Norsesquiterpenes from the Latex of Euphorbia dentata and Their Chemical Defense Mechanisms against Helicoverpa armigera

Euphorbia dentata (Euphorbiaceae), an invasive weed, is rarely eaten by herbivorous insects and could secrete a large amount of white latex, causing a serious threat to local natural vegetation, agricultural production and human health. In order to prevent this plant from causing more negative effects on humans, it is necessary to understand and utilize the chemical relationships between the latex of E. dentata and herbivorous insects. In this study, three new norsesquiterpenes (1–3), together with seven known analogues (4–10), were isolated and identified from the latex of E. dentata. All norsesquiterpenes (1–10) showed antifeedant and growth-inhibitory effects on H. armigera with varying levels, especially compounds 1 and 2. In addition, the action mechanisms of active compounds (1–3) were revealed by detoxifying enzyme (AchE, CarE, GST and MFO) activities and corresponding molecular docking analyses. Our findings provide a new idea for the development and utilization of the latex of E. dentata, as well as a potential application of norsesquiterpenes in botanical insecticides.


Introduction
Plants and herbivorous insects are important components of terrestrial ecosystems.Herbivorous insects need to adapt and depend on the host plants for survival, and these host plants affect the feeding of herbivorous insects through their chemicals.The two form many complex adaptive mechanisms in the process of long-term co-evolution [1].Plant latex, usually stored in laticifers, is an opaque viscous liquid that can be secreted immediately when plants are subjected to herbivorous feeding insects or mechanical damage [2,3].Plant latex contains a large number of secondary metabolites with diverse types and complex structures.Most of the metabolites have certain antifeedant and growthinhibitory effects on herbivorous insects, acting as a chemical defense in resisting the feeding of herbivorous insects [4,5].However, the secondary metabolites of most plant latexes against herbivorous insects have not been systematically studied, and their defense mechanisms against herbivorous insects are less researched.More research findings are needed to support it.
Euphorbia is the largest genus in the Euphorbiaceae family, with more than 2000 species worldwide.It is mainly distributed in tropical and subtropical regions of Africa and America [6].In addition, Euphorbia is the largest genus of plants with latex.Some of the latexes of this genus have been used to kill fish or large animals [7].The main characteristic of Euphorbia is that their latexes are rich in secondary metabolites, especially terpenoids with complex structural frameworks, including monoterpenes, sesquiterpenes, diterpenes, and triterpenes [6].Among them, sesquiterpenes are naturally defensive compounds in Molecules 2023, 28, 7681 2 of 12 plants, which have significant antifeedant and growth-inhibitory effects on herbivorous insects [8].
Euphorbia dentata Michx. is an annual plant of the genus Euphorbia in the family Euphorbia.It originated in North America and has spread to many countries in Europe, Australia, and Asia [9].In recent years, the plant has grown rapidly in China and has a strong adaptability to new habitats.Some single-dominant communities have formed in Changsha of Hunan Province, Shijiazhuang and Baoding of Hebei Province, and Guanling of Guizhou Province, causing great harm to local natural vegetation, agricultural production, and human health.Field observations found that the whole plant of E. dentata contained white latex, which will flow out of the plant, especially when the plant is damaged by herbivorous insects.Therefore, we speculated that the strong invasive ability of the plant may be closely related to the successful chemical defense mechanisms of its latex against herbivorous insects.However, studies on secondary metabolites of E. dentata have not been conducted.Since E. dentata belongs to the genus Euphorbia, we hypothesized that E. dentata is also rich in sesquiterpenes.
Helicoverpa armigera (Lepidoptera: Noctuidae) is a herbivorous insect widely distributed in China and other areas of the world.The species is a euryphagous insect, and its host plants have more than thirty families [10].In recent years, with the adjustment of agricultural planting structures, the occurrence and harm of H. armigera have become increasingly serious, causing great economic losses to local agriculture [11].To effectively control H. armigera, extensive research has been carried out in chemical pesticides.Although some progress has been made, it has not stopped the large-scale trend of worldwide harm that H. armigera is causing.In addition, due to the long-term dependence on chemical pesticides, H. armigera has developed a strong resistance, and the ecological environment damage caused by chemical pesticides has become increasingly serious [12].Therefore, it is vital to search for safe and effective defensive substances from chemical interactions between plant and herbivorous insects.
In the process of adapting to external stress, insects could produce a class of detoxifying enzymes that could metabolize a large number of foreign substances.Acetylcholinesterase (AchE), carboxylesterase (CarE), glutathione-S-transferase (GST), and mixed-functionoxidase (MFO) are important detoxifying enzymes in insects, playing an important role in metabolizing toxic compounds and maintaining normal physiological activities [13,14].In addition, molecular docking analysis has become an important technique for exploring the interactions between molecules, providing visual analyses to explore the binding sites and modes of action of enzymes and compounds [15].Therefore, it is necessary to study the defense mechanisms of compounds by detoxifying enzyme activities and corresponding molecular docking analyses.
Field investigations found that H. armigera could feed on a small amount of E. dentata.At the same time, E. dentata secretes white latex from its damaged area.In addition, the latex of E. dentata and the methanol extract of latex have significant antifeedant effects on H. armigera (Figures S31 and S32), which also proved that chemical defense is the main form of defense against H. armigera.These phenomena suggest that the latex of E. dentata has a chemical defense mechanism against H. armigera.However, the secondary metabolites from the latex of E. dentata and their defense mechanisms against H. armigera are still unknown.In this study, we isolated and identified norsesquiterpenes (Figure 1) from the latex of E. dentata.Additionally, the antifeedant and growth-inhibitory effects of norsesquiterpenes on H. armigera were also investigated.In addition, the action mechanisms of active norsesquiterpenes were revealed by detoxifying enzyme activities and corresponding molecular docking analyses.

