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Abstract

Background: Aralia armata (Araliaceae) is considered to exhibit effective molluscicidal activity, however, the relationship between the chemical components and molluscicidal activity has not been clearly elucidated. This research attempts to decipher these correlations among the 15 compounds isolated from Vietnam-grown A. armata roots against the freshwater snail, Pomacea canaliculata, a gastropod causing severe damage in agricultural production. Methods: Fifteen saponins were isolated from the methanol root extract of A. armata using chromatographic methods and were identified using spectroscopic techniques. The compounds were screened for molluscicidal activity against P. canaliculata, as well as toxicity against brine shrimp (Artemia sp.) and phytotoxicity against rice germination and growth. Results: The saponin compounds exhibited extraordinary inhibition of P. canaliculata with LC₅₀ values ranging from 7.90 to 17.50 µg/mL. Notably, the active compounds from A. armata exhibit safety for both nontarget aquatic animals, specifically Artemia sp. with LC₅₀ values between 148.55 and 193.22 µg/mL, and the growth and development of Oryza sativa L. plants showed very little difference compared with the negative control. A molecular docking analysis indicated P. canaliculata acetylcholinesterase (PcAChE) and the actin-binding protein villin (PcVillin) to be potential biomolecular targets of the A. armata saponins. Conclusion: The present experimental and in silico data illustrate the potential of A. armata in agricultural applications.

Keywords

eco-friendly molluscicidal activity saponin isolation molecular docking

Introduction

The genus Aralia is a large genus in the family Araliaceae. Up to now, scientists have discovered about 30 different species, popularly distributed in Asia and America.¹ Aralia armata, a common herb in China and Vietnam, is known as a folk medicine reported to show anti-inflammatory (arthritis, hepatitis), immunostimulating, and antimalarial activities.² Previous studies have shown that major constituents of the Aralia genus include saponins (triterpenoid glycosides), flavonoids, steroids, and acetylenic lipids,^3,4 among which saponins are characteristic of A. armata in particular and the Aralia genus in general.^5,6 The saponin compounds in A. armata have exhibited various activities, including antibacterial,^7–10 antifungal,^11–14 antioxidant,^15–17 anti-inflammatory,^18–22 in vitro inhibition of cancer cells,^23–27 and anti-HIV activities.^28–30 Moreover, saponins are also utilized as treatments against mollusks that caused crop damage, namely the species Pomacea canaliculata, Pomacea maculata, Gyraulus convexiusculus, and Tarebia granifera.^31–35

Currently, diseases related to parasites are a concern in many countries around the world.^36,37 The freshwater snail P. canaliculata (also known as the golden apple snail) is known to be an intermediate host for several parasites that are pathogenic to humans. For example, the parasitic worm Angiostrongylus cantonensis is the cause of filariasis in human beings, which is characterized by eosinophilic meningitis.^38–40 Gnathostoma spinigerum causes gnathostomiasis, an infection that human acquires by ingesting the third-stage larvae of this worm,^41,42 which can lead to blindness, paralysis, and even death.⁴³ Notably, P. canaliculata also seriously affects agricultural production, especially rice,^44–46 and it is one of the most invasive species in the world.⁴⁷ A significant portion of agricultural land in Asia has been invaded and damaged by the golden apple snail.⁴⁴ The large-scale increase in the number of golden apple snails has affected rice cultivation throughout tropical and subtropical regions as well as southern Europe and the United States of America.^48,49 One of the common solutions employed is pesticide application, which contains many toxic chemicals that can seriously affect human health, the ecosystem, and the environment. Alternatively, natural biological agents that could control the snail population effectively and reduce the risk of development of resistance to chemical pesticides would be welcome. To the best of our knowledge, no studies on the molluscicidal activity and chemical composition of A. armata grown in Vietnam have been published. Driven by those challenges, the chemical composition from the roots of A. armata and their activity against P. canaliculata is reported for the first time, simultaneously exploring the relationship between the chemical structures and molluscicidal activity. This study also illustrates the safety of components from A. armata for the aquatic environment in general and nontarget aquatic animals in particular.

Results and Discussion

Isolation

Eight oleanolic acid saponins, pseudoginsenoside RT₁ (AR-01),⁵⁰ chikusetsusaponin IV (AR-02),⁵¹ narcissiflorine (AR-03),⁵² stipuleanoside R₂ methyl ester (AR-04),⁵⁰ stipuleanoside R₁ (AR-05),⁵³ stipuleanoside R₂ (AR-06),⁵⁰ 3-O-{β-D-glucopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-galactopyranosyl-oleanolic acid (AR-09),⁵⁰ and 3-O-{β-D-glucopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-galactopyranosyl}-oleanolic acid-28-O-β-D-glucopyranoside (AR-10)⁵⁰; 5 oleanane-type triterpene glycosides, AR-07,⁵⁴ AR-08,⁵⁴ 3-O-β-D-glucuronopyranosyl-oleanolic acid-28-O-β-D-glucopyranoside (AR-13),⁵⁰ 3-O-β-D-glucuronopyranosyl-23-hydroxyoleanolic acid-28-O-β-D-glucopyranoside (AR-14),⁵⁵ and chikusetsusaponin IVa methyl ester (AR-15)⁵⁶; and 2 monoterpene glycosides, linalool 3-O-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranoside (AR-11)⁵⁷ and linalool 3-O-α-L-arabinopyranosyl-(1→6)-O-β-D-glucopyranoside (AR-12),⁵⁸ were characterized from the methanol extract of the roots of A. armata. The identifications of these compounds are first based on the comparison of their NMR data with the data of suggested compounds previously reported^50–58 and further confirmed by HSQC and HMBC spectra when necessary. Notably, to the best of our knowledge, among these isolated compounds, six (AR-07, AR-08, AR-14, AR-15, AR-09, AR-10) are reported for the first time from the A. armata roots, and two (AR-11, AR-12) are reported for the first time from the genus Aralia. In particular, compounds AR-09 and AR-10 were previously detected only in the leaves of A. armata, but are now observed in the roots of this species.

