Current Topics: Regular Article
Isolation and Identification of Three New Iridoid Glucosides from the Aerial Parts of Paederia scandens
2025 Volume 73 Issue 3 Pages 156-161
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2025 Volume 73 Issue 3 Pages 156-161
Sulfur-containing compounds are found in various medicinal products, where they play crucial roles in biological activities such as antimicrobial, anticancer, and other therapeutic effects. These compounds are commonly found in species of Allium, especially onions and garlic; however, there is little evidence of their presence in other plants. In particular, sulfur-containing iridoid glycosides with anticancer properties, which are very promising compounds as pharmaceutical seeds, have been isolated from Paederia scandens (Rubiaceae), also known as the skunk vine because of its strong smell caused by methyl mercaptan. Herein, we describe the isolation and structural elucidation of 3 new iridoid glycosides from the aerial parts of P. scandens. Their biosynthetic pathways are also discussed.
Sulfur-containing compounds are of relevance in the field of pharmaceuticals, representing over 10% of all drugs. In particular, sulfonylureas and acetazolamide, which function as hypoglycemic agents derived from sulfa drugs and as a diuretic drug, respectively, are among the most widely used beneficial agents. Additionally, sulfur is essential for the functioning of proton pump inhibitors.1) Sulfur-containing compounds are widely present in plants of the genus Allium (Amaryllidaceae), including onions and garlic2–4); however, reports on sulfur-containing compounds in other plants are scarce and mainly limited to Salacia reticulata (Celastraceae)5) and Nuphar pumilum (Nymphaeaceae).6–8) In the field of natural product chemistry, these compounds are interesting research targets because of their various biological activities.3,7,8) Sulfur-containing compounds are also present in Paederia scandens (Lour.) Merr. (Rubiaceae), a widely known folk medicinal plant that is distributed throughout South, East, and Southeast Asia. The extracts obtained from the dried leaves and fruits of this species are used for treating diarrhea, and the juice from the fruits is applied as a topical remedy for skin conditions.9) In English-speaking countries, P. scandens is sometimes called “skunk vine” owing to the indescribable stench that its fruits and leaves produce when wounded. This odor is caused by methyl mercaptan, which is produced by the enzymatic breakdown of the main component, paederosidic acid, a sulfur-containing iridoid glycoside.10,11) P. scandens contains paederoside10) and its dimers,11) which possess anti-inflammatory and anticancer activities.12,13) Our group was interested in investigating the presence of additional sulfur-containing compounds in P. scandens, aiming to identify new candidates against cancer and other diseases. Herein, we describe the isolation and structural elucidation of a total of 6 iridoid glycosides, that is, 3 novel compounds [paescanosides A–C (1–3)] (Fig. 1) and 3 known compounds [paederoside (4), paederosidic acid (5), and saprosmoside D (6)]. These compounds were isolated from the 1-BuOH-soluble fraction of the aerial parts of P. scandens, purified by column chromatography (CC) using silica gel, octadecyl silica (ODS), and Sephadex LH-20 as the stationary phase and preparative HPLC, and structurally characterized using NMR and other analytical techniques. Furthermore, we propose a biosynthetic pathway for sulfur-containing iridoid glycosides, whose biosynthesis is still unknown.