Structural Elucidation
Norsescycldione A (1) was obtained as a yellowish oil with C13H14O6 (seven degrees of unsaturation) using its HR-ESIMS data (m/z 289.0628 [M + Na] + , calculated for C13H14O6Na, 289.0688).Its 1D-NMR spectra (Table 1 Compared with the literature, it was found that compound 1 possessed an ionone-type norsesquiterpene skeleton [16].In addition, the computed ECD curve of (6S)-1a matched well with the experimental result of 1 (Figure 3), establishing its stereochemical structure.Thus, the structure of 1 was confirmed and named Norsescycldione A (Figure 1).

Structural Elucidation
Norsescycldione A (1) was obtained as a yellowish oil with C 13 H 14 O 6 (seven degrees of unsaturation) using its HR-ESIMS data (m/z 289.0628 [M + Na] + , calculated for C 13 H 14 O 6 Na, 289.0688).Its 1D-NMR spectra (Table 1) showed four carbonyl groups Compared with the literature, it was found that compound 1 possessed an ionone-type norsesquiterpene skeleton [16].In addition, the computed ECD curve of (6S)-1a matched well with the experimental result of 1 (Figure 3), establishing its stereochemical structure.Thus, the structure of 1 was confirmed and named Norsescycldione A (Figure 1).Norsescycldione B (2) was obtained as a yellowish oil with C13H14O5 (seven degrees of unsaturation) using its HR-ESIMS data (m/z 273.0736 [M + Na] + , calculated for C13H14O5Na, 273.0739).Its 1D-NMR spectra (Table 1 .A comparison of its 1D NMR data with Norsescycldione A indicated that they were very similar except for one less hydroxyl group, which showed that compound 2 also possessed a norsesquiterpene skeleton similar to Norsescycldione A. In the HMBC spectrum (Figure 2), the key correlations of H-4/C-2, C-5, C-11; H-7/C-1, C-5, C-9; H-10/C-8, C-9; H-12/C-2, C-13 established its planar structure, which also proved that the missing hydroxyl group was at the position of C-6.In addition, the computed ECD curve of (6R)-2a matched well with the experimental result of 2 (Figure 3), establishing its stereochemical structure.Thus, the structure of 2 was confirmed and named Norsescycldione B (Figure 1).
Norsescycldione C (3) was obtained as a yellowish oil with C13H16O4 (six degrees of unsaturation) using its HR-ESIMS data (m/z 259.0949 [M + Na] + , calculated for C13H16O4Na, 259.0946).Its 1D-NMR spectra (Table 1)   .A comparison of its 1D NMR data with Norsescycldione A indicated that they were very similar except for one less hydroxyl group, which showed that compound 2 also possessed a norsesquiterpene skeleton similar to Norsescycldione A. In the HMBC spectrum (Figure 2), the key correlations of H-4/C-2, C-5, C-11; H-7/C-1, C-5, C-9; H-10/C-8, C-9; H-12/C-2, C-13 established its planar structure, which also proved that the missing hydroxyl group was at the position of C-6.In addition, the computed ECD curve of (6R)-2a matched well with the experimental result of 2 (Figure 3), establishing its stereochemical structure.Thus, the structure of 2 was confirmed and named Norsescycldione B (Figure 1).
Norsescycldione C (3) was obtained as a yellowish oil with C 13 H 16 O 4 (six degrees of unsaturation) using its HR-ESIMS data (m/z 259.0949 [M + Na] + , calculated for C 13 H 16 O 4 Na, 259.0946).Its 1D-NMR spectra (Table 1 In addition, the computed ECD curve of (6S)-3a matched well with the experimental result of 3 (Figure 3), establishing its stereochemical structure.Thus, the structure of 3 was confirmed and named Norsescycldione C (Figure 1).