Molluscicidal Activity

The molluscicidal activity of fractions and compounds isolated from A. armata roots against golden apple snail (P. canaliculata) after 24 h is represented by LC₅₀ and LC₉₀ values (µg/mL). A comparison of the molluscicidal activity of the fractions, the active compounds from A. armata, and the positive control (tea saponin) is illustrated in Table 1. The experimental results indicate that the molluscicidal activity is directly proportional to the concentration of the active ingredients. It can be seen that the molluscicidal activity of the fractions is particularly noteworthy, especially the aqueous fraction (LC₅₀ 18.59 µg/mL), which is the basis for choosing the aqueous fraction to further separate and search for active compounds with molluscicidal activity from the roots of A. armata. Indeed, the compounds isolated from A. armata roots exhibit remarkably robust activity against the P. canaliculata snail, the LC₅₀ values of the compounds are in the range of 7.90 to 17.50 µg/mL. Furthermore, the molluscicidal capacities of several compounds from A. armata are higher than that of tea saponin (LC₅₀ = 11.02 µg/mL), an agent known to have powerful molluscicidal capacity.⁵⁹ Thus, the contents of saponin compounds separated from A. armata roots demonstrated significant molluscicidal activity.

Table 1.

The Molluscicidal Activity of Compounds Isolated from Aralia armata.

Test agent	LC₅₀ (95% limits)	LC₉₀ (95% limits)	χ²	p
CH₂Cl₂ fraction	51.35 (43.57–60.03)	186.90 (152.12–241.27)	11.91	0.036
EtOAc fraction	41.43 (34.54–50.17)	276.66 (197.56–434.10)	9.06	0.107
Aqueous fraction	18.59 (15.78–21.84)	89.26 (69.47–123.41)	5.65	0.342
CAA2A2	14.40 (12.46–16.56)	52.42 (43.01–67.02)	7.47	0.188
CAA2C2	11.64 (10.08–13.38)	40.71 (33.57–51.79)	10.00	0.075
CAA4B2	12.45 (10.75–14.34)	45.66 (37.44–58.48)	4.69	0.455
CAA2A4	13.02 (11.34–14.90)	43.06 (35.68–54.42)	4.00	0.550
CAA2A3	17.88 (15.36–20.50)	49.92 (42.55–60.68)	7.10	0.213
CAA4B3	11.54 (9.89–13.39)	45.44 (36.71–59.59)	7.76	0.101
AR-01	17.33 (15.44–19.60)	33.15 (29.47–38.35)	5.52	0.356
AR-02	7.90 (6.83–9.11)	27.89 (22.61–36.49)	10.22	0.069
AR-03	17.50 (15.72–19.61)	31.32 (28.04–35.89)	6.68	0.245
AR-04	8.00 (6.93–9.18)	26.78 (21.85–34.77)	7.15	0.210
AR-05	17.33 (15.34–19.71)	34.70 (30.70–40.37)	6.88	0.230
AR-06	8.34 (7.21–9.62)	29.81 (24.13–39.08)	7.52	0.185
AR-07	9.83 (8.20–11.75)	61.19 (46.14–90.83)	22.22	0.000
AR-09	16.17 (14.02–18.69)	57.88 (46.52–76.68)	8.76	0.119
AR-08	9.40 (7.89–11.17)	53.54 (40.61–76.74)	9.18	0.102
AR-10	15.03 (13.11–17.22)	47.78 (39.08–61.84)	7.39	0.194
AR-11	15.59 (13.87–17.64)	30.29 (26.92–35.00)	4.85	0.434
AR-12	10.30 (8.96–11.82)	33.90 (27.69–43.90)	6.60	0.252
AR-13	7.59 (6.64–8.67)	22.70 (18.77–28.99)	5.62	0.345
AR-14	8.73 (7.57–10.06)	30.37 (24.66–39.64)	9.86	0.079
AR-15	7.61 (6.70–8.64)	20.74 (17.33–26.15)	9.49	0.091
Tea saponin (positive control)	11.02 (9.35–12.79)	36.20 (30.00–45.84)	11.75	0.038

AChE Enzyme Inhibitory Activity

Several isolated compounds from A. armata were screened for inhibitory activity against acetylcholinesterase (AChE, see Table 2).

Table 2.

AChE Inhibitory Activity of Some Isolated Compounds.

Concentration (µg/mL)	AR-01		AR-02		AR-03		AR-04		AR-05
Concentration (µg/mL)	% inhibition	SD	% inhibition	SD	% inhibition	SD	% inhibition	SD	% inhibition	SD
500	88.39	1.44	86.57	2.26	84.91	2.05	83.45	1.44	84.69	3.80
100	54.28	1.64	54.57	1.64	53.70	1.23	45.86	2.46	48.62	2.26
20	33.38	1.23	23.15	0.31	19.52	1.95	16.18	1.16	18.43	1.23
4	9.07	0.95	11.47	0.95	7.40	0.21	7.55	0.21	4.72	0.33
IC₅₀	68.62 ± 6.48		91.49 ± 5.78		100.03 ± 8.13		138.84 ± 11.81		118.05 ± 10.76
Concentration (µg/mL)	AR-07		AR-09		AR-10		AR-12		Galantamine*
Concentration (µg/mL)	% inhibition	SD	% inhibition	SD	% inhibition	SD	% inhibition	SD	% inhibition	SD
500	88.39	2.87	86.57	2.98	82.73	2.26	84.47	3.28	89.29	1.85
100	52.76	2.57	49.42	1.33	47.24	2.77	54.64	1.95	54.93	2.36
20	32.15	0.31	19.59	1.03	27.87	0.41	29.32	1.13	23.01	1.23
4	7.04	0.54	6.89	0.72	3.19	0.21	11.39	0.62	8.35	0.62
IC₅₀	74.05 ± 7.38		113.22 ± 7.05		112.67 ± 11.10		78.24 ± 5.64		1.75 ± 0.15

*Galantamine was tested in the concentration range of 10, 2, 0.4, and 0.08 µg/mL.

