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Abstract

The monoterpenoid glucoindole alkaloids comprise a large class of structurally complex natural products that have attracted widespread attention owing to their biological activities and structural diversity. In the present study, we investigated the constituents of a methanol extract of Dipsacus asper roots and obtained a new monoterpenoid glucoindole alkaloid, (3R,5S)-5-carboxyvincosidic acid 22-loganin ester (1), together with the known alkaloid (3S,5S)-5-carboxystrictosidic acid 22-loganin ester (dipsaperine) (2). We herein report our structural elucidation of 1 by spectroscopic analysis and its anti-inflammatory activity as revealed by its inhibition of nitric oxide production in lipopolysaccharide-activated RAW264.7 cells.

Keywords

monoterpenoid glucoindole alkaloid NO production inhibitory activity

Dipsacus asper Wall (Dipsacaceae) is a perennial herb that is widely distributed in the mountainous regions of China, Korea, and Japan.¹ Dipsacus asper root is a well-known traditional medicine and has been used as a tonic, an analgesic, and an anti-inflammatory agent in the treatment of spermatorrhea and pain.² It is also a common ingredient in formulations used to treat bone fractures and promote liver and kidney function.² It contains iridoid glycosides, triterpenoid saponins, alkaloids, and phenolic compounds,¹ imbuing it with a range of biological activities such as anti-inflammatory,³ antioxidant,⁴ anticomplementary,⁵ apoptosis-inducing,⁶ cytotoxic,⁷ inhibition of Aβ-induced cytotoxicity,^8,9 antinociceptive,¹⁰ cardioprotective,¹¹ and osteoprotective effects.^12,13

In a previous study on the anti-inflammatory constituents of traditional medicinal plants, we analyzed the water extract of D. asper roots, which showed significant half-maximal inhibitory activity (IC₅₀ = 45.1 µg/mL) against the production of nitric oxide (NO) in lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells. Through bioassay-guided fractionation of the water extract, we isolated a new pyridine alkaloid-coupled iridoid glucoside (dipasperoside A), a new trisiridoid glucoside (dipasperoside B), and a known monoterpene indole alkaloid (3β,5α-tetrahydrodesoxycordifoline lactam), as well as 8 known iridoids (cantleyoside, dipsanosides A and B, loganic acid, loganin, sweroside, sylvestroside I, triplostoside A), 4 phenolics (3,4-dihydroxybenzaldehyde, 3,4-dihydroxybenzoic acid, caffeic acid, and vanillic acid), 3 known quinic acid derivatives (3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid), and 4 known saponins (akebia saponin D, 4′-O-acetyl-akebia saponin D, dipsacus saponin A, and 3-O-α-l-arabinopyranosyl-28-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyloleanolic acid).^14,15 Thereafter, we analyzed their NO production inhibitory activity against LPS-activated RAW264.7 cells and identified dipasperoside A (IC₅₀ = 15.2 µM) and akebia saponin D (IC₅₀ = 12.7 µM) as active constituents.¹⁴

In the present study, as part of our ongoing phytochemical investigation of this plant, we isolated 2 monoterpenoid glucoindole alkaloids from the methanol (MeOH) extract of D. asper roots and identified therein a new alkaloid, (3R,5S)-5-carboxyvincosidic acid 22-loganin ester (1), and the known alkaloid, (3S,5S)-5-carboxystrictosidic acid 22-loganin ester (dipsaperine¹⁶) (2) (Figure 1). Accordingly, we herein report the structure elucidation of 1 and its NO production inhibitory activity in LPS-activated RAW264.7 cells.

Figure 1

Structures of compounds 1 and 2.