Paescanoside A (1) was obtained as a brown oil and exhibited negative optical rotation (
| Position | 1 | 2 | 3 | |||
|---|---|---|---|---|---|---|
| δH | δC | δH | δC | δH | δC | |
| 1 | 5.06 (d, J = 9.2) | 101.2 | 5.97 (d, J = 1.6) | 93.3 | 5.97 (s-like) | 93.4 |
| 2 | ||||||
| 3 | 7.65 (d, J = 0.8) | 155.2 | 7.30 (d, J = 2.0) | 150.3 | 7.28 (s-like) | 150.3 |
| 4 | 108.3 | 106.2 | 106.2 | |||
| 4a | 3.04 (dd, J = 1.6, 8.4) | 42.4 | 3.69 (m) | 37.4 | 3.67 (m) | 37.5 |
| 5 | 4.80 (dd, J = 2.4, 7.0) | 75.4 | 5.56 (m) | 86.3 | 5.56 (d-like, J = 6.4) | 86.3 |
| 6 | 6.03 (d, J = 1.6) | 132.4 | 5.73 (dt, J = 2.0, 6.4) | 128.9 | 5.69 (s-like) | 128.9 |
| 7 | 145.4 | 144.4 | 144.4 | |||
| 7a | 2.63 (t, J = 8.0) | 46.2 | approx. 3.3* | 45.3 | approx. 3.3* | 45.3 |
| 8 | 4.95 (d, J = 14.8) 5.10 (d, J = 14.8) |
66.2 | 4.65 (m) 4.86 (m) |
61.9 | 4.73 (d, J = 20) 4.83** |
61.5 |
| 9 | 168.9 | 172.6 | 172.6 | |||
| 10 | 172.8 | 175.5 | ||||
| 11 | 2.35 (s) | 13.5 | 2.39 (q, J = 8.0) | 28.1 | ||
| 12 | 1.13 (t, J = 7.2) | 9.4 | ||||
| 1' | 4.72 (d, J = 7.6) | 100.6 | 4.68 (d, J = 8.4) | 100.0 | 4.63 (d, J = 8.0) | 100.0 |
| 2' | 3.25 (m) | 74.9 | 3.19 (m) | 74.6 | 3.16 (t, J = 8.8) | 74.7 |
| 3' | 3.28 (m) | 77.9 | approx. 3.3* | 77.9 | 3.2 (m) | 77.9 |
| 4' | 3.28 (m) | 71.5 | 3.27 (m) | 71.6 | 3.3 (m) | 71.5 |
| 5' | 3.36 (m) | 78.6 | 3.38 (m) | 78.4 | 3.35** | 78.3 |
| 6' | 3.64 (dd, J = 6.4, 12.0) 3.85 (dd, J = 1.2, 12.0) |
63.0 | 3.65 (m) 3.91 (dd, J = 1.2, 12.0) |
62.8 | 3.66 (m) 3.90 (m) |
62.7 |
| 1" | 4.16 (t, J = 8.4) | 65.1 | 167.5 | |||
| 2" | 1.68 (quintet, J = 8.4) | 31.9 | 5.82 (d, J = 12.8) | 115.9** | ||
| 3" | 1.43 (sextet, J = 7.2) | 20.4 | 6.92 (d, J = 12.8) | 146.0 | ||
| 4" | 0.970 (t, J = 7.2) | 14.1 | 127.6 | |||
| 5" (9") | 7.62 (d, J = 8.0) | 133.7 | ||||
| 6" (8") | 6.75 (J = 8.0) | 115.9** | ||||
| 7" | 160.2 | |||||
*The signal overlapped with the solvent peak. The HMQC cross-peaks were confirmed. **The peaks were overlapping.
Paescanoside B (2) was obtained as a yellow oil with positive optical rotation (
Paescanoside C (3) was obtained as a brown oil exhibiting negative optical rotation (
Finally, since the 1D NMR spectra suggested that the sugar structure was d-glucose, the retention times of authentic d-glucose were compared with those obtained after hydrolyzing 1–3 using an evaporative light-scattering detector-HPLC (ELSD-HPLC). The retention time of the d-glucose standard was consistent with that obtained after hydrolysis (7.2 min), which provided further evidence that the sugar bound to 1–3 was d-glucose (Supplementary Materials).
