraction 2 was subjected to ODS CC eluting with CH₃OH–H₂O (0 : 1–1 : 0 and silica gel with CHCl₃–MeOH–H₂O (8.5 : 1.5 : 0.15–7 : 3 : 0.3) to give compounds 4 (28 mg), 5 (19 mg), and 6 (25 mg). Fraction 3 was subjected to ODS CC eluting with CH₃OH–H₂O (7 : 3) and silica gel with CHCl₃–MeOH–H₂O (8 : 2 : 0.2–7 : 3 : 0.3) to give compound 1 (24 mg) and 2 (19 mg).

Rotunduside G (1)

White amorphous powder; [α]_D²⁵ −69.6 (c=1.0, MeOH); IR ν_max (KBr): 3200–3445, 1601, and 1697 cm⁻¹. UV (MeOH) λ_max (log ε): 326 (4.68), 305 (4.47), and 238 (4.51) nm; HR-ESI-MS m/z: 995.3009 [M+Na]⁺ (Calcd for C₄₃H₅₆O₂₅Na, 995.3006). ¹H-NMR (400 MHz, C₅D₅N) δ: 5.27 (1H, d, J=7.5 Hz, H-1), 7.63 (1H, s, H-3), 3.16 (1H, dt, J=8.0, 8.0, 2.4 Hz, H-5), 2.39 (1H, ddd, J=18.4, 2.4, 2.1 Hz, H-6a), 2.80 (1H, ddd, J=18.4, 8.0, 2.0 Hz, H-6b), 5.77 (1H, dq, J=2.1, 2.0, 2.0, 2.0 Hz, H-7), 3.04 (1H, t, J=7.7 Hz, H-9), 4.24, 4.19 (each 1H, d, J=14 Hz, H-10), 3.58 (3H, s, H-12), 4.79 (1H, d, J=7.8 Hz, H-1′), 3.11–3.68 (4H, m, H-2′,3′,4′,5′), 4.17 (1H, dd, J=12.0, 2.0 Hz, H-6′a), 3.86 (1H, m, H-6′b), 4.48 (1H, d, J=7.6 Hz, H-1″), 3.30–3.57 (4H, m, H-2″,3″,4″,5″), 4.53 (1H, dd, J=12.1, 2.0 Hz, H-6″a), 4.26 (1H, dd, J=12.1, 6.1 Hz, H-6″b), 6.69 (2H, s, H-2‴, H-6‴), 7.55 (1H, d, J=16 Hz, H-α), 6.29 (1H, d, J=16 Hz, H-β), 4.88 (1H, d, J=7.6 Hz, H-1″″), 4.29 (1H, d, J=8.1 Hz, H-5″″), 4.56 (2H, d, J=6.8 Hz, H-1″‴), 5.37 (1H, t, J=6.8 Hz, H-2″‴), 1.68 (3H, s, H-4″‴), 1.77 (3H, s, H-5″‴). ¹³C-NMR (100 MHz, C₅D₅N) δ: 98.0 (C-1), 150.3 (C-3), 111.6 (C-4), 35.5 (C-5), 39.3 (C-6), 127.1 (C-7), 147.2 (C-8), 46.7 (C-9), 60.9 (C-10), 167.7 (C-11), 51.9 (C-12), 100.5 (C-1′), 75.3 (C-2′), 78.4 (C-3′), 71.8 (C-4′), 78.0 (C-5′), 70.1 (C-6′), 103.9 (C-1″), 75.3 (C-2″), 77.9 (C-3″), 71.2 (C-4″), 74.6 (C-5″), 64.4 (C-6″), 128.6 (C-1‴), 107.1(2C, C-2‴, C-6‴), 154.4 (C-3‴), 141.3 (C-4‴), 154.4 (C-5‴), 168.6 (C=O), 115.3 (C-α), 147.8 (C-β), 102.8 (C-1″″), 72.9 (C-2″″), 75.3 (C-3″″), 71.6 (C-4″″), 74.8 (C-5″″), 171.7 (C-6″″), 60.8 (C-1″‴), 121.7 (C-2″‴), 138.4 (C-3″‴), 25.9 (C-4″‴) 18.1 (C-5″‴).

Rotunduside H (2)