Materials and Methods

General Experimental Procedures

Optical rotation was recorded on a JASCO DIP-140 digital polarimeter. Infrared (IR) spectra were measured with a Shimadzu IR-408 spectrophotometer. Nuclear magnetic resonance (NMR) spectra were obtained using a JEOL JNM-LA400 spectrometer with tetramethylsilane (TMS) as an internal standard, and chemical shifts are expressed in δ values (ppm). High-resolution mass spectrometry (HR-MS) measurement was performed on a Shimadzu time of flight (TOF) mass spectrometer equipped with an electrospray ionization (ESI) interface. Column chromatography was performed using silica gel (silica gel 60N, spherical, neutral, 40-50 µm, Kanto Chemical Co., Inc.) and reversed-phase silica gel (Cosmosil 75C₁₈-OPN, Nacalai Tesque Inc.). Medium-pressure liquid chromatography (MPLC) was performed using a Büchi double pump module C-605 system. Preparative high performance liquid chromatography (HPLC) was performed on a Discovery C18 column (10 × 250 mm i.d., 5 µm, Supelco, USA; flow rate: 2 mL/min) using a Waters 600 pump (Waters, USA) and a Waters 2998 photodiode array detector (Waters, USA).

Plant Specimens

The roots of D. asper Wall (collected in China, Lot No. 120204) were purchased from and authenticated by Uchida Wakanyaku, Ltd. (Tokyo, Japan) in February 2011. A voucher specimen (TMPW 27214) was deposited at the Museum of Materia Medica, Research Center for Ethnomedicines, Institute of Natural Medicine, University of Toyama, Japan.

Extraction and Isolation

Commercially purchased D. asper roots (400 g) were extracted with MeOH (3 L) under reflux for 50 minutes twice to yield 72.8 g of extract. The extract (70 g) was suspended in water (H₂O, 500 mL) and successively partitioned with hexane, ethyl acetate (EtOAc), and n-butanol (n-BuOH, each 400 mL × 2) to give hexane (5.6 g), EtOAc (22.3 g), n-BuOH (28.4 g), and H₂O (13.3 g) fractions. The n-BuOH fraction (28 g) was chromatographed on silica gel with MPLC (5 × 25 cm, flow rate: 25 mL/min) using a MeOH/trichloromethane (CHCl₃) solvent system (5%-40% linear gradient, 100% for 4 hours), and every 150 mL of the eluate was collected to give 40 fractions. Fraction 39 (560 mg) was then subjected to preparative HPLC eluted with H₂O/acetonitrile (79:21) to yield compounds 1 (2.3 mg, t _R 26.3 minutes) and 2 (6.8 mg, t _R 22.6 minutes).

(3R,5S)-5-Carboxyvincosidic Acid 22-Loganin Ester (1)

Colorless amorphous solid.

_{$[α]_{D}^{22}$}: –107.3 (c 0.1, CH₃OH).

IR (KBr): 3406 (br), 2925, 1685, 1631, 1437 cm^–1.

¹H-NMR (400 MHz, methanol-d4 [CD₃OD]): see Table 1 (supplemental Figure S1).

Table 1

¹H and ¹³C Nuclear Magnetic Resonance Data for Compounds 1 and 2 in Methanol-d4 (J Values in Parentheses).

	1		2
No	δ _C	δ _H	δ _C	δ _H
2	129.9		130.3
3	52.8	4.88 m	53.2	4.52 br d (11.6)
5	55.0	4.13 dd (8.8, 5.6)	59.6	3.87 dd (12.8, 4.8)
6	23.7	3.40 m3.17 m	24.1	3.45 m^a 3.04 t (12.8)
7	107.2		108.4
8	127.4		127.5
9	119.2	7.47 d (8.0)	119.2	7.46 d (8.0)
10	120.6	7.04 t (8.0)	120.6	7.04 t (8.0)
11	123.5	7.14 t (8.0)	123.4	7.12 t (8.0)
12	112.4	7.36 d (8.0)	112.3	7.32 d (8.0)
13	138.3		138.5
14	35.2	2.35 m2.16 m	34.6	2.46 br t (14.0)2.26 ddd (14.0, 11.6, 4.4)
15	32.7	3.19 m	32.6	3.11 m^a
16	110.8		108.9
17	154.9	7.64 s	157.2	7.87 s
18	120.8	5.34 d (17.6)5.26 d (10.8)	119.8	5.40 d (17.6)5.27 d (10.8)
19	134.9	5.92 ddd (17.6, 10.8, 7.6)	135.2	5.86 ddd (17.6, 10.8, 7.6)
20	45.6	2.82 dt (7.6, 5.2)	45.3	2.80 ddd (9.2, 7.6, 4.4)
21	97.8	5.63 d (7.6)	97.6	5.93 d (9.2)
22	169.3		171.1
23	173.6		173.5

