Plant Growth Inhibitory Activity of the Extracts of Acmella oleracea and its Growth Inhibitory Substances

Abstract

Aqueous methanol extracts of Acmella oleracea inhibited the growth of roots and shoots of cress (Lepidum sativum), lettuce (Lactuca sativa), Italian ryegrass (Lolium multiflorum), and barnyard grass (Echinochloa crus-galli) with the extract concentration-dependent manner. The extracts were then purified and 2 growth inhibitory substances were isolated and identified by spectral data as (E,E)-2,4-undecadien-8,10-diynoic acid isobutylamide (compound 1) and nona-(2Z)-en-6,8-diynoic acid 2-phenylethylamide (compound 2). Compound 1 inhibited the growth of cress roots and shoots at concentrations greater than 0.3 mM and that of barnyard grass root at concentrations greater than 0.03 mM. Compound 2 inhibited the growth of cress shoots and barnyard grass roots at concentrations greater than 1 mM. The inhibition on the seedling growth indicates that both compounds had growth inhibitory activity and may contribute to the inhibitory effect of A. oleracea extracts. This is the first report of having growth inhibitory activity of compounds 1 and 2 on plants.

Acmella oleracea (L.) R.K. Jansen (synomym: Spilanthes acmella Murr.) is herb species in the Asteraceae family and distributed worldwide throughout tropical and subtropical areas. ¹ The family Asteraceae is one of the largest families of vascular plants. Acmella oleracea is known as a toothache plant because it relieves toothache symptoms. ² It has also been used in Ayurveda and folklore medicinal treatments for headache, asthma, rheumatism, fever, sore throat, and hemorrhoids. ^{2 -4} Biological properties of A. oleracea mainly depend on N-alkylamides and the major constituent of the N-alkylamides group is spilanthol. ⁵ Spilanthol is responsible for strong local anesthetic, analgesic, ^6,7 and insecticidal activities. ⁸ In addition, the extracts of A. oleracea has been shown to possess antioxidant activity in vitro conditions. ^9,10 We also found growth inhibitory activity of the extracts of A. oleracea on several plants. However, there has been no report on the compounds with growth inhibitory activity in A. oleracea. Therefore, in this study 2 plant growth inhibitory substances in A. oleracea were isolated and characterized and their biological activities were determined.

Acmella oleraces extracts showed the growth inhibitory activity on the root and shoot growth of cress, lettuce, Italian ryegrass, and barnyard grass in an extract-concentration dependent manner (Figure 1). The extract obtained from 30 mg of A. oleracea inhibited cress, lettuce, Italian ryegrass, and barnyard grass resulting in root growth of 4.7%, 0%, 21.3%, and 57.1% compared with that of control root growth, respectively, and inhibited cress, lettuce, Italian ryegrass, and barnyard grass resulting in shoot growth of 7.8%, 0%, 43.4%, and 98.9% compared with that of control shoot growth, respectively.

Figure 1

Effects of aqueous methanol extracts of A. oleracea on root and shoot growth of cress, lettuce, Italian ryegrass, and barnyard grass. Means ± SE from 4 independent experiments with 10 seedlings for each determination are shown. Asterisks indicate significant difference between control and treatment: *P < 0.05, **P < 0.01, ***P < 0.001.

IC₅₀ values of the extracts on roots of cress, lettuce, Italian ryegrass, and barnyard grass were 5.9, 0.4, 25.9, and 7.8 mg dry weight equivalent extract/mL, respectively, and those on shoots of cress, lettuce, Italian ryegrass, and barnyard grass were 4.3, 0.8, 126.0, and 15.1 mg dry weight equivalent extract mL^–1.

The extract of A. oleraces therefore had an inhibitory effect on both the dicotyledonous (cress and lettuce) and monocotyledonous (Italian ryegrass and barnyard grass) plants. These results suggest that A. oleraces may possess growth inhibitory activity and contain growth inhibitory substances.

The extracts of A. oleracea were separated with a silica gel column and the biological activity of all separated fractions was determined by cress bioassay. The active fraction was further purified by Sephadex LH-20, reverse-phase C₁₈ Sep-Pak cartridges, and high-performance liquid chromatography (HPLC), and 2 active compounds 1 and 2 were isolated.