Acute Toxicity for Brine Shrimp (Artemia sp.)

To evaluate the impact of active ingredients from A. armata on the aquatic environment, acute toxicity experiments on adult brine shrimp (Artemia sp.) were conducted. The fractions closest to the pure compounds were put through trials. Experimental data indicate that the higher the sample concentrations, the higher the toxicity to brine shrimp (Figure 1). At concentrations of 25 to 300 µg/mL, most of the fractions were strongly toxic for Artemia sp., with mortality rates higher than 80%. The fraction AA2A2 shows the highest toxicity with 100% of mortality at 250 µg/mL concentration.

Figure 1.

Schematic demonstration of the mortality of Artemia sp. depending on the concentration of fractions.

The LC₅₀ values (µg/mL) for brine shrimp ranged from 148.55 to 193.22 µg/mL after 24 h of exposure. Compared with the toxicity to P. canaliculata, which has LC₅₀ values in the range of 11.54 to 17.88 µg/mL, the toxicity of the fractions from A. armata to the golden apple snail is much higher than that of the brine shrimp (10–15 times) under the same experimental conditions (Table 3). Over the LC₉₀ concentration range of the fractions CAA2A2, CAA4A3, CAA2A4, CAA4B2, CAA4B3, AA2C2 (40.71–52.42 µg/mL) killed <8% of Artemia sp. Thus, it can be seen that the molluscicidal components of A. armata roots are virtually safe for aquatic animals at the median lethal concentrations.

Table 3.

Comparison of Acute Toxicity to Artemia sp. and Molluscicidal Activity of Fractions Isolated from Aralia armata.

Fraction	LC₅₀ (95% limits)	LC₉₀ (95% limits)	χ²	p	SI*
CAA2A2	148.55 (138.53–158.39)	236.50 (222.13–254.77)	18.07	0.001	10.32
CAA4A3	178.36 (168.00–188.82)	274.27 (258.30–294.69)	8.40	0.078	9.98
CAA2A4	193.22 (171.02–221.94)	711.87 (531.28–1120.56)	3.82	0.431	14.84
CAA4B2	155.41 (143.89–167.07)	310.50 (278.37–358.14)	4.50	0.343	12.48
CAA4B3	164.48 (150.52–178.11)	303.59 (279.18–337.50)	5.22	0.265	14.25
AA2C2	162.11 (152.10–172.08)	251.77 (237.10–270.33)	8.37	0.079	13.93

*SI = selectivity index = LC₅₀ (Artemia sp.)/LC₅₀ (P. canaliculata).

Phytotoxic Activity of Aqueous Fraction

This study aimed to evaluate the control effect against P. canaliculata of the water-soluble fraction of the roots of A. armata. Additionally, the phytotoxicity of the aqueous fraction was evaluated on rice (Oryza sativa L.), the results of which are presented in Table 4. Compared with the negative control (water), the aqueous fraction did not exhibit detrimental effects on the germination or growth of rice.

Table 4.

Phytotoxic Activity Results of the Aqueous Fraction.

Concentration (μg/mL)	N	Shoot length (cm)				Root length (cm)	Germination rate (%)
		7 days	14 days	21 days	28 days	28 days
230	100	9.4 ± 1.2	19.5 ± 2.3	34.9 ± 4.9	44.1 ± 8.0	15.6 ± 3.0	100
23	100	9.3 ± 1.2	19.3 ± 3.1	33.2 ± 4.7	43.3 ± 8.5	15.0 ± 3.4	99 ± 2
Control	100	8.6 ± 1.5	19.0 ± 3.3	33.3 ± 4.6	43.1 ± 9.1	14.7 ± 3.8	99 ± 2

In silico Evaluation of A. armata Saponins

There are apparently no P. canaliculata protein structures available in the Protein Data Bank.⁶⁰ There are, however, several P. canaliculata protein sequences available from the Protein Database.⁶¹ Homology models for several P. canaliculata proteins, acetylcholinesterase (PcAChE), alanine aminotransferase (PcALT), aspartate aminotransferase (PcAST), heat-shock protein 70 (PcHsp70), phenylalanine-4-hydroxylase (PcPheOH), and actin-binding protein villin-1 (PcVillin), were constructed from crystal structure templates available in the Protein Data Bank using the SWISS MODEL server (https://swissmodel.expasy.org/).

Molecular docking was carried out using Molegro Virtual Docker v 6.0.1 (Molegro ApS). From the docking scores (Table 5), it is apparent that the A. armata saponins preferentially target P. canaliculata acetylcholinesterase (PcAChE) and actin-binding protein (PcVillin).

Table 5.

Normalized Docking Scores (DS_norm, kJ/mol) of Aralia armata Saponins with Pomacea canaliculata Target Proteins.