Antifeedant and Growth-Inhibitory Effects
It is well known that sesquiterpenes are naturally defensive compounds in plants and have significant antifeedant and growth-inhibitory effects on herbivorous insects [8].From this, we speculated that the norsesquiterpenes (1-10) may be the potential defense substances for the latex of E. dentata against herbivorous insects.Field investigations found that H. armigera (a model herbivorous insect for evaluating chemical defense function) [23,24] could feed on a small amount of E. dentata.At the same time, E. dentata secretes white latex from its damaged area.These phenomena suggest that the latex of E. dentata has a defense mechanism against H. armigera.However, the secondary metabolites from the latex of E. dentata and their defense mechanisms against H. armigera are still unknown.Therefore, we studied their chemical defense functions (antifeedant and growth).As shown in Table 2, norsesquiterpenes (1-10) had antifeedant and growth-inhibitory effects with varying levels (100, 50, 25 µg/mL).In general, compounds 1-3 exhibited a stronger chemical defense function (antifeedant and growth-inhibitory effects) compared with other compounds (4-10).Among these, compounds 1 and 2 exhibited significant antifeedant effects at 100 µg/mL (85.16 ± 7.44% and 80.62 ± 6.55%, respectively), which was even comparable to neem oil (92.28 ± 7.11%).Moreover, compounds 1 and 2 also showed potent growth-inhibitory effects at 100 µg/mL (74.28 ± 8.35% and 78.11 ± 6.26%, respectively), and interestingly, they did not cause the death of H. armigera compared with the positive control.

Detoxifying Enzymes Effects
In the process of adapting to external stress, insects could produce a class of detoxifying enzymes that could metabolize a large number of foreign substances.AchE, CarE, GST, and MFO are important detoxifying enzymes in insects, playing an important role in metabolizing toxic compounds and maintaining normal physiological activities [13,14].Therefore, the action mechanisms of active norsesquiterpenes (1-3) were revealed by detoxifying enzyme activities.The effects of compounds 1-3 on AchE, CarE, GST, and MFO activities of H. armigera at different times (0, 6, 12, 24, 48 h) are shown in Figure 4.In general, after treatment with different compounds (1-3), the AchE and GST activities of H. armigera decreased with the extension of time, MFO activities of H. armigera increased with the extension of time, and CarE activities of H. armigera increased first and then decreased with the extension of time.Among these, the changes in AchE, CarE, and MFO activities in each treatment group were not significantly different from those in the blank control.It is worth noting that GST activities significantly decreased by 35.54%, 18.41%, 41.25%, and 56.79% under compound 1 treatment; 13.99%, 35.60%, 54.16%, and 44.15% under compound 2 treatment; and 15.49%, 33.63%, 33.12%, and 31.42%under compound 3 treatment compared with the blank controls at 0-6, 6-12, 12-24 and 24-48 h, respectively.
Therefore, the action mechanisms of active norsesquiterpenes (1-3) were revealed by detoxifying enzyme activities.The effects of compounds 1-3 on AchE, CarE, GST, and MFO activities of H. armigera at different times (0, 6, 12, 24, 48 h) are shown in Figure 4.In general, after treatment with different compounds (1-3), the AchE and GST activities of H. armigera decreased with the extension of time, MFO activities of H. armigera increased with the extension of time, and CarE activities of H. armigera increased first and then decreased with the extension of time.Among these, the changes in AchE, CarE, and MFO activities in each treatment group were not significantly different from those in the blank control.It is worth noting that GST activities significantly decreased by 35.54%, 18.41%, 41.25%, and 56.79% under compound 1 treatment; 13.99%, 35.60%, 54.16%, and 44.15% under compound 2 treatment; and 15.49%, 33.63%, 33.12%, and 31.42%under compound 3 treatment compared with the blank controls at 0-6, 6-12, 12-24 and 24-48 h, respectively.

