Nematocidal Constituents from the Ethanol Extract of Evodia rutaecarpa Hort Unripe Fruits

e ethanol extract of Chinese medicinal herb, Evodia rutaecarpa Hort unripe fruits, was found to possess nematocidal activity against the root-knot nematodes, Meloidogyne incognita, during the screening program for new agrochemicals from local wild plants and Chinese medicinal herbs. Bioactivity-guided chromatographic separation of the ethanol extract of E. rutaecarpa on repeated silica gel columns led to isolate �ve constituent components (two limonoids, evodol and limonin; three alkaloids, evodiamine, rutaecarpine, and wuchuyuamide I). Evodiamine (LC50 = 73.55 μμg/mL) and rutaecarpine (LC50 = 120.85 μμg/mL) exhibited stronger nematocidal activity againstM. incognita than the crude ethanol extract of E. rutaecarpa (LC50 = 131.54 μμg/mL). Wuchuyuamide I, evodol, and limonin also possessed nematocidal activity againstM. incognita with LC50 values of 147.87 μμg/mL, 155.02 μμg/mL, and 197.37 μμg/mL, respectively, but weaker than the crude ethanol extract of E. rutaecarpa.


Introduction
Root-knot nematodes are important agricultural pests worldwide.Possible control strategies include chemical nematocides, crop rotation, and use of resistant cultivars, when available.e broad host range of Meloidogyne species, however, makes crop rotation difficult, and the use of nematocides, in spite of their efficacy for controlling nematodes, has negative side effects that have led to banning or restricting their use [1].On the other hand, resistance-breaking populations of Meloidogyne are challenging the use of resistant cultivars [2].ose are now resulting in a strong interest in nematicides of natural origin [3].One alternative is to screen naturally occurring compounds in plants, which are known as plant secondary compounds.Many plant constituents and metabolites have been investigated for activity against plantparasitic nematodes [4][5][6][7].A series of nematicidal substances of plant origin such as triglycerides, sesquiterpenes, alkaloids, steroids, diterpenes, and �avonoids have been identi�ed [3].During our screening program for new agrochemicals from local wild plants and Chinese medicinal herbs, ethanol extract of Evodia rutaecarpa Hort unripe fruits (Family: Rutaceae) was found to possess strong nematocidal activity against the root-knot nematodes, Meloidogyne incognita (Kofoid and White) Chitwood.M. incognita is the most economically important and widely distributed nematode throughout China, and a considerable crop loss is caused by this nematode [7].
E. rutaecarpa unripe fruit is a commonly used Chinese medicinal herb and has been recommended for the treatment of abdominal pain, acid regurgitation, nausea, diarrhea, hernia, and dysmenorrhea and is also used externally for treatment of aphthous stomatitis [8].E. rutaecarpa is also documented as insecticidal/antiparasitic in the literature [9].e essential oil of E. rutaecarpa unripe fruits possessed strong fumigant toxicity against two grain storage insects, Tribolium castaneum and Sitophilus zeamais, and three active constituent compounds were isolated from the essential oil of E. rutaecarpa [10,11].Two quinolone alkaloids isolated from the herb exhibited toxicity to brine shrimp (Artemia salina) [12].Moreover, three alkaloids, evodiamine, rutaecarpine, and rhetsinine, isolated from E. rutaecarpa by bioassayguided fractionation showed insecticidal activity against fruit �ies (Drosophila melanogaster) [13].e ethanol extract of E. rutaecarpa also possessed strong antifeedant activity against the two stored product insect pests (T.castaneum and S. zeamais) [14].However, as far as we know, there are no reports about isolation of active components against nematodes from this Chinese medicinal herb.In this paper, the ethanol extract of E. rutaecarpa was evaluated for toxicity against M. incognita and �ve active constituent compounds (Figure 1) were isolated and identi�ed from the ethanol extract of E. rutaecarpa by bioassay-directed fractionation.

Chinese Medicinal Herb and Extraction. E. rutaecarpa
(5 kg, dried unripe fruits), purchased from Anguo Chinese Herbs Market, Hebei province, China, were ground to a powder and extracted with 95% ethanol (3 × 10 L) at room temperature over a period of three weeks.e extracts were concentrated using a vacuum rotary evaporator to afford a syrupy gum (454 g).is syrup was partitioned between methanol water and n-hexane (3 × 1,000 mL).e nhexane extracts were evaporated off to give a residue (232 g).e aqueous layer was repartitioned with chloroform (3 × 1,000 mL) to provide a residue (44 g) aer evaporation of chloroform.Further partitioning with ethyl acetate (3 × 1,000 mL) gave a residue (65 g) aer evaporation of the solvent.