The molecular formula of compound 1 was determined to be C₁₅H₁₉NO by using HRESIMS at m /z 230.1542 [M + H]⁺ (calcd. for C₁₅H₂₀NO, 230.1545, Δ = −0.3 mmu). ¹H nuclear magnetic resonance (NMR) (400 MHz, CDCl₃) of compound 1 showed δ_H 7.19 (dd, J = 15.2, 10.8 Hz, 1 H, H-3), 6.20 (dd, J = 14.4, 10.8 Hz, 1 H, H-4), 6.05 (dt, J = 14.4, 6.8 Hz, 1 H, H-5), 5.81 (d, J = 15.2 Hz, 1 H, H-2), 5.51 (brs, 1 H, NH), 3.17 (dd, J = 6.8, 6.0 Hz, 2 H, H-1′), 2.39 (m, 4 H, H-6, 7), 1.98 (s, 1 H, H-11), 1.80 (m, 1 H, H-2′), and 0.93 (d, J = 6.8 Hz, 6 H, H-3′). The ¹³C NMR (100 MHz, CDCl₃) showed δ_C 166.2 (C-1), 140.6 (C-3), 139.2 (C-5), 129.9 (C-4), 123.3 (C-2), 76.8 (C-8), 68.6 (C-11), 65.9 (C-9 or 10), 65.2 (C-10 or 9), 47.1 (C-1′), 31.4 (C-6), 28.8 (C-2′), 20.3 (C-3′), and 19.0 (C-7). Comparison of the spectral analysis of the compound with data ^11,12 indicates compound 1 was (E,E)-2,4-undecadien-8,10-diynoic acid isobutylamide (anacycline, Figure 2). This is the first time that this alkamide with diene-diyne structure was isolated from A. oleracea leaves, but it has been previously isolated from Artemisia dracunculus, ¹¹ Achillea ptarmica, and Anacyclus pyrethrum. ^12,13

Figure 2

Chemical structure of (E,E)-2,4-undecadien-8,10-diynoic acid isobutylamide (compound 1) and nona-(2Z)-en-6,8-diynoic acid 2-phenylethylamide (compound 2).

The molecular formula of compound 2 was determined to be C₁₇H₁₇NO by using HRESIMS at m/z 252.1385 [M + H]⁺ (calcd. for C₁₇H₁₈NO, 252.1388, Δ = −0.3 mmu). The ¹H NMR (400 MHz, CDCl₃) of compound showed δ_H 7.20-7.34 (m, 5 H, H-4′, 5′, 6′), 6.03 (dt, J = 11.2, 7.2 Hz, 1 H, H-3), 5.71 (brd, J = 11.2 Hz, 1 H, H-2), 5.52 (brs, 1 H, NH), 3.57 (dt, J = 7.2, 6.8 Hz, 2 H, H-1′), 2.84-2.89 (m, 4 H, H-4, 5), 2.41 (t, J = 6.8 Hz, 2 H, H-2′), 1.98 (s, 1 H, H-9). The ¹³C NMR (100 MHz, CDCl₃) showed δ_C 166.1 (C-1), 142.4 (C-3), 139.0 (C-3′), 128.9 (C-4′), 128.8 (C-5′), 126.7 (C-6′), 124.0 (C-2), 65.6 (C-7), 65.0 (C-9), 40.5 (C-2′), 35.8 (C-6), 27.1 (C-4), and 19.1 (C-5). Comparison of the spectral analysis of the compound with data ¹⁴ indicates that compound 2 was nona-(2Z)-en-6,8-diynoic acid 2-phenylethylamide (Figure 2). Compound 2 has been isolated as an amide mixture with deca-(2Z)-en-6,8-diynoic acid 2-phenylethlylamide from A. oleracea. ¹⁴

Compound 1 significantly inhibited the growth of cress roots and shoots at concentrations greater than 0.3 mM and that of barnyard grass roots at concentrations greater than 0.03 mM (Figure 3). No significant inhibition was found on the growth of barnyard grass shoots. IC₅₀ values of compound 1 were 0.49 and 0.49 mM for cress roots and shoots, and 0.12 mM for barnyard grass roots. Compound 1 was found inhibitory activity on the melanin production in mouse melanoma cells ¹¹ and antiprotozoal activity. ¹² Compound 2 significantly inhibited the shoot growth of cress and root growth of barnyard grass at concentrations greater than 1 mM (Figure 4). IC₅₀ values of compound 2 were 0.95 and 2.2 mM for cress shoots and barnyard grass roots, respectively. A mixture of compound 2 with deca-(2Z)-en-6,8-diynoic acid 2-phenylethlylamide has been found larvicidal activity on Aedes aegypti. ¹⁴ However, both compounds 1 and 2 have not been reported on the growth inhibitory activity on plants. Therefore, this is the first report of having growth inhibitory activity of compounds 1 and 2 on plants. The extracts of A. oleracea showed a growth inhibitory activity of cress, lettuce, Italian ryegrass and barnyard grass. Compounds 1 and 2 may contribute to the growth inhibitory effect of A. oleracea.

Figure 3

Effects of (E,E)-2,4-undecadien-8,10-diynoic acid isobutylamide on the growth of roots and shoots of cress and barnyard grass. Means ± SE from 4 independent experiments with 10 seedlings for each determination are shown. Asterisks indicate significant difference between control and treatment: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Effects of nona-(2Z)-en-6,8-diynoic acid 2-phenylethylamide on the growth of roots and shoots of cress and barnyard grass. Means ± SE from 4 independent experiments with 10 seedlings for each determination are shown. Asterisks indicate significant difference between control and treatment: *P < 0.05, **P < 0.01, ***P < 0.001.