White amorphous powder; [α]_D²⁵ −71.3 (c=1.0, MeOH); IR ν_max (KBr): 3200–3445, 1600, 1698 cm⁻¹; UV (MeOH) λ_max (log ε): 325 (4.63), 306 (4.44), and 236 (4.49) nm; HR-ESI-MS m/z: 1011.2954 [M+Na]⁺ (Calcd for C₄₃H₅₆O₂₆Na, 1011.2958). ¹H-NMR (400 MHz, C₅D₅N) δ: 5.28 (1H, d, J=7.5 Hz, H-1), 7.63 (1H, s, H-3), 3.15 (1H, dt, J=8.0, 8.0, 2.4 Hz, H-5), 2.40 (1H, ddd, J=18.4, 2.4, 2.1 Hz, H-6a), 2.82 (1H, ddd, J=18.4, 8.0, 2.0 Hz, H-6b), 5.79 (1H, dq, J=2.1, 2.0, 2.0, 2.0 Hz, H-7), 3.06 (1H, t, J=7.8 Hz, H-9), 4.23, 4.17 (each 1H, d, J=13.8 Hz, H-10), 3.60 (3H, s, H-12), 4.81 (1H, d, J=7.5 Hz, H-1′), 3.13–3.69 (4H, m, H-2′,3′,4′,5′), 4.15 (1H, dd, J=12.4, 2.2 Hz, H-6′a), 3.87 (1H, dd, J=12.4, 6.6 Hz, H-6′b), 4.49 (1H, d, J=7.8 Hz, H-1″), 3.31–3.58 (4H, m, H-2″,3″,4″,5″), 4.52 (1H, dd, J=12.0, 2.0 Hz, H-6″a), 4.27 (1H, dd, J=12.0, 6.1 Hz, H-6″b), 6.70 (2H, s, H-2‴, H-6‴), 7.54 (1H, d, J=16 Hz, H-α), 6.29 (1H, d, J=16 Hz, H-β), 4.89 (1H, d, J=7.4 Hz, H-1″″), 4.31 (1H, d, J=8.0 Hz, H-5″″), 5.66 (2H, s, H-1″‴), 2.87 (1H, m, H-3″‴), 1.23 (6H, d, J=8.0 Hz, H-4″‴, H-5″‴). ¹³C-NMR (100 MHz, C₅D₅N) δ: 98.1 (C-1), 150.3 (C-3), 111.4 (C-4), 35.3 (C-5), 39.1 (C-6), 127.3 (C-7), 147.1 (C-8), 46.6 (C-9), 60.9 (C-10), 167.8 (C-11), 51.8 (C-12), 100.7 (C-1′), 75.4 (C-2′), 78.6 (C-3′), 71.7 (C-4′), 78.3 (C-5′), 70.1 (C-6′), 104.1 (C-1″), 75.5 (C-2″), 77.8 (C-3″), 71.1 (C-4″), 74.5 (C-5″), 64.6 (C-6″), 128.8 (C-1‴), 107.3 (2C, C-2‴, C-6‴), 154.5 (C-3‴), 141.4 (C-4‴), 154.5 (C-5‴), 168.8 (C=O), 115.5 (C-α), 147.9 (C-β), 102.9 (C-1″″), 73.0 (C-2″″), 75.3 (C-3″″), 71.6 (C-4″″), 74.9 (C-5″″), 171.9 (C-6″″), 68.4 (C-1″‴), 205.8 (C-2″‴), 36.7 (C-3″‴), 17.4 (2C, C-4″‴, C-5″‴).

Animals and Drug Administration

Male NIH mice (18–25 g) were obtained from the Medical Animal Center, Guangzhou University of Chinese Medicine, Guangdong Province, China. The animals were kept in a 12 h light/dark cycle at ambient temperature of 23±2°C with free access to standard laboratory food and tap water. The extract and standard drug were suspended in 0.5% carboxymethyl cellulose aqueous solution (vehicle), and were given orally 1 h before the experiments in 15 mL/kg. Control animals received vehicle under the same conditions. Compounds were given i.g. at dose of 50 mg/kg. Fluoxetine at dose of 20 mg/kg was given intraperitoneally (i.p.) in the forced swimming test (FST) and tail suspension test (TST). Experiments were conducted between 8:00–16:00. The mice were just used once. Each group in the behavioral experiment was consisted of 8 animals. Experiments were carried out in compliance with the Experimental Animal Management Bill of the November 14th 1988 Decree No. 2 of National Science and Technology Commission, China.

FST

The test was conducted according to the reported methodology originally described by porsolt with minor modification.¹⁸⁾ Briefly, mice were individually forced to swim in an open cylindrical container (diameter 14 cm, height 20 cm), with a depth of 15 cm of water at 25±1°C. The immobility time was observed by unaided eyes of a trained observer, who was blind to the experimental conditions, during the last 4 min of a single 6-min test session. Mice were considered immobile when they did not further attempt to escape except the movements necessary to keep their heads above the water.

TST

The mice were suspended by their tails, and the cumulative period of immobility during an observation period of 6 min was measured.¹⁹⁾ Mice both acoustically and visually isolated were suspended 50 cm above the floor by adhesive tape placed approximately 1 cm from the tip of the tail. Immobility was defined as the absence of any limb or body movements, except for those caused by respiration or when they hung passively and completely motionless. The parameter obtained was the number of seconds spent immobile. Parameter used was the number of seconds spent immobile. The total immobility period was scored manually during 6 min test session with the help of stop-watch. % Reduction=100−(Mean immobility time of test mouse/Mean immobility time of control×100).

Acid Hydrolysis of Compounds 1 and 2

Each compound (1.0 mg) was heated at 95°C with dioxane (0.5 mL) and 5% H₂SO₄ (0.5 mL) for 1 h. After neutralization with Amberlite IRA-400 (OH⁻ form), each reaction mixture was concentrated and the residue was passed through a Sep-Pak C₁₈ cartridge with H₂O. The eluate was concentrated and the residue was treated with L-cysteine methyl ester hydrochloride (1 mg) in pyridine (0.125 mL) at 60°C for 1 h. The solution was then treated with N,O-bis(trimethylsilyl)trifluoroacetamide (0.05 mL) at 60°C for 1 h. The supernatant was applied to GC; GC conditons: column, Supelco SPB™-1, 30 m×0.25 mm; column temperature, 230°C; N₂ flow rate, 0.8 mL/min; t_R, 22.80 min (D-glucose), 22.01 min (L-glucose), 22.87 min (D-glucuronic acid), 22.09 min (L-glucuronic acid).^20,21) D-Glucose and D-glucuronic acid were all detected from compounds 1 and 2.

Statistical Analysis

Data analysis was performed by one-way analysis of variance with the Dunnett’s post-hoc test for multiple comparisons by SPSS 10.0 software. Data were expressed as the mean±standard error of the mean (S.E.M.). The level of statistical significance was set at p<0.05.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (31400295), the China Spark Program (2014GA780014), Science and Technology Planning Project of Guangdong Province (2014A020221057).

Conflict of Interest

The authors declare no conflict of interest.

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