Ligand	PcAChE		PcALT		PcAST		PcHsp70		PcPheOH		PcVillin
Ligand	(6ARX)	(6ARY)	(3IHJ)	(1AKA)	(1AMA)	(1TAR)	(7O6R)	(7SQC)	(5DEN)	(6HYC)	(3FFN)	(2FGH)
AR-01	−91.9	−100.3	−85.4	−82.0	−69.6	−90.8	−94.6	−92.1	−59.0	no dock	−107.7	−110.3
AR-02	−63.6	−106.5	−73.7	−67.0	−20.8	−99.4	−96.8	−65.5	−73.9	−62.0	−85.6	−100.5
AR-03	−103.8	−102.5	−70.9	−77.2	−54.8	−44.3	−98.5	−56.1	−67.5	−91.0	−104.4	−107.2
AR-04	−94.3	−115.7	−56.8	−72.0	−58.2	−71.1	−82.8	−39.3	−36.0	no dock	−85.3	−86.6
AR-05	−118.5	−102.5	−76.5	−78.9	−59.3	−62.0	−80.5	−13.4	−26.3	−18.5	−91.6	−113.2
AR-06	−55.0	−105.0	−75.7	−63.8	−54.2	−65.2	−63.2	−49.7	no dock	−78.7	−100.8	−110.8
AR-07	−74.8	−43.6	−75.6	−83.3	−24.0	−29.1	−99.1	no dock	−1.8	−87.6	−97.2	−101.0
AR-08	−118.2	−45.3	−82.6	−66.9	−30.8	−78.6	−87.4	no dock	−23.8	−68.5	−93.3	−86.2
AR-09	−99.6	−97.3	−75.6	−73.2	−67.5	−69.5	−96.8	−24.6	no dock	−42.7	−100.1	−106.1
AR-10	−52.6	−90.1	−67.1	−76.0	−33.4	−80.1	−95.1	−48.0	no dock	no dock	−96.0	−93.1
AR-11	−118.3	−124.6	−86.2	−92.4	−101.9	−99.3	−94.4	−113.5	−119.9	−118.7	−127.4	−110.7
AR-12	−111.2	−117.7	−91.8	−94.6	−87.5	−90.3	−102.2	−106.2	−117.1	−119.1	−122.7	−111.5
AR-13	−111.5	−104.1	−94.1	−71.6	−83.9	−54.2	−76.3	−41.5	no dock	−62.3	−90.2	−99.6
AR-14	−103.8	−107.0	−80.0	−68.2	−90.9	−81.1	−87.3	−40.2	−28.5	−65.2	−94.3	−64.0
AR-15	−90.8	−106.4	−73.5	−83.3	−78.0	−76.4	−92.3	−8.7	no dock	−49.9	−102.6	−81.4

Lowest-energy docking scores for each ligand are highlighted in bold. “no dock” = positive docking energy.

Acetylcholinesterase catalyzes the hydrolysis of the neurotransmitter acetylcholine, which serves to terminate synaptic transmission. Inhibition of AChE leads to paralysis and possibly death of the organism. Thus, PcAChE inhibition by A. armata saponins may account for the observed toxicity of these compounds to P. canaliculata. The lowest-energy docking scores for ligands against PcAChE were AR-05 and AR-08 (−118.5 and −118.2 kJ/mol, respectively). The key intermolecular contacts of AR-05 and AR-08 in the binding site of PcAChE are summarized in Table 6.

Table 6.

Key Intermolecular Contacts Between the Aralia armata Ligands With the Best Docking Scores and Their Pomacea canaliculata Protein Targets.

Ligand	Target	Key intermolecular contacts
AR-05	Acetylcholinesterase (PcAChE)	Tyr361 (H-bond), Tyr150 (H-bond), Asp99 (H-bond), Gln305 (vdW), Trp357 (H-bond), Phe308 (vdW), Pro97 (H-bond), Phe358 (vdW)
AR-08	Acetylcholinesterase (PcAChE)	Tyr361 (H-bond), Asp99 (H-bond), Trp357 (H-bond), Trp111 (vdW), Gln305 (vdW), Trp98 (H-bond), Phe308 (vdW), Phe358 (vdW)
AR-11	Villin, actin-binding protein (PcVillin)	Lys619 (vdW), Glu617 (H-bond), Trp356 (vdW), Pro357 (H-bond), Leu620 (H-bond), Asp355 (vdW)
AR-12	Villin, actin-binding protein (PcVillin)	Lys619 (H-bond), Glu617 (H-bond), Arg618 (H-bond), Trp356 (vdW), Leu620 (H-bond)

H-bond = hydrogen-bonding interaction; vdW = van der Waals (hydrophobic) interaction.

Villin is a member of the gelsolin family of actin-modulating proteins.⁶² These proteins block the ends of actin causing the depolymerization of actin filaments, but also catalyzing the bundling activity of actin, thereby influencing the polymeric state of actin. As important components of the cytoskeleton, actin-binding proteins play a central role in cell locomotion and motility, and inhibition of actin-binding proteins would likely have serious consequences. The compounds with the best docking scores with PcVillin were AR-11 and AR-12 (−127.4 and −122.7 kJ/mol, respectively).

Experimental

General

The mass spectra were recorded on an Agilent 6530 accurate-mass Quadrupole Time-of-Flight liquid chromatography-mass spectrometry system. NMR spectroscopic analyses were carried out by the Bruker 500 MHz spectrometer. The Jasco P2000 polarimeter is utilized for measuring the optical rotation of pure compounds. Silica gel (43–60 μm), reversed-phase C18 gel, and diaion HP-20 resins were used in the column chromatography. In addition, the Agilent 1100 HPLC system was utilized for HPLC analysis. The compounds were examined by thin-layer chromatography using the precoated silica gel 60 F₂₅₄, RP-18 F_254S sheets. The spots were visualized by dipping thin-layer chromatography sheets into 5% H₂SO₄ solution in ethanol followed by heating at 110°C for 10 min.

Plant Material

The roots of A. armata were collected in Hoa Vang district, Danang city (16°00′55″N 108°07′8″E) in October 2020. Plant samples were authenticated by Nguyen The Cuong (The Institute of Marine Biochemistry, VAST, Hanoi, Vietnam). The voucher specimen (Code: NCCT-P71R) was deposited in the Institute of Marine Biochemistry, VAST, Hanoi, Vietnam.