Results and Discussion
Five isolated constituent compounds and the crude ethanol extract of E. rutaecarpa unripe fruits exhibited nematocidal toxicity against the root-knot nematodes, M. incognita (Table 1).e two alkaloids, evodiamine (LC 50 = 73.55g/mL) and rutaecarpine (LC 50 = 120.85g/mL), exhibited stronger nematocidal activity against M. incognita than the crude ethanol extract (LC 50 = 131.54g/mL).It indicated that the nematocidal activity of ethanol extract of E. rutaecarpa unripe fruits may be attributed to the present of the two alkaloids.Moreover, evodiamine shows the same level of nematocidal activity against M. incognita as the positive control, carbofuran (LC 50 = 72.29 g/mL), while the ethanol extract of E. rutaecarpa unripe fruits and rutaecarpine exhibit only half level of nematocidal activity, compared with carbofuran.In the previous studies, evodiamine was demonstrated to possess anticancer activities both in vitro and in vivo by inhibiting proliferation, invasion, and metastasis, inducing apoptosis of a variety of tumor cell lines [20].Moreover, evodiamine has been shown to reduce fat uptake in mouse studies.It is suspected that its mechanism of action is similar to that of capsaicin [21].As such, it has been included in some dietary supplements.Rutaecarpine has shown a variety of intriguing biological properties such as antithrombotic, anticancer, anti-in�ammatory and analgesic, antiobesity and thermoregulatory, vasorelaxing activity, as well as effects on the cardiovascular and endocrine systems [22].Moreover, the two isolated alkaloids, evodiamine and rutaecarpine, exhibited insecticidal activity against the fruit �ies (D. melanogaster) [13].However, the two isolated constituent compounds were �rst time to be evaluated for nematocidal activity against the root-knot nematodes.e other three isolated constituents, wuchuyuamide I, evodol, and limonin, also possessed nematocidal activity against M. incognita with LC 50 values of 147.87 g/mL, 155.02 g/mL, and 197.37 g/mL, respectively, but weaker than the ethanol extract of E. rutaecarpa (Table 1).In the previous reports, two limonoids, evodol and limonin, were demonstrated to possess antifeedant/feeding deterrent activity against Colorado potato beetle (Leptinotarsa decemlineata) and the fall armyworm larvae (Spodoptera frugiperda) [23][24][25] and also exhibited mosquito larvicidal activity against Aedes albopictus and A. aegypti [26,27].Several tirucallane triterpenoids derived from Melia azedarach fruits, for example, 3--tigloylmelianol, melianone, 21-acetoxy-melianone, and methyl kulonate as well as limonin were evaluated for their nematocidal activity against M. incognita, and no signi�cant effect on M. incognita was observed at a dose range of 31.2-500g/mL [28].However, the two other isolated constituent compounds were �rst time to be evaluated for nematocidal activity against the root-knot nematodes.
Considering the currently used nematocides are synthetic and usually possess high toxic to nontarget organisms, nematocidal activity of the ethanol extract of E. rutaecarpa unripe fruits and the �ve isolated constituent compounds are quite promising and they show potential to be developed as possible natural nematocides for control of the root-knot nematodes.However, little has been done on mechanisms of action of these �ve compounds (two limonoids and three indole alkaloids) against nematodes.In addition, further testing is necessary to evaluate the spectrum of nematocidal activity against other plant parasitic and free-living nematodes and their phytotoxicity to crops and to develop formulations to improve the efficacy and stability and to reduce cost.Moreover, for the practical use of the ethanol extract of E. rutaecarpa unripe fruits and its constituents as novel nematocides to proceed, further research is needed to establish their human safety and environmental safety.However, in traditional Chinese medicine, the unripe fruits of E. rutaecarpa were commonly used for the treatment of abdominal pain, acid regurgitation, nausea, diarrhea, hernia, and dysmenorrhea and were also used externally for treatment of aphthous stomatitis [8].However, no experimental data about the safety of ethanol extract of this medicinal herb and the four other isolated constituents is available so far.erefore, any attempt to develop an alkaloid-derived agrochemical must be carefully evaluated for harmful effects.
T 1: Nematocidal toxicity of the ethanol extract of E. rutaecarpa and isolated compounds against Meloidogyne incognita.
[19]essential oil were subjected to probit analysis using the PriProbit Program V1.6.3 to determine LC 50 (median lethal concentration) values and their 95% con�dence intervals (CI 95%)[19].Carbofuran was purchased from National Center of Pesticide Standards (8 Shenliao West Road, Tiexi District, Shenyang 110021, China) and used as a positive control.