1′	97.6	5.25 d (5.6)	97.6	5.25 d (5.6)
3′	152.7	7.43 s	152.7	7.42 s
4′	113.0		113.0
5′	32.7	3.11 br q (8.0)	32.8	3.10 m^a
6′	40.4	2.34 dd (14.0, 8.0)1.76 ddd (14.0, 8.0, 5.2)	40.5	2.35 dd (14.0, 7.6)1.75 ddd (14.0, 6.0, 5.2)
7′	79.1	5.28 br t (5.2)	79.8	5.29 br t (5.2)
8′	41.1	2.13 dqd (8.8, 6.8, 5.2)	41.2	2.15 dqd (8.8, 6.8, 5.2)
9′	47.0	2.06 ddd (8.8, 8.0, 5.6)	46.9	2.07 ddd (8.8, 8.4, 5.6)
10′	13.9	1.02 d (6.8)	14.1	1.08 d (6.8)
11′	169.3		169.3
11′-OCH₃	51.8	3.69 s	51.8	3.68 s
21-O-Glc
1″	100.3	4.76 d (8.0)	100.5	4.85 d (8.0)
2″	74.7	3.26 m	74.7	3.25 m
3″	78.0	3.39 m	77.8	3.39 t (9.2)
4″	71.6	3.29 m	71.7	3.29 m
5″	78.3	3.35 m	78.3	3.34 m
6″	62.8	3.93 dd (12.0, 2.4)3.66 m	63.1	4.02 dd (11.6, 2.0)3.68 m
1′-O-Glc
1″′	100.2	4.66 d (7.6)	100.2	4.66 d (7.6)
2″′	74.7	3.19 dd (9.2, 7.6)	74.6	3.19 dd (9.2, 7.6)
3″′	78.0	3.39 m	77.8	3.39 t (9.2)
4″′	71.6	3.29 m	71.6	3.29 m
5″′	78.5	3.41 m	78.8	3.42 m
6″′	62.8	3.89 dd (12.0, 2.0)3.65 m	62.8	3.88 dd (11.6, 2.0)3.63 m

^aChemical shifts were deduced on the basis of cross peaks in correlation spectroscopy and heteronuclear multiple-quantum correlation spectra.

¹³C-NMR (100 MHz, CD₃OD): see Table 1 (supplemental Figure S2).

HR-TOF-ESI-MS: m/z [M + H⁺] calcd for C₄₄H₅₆N₂O₂₀: 933.3505; found: 933.3521.

(3S,5S)-5-Carboxystrictosidic Acid 22-Loganin Ester (2)

Colorless amorphous solid.

_{$[α]_{D}^{22}$}: –166.3 (c 0.1, CH₃OH).

IR (KBr): 3401 (br), 2927, 1685, 1630, 1442 cm^–1.

¹H-NMR (400 MHz, CD₃OD): see Table 1 (supplemental Figure S7).

¹³C-NMR (100 MHz, CD₃OD): see Table 1 (supplemental Figure S8).

HR-TOF-ESI-MS: m/z [M + H⁺] calcd for C₄₄H₅₆N₂O₂₀: 933.3505; found: 933.3519.

NO Production Inhibition Assay

RAW264.7 cells were seeded in 96-well plates at a density of 1 × 10⁵ cells/well and allowed to adhere at 37°C for 4 hours in a humidified atmosphere containing 5% carbon dioxide and 95% air. Then, the cells were treated with Escherichia coli LPS (100 ng/mL, Sigma-Aldrich Co., St. Louis, MO, U.S.A.) with or without the test samples at 5 different concentrations for 24 hours. NO production was determined by measuring the accumulation of nitrite in the supernatant of the RAW264.7 cells using Griess reagent. Briefly, 50 µL of the cell culture supernatant was mixed with 50 µL of Griess reagent (1:1 of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-1-naphthylethylenediamine dihydrochloride in H₂O, Wako Pure Chemical Industries, Ltd, Osaka, Japan) in a 96-well plate and allowed to react for 10 minutes at room temperature. The absorbance of the solution at 550 nm was then measured with a Multiskan FC microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the nitrite concentration in the supernatant was calculated using a standard curve prepared using different known concentrations of sodium nitrite (Wako Pure Chemical Industries, Ltd, Osaka, Japan). The cytotoxicities of the compounds against RAW264.7 cells were assessed by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT; Sigma-Aldrich Inc., St. Louis, USA) assay method. L-NMMA (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) was used as the positive control in this study, and all experiments were performed in triplicate.