Snails (P. canaliculata)

Eggs of wild P. canaliculata were collected from a field in Dien Ban District, Quang Nam province, Vietnam (15°55′58″N 108°11′46″E) in November 2021, the time to suspend rice seeding from the rice fields in the central provinces of Vietnam. Snail eggs after harvest were incubated in the laboratory at 25 ± 2°C, 70 ± 5% humidity. After hatching, snails were fed water spinach leaves (Ipomoea aquatica) and raised to a size of 3 to 5 mm (length) in about 2 to 3 weeks. P. canaliculata snail was identified by Dr Nguyen Huy Hung.

Brine Shrimp (Artemia sp.)

Juvenile brine shrimps (3–4 mm) were collected at Danang city, Vietnam. Brine shrimps after collection were cultured at salinity 3.5%, pH 7.5 to 8.0, temperature 25 ± 2°C, humidity 70 ± 5%, cycle 12 h dark: 12 h light, fed algae during growth. Adult brine shrimps were 9 to 10 mm long (10–12 days from the collection date).

Rice

The original 13/2 rice provided by Quang Nam Agricultural Seed Joint Stock Company in Vietnam was used for germination assay and seedling toxicity test. The soil used for the seedling rice toxicity test was collected in Dien Ban District, Quang Nam province, Vietnam (15°55′58″N 108°11′46″E), in the rice field where golden apple snails were collected. After collection, the soil was loosened before testing.

Extraction and Isolation

Fresh roots of A. armata (5 kg) were dried and pulverized and then extracted with methanol (MeOH) in an ultrasonic bath 4 times (30 min each). The combined extracts were evaporated to obtain MeOH extracts (135 g). The MeOH extracts were partitioned by the liquid–liquid extraction with dichloromethane and ethyl acetate successively. The afforded aqueous fraction was chromatographed on a diaion column (HP-20) with an aqueous solvent to remove salts and oligosaccharides. Next, the collected sample was eluted on a chromatographic column with MeOH/water (m/w) solvents (25%, 50%, 75%, and 100% volume of MeOH), obtaining four major fractions (AA1–AA4).

Fraction AA2 (12.0 g) was chromatographed on a silica gel column, eluting with dichloromethane/methanol (D/M) (1/0–0/1, v/v) to obtain four fractions, AA2A–AA2D. The AA2A fraction (6.3 g) was eluted by the reversed-phase C18 gel chromatographic column using the solvent system MeOH/water (3/4, v/v) to obtain five subfractions, AA2A1–AA2A5. Subfraction AA2A2 (521 mg) was purified on an HPLC (J'sphere ODS-H-80 column 250 mm × 20 mm, eluting with 20% acetonitrile in water, a flow rate of 3 mL/min) to obtain three compounds AR-01 (19.6 mg), AR-02 (7.6 mg), and AR-03 (8.2 mg). The AA2A4 subfraction (465 mg) was purified on HPLC (J'sphere H-80 column 250 mm × 20 mm, using 15% acetonitrile in water, a flow rate of 2 mL/min) to produce three compounds AR-04 (11.5 mg), AR-05 (9.0 mg), and AR-06 (8.4 mg). Fraction AA2C (3.0 g) was separated by reversed-phase C18 gel column chromatography with MeOH/water (3/5, v/v) to obtain four subfractions, AA2C1–AA2C4. The AA2C2 subfraction (418 mg) was purified on HPLC (J'sphere H-80 column 250 mm × 20 mm, eluting with 16% acetonitrile in water, a flow rate of 2 mL/min) to yield two compounds AR-07 (9.7 mg) and AR-08 (11.3 mg).

Fraction AA4 (15.0 g) was chromatographed on a silica gel column, eluting with dichloromethane/methanol (1/0–0/1, v/v) to give four fractions AA4A–AA4D. Fraction AA4A (3.5 g) was chromatographed on a reversed-phase C18 column, eluting with methanol/water (3/3, v/v) to give four subfractions AA4A1–AA2A4. Subfraction AA4A3 (890 mg) was further purified by HPLC (J'sphere H-80 column, 250 mm length × 20 mm ID, eluting with 20% acetonitrile in water, a flow rate of 2 mL/min) to give compounds AR-09 (7.2 mg) and AR-10 (7.5 mg). Fraction AA4B (5.7 g) was isolated by a reversed-phase chromatography column, eluting with MeOH/water (3/2, v/v) to obtain five subfractions, AA4B1–AA4B5. The AA4B2 fraction (450 mg) was further purified by HPLC (J'sphere H-80 column, 250 mm length × 20 mm ID, eluting with 16% acetonitrile in water, a flow rate of 3 mL/min), yielding two compounds AR-11 (8.2 mg) and AR-12 (7.5 mg). Finally, three compounds AR-13 (14.5 mg), AR-14 (12.0 mg), and AR-15 (18.0 mg) were collected on HPLC purification (J'sphere H-80 column, 250 mm length × 20 mm ID, eluting with 20% acetonitrile in water, a flow rate of 3 mL/min) from AA4B3 fraction (640 mg).

Molluscicidal Activity

Molluscicidal activity tests of pure compounds were performed according to the procedure of Ding et al⁶³ with slight modifications. For the assay, the prepared samples were dissolved in DMSO (1% stock solution), placed in 150 mL distilled water, and added 20 snails into each beaker. The sets of controls using tea saponin and DMSO were treated as positive and negative controls, respectively. Tests were performed with concentrations of 100, 50, 25, 12.5, 6.0, 3.0, and 1.5 μg/mL; and concentrations of 50, 25, 12.5, 6.0, 3.0, and 1.5 μg/mL for the positive control (tea saponin), repeating the experiment 4 times at each concentration. The experiment was conducted at a temperature of 25 ± 2 °C with a photoperiod of 12 h:12 h (light/dark). After 24 h, the treated snails were transferred to beakers containing 150 mL of distilled water for recovery. After the next 24 h, the snails were assessed for mortality (immobility, does not attach to the wall of the beaker, and retraction inability of the head inside the shell in response after being lightly pushed by a blunt object). The number of dead/live snails was recorded.