Discussion
Recently, researchers have paid more attention to the chemical relationships between invasive plants and native herbivorous insects [25,26].This will help researchers to better reveal the invasion mechanism of alien plants from the perspective of chemical defense, and provides a new idea for the development and utilization of invasive plants.In our study, three new norsesquiterpenes (1-3), together with seven known analogues (4-10), were isolated from the latex of E. dentata and their structures were determined via HR-ESIMS verifications, NMR analyses, and ECD calculations.Among these, compounds 4, 8, and 9 were obtained from the Euphorbia genus for the first time.Moreover, this was also the first systematic study of the chemical composition of this plant.All norsesquiterpenes (1-10) showed chemical defense functions in H. armigera with varying levels (100, 50, 25 µg/mL), especially compounds 1 and 2. These results indicate that the norsesquiterpenes

Discussion
Recently, researchers have paid more attention to the chemical relationships between invasive plants and native herbivorous insects [25,26].This will help researchers to better reveal the invasion mechanism of alien plants from the perspective of chemical defense, and provides a new idea for the development and utilization of invasive plants.In our study, three new norsesquiterpenes (1-3), together with seven known analogues (4-10), were isolated from the latex of E. dentata and their structures were determined via HR-ESIMS verifications, NMR analyses, and ECD calculations.Among these, compounds 4, 8, and 9 were obtained from the Euphorbia genus for the first time.Moreover, this was also the first systematic study of the chemical composition of this plant.All norsesquiterpenes (1-10) showed chemical defense functions in H. armigera with varying levels (100, 50, 25 µg/mL), especially compounds 1 and 2. These results indicate that the norsesquiterpenes (1-3) from the latex of E. dentata could function as chemical defense substances against H. armigera, which may help E. dentata to gain a competitive advantage over other plants, as well as supporting theory for the defensive functions of sesquiterpenes against herbivorous insects [8].By analyzing the structure-activity relationships of norsesquiterpenes (1-10), the presence of one carboxyl group at the position of C-3 may have a positive effect on chemical defense function.However, more studies of norsesquiterpene analogues are needed to confirm this.
In addition, in the detoxifying enzyme activities, the GST activities significantly decreased under treatment with compounds 1-3 compared with a blank control with an extension of time.This indicated that GST may be a key target of H. armigera for compounds 1-3 to exert their chemical defense functions.GST is an important detoxifying enzyme in insects, which could catalyze the binding of harmful substances and reduce glutathione, thereby increasing the water solubility of harmful substances and excreting them [13].Norsesquiterpenes (1-3) can significantly inhibit the GST activity of H. armigera, which indicates that compounds 1-3 may block the excretion of harmful substances, resulting in the inhibition of the feeding and growth of H. armigera.On this basis, we explored the binding sites and modes of action of GST, as well as compounds 1-3, using molecular docking analyses.The binding energies of compounds 1 and 2 with GST of H. armigera were lower than those of compound 3, indicating that compounds 1 and 2 bound to GST more stably than compound 3.This was essentially consistent with the above results (antifeedant and growth-inhibitory effects of compounds 1-3).It is well-known that hydrogen bonds play a more important role than other forces in interactions between molecules [27].Compounds 1 and 2 formed more hydrogen bonds with amino acids than compound 3, which not only verified that more hydrogen bonds led to a more stable binding between enzyme and compound, but also indicated that the common amino acids (ARG68, GLU66, and SER67) could be key active sites of GST interacting with compounds 1-3.

Plant Material
E. dentata was identified by Professor Bo Qu and its voucher specimen (NO.ZW-2020-0073) and was kept in Shenyang Agricultural University.The latex of E. dentata (aerial part) was collected from Beijing Botanical Garden, China (40 • 01 E, 116 • 21 N) in August 2020.

Insect Material
H. armigera was purchased from Henan Keyun Bio-Pesticide Co., Ltd.(Henan, China) and identified by Associate Professor Lu Jiang (Shenyang Agricultural University).

ECD Calculations
The ECD calculations were carried out by a previously reported method [25].The conformational analyses of compounds 1-3 were performed using Spartan 14.0 software under the MMFF94 force field.The obtained conformations were optimized using Gaussian 09 software at the B3LYP/6-31G (d) level.Theoretical calculations were performed using TDDFT at the B3LYP/6-311+G (2d, p) level in methanol.The final ECD spectra were generated using SpecDis 1.60 software based on the Boltzmann weighting of each conformation.