Figure 3 shows a plausible, but as yet unelucidated, biosynthetic pathway for sulfur-containing iridoid glycosides. The sulfur-containing compounds from P. scandens are characterized by a methylsulfanyl group attached to the hydroxy group at C-8 (Supplementary Materials). Possible sources of sulfur atoms include the sulfur-containing amino acids cysteine and methionine, from which sulfur-containing compounds are biosynthesized as secondary metabolites in the Allium genus and Brassica family by enzymatic action.15–17) The limited reports of compounds with methylsulfanyl structures in plants other than P. scandens suggests that this structure is produced by a specific enzymatic reaction. Accordingly, we propose the following biosynthetic pathways (Fig. 3): In Pathway 1, geraniol (A), a monoterpene, undergoes oxidation and cyclization to form iridodial (B), followed by further oxidation to form keto iridodial (C). Subsequently, C is converted from the keto form to the hemiacetal form C′, which undergoes oxidation into D and glucosylation to produce deacetyl asperuloside (F) via highly oxidized methyl-free loganin (E).18,19) As shown in Pathway 2, sulfur-containing amino acids (G), including cysteine, methionine, and glutathione, are generally synthesized from sulfate ions obtained from the environment. These compounds could be metabolized by unidentified enzymes into methyl mercaptans (H). A nucleophilic reaction of H with carbon dioxide, probably obtained from the atmosphere during photosynthesis, would result in the production of S-methyl-O-hydrocarbonothioate (I) by P. scandens. This reaction may be analogous to the carbonate fixation reaction and could be catalyzed by a specific enzyme. Finally, 4 could be biosynthesized via ester formation through the reaction of I with the hydroxy group at C-8 of deacetyl asperuloside (F).
In summary, 3 new iridoid glycosides were obtained from the aerial parts of P. scandens, and their structures were identified. One compound was identified as a derivative of 5 containing a sulfur atom. A plausible biosynthetic pathway for sulfur-containing iridoid glycosides involves the formation of ester bonds between iridoid glycosides biosynthesized via the mevalonic acid pathway and S-methyl-O-hydrocarbonothioate, which is enzymatically obtained from sulfur-containing amino acids. The biological activities of 4 and other sulfur-containing iridoid glycosides from P. scandens were evaluated to determine their anticancer activity; however, insufficient data were obtained to elaborate a systematic comparison between sulfur-containing and non-sulfur-containing iridoid glycosides, which would allow elucidating the role of the sulfur atoms in the activities of these compounds. This will be the subject of future studies.
The specific rotations were determined using a Horiba SEPA-500 digital polarimeter (l = 5 cm). The IR spectra were recorded on a Shimadzu IR Affinity-1S (ATR) spectrometer. ESI-MS and HR-ESI-MS spectra were obtained using a Shimadzu LCMS-9030 spectrometer. 1H-NMR and 13C-NMR spectra were acquired using JEOL (Tokyo, Japan) JNM-ECZ 400S (400 MHz) and JEOL JNM-ECZ 400S (100 MHz) spectrometers, respectively. HPLC was performed using Shimadzu (Kyoto, Japan) HPLC and SPD-10Avp UV–VIS detectors. YMC Co., Ltd. (Kyoto, Japan) Triart C18 (150 × 2.0 and 250 × 10 mm i.d.) columns were used for analytical and preparative purposes. The following materials were used for chromatography: normal-phase silica gel CC, silica gel AP-350S (Daiko Trading Co., Ltd., Tokyo, Japan, 200–400 mesh); reversed-phase silica gel CC, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., Aichi, Japan, 100–200 mesh), gel filtration CC Sephadex LH-20 (Cytiva, Tokyo, Japan), TLC plates precoated with silica gel 60F254 (Merck, Darmstadt, Germany, 0.25 mm) and silica gel RP-18 F254S (Merck, 0.25 mm) (reversed phase), and TLC plates precoated with silica gel 60 F254S (Merck, 0.25 mm) for normal-phase HPTLC. TLC detection was performed by spraying with 1% Ce(SO4)2–10% aqueous H2SO4 followed by heating.
Plant MaterialP. scandens was collected from the wild on the campus of Gifu University of Medical Science in October 2022. The plant material was identified to be P. scandens upon examination by M.F.