Acute 48 h Toxicity Test for Artemia sp.

The experimental method with Artemia sp. was referenced based on the procedure of Cong et al.⁶⁴ Test samples were prepared by dissolving in DMSO (1% stock solution). The prepared sample was then dissolved in a distilled water solution containing 3.5% NaCl (solution 1). Twenty adult brine shrimps were placed in a beaker containing 150 mL of solution 1 with a certain concentration, and the same was done for the blank control (only brine shrimps with 3.5% NaCl solution and 1% DMSO). The individuals were not fed during the experiment. The experiment was carried out at concentrations of 300, 250, 200, 150, 100, 50, and 25 μg/mL, repeated 4 times at each concentration with the condition of temperature 25 ± 2 °C, humidity 70 ± 5%, cycle 12 h dark: 12 h light. After 24 and 48 h, the number of dead/living individuals was recorded. The individuals that stopped moving within 10 s were determined to be dead.

Germination Assay

Germination assay was performed based on the method of Abedin and Meharg⁶⁵ with some modifications. The test was performed using the aqueous fraction of A. armata root extract, near the mean lethal concentration of the aqueous fraction and tea saponin for the golden apple snail, of 23 µg/mL and at the concentration 10 times higher than that value of 230 μg/mL. Twenty-five rice seeds were placed in a petri dish 4 cm in diameter; the experiment was repeated 4 times at each concentration. Rice seeds were washed 3 times with distilled water before soaking with 2 mL of distilled water for 24 h. Next, filter papers (9 cm in diameter) moistened with distilled water were used to wrap the rice seeds and placed in a petri dish with a lid. The experiments were conducted in the condition of temperature 25 ± 2 °C, humidity 70 ± 5%, cycle 12 h dark:12 h light. After 2 days, the percentage of germinated and nongerminated rice seeds was recorded.

Seedling Toxicity Test

Toxicity to rice at the seedling stage was carried out based on the method of Khan et al⁶⁶ with a few modifications. Rice seeds in the germination assay continued to be used for this trial. The trial was performed on the aqueous fraction of A. armata root as above (23 and 230 µg/mL). The blank sample considered as a negative control was also performed using water without any agent. The experiment was conducted at the Medicinal Garden, Duy Tan University, Vietnam. The experiment was carried out in conditions of temperature 28 °C to 33 °C, humidity 65% to 85%, and cycle 12 h dark:12 h light. The pots (20 cm high, 10 cm diameter) were filled with soil, and 300 mL of the test solution was added to soften the soil, which is favorable for sowing. Five seeds were sown in each pot, and the experiment was repeated 10 times at each concentration. The heights of the rice plants were measured after 7, 14, 21, and 28 days of growth. On day 28, the roots of rice plants were measured. The height of the rice plant was measured from the culm base to the tip of the longest leaf, and the root length was measured from the junction between the stem and the base of the plant to the tip of the longest root. Mean values of rice plant height and root length were recorded.

AChE Enzyme Inhibitory Test

Acetylcholinesterase (AChE) inhibitory activity of essential oil was performed according to the method described by Ellman et al⁶⁷ and our previous study.³⁵ The stock solution was obtained by dissolving the essential oil in DMSO (Merck), which was then diluted with H₂O (deionized distilled water) to obtain different experimental concentrations. Each solution mixture consisted of 140 μL of phosphate buffer solution (pH 8), 20 μL of essential oil at concentrations of 500, 100, 20, and 4 μg/mL) and 20 μL of the enzyme AChE (0.25 IU/mL). The reaction mixtures were transferred to the test wells of a 96-well microtiter plate and incubated at 25 °C for 15 min. Then, solutions of 10 μL dithiobisnitrobenzoic acid (DTNB; 2.5 mM) and 10 μL acetylthiocholine iodide (ACTI; 2.5 mM) were added to each of the test wells and incubation continued for 10 min at 25 °C. At the end of the experiment, the absorbance of each solution was measured at 405 nm. Galantamine was used as a positive control. The negative control well did not contain the test sample. Each test was carried out in triplicate.

Data Analysis

Median lethal concentration (LC₅₀) values were computed via log-probit analysis⁶⁸ using Minitab® version 19.2020.1 (Minitab, LLC) with 95% confidence limits. The differences between treatments were controlled by the Euclidean distance.

Homology Modeling

Homology models for several P. canaliculata proteins, available from the Protein Database,⁶¹ were constructed from crystal structure templates available in the Protein Data Bank⁶⁰ using the SWISS MODEL server.⁶⁹ Three-dimensional structural models of P. canaliculata acetylcholinesterase (PcAChE), P. canaliculata alanine aminotransferase (PcALT), P. canaliculata aspartate aminotransferase (PcAST), P. canaliculata heat-shock protein 70 (PcHsp70), P. canaliculata tyrosine hydroxylase (PcPhOH), and P. canaliculata actin-binding protein (PcVillin) were generated based on multiple-threading alignments; the global model quality estimation was used to rank models (Table 7).

Table 7.

Pomacea canaliculata Target Protein Structures from Homology Modeling.