Antifeedant and Growth-Inhibitory Assay
The antifeedant assay was carried out according to a previously reported method [26].The leaf discs (1 cm in diameter from the leaves of Brassica chinensis) of the treatment group were painted with latex (50 µL) of E. dentata, methanol extract of latex (50 µL, 0.5 mg/mL), or methanol solution (20 µL) containing different concentrations of compounds (100, 50, and 25 µg/mL).The blank control group was painted with the same amount of methanol.The positive control group was painted with the same amount of commercial insecticide (neem oil).After natural drying, four discs (two treated and two blank controls) were placed in a Petri dish at a crosswise position.Two third-instar H. armigera (starved for 6 h) were placed in each Petri dish.When about 80% of the leaf discs (blank control) were consumed, the H. armigera was removed from the Petri dish, and the consumed area of the leaf discs was measured.Each assay was repeated at least five times.Antifeedant rate (%) was calculated as (A C − A T )/Ac × 100 (A C , the consumed area of leaf discs in the blank control group; A T , the consumed area of leaf discs in the treatment group).
The growth-inhibitory assay was carried out according to a previously reported method [28].The leaf discs (1 cm in diameter from the leaves of B. chinensis) of the treatment group were painted with methanol solution (20 µL) containing 100 µg/mL compounds.The blank control group was painted with the same amount of methanol.The positive control group was painted with the same amount of commercial insecticide (neem oil).After natural drying, ten discs (ten treated or ten controls) were placed in a Petri dish.A weighed third-instar H. armigera (starved for 6 h) was placed in each Petri dish.The H. armigera were accurately weighed at 24, 48, and 72 h, respectively.Each assay was repeated at least five times.The growth-inhibitory rate (%) was calculated as (W C − W T )/Wc × 100 (W C , the gained weight of H. armigera in the blank control group; W T , the gained weight of H. armigera in the treatment group).
The leaf discs were pretreated as the growth-inhibitory assay above.After natural drying, ten discs (ten treated or ten controls) were placed in a Petri dish.A third-instar H. armigera (starved for 6 h) was placed in each Petri dish.The activities of detoxifying enzymes were measured at 0, 6, 12, 24, and 48 h, respectively.The extraction of enzymes was carried out according to a previously reported method [29].Third-instar H. armigera with similar sizes were weighed, and nine volumes of normal saline were added.The obtained homogenate was then centrifuged at 12,000 rpm for 10 min, and the supernatant was used for the follow-up enzyme solution.

Molecular Docking Analyses
The molecular docking analyses were carried out according to a previously reported method [26].The 3D structure of the enzyme (GST of H. armigera) was constructed via homology modeling using the EasyModeller 4.0 software.The GST sequence of H. armigera was obtained from the NCBI database in FASTA format (GenBank NO.BK40535.1).The optimal template (PDB code: 3VK9) was searched using the BLAST server based on E value, sequence identity, and query coverage (Table S1).The 3D structures of compounds 1-3 were optimized by the ChemDraw-3D 14.0 software to minimize energy.The Molegro Virtual Docker 4.0 software was carried out for further docking analyses.An active pocket (radius = 15.0)centered on x (39.90), y (33.15), and z (39.55) was formed based on the binding sites of the original ligand (glycerol) and enzyme (3VK9).

Figure 4 .
Figure 4.The effects of compounds 1-3 (100 µg/mL) on the detoxifying enzymes of H. armigera: (A) the effects of compounds 1-3 on AchE; (B) the effects of compounds 1-3 on CarE; (C) the effects of compounds 1-3 on MFO; and (D) the effects of compounds 1-3 on GST.CK = Control check.

Figure 4 .
Figure 4.The effects of compounds 1-3 (100 µg/mL) on the detoxifying enzymes of H. armigera: (A) the effects of compounds 1-3 on AchE; (B) the effects of compounds 1-3 on CarE; (C) the effects of compounds 1-3 on MFO; and (D) the effects of compounds 1-3 on GST.CK = Control check.

Figure 5 .
Figure 5. Molecular docking analyses of compounds 1-3 with GST: (A) 3D diagram of the interactions between compound 1 and the active pocket of GST; (B) 2D diagram of the interactions between compound 1 and amino acids of GST; (C) 3D diagram of the interactions between compound 2 and the active pocket of GST; (D) 2D diagram of the interactions between compound 2 and amino acids of GST; (E) 3D diagram of the interactions between compound 3 and the active pocket of GST; and (F) 2D diagram of the interactions between compound 3 and amino acids of GST.

Figure 5 .
Figure 5. Molecular docking analyses of compounds 1-3 with GST: (A) 3D diagram of the interactions between compound 1 and the active pocket of GST; (B) 2D diagram of the interactions between compound 1 and amino acids of GST; (C) 3D diagram of the interactions between compound 2 and the active pocket of GST; (D) 2D diagram of the interactions between compound 2 and amino acids of GST; (E) 3D diagram of the interactions between compound 3 and the active pocket of GST; and (F) 2D diagram of the interactions between compound 3 and amino acids of GST.
a : H. armigera were dead within 24 h, b : the content of compound was not enough).