Results and Discussion

Structures of the Monoterpenoid Glucoindole Alkaloids

Compounds 1 and 2 were obtained as amorphous solids with $[α]_{D}^{22}$ –107.3 (c 0.1, CH₃OH) and $[α]_{D}^{22}$ –166.3 (c 0.1, CH₃OH), respectively, and their IR spectra show the presence of hydroxyl and α,β-unsaturated ester carbonyl groups. The ¹H-NMR and ¹³C-NMR spectra of 1 and 2 are similar, both presenting signals similar to those for iridoid/secoiridoid glucosides (cantleyoside, dipsanosides A and B, loganic acid, loganin, sylvestroside I, and triplostoside A) isolated from the same extract.¹⁴ HR-TOF-ESI-MS analysis indicated they have the same molecular formula (C₄₄H₅₆N₂O₂₀). The presence of 2 nitrogen atoms indicated that 1 and 2 are alkaloid-coupled iridoid glycosides.

The ¹H-NMR and ¹³C-NMR spectra of 1 and 2 (supplemental Figures S1, S2, S7 and S8), analyzed using ¹H–¹H shift correlation spectroscopy (COSY), heteronuclear multiple-quantum correlation (HMQC) spectroscopy, and heteronuclear multiple-bond correlation (HMBC) spectroscopy (supplemental Figures S3–S5 and S9–S11) confirm the presence of both loganin glycoside and secologanin glycoside moieties (Figure 2). Furthermore, the ¹H-NMR and ¹³C-NMR spectra present signals evidencing a 2,3-disubstituted indole ring, a methylene group, 2 methine groups, and a carboxyl unit (Table 1). The data (other than that for the loganin moiety) closely resemble those of 5(S)-5-carboxyvincosidine and 5(S)-5-carboxystrictosidine,¹⁷ which are C-3 epimers of secoiridoid glucoindole alkaloids, except for the absence of the O-methyl group in 1 and 2. Taken together, these data indicate that 1 and 2 are monoterpenoid glucoindole alkaloids consisting of a 5-carboxyvincosidine/5-carboxystrictosidine moiety and a loganin moiety. The linkage site between 5-carboyvincosidine/5-carboxystrictosidine and the loganin moiety was established based on the HMBC correlations for H-7′ (δ _H 5.28 for 1; δ _H 5.29 for 2) with an ester carbonyl carbon (δ _C 169.3 for 1; δ _C 171.1 for 2, C-22). Accordingly, 1 and 2 were concluded to be 5-carboyvincoside-22-loganin ester and 5-carboxystrictoside-22-loganin ester.

Figure 2

Correlation spectroscopy (bold lines) and significant heteronuclear multiple-bond correlation spectroscopy (red arrows: ¹H→¹³C) correlations in both compounds 1 and 2.

The monoterpenoid glucoindole alkaloid (3S,5S)-5-carboxystrictosidic acid 22-loganin ester (dipsaperine) was recently isolated from the roots of D. asper.¹⁶ The ¹H-NMR and ¹³C-NMR data for 2 in the present study are identical with those reported for dipsaperine, confirming the structure of 2. Thus, the structure of 1 is assumed to be its 3-epimer, (3R,5S)-5-carboxyvincosidic acid 22-loganin ester.