P canaliculata protein target	Protein sequence	Template PDB structure	Sequence identity	GMQE
Acetylcholinesterase (PcAChE)	1 mrqmmedhfa tlmlaavlvl llqearlvsg fgpivdtdkg qvqgktifvn gkkldvfygi	6ARX	53.96	0.70
	61 pyakpplkey rfrhpeqidr wegvlnatvl pksciqpwdd mfqnfsgsqm wnpntpised	6ARX	53.96	0.70
	121 clylnvwvpr vnlpyrhkav mvwiyggcfy sgtstldvyd pkylavendv ivvsmnyrlg	6ARY	53.96	0.70
	181 algflvmntp eargnaglld qrmalewvqr nienfggsph nvtlfgesag aasvglhlvs
	241 slsrglfnra ilqsgapqas wvtytpsegh srsrrlastl gcdgnlsdae vvqclrlvna
	301 hvffqnefsv vkgivqfpfl pvidgfflte spseylrrgr fkktplllgs nanegtwflv
	361 yedtenfsih enkqisdemt ghligslfyy hpqyptplnk fgkeaimfey tdwarpkdpw
	421 srrdhiaqav gdlhfvcpvn dladhyaqng qrvynywfeh ryslnpwpaw mgtlhgdeif
	481 fvfgkpldpa lgftdeekql srkimrywtn faktgdpnrg pgepsiqewp eyrhhernyl
	541 nlslsllktg yvdthkavra rqcafwhlyl pslvagtsdi savekewkee fhewktryiv
	601 dwkhqfdnfl nnyekedkdl reslressft plnvinvekt ivinnrehas yitlthslth
	661 slthslthsl taaeklptny lqyiv
Alanine aminotransferase (PcALT)	1 mfrservvtr tarkllqlps svepplcrrw kvltaaninp hvknmeyavr gpiviragei	3IHJ	63.21	0.85
	61 eqelkkgiqk pfedvirani gdchatgqrp ltfirqvial ctypdlmkss dfpsdaknra
	121 qrildgcggk sigaysdsag vrviredvar yiskrdgfps dadniflstg asdgiksilk
	181 lamtgesgnk ragvmipipq yplytatiae ynaypigyfl tesnnwaldm telrraitaa
	241 rpncqpraiv vinpgnptgq vltkeniqdi irfakeenll imadevyqhn vyaegcafhs
	301 fkkvlmemgp eystmemasf mstskgymge cgfrggyaea infdpdvkam lvksisaklc
	361 ssvsgqavmd vvvnppqpge psyevfkkeh dtvlgqlaek akmvtetfns ipgitcntvq
	421 gamyafprih lpkkaidaak vkgqapdafy cynlleetgi cvvpgsgfgq vegtyhfrtt
	481 ilppveklge mlarfrdfhv rfmdkys
Aspartate aminotransferase (PcAST)	1 mavqvlrssc sfsrllsknv psagtvsrna awwsnvemgp pdailgvtea ykrdpnpnkv	1AKA	69.58	0.88
		1AMA	70.00	0.87
		1TAR	70.00	0.88
	61 nlgvgayrdd ngkpyvlscv lkaeqeimas kldkeyagia gipeftkaaa elafgadspv
	121 ttgglnvtsq cisgtgslrv gaaffskwya kskdfwvptp twgnhtpifk hsglevkqyr
	181 yydpktcgfd fkgamedfsk ipagsvvvlh acahnptgvd pkpeqwkeis qlfkkqglyp
	241 ffdmayqgfa sgdidrdafa vrqfvkdgqq vvlaqsfakn mglygervga ftvvcdskee
	301 adrvmsqiki lirpmysnpp ihgariaari lttpalrsew lkevkgmadr iismrkqlre
	361 glqkegstrn wqhitdqigm fcftglnptq verltkefsv yltkdgrisv aglssknvgy
	421 lakamhevtk
Heat-shock protein 70 (PcHsp70)	1 makapaigid lgttyscvgv frhgqveiia ndqgqrttps yvaftdterl vgdaaknqaa	7O6R	79.60	0.81
	61 mnpentvfda krligrrfdd ssvqadmkhw pfkvvndggk pkiqvqykge tktfapeeis	7SQC	67.55	0.86
	121 smvlvkmket aeaylgqrvk davitvpayf ndsqrqatkd agniaglnvl riineptaaa
	181 laygldknak geknvlifdl gggtfdvsil qisegslfev kstagdthlg gedfdgrmvq
	241 hfcqefqrky kkdisknara irrlltacer akrtlsssae asieidslye gidfytritr
	301 arfeelcadl frktlepveq alrdakldkr qihevvlvgg stripriqkm lsefmggkel
	361 nksihpdeav aygaavqaav lsggtsdsir dvllvdvapl slgietaggv mtklverntt
	421 ipnktsqift tyadnqpavt iqvyegeram tkdnnllgkf eltgippapr gvpqievtfd
	481 idangimtvt aqdkstgkss nitiknekgr lskedidrmv ndaerfreed ekqrqrvear
	541 nrlesylfnv rqavgdagdk lssadkdtvn kacddalhwl dnnslaekee fedklkevqk
	601 vcspvmarmh gagqggagqp gagqgypsgg gghaggptve evd
Phenylalanine-4-hydroxylase (PcPheOH)	1 mqstenmgvr grqcvmcgci vgiteiatkc pqsntyqhiy vqdyvdhegs tsiifslpek	5DEN	64.68	0.84
		5HYC	65.89	0.83
	61 vgalaealkv fetnnvnlhh iesrpsledk dcyeffvscd nvrgglyeai dilkgrtknl
	121 qvlsrnpenk kdevpwfprk irdldrfanq ilsygselda dhpgfkdpvy rtrrkefadi
	181 afnykhgepi prvqytdeev ktwgtifqel tklypthacr efnhvfplli dncgfrenni
	241 pqlqdisdfl kdctgfqlrp vagllssrdf laglafrvfh stqyirhgsk pmytpepdvc
	301 hevlghvplf adpsfaqfsq eiglaslgap deyiqklatl ywftvefglc rqeghlkayg
	361 agllssfgel qyaltdkpev rpfdpqktst qeypitsfqp vyfvadsfdd akeklkaysl
	421 tiprpfsvry naytqsvevi nnkeqiinlt rtikgdlsll edalqkvnrl
Villin-1 (PcVillin)	1 makldpafsr idrnragfyi wrienlevvp vpkdyygeff rgdsyivlsi keirsstlds	2FGH	42.23	0.71
	61 hvhfwlgaet sqdeagvaay ktvelddllg gspvqhreve nhesqrflsy fphgikikag	2FGH	42.23	0.71
	121 gvksgfnhve rgvfqprliq vkgkrnprfs elpsidwefm nhgdcfildl gnlifpwlga	3FFN	42.80	0.72
	181 ssnrtekmkt lehcrrlrde rgggqkasii ivedgkeesl dadckpifek flsirerhqv
	241 qspemggvde svesalftai klfscreedg tlkiqevkag plskkdllse dsfiidngpn
	301 gawvwigkra stkekkeamr navgflrkkg ypsdtkvtrv vegaepsefk clfrdwpqtp
	361 ptgkvyiqsk iaktvqtrfd aatlhtnhal aaetqmvddg sgkvevwrve dfdlkpvdks
	421 vvgefysgdc yviqytyevh grphhliyyw lgskatsdek gtaalkavel ddklggtavq
	481 vrivqykepp hfmalfggrm iifqggksgw asssngdksa egpgdrymlh vrgtsqldtk
	541 avqvdmrass lnsndtfvif tktltyiwyg kgctgderem akhvaarspr sqigivegqe
	601 kpefwevlgg keeyaserkl qdegshpprl fqisnasgsv kaeelydfvq edlipedvmm
	661 ldcwdciylw vgkgsnkveq eeaqhlaiey lksdpagrdh dtpiykvsqg weppsftgff
	721 gvwdrdlwsk alsyeeakrr indenvplsl vkenvangiq dfsdmpkysy eeltvevdql
	781 psgvdpaare ihlreedfkr ifsmtypefs klpiwkqknm kkqknlf

