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Abstract

Two new fatty acid derivatives identified as 5,7-dihydroxy-6-oxoheptadecanoic acid (1) and ethyl-5,7-dihydroxy-6-oxooctadecanoate (2) together with four known compounds, ethyl linolenate (3), ethyl linoleate (4), ethyl pheophorbide A (5), and pheophorbide A (6), were isolated from the aerial parts of Tiliacora triandra. All the compounds were isolated from T. triandra for the first time. The structures of the compounds were elucidated using high-resolution electrospray ionization mass spectrometry, 1-dimensional and 2-dimensional nuclear magnetic resonance spectroscopy, and comparison with literature data. All the isolated compounds were evaluated for their in vitro inhibitory activity against α-glucosidase and α-amylase. Compounds 1-6 exhibited α-glucosidase inhibitory activity with half-maximal inhibitory concentration values (IC₅₀) values in the range of 11.58-424.06 μM, while only compound 1 displayed inhibitory activity against α-amylase at an IC₅₀ value of 26.27 μM.

Tiliacora triandra (Colebr.) Diels (Menispermaceae) is a flowering plant native to Southeast Asian countries, particularly Thailand and Laos. The leaf of the plant is used as a popular cuisine in the northern part of Thailand and is used traditionally as treatment for fever, diabetes, and malaria.^1,2 T. triandra is known for its anticancer, antibacterial, antidiabetic, and acetylcholinesterase inhibitory activities.^3

-6 Moreover, T. triandra root is a rich source of antimalarial bisbenzylisoquinoline alkaloids.^1,7 In our ongoing research for the discovery of new secondary metabolites from Thai medicinal plants, the separation of the ethyl acetate extract from the aerial part of T. triandra led to the isolation of two new fatty acid derivatives (1 and 2), along with four known compounds: ethyl linolenate (3), ethyl linoleate (4) ethyl pheophorbide A (5), and pheophorbide A (6) (Figure 1). We thus describe the structural elucidation of the new compounds and the α-glucosidase and α-amylase inhibitory activities of the isolated compounds.

Figure 1

Structures of compounds 1-6 from Tiliacora triandra (Colebr.) Diels.

Compound 1 was isolated as a light yellow oil with a molecular formula of C₁₇H₃₂O₅ based on high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) ion peak at m/z 317.2090 [M + H]⁺. The 1-dimensional nuclear magnetic resonance (1D NMR) spectra data (Table 1) of 1 revealed the presence of 17 carbons consisting of 1 carboxylic carbon at δ _C 180.2 (C-1), 1 ketone at δ _C 204.0 (C-6), 2 oxygenated methine at δ _C 89.6 and 91.5 (C-5 and C-7), 1 methyl carbon at δ _C 14.0 (C-17), and 12 methylenes resonating between δ _C 22.6 and 33.3 (C-2 to C-4 and C-8 to C-16). The ¹H NMR spectrum revealed the presence of signals attributed to 1 terminal methyl at δ _H 0.88, 2 oxygenated methine proton signals at δ _H 5.09 and 5.05, and methylene protons chain at δ _H 1.26-1.75, suggesting the existence of a fatty acid core in the structure. Furthermore, 3 methylene protons at δ _H 2.39 (H-2), 2.04 (H-4), and 1.97 (H-8) were assigned to methylenes adjoined to the carboxylic (C-1) and oxymethine carbons (C-5 and C-7), respectively. The heteronuclear multiple bond correlations (HMBC) from the proton signals H-2 (δ _H 2.39) to C-1 (δ _C 180.2), C-3 (δ _C 23.9) and C-4 (δ _C 28.1), cross-peaks between the proton signals H-3 (δ _H 1.75) to C-1/C-2/C-4/C-5, H-4 (δ _H 2.04) to C/2/C-3/C-5, and H-5 (δ _H 5.05) to C/3/C-4/C-6/C-7 indicated the existence of a butyl group between the carboxyl (C-1) and ketone (C-6) carbons (Figure 2). Additional HMBC was observed from the methine proton at H-7 (δ _H 5.09) to C-5/C-8/C-9; H-8 (δ _H 1.97) to C-6/C-7/C-9/C-10; H-9 (δ _H 1.39) to C-7/C-8/C-10, and the methyl signal at H-17 (δ _H 0.88) to C-15 (δ _C 31.9) and C-16 (δ _H 22.6). The structure of 1 was further verified by comparison of the NMR data with other closely related previously described compounds.^8,9 Thus, the structure of 1 was assigned as 5,7-dihydroxy-6-oxoheptadecanoic acid.

Table 1

¹H (500 MHz, CDCl₃) and ¹³C (125 MHz, CDCl₃) NMR Data of Compounds 1 and 2 .