Experimental

Plant Material

The whole plants of A. oleracea was collected in Chiang Mai province, Thailand, in August 2012. The plant was dried, ground into a powder, and kept at 4°C until extraction. Biological activity was determined using cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), Italian ryegrass (Lolium moltiflorum Lam.), and barnyard grass (Echinochloa crus-galli (L.) P. Beauv.).

Extraction and Bioassay

Powder of A. oleracea (100 g) was extracted twice with 700 mL of 70% (v/v) aqueous methanol for 2 days and filtered using filter paper (No. 2; Toyo Ltd., Japan). The residue was extracted again with 1 L of methanol for 1 day and filtered. Two filtrates of the extracts were combined and evaporated to dryness at 40°C. The extract was dissolved with 200 mL of methanol, and an aliquot of the extract at 5 concentrations (3, 10, 30, 100, and 300 mg dry weight equivalent extract mL^–1) was added to a sheet of filter paper in Petri dishes (28 mm) and the solvent was dried in a fume hood. The filter paper was moistened with 0.6 mL of 0.05% (v/v) aqueous solution of polyoxyethylenesorbitan monolaurate (Tween 20; Nacalai, Kyoto, Japan) and 10 seeds of cress, lettuce, and 10 pre-germinated seeds of Italian ryegrass and barnyard grass were then placed on it. Only Tween 20 solution was used in control treatment. All treatments were incubated in the dark at 25°C for 48 hours. The length of roots and shoots of those seedlings were measured and calculated comparing with the length of control seedlings. Concentrations required for 50% growth inhibition (IC₅₀) on the roots and shoots of the test plants were determined using a logistic regression function based on the bioassay.

Isolation of Active Compounds

Powder of A. oleracea was extracted with 70% (v/v) aqueous methanol and concentrated to produce an aqueous residue as described above. The aqueous residue was adjusted to pH 7.0 with 1 M phosphate buffer and partitioned 3 times against an equal volume of ethyl acetate. The ethyl acetate fraction was evaporated to dryness after drying over anhydrous Na₂SO₄. The residue of ethyl acetate fraction was subjected to a column of silica gel (60 g, silica gel 60, 70-230 mesh; Nacalai, Kyoto, Japan) and eluted with 20%, 30%, 40%, 50%, 60%, 70%, and 80% ethyl acetate in n-hexane (v/v; 100 mL per step), ethyl acetate (100 mL), and methanol (300 mL). The biological activity of all separated fractions was determined using a cress bioassay. A growth inhibitory active fraction was obtained by elution with 60% ethyl acetate in n-hexane. After evaporation of the active fraction, the residue was subjected to a column of Sephadex LH-20 (100 g; Amersham Pharmacia Biotech, Buckinghamshire, UK) and eluted with 20%, 40%, 60%, and 80% (v/v; 150 mL per step) aqueous methanol and methanol (200 mL per step). The active fraction obtained by elution with 80% aqueous methanol was evaporated to dryness and subjected to a reverse phase C₁₈ cartridge (1.2 × 6.5 cm; YMC Ltd., Kyoto,) eluted with 20%, 30%, 40%, 50%, 60%, 70%, and 80% (v/v; 15 mL per step) and methanol (30 mL). The active fractions were eluted with 60% aqueous methanol and evaporated to dryness. The residue was finally separated using reverse-phase HPLC (ODS AQ-325, 10 mm i.d. × 50 cm; YMC Ltd.) and eluted at a flow rate of 1.5 mL min⁻¹ with 60% aqueous methanol and detected at 220 nm. Inhibitory activity was found in peak fractions eluted between 108 to 112 minutes (compound 1) and 113 to 119 minutes (compound 2). The isolated compounds were determined using HRESI mass, ¹H NMR (400 MHz, CDCl₃), and ¹³C NMR spectra (100 MHz, CDCl₃).

Biological Activity of the Isolated Compounds

Compounds 1 and 2 were dissolved in methanol and an aliquot of solution (0.03, 0.1, 0.3, 1, 1300, and 2 mM) was added to a sheet of filter paper in the Petri dishes as described above, and 10 seeds of cress or 10 pregerminated seeds of barnyard grass were placed on the filter paper. All the treatments were incubated in the dark at 25°C for 48 hours. The length of seedling shoots and roots were measured and calculated by comparing with the growth of control seedlings.

Statistical Analysis

All experiments were performed with 3 replicates (10 seeds/replicate) and repeated twice (n = 60). The bioassay data were analyzed by SPSS (ver. 21) using one-way ANOVA and the differences between treatments were evaluated by Tukey’s test.