Molecular Docking

Molecular docking of the A. armata saponin ligands (AR-01–AR-15) was carried out as previously described.⁷⁰ Each saponin structure was built using Spartan ’18 for Windows, v 1.4.4 (Wavefunction, Inc.), and the lowest-energy conformation was determined using molecular mechanics (Merck Molecular Force Field⁷¹). Molegro Virtual Docker v 6.0.1 (Molegro ApS) was used for the molecular docking on the 12 protein structures (Table 4). The ligand orientations with each target protein were ranked based on MolDock “rerank” energy values (E_dock). The docking energies were corrected to account for molecular weight bias to give normalized docking scores (DS_norm).⁷⁰ The lowest-energy docking scores are summarized in Table 5.

Conclusions

The molluscicidal activity of 15 purified saponins separated from A. armata roots grown in Vietnam against golden apple snail P. canaliculata has been investigated in this study. The structures of the compounds are determined by spectroscopic and spectrometric analyses as well as comparisons with the previously published data. The correlation between the saponins and their molluscicidal activity is also unveiled. The robust molluscicidal activity of these isolated saponins exhibited LC₅₀ values ranging from 7.90 to 17.50 µg/mL. Experiments show that at the median lethal concentration for P canaliculata, the active ingredients have no significant impact on the aquatic environment. Specifically, the LC₅₀ values of the compounds for brine shrimp (Artemia sp.) range from 148.55 to 193.22 μg/mL. In addition, the data obtained from the experiment also demonstrate the safety of the A. armata root extract. Based on molecular docking, P. canaliculata acetylcholinesterase and villin may be considered molecular targets of the A. armata saponins isolated and characterized in this work. There is much that is still unknown about the proteomics of P. canaliculata, however, and there may be other, yet uncharacterized, protein targets accounting for the molluscicidal activity of A. armata components. The obtained results illustrate that the saponin compounds from A. armata roots in Vietnam turn out to be potential compounds with excellent efficacy and safety for agricultural applications.

Footnotes

Author Contributions

THCN, GTKL, PHY, T-TH, DTTV, PVK, NHH_, P-CK_, and WNS contributed to conceptualization; THCN and NHH contributed to formal analysis; THCN and PVK contributed to writing‒original draft preparation; THCN, NHH_, P-CK, and WNS contributed to visualization; GTKL, PHY, T-TH, and DTTV contributed to writing‒review and editing. All authors have read and agreed to the published version of the manuscript.

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.

Ethical Approval

Ethical approval is not applicable to this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or vertebrate animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iDs

Phan Van Kiem

William N. Setzer

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Molluscicidal Activity of Compounds From the Roots of Aralia armata Against the Golden Apple Snail ( Pomacea canaliculata )

Abstract

Keywords

Introduction

Results and Discussion

Isolation

Molluscicidal Activity

AChE Enzyme Inhibitory Activity

Acute Toxicity for Brine Shrimp (Artemia sp.)

Phytotoxic Activity of Aqueous Fraction

In silico Evaluation of A. armata Saponins

Experimental

General

Plant Material

Snails (P. canaliculata)

Brine Shrimp (Artemia sp.)

Rice

Extraction and Isolation

Molluscicidal Activity

Acute 48 h Toxicity Test for Artemia sp.

Germination Assay

Seedling Toxicity Test

AChE Enzyme Inhibitory Test

Data Analysis

Homology Modeling

Molecular Docking

Conclusions

Footnotes

Author Contributions

Declaration of Conflicting Interests

Funding

Ethical Approval

Statement of Human and Animal Rights

Statement of Informed Consent

ORCID iDs

References