The stereochemistry of 1 was determined based on the analysis of coupling constants and rotating frame nuclear Overhauser effect spectroscopy (ROESY) correlations (see supplemental Figures S6 and S12). The ¹H-NMR COSY, HMQC, and HMBC data for 1 indicate that the chemical shifts for H-3 (δ _H 4.88, m) and H-5 (δ _H 4.13, dd, J = 8.8, 5.6 Hz) are identical to those of the corresponding protons in 5(S)-5-carboxyvincosidine (H-3: δ _H 4.65, m; H-5: δ _H 4.14, dd, J = 8.4, 6.4 Hz), which are arranged with a 3,5-trans relationship, rather to those in 5(S)-5-carboxystrictosidine (H-3: δ _H 4.51, d, J = 11.3 Hz; H-5: δ _H 3.83, ddd, J = 11.8, 4.2, 1.5 Hz), which are arranged in a 3,5-cis relationship.¹⁷ The 3,5-trans relationship in 1 was further confirmed by the fact that a H-3/H-5 ROESY correlation is only observed for 2 (3,5-cis relationship) (Figure 3), while a H-3/H-15 ROESY correlation is only observed in 1 (Figure 3(a)), confirming the stereochemistry at C-3 in 1. Likewise, the ROESY correlations H-15/H-20 in 1 (Figure 3(a)) and H-15/H-20 and H₂-18/H-21 in 2 (Figure 3(b)) confirm the stereochemistry of the secologanin moiety. Moreover, the ROESY correlations H-5′/H-9′, H-9′/H₃-10′, H-7′/H-8′, and H-1′/H-8′ in both 1 and 2 (Figure 3(c)) confirm the stereochemistry of the loganin moiety.

Figure 3

Significant rotating frame nuclear Overhauser effect spectroscopy correlations (red arrows) in compounds 1 and 2. (a) The 5-carboxyvincosidine moiety in 1, (b) the 5-carboystrictosidine moiety in 2, and (c) the loganin moiety in compounds 1 and 2.

Thus, compound 1 is determined to be (3R,5S)-5-carboxyvincosidic acid 22-loganin ester. Interestingly, this structure had been previously given to pterocephaline, a monoterpenoid glucoindole alkaloid isolated from Pterocephalus pinardii.¹⁸ However, the structure of pterocepharine was revised to (3S,5S)-5-carboxystrictosidic acid 22-loganin ester (dipsaperin, 2) by Yu et al.¹⁶ Thus, 1 is a new compound.

NO Production Inhibition Activity

The inhibitory activity of compounds 1 and 2 against NO production was examined in LPS-activated RAW264.7 macrophage cells. Both compounds inhibit NO production in LPS-activated RAW264.7 macrophage cells, with 1 exhibiting an IC₅₀ value of 21.3 µM and 2 exhibiting an IC₅₀ value of 20.5 µM. These activities are very similar to that of the positive control L-NMMA (22.6 µM), a non-selective NO synthase (NOS) inhibitor,¹⁹ but weaker than those of dipasperoside A (IC₅₀ = 15.2 µM) and akebia saponin D (IC₅₀ = 12.7 µM), both of which are obtained from the water extract of D. asper roots.^14,15 Furthermore, an MTT assay revealed that compounds 1 and 2 exhibit no significant cytotoxicity against LPS-activated macrophage RAW264.7 cells at concentrations of up to 50 µM.

Conclusion

We analyzed a MeOH extract of the traditional medicine D. asper and isolated 2 monoterpenoid glucoindole alkaloids. They were determined by exhaustive spectroscopic analysis to be a new alkaloid, (3R,5S)-5-carboxyvincosidic acid 22-loganin ester (1), and the known alkaloid (3S,5S)-5-carboxystrictosidic acid 22-loganin ester (dipsaperin, 2). Their inhibitory activities against NO production in LPS-activated RAW264.7 macrophage cells were determined to be comparable to that of a nonselective NOS inhibitor, N^G -monomethyl-l-arginine. Thus, our study further demonstrates the value of D. asper as a potential source of medicinally important compounds and has identified a new compound that exhibits potential as an anti-inflammatory agent.

Supplemental Material

Supplementary material - Supplemental material for A New Monoterpenoid Glucoindole Alkaloid From Dipsacus asper

Supplemental material, Supplementary material, for A New Monoterpenoid Glucoindole Alkaloid From Dipsacus asper by Feng Li, Yuto Nishidono, Ken Tanaka, Shiro Watanabe and Yasuhiro Tezuka in Natural Product Communications

Footnotes

Acknowledgments

The authors would like to thank Enago () for the English language review.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Shiro Watanabe

Yasuhiro Tezuka

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