Extraction and IsolationDried aerial parts of P. scandens (2.1 kg) were chopped in a mixer and soaked overnight in 80% aqueous acetone 3 times. The acetone extract (200 g), which was obtained after evaporation, was separated into ethyl acetate (EtOAc), 1-BuOH, and water fractions. The EtOAc and 1-BuOH fractions were evaporated in vacuo to obtain EtOAc (39 g) and 1-BuOH (44 g) residues. The 1-BuOH-soluble fraction was subjected to normal-phase silica gel CC [CHCl3–MeOH (1 : 0 → 50 : 1 → 10 : 1 → 7 : 1 → 5 : 1 → 2 : 1 → 1 : 1 → 0 : 1, v/v)] to give 8 fractions (fr. 1, 0.062 g; fr. 2, 2.5 g; fr. 3, 4.1 g; fr. 4, 9.3 g; fr. 5, 17.8 g; fr. 6, 4.9 g; fr. 7, 1.3 g; fr. 8, 2.0 g). Fr. 3 was further separated by reversed-phase silica gel CC [MeOH–H2O (6 : 4 → 7 : 3 → 8 : 2 → 9 : 1 → 1 : 0, v/v)] to give 7 subfractions (subfr.). Subfr. 3-5 was further subjected to reversed-phase silica gel CC [MeOH–H2O (6 : 4, v/v)] to give 8 fractions. Finally, subfr. 3-5-2 was purified using preparative HPLC [mobile phase : MeOH–H2O (50 : 50, v/v)] to yield paescanoside A (1, 10.1 mg). Fr. 4 was further separated by reversed-phase silica gel CC [MeOH–H2O (6 : 4 → 7 : 3 → 8 : 2 → 9 : 1 → 1 : 0, v/v)] to give 9 fractions. Subfr. 4-2 was separated by gel filtration CC with Sephadex LH-20 [MeOH–H2O (40 : 60, v/v)] to give 9 fractions. Subfr. 4-2-4 was separated using Sephadex LH-20 [MeOH–H2O (40 : 60, v/v)] to give 6 fractions. Finally, subfr. 4-2-4-6 was purified using preparative HPLC [mobile phase: MeOH–H2O (60 : 40, v/v)] to yield paescanoside B (2, 4.2 mg). Subfr. 4-4 was separated using Sephadex LH-20 [MeOH–H2O (40 : 60, v/v)] to give 13 fractions. Subfr. 4-4-6 was purified using preparative HPLC [mobile phase: MeOH–H2O (25 : 75, v/v)] to yield paescanoside C (3, 1.8 mg). Subfractions 4-5 and 4-6 were combined and separated using Sephadex LH-20 [MeOH–H2O (50 : 50, v/v)] to give 8 fractions. Subfr. 4-5,6-3 was separated by reversed-phase silica gel CC [MeOH–H2O (20 : 80 → 40 : 60, stepwise, v/v)] to give 15 fractions. Subfr. 4-5,6-3-2 was purified using preparative HPLC [mobile phase: MeOH–H2O (25 : 75, v/v)] to yield paederoside (4, 24.2 mg) and paederosidic acid (5, 0.4 mg). Subfr. 4-5,6-3-10 was purified using preparative HPLC [mobile phase: MeOH–H2O (40 : 60, v/v)] to yield saprosmoside D (6, 11.7 mg).
Paescanoside A (1)Brown oil;
Brown oil;
Brown oil;
Compounds 1–3 were dissolved in distilled water (1.0 mL). The samples were separated (1.0 mg) and treated with 2 M HCl (1.0 mL) and distilled water (2.0 mL). Each mixture was heated at 80°C for 1 h. The reaction mixture was partitioned with EtOAc. The aqueous layers were neutralized with Amberlite IRA67 (Organo Co.), evaporated under reduced pressure, and analyzed by HPLC using ELSD. The analytical conditions for the monosaccharide were as follows: column, XBridge Amide 3.5 µm 2.1 × 150 mm (Waters); solvent A, water; solvent B, acetonitrile; detector, UV–Vis (210 nm); ELSD (gain, 6; temperature, 40°C; press, 350 kPa); flow rate, 0.2 mL/min. The linear gradient was 0 min; B 80%, 0–20 min; B 80–65%, 20 min; B 80%, 20–25 min. Authentic d-glucose was procured from Nacalai Tesque, Inc. (Kyoto, Japan).
This work was supported by JSPS KAKENHI (Grant Number: 20K22727 to M.F.).
The authors declare no conflict of interest.
This article contains supplementary materials.
