Isolation of Nematicidal Triterpenoid Saponins from Pulsatilla koreana Root and Their Activities against Meloidogyne incognita

Pulsatilla koreana, a species endemic to Korea, is an important herb used in traditional medicine to treat amoebic dysentery and malaria. In the present study, 23 oleanane-type triterpenoid saponins 1–23 and eight lupane-type triterpenoid saponins 24–31 were isolated from the roots of P. koreana. Their structures were elucidated on the basis of spectroscopic data. The methanol extract and isolated compounds were next assessed for nematicidal activity against the root-knot nematode (Meloidogyne incognita). The methanol extract showed strong nematicidal activity after 48 h, with a LC50 value of 92.8 μg/mL. Compounds 2, 5, 9, 20, and 21 showed significant effects, with LC50 values ranging from 70.1 to 94.7 μg/mL after 48 h. These results suggest that triterpenoid saponins from P. koreana should be explored as potential natural nematicides for developing new agents to control root-knot nematode disease.


Introduction
Plant parasitic nematodes inflict serious damage on agricultural crops and plants. The primary pathogen, the root-knot nematode (Meloidogyne incognita), is known to predispose the host to attacks from soil borne fungal pathogens (secondary pathogens), resulting in a synergistic effect. In this OPEN ACCESS phenomenon, M. incognita, by causing wounds in the roots of a plant through penetration of a stylet, provides a way for other fungal pathogens to enter and thus result in disease complex incidences [1][2][3]. In the past, synthetic compounds have mainly been used for plant protection, but many of these pesticides have side effects including residues in plants, contamination of groundwater, the potential for adverse ecological impacts from pesticide use, and the creation of a continuing need for the development of new nematode control strategies and products [4]. Recently, one alternative used has been to screen naturally occurring plant secondary compounds for appropriate nematicidal activity. Various nematicidal substances of plant origin such as triglycerides, sesquiterpenes, alkaloids, steroids, diterpenes, and flavonoids, have been identified in this way [5]. These compounds can be developed for use as nematicides themselves, or can serve as model compounds for the development of chemically synthesized derivatives with enhanced activity and reduced environmental impacts [6].
During our search for botanical pesticides from natural plants, we found that the methanol extract of Pulsatilla koreana Nakai (Ranuculaceae) has high nematicidal activity against M. incognita. Pulsatilla koreana, a hairy, tufted, perennial herb that grows in Korea, and is used as a traditional medicine to treat various maladies such as amoebic dysentery and malaria [7]. Several Pulsatilla species, including P. ambigua, P. chinensis, P. dahurica and P. turczaninovii have been employed to treat diarrhea, vaginal trichomoniasis, and bacterial infections. Pharmacological investigations have suggested that triterpene saponins are important bioactive components [8][9][10][11][12]. However, the composition of nematicidally active compounds in this plant has not been previously reported. In this study, 31 triterpenoid saponins were isolated ( Figure 1) and their nematicidal activities against the root-knot nematode were evaluated.

Nematicidal Activity
An investigation on the nematicidal activity of the methanol extract and isolated compounds of P. koreana root against M. incognita was conducted. The activity of all samples was also examined separately. The internal structures of the nematodes were disintegrated and many vacuoles formed within their bodies during the experiments. After 72 h, only residual, empty somatocysts of the dead nematodes could be observed ( Figure 2E,F). The methanol extract of P. koreana showed strong nematicidal activity after 48 h, with a LC 50 value of 92.8 μg/mL compared with positive control (72.7 μg/mL) ( Table 1). Moreover, the nematicidal activity of methanol extract dramatically increased 24 h after the treatment.   (Table 1). After 72 h, the nematicidal activity effects of compounds 2, 5, 9, 20, and 21 showed no significant progress, suggesting that the isolated compounds exhibited activity within 48 h after treatment. A previous study reported that P. koreana showed significant activity against the root-knot nematode, but the active constituents were not determined [32]. To the best of our knowledge, this is the first report of nematicidal active constituents from P. koreana. In the structure-activity relationships of oleanane-type triterpenoid saponins 1-22, compounds 9, 20 and 21 showed the strongest nematicidal activity against M. incognita, and their structures were similar. When the sugar unit at C-3 of the aglycone was linked to a rhamnose-arabinose or rhamnose-(glucose-)arabinose chain, the nematicidal activity increased significantly when compared to other sugar chains. Although compounds 2, 5, 12, and 14 also link a rhamnose-arabinose or rhamnose-(glucose-)arabinose chain, their effects were weaker than those of compounds 9, 20 and 21, suggesting that a carboxyl group at position C-28 of aglycone is a key functional element. This suggestion was supported by comparing with previous work [9,33,34]. These data may be useful to evaluate the structure-activity relationships of other triterpenoid saponins, and to develop nematicidal activity against M. incognita.

Extraction and Isolation
Dried roots of P. koreana (2.0 kg) were extracted with MeOH under reflux for 10 h (7 L × 3 times) to yield 500.0 g of extract. This extract was suspended in water and partitioned with ethyl acetate to yield the corresponding ethyl acetate (37.0 g) and water (463.0 g) extracts. The water extract was partitioned with n-BuOH to yield the n-BuOH (130.0 g) extract. The ethyl acetate extract was subjected to silica gel column chromatography with a gradient of CHCl 3 /MeOH (50:1, 20:1,10: 1 and 1:1; 2 L for each step) to give 6 fractions (Fr. E1-E6). The fraction E4 was separated using an YMC column with a MeOH/actone/H 2 O (0. 25

Nematicidal Assay
A soil sample was collected from a pure culture of M. incognita and maintained on muskmelon roots in a greenhouse in Gimcheon (Gyeongbuk, Korea). Emerged larvae were collected from the soil using the Baermann funnel technique. Larvae were placed in a cavity block with water for a bioassay after they had been counted in a counting chamber. MeOH extract and the compounds isolated from P. koreana (5.0 mg) were prepared in 0.1 mL dimethyl sulfoxide (DMSO) and then diluted in water to obtain various concentration preparations (50-200 μg/mL). The standard nematicide fosthiazate was used for the comparison [35]. As a negative control of nematicidal activity, 5% DMSO was used. Approximately 40-60 freshly hatched second-stage juveniles in 450 μL of water and 50 μL of each of the 12 compounds at different concentrations were introduced into 24-well plates with three replicates performed. Plates were kept at room temperature (23-25 °C) under laboratory conditions. Inactive nematodes (dead nematodes) counted after 24, 48, and 72 h [36]. After the last count, inactive juveniles were maintained in distilled water for 72 h to observe their revival ( Figure 2). Five repetitions for each treatment were performed using water as a control.

Conclusions
In present study, bioassay-guided chromatographic fractionation and isolation were successfully used to yield 31 triterpenoid saponins from P. koreana roots. Three of the isolated compounds, raddeanoside R13 (9), hederoside C (20), and pulsatilla saponin D (21) may have potential as natural nematicides or as lead molecules for developing new nematicides to control root-knot nematode disease caused by M. incognita.