Position	Compound 1		Compound 2
	δ _C (ppm)	δ _H ppm (Mult., J, in Hz)	δ _C (ppm)	δ _H ppm (Mult., J, in Hz)
1	180.2		173.7
2	33.3	2.39 (t, 7.5)	33.6	2.34 (t, 7.5)
3	23.9	1.75 (qt, 7.5)	24.3	1.75 (qt, 7.5)
4	28.1	2.04 (dq, 7.5; 3)	28.3	2.03 (dq, 7.5; 3)
5	89.6	5.05 (overlap)	89.8	5.05 (overlap)
6	204.0		204.0
7	91.5	5.09 (overlap)	91.4	5.09 (overlap)
8	28.9	1.97 (dq, 7.5; 3)	28.9	1.97 (dq, 7.5; 3)
9	29.1	1.39 (qt, 7.5)	29.1	1.39 (qt, 7.5)
10-14	29.2-29.6	1.26 (overlap)	29.2-29.6	1.26 (overlap)
15	31.9	1.26 (overlap)	29.6	1.26 (overlap)
16	22.6	1.28 (overlap)	31.9	1.26 (overlap)
17	14.0	0.88 (t, 7.0)	22.7	1.26 (overlap)
18			14.1	0.88 (t, 7.0)
1’			60.2	4.12 (q, 7.0)
2’			14.2	1.25 (q, 7.0)

Figure 2

Key ¹H-¹H COSY and HMBC correlations of compounds 1-2.

The molecular formula of compound 2 was deduced as C₂₀H₃₈O₅ according to the HR-ESI-MS peak at m/z 391.2859 [M + CH₃OH + H]⁺. A detailed comparison of the NMR data of 2 with 1 suggested a close similarities in their core skeleton. The main difference between compounds 2 and 1 is the presence of an ethyl ester moiety (δ _C 14.2, 60.2 and 173.7; δ _H 1.25 and 4.12) which replaces the carboxylic acid in 1 as well as 1 additional methylene group. The HMBC spectrum showed crosspeaks from H-2′ (δ _H 1.25) to C-1 (δ _C 60.2), H-1′ (δ _H 4.12) to C-2′ (δ _C 14.2) and C-1 (δ _C 173.7), H-2 (δ _H 2.34) to C-1 (δ _C 173.7), C-3 (δ _C 24.3) and C-4 (δ _C 28.4), H-3 (δ _H 1.75) to C-1 (δ _C 173.7), C-2 (δ _C 33.6), C-4 (δ _C 28.4), and C-5 (δ _C 89.8) (Figure 2). These connectivities led to the positioning of the ethyl ester moiety. Therefore, the structure of 2 was determined as ethyl-5,7-dihydroxy-6-oxooctadecanoate.

The structures of the isolated known compounds, ethyl linolenate (3), ethyl linoleate (4), ethyl pheophorbide A (5), and pheophorbide A (6), were identified by comparing their spectroscopic data with previous report.^10

-13 Compounds 1 -6 exhibited α-glucosidase inhibitory activity with half-maximal inhibitory concentration (IC₅₀) values in the range of 11.58-424.06 μM, while only compound 1 displayed inhibitory activity against α-amylase at an IC₅₀ value of 26.27 µM (Table 2).

Table 2

α-Glucosidase and α-Amylase Inhibition of Compounds 1-4.

Compound	α-Glucosidase inhibition (IC₅₀; µM)	α-Amylase inhibition (IC₅₀; µM)
1	11.58 ± 0.32^a	26.27 ± 1.11^a
2	105.61 ± 6.12^b	NA
3	104.77 ± 4.56^b	NA
4, 5, 6	424.06 ± 15.25^c, 22.11 ± 1.01^d, 28.20 ± 2.41^d	NA
Acarbose	500.91 ± 10.26^e	177.65 ± 0.88^b

NA, not active; IC₅₀, half-maximal inhibitory concentration.

Data were expressed as mean ± SD (n=5). Values having different lowercase letters as superscript across the same column indicates significant differences (P < 0.05).

Experimental

General Experimental Procedures

NMR spectra data were recorded on a Fourier Transform NMR spectrometer 500 MHz Unity Inova (Varian, Germany). IR spectra were obtained using a Perkin Elmer FT-IR spectrophotometer Gx (Massachusetts, USA). Optical rotation analysis was conducted using a Kruss Optronic polarimeter (Kruss, Germany), while the mass spectra were performed on a 1290 Infinity II LC-6545 Q-TOF spectrometer (Agilent Technologies, USA). Column chromatography was carried out with VertiFlash silica gel 60-200 µm (Vertical Chromatography, Bangkok, Thailand). Semipreparative reverse-phase high performance liquid chromatography (RP-HPLC) was performed on a Jasco HPLC-4000 system using a semipreparative Vertisep GES C18 Column (10 × 250 mm, 5 µm; Vertical Chromatography, Bangkok, Thailand).

Plant Material

The leaves and twigs of T. triandra were collected in August 2017 from Ban Prang Chai, Phattalung Province, Thailand. The plant was authenticated at the Faculty of Traditional Thai Medicine, Prince of Songkla University, Thailand, where a voucher specimen (TTM/TT/001) was stored.

Extraction and Isolation

The dried and ground aerial part of the plant (leaves, 800 g and twigs, 500 g) were exhaustively macerated with 95% ethanol (4 × 5 L). The extract was filtered and evaporated under reduced pressure to yield crude ethanol extract (84.2 g). The crude ethanolic extract was suspended in water (H₂O) and partitioned between hexane and ethyl acetate to afford hexane soluble (TT-001; 20.4 g) and ethyl acetate soluble (TT-002; 16.1 g) fractions.

Fraction TT-002 (1.5 g) was subjected to silica gel column chromatography, eluting with a gradient solvent system of petroleum ether–acetone (4:1 to 1:4). This yielded 9 subfractions: F-1 (284.3 mg), F-2 (132.5 mg), F-3 (151.5 mg), F-4 (48.7 mg), F-5 (26.7 mg), F-6 (15.4 mg), F-7 (76.1 mg), F-8 (18.0 mg), and F-9 (33.7 mg). Subfraction F-3 was further purified by RP-HPLC using a mobile phase of acetonitrile–H₂O (87:13) at a flow rate of 3.5 mL/min to obtain compound 1 (6.7 mg; 18.34 minutes). Subfractions F5 (26.7 mg) and F6 (15.4 mg) were also subjected to RP-semipreparative HPLC using acetonitrile–H₂O (90:10), at a flow rate of 5.5 mL/min to afford compounds 5 (8.8 mg; 39.66 minutes) and 6 (2.6 mg; 46.73 minutes).

Another portion of TT-002 (1.8 g) was subjected to silica gel column chromatography and eluted with a gradient solvent system of petroleum ether–acetone (4:1 to 1:4) to afford 9 subfractions: F-10 (268.4 mg), F-11 (337.8 mg), F-12 (152.6 mg), F-13 (223.0 mg), F-14 (128.5 mg), F-15 (31.5 mg), F-16 (39.4 mg), F-17 (145.9 mg), and F-18 (240.2 mg). Subfractions 11 (337.8 mg), 12 (152.6 mg), and 13 (223.0 mg) were further purified by RP-HPLC (acetonitrile–H₂O; 87: 13; flow rate of 3.5 mL/min) to obtain compounds 1 (98.6 mg; 18.41 minutes), 2 (47.7 mg; 56.34 minutes), 3 (9.4 mg; 28.16 minutes), and 4 (27.1 mg; 38.64 minutes).

5,7-Dihydroxy-6-Oxoheptadecanoic Acid (1)

Yellow oil; ${[α]}_{D}^{25}$ −15 (c 0.02, EtOH)

¹H NMR (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃): see Table 1.

HR-ESI-MS m/z 317.2090 [M + H]⁺ (Calcd. for C₁₇H₃₃O₅, 317.2322).

Ethyl-5,7-Dihydroxy-6-Oxooctadecanoate (2)

Yellow oil; ${[α]}_{D}^{25}$ -24 (c 0.01, EtOH)

¹H NMR (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃): see Table 1.

HR-ESI-MS m/z 391.2859 [M + CH₃OH + H]⁺ (Calcd. for C₂₁H₄₃O₆, 391.3054).

Determination of α-Amylase Inhibition

See Supplemental Material.

Determination of α-Glucosidase Inhibition

See Supplemental Material.

Supplemental Material

Supplementary material - Supplemental material for Chemical Constituents From the Aerial Part of Tiliacora triandra (Colebr.) Diels and Their α-Glucosidase and α-Amylase Inhibitory Activity

Supplemental material, Supplementary material, for Chemical Constituents From the Aerial Part of Tiliacora triandra (Colebr.) Diels and Their α-Glucosidase and α-Amylase Inhibitory Activity by Emmanuel Ayobami Makinde, Chitchamai Ovatlarnporn, Chonlatid Sontimuang, Gaëtan Herbette and Opeyemi Joshua Olatunji in Natural Product Communications

Footnotes

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) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the Annual Income Budget of Prince of Songkla University (Grant number: TTM610424S). EAM is grateful to the Thailand Education Hub for ASEAN Countries Masters Award 2017 of the Prince of Songkla University, Thailand.

ORCID ID

Gaëtan Herbette

Opeyemi Joshua Olatunji

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Supplementary Material

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