Insecticidal Triterpenes in Meliaceae: Plant Species, Molecules and Activities: Part Ⅰ (Aphanamixis-Chukrasia)

Plant-originated triterpenes are important insecticidal molecules. The research on insecticidal activity of molecules from Meliaceae plants has always received attention due to the molecules from this family showing a variety of insecticidal activities with diverse mechanisms of action. In this paper, we discuss 102 triterpenoid molecules with insecticidal activity of plants of eight genera (Aglaia, Aphanamixis, Azadirachta, Cabralea, Carapa, Cedrela, Chisocheton, and Chukrasia) in Meliaceae. In total, 19 insecticidal plant species are presented. Among these species, Azadirachta indica A. Juss is the most well-known insecticidal plant and azadirachtin is the active molecule most widely recognized and highly effective botanical insecticide. However, it is noteworthy that six species from Cedrela were reported to show insecticidal activity and deserve future study. In this paper, a total of 102 insecticidal molecules are summarized, including 96 nortriterpenes, 4 tetracyclic triterpenes, and 2 pentacyclic triterpenes. Results showed antifeedant activity, growth inhibition activity, poisonous activity, or other activities. Among them, 43 molecules from 15 plant species showed antifeedant activity against 16 insect species, 49 molecules from 14 plant species exhibited poisonous activity on 10 insect species, and 19 molecules from 11 plant species possessed growth regulatory activity on 12 insect species. Among these molecules, azadirachtins were found to be the most successful botanical insecticides. Still, other molecules possessed more than one type of obvious activity, including 7-deacetylgedunin, salannin, gedunin, azadirone, salannol, azadiradione, and methyl angolensate. Most of these molecules are only in the primary stage of study activity; their mechanism of action and structure–activity relationship warrant further study.


Introduction
Pesticides provide tremendous benefit to modern agriculture. It is well known that the increase of crop yields largely depends on synthetic pesticides. However, it is also recognized that synthetic pesticides have some negative impacts and the indiscriminate application of synthetic pesticides has resulted in contamination of water, soil, air, and crop products, etc. The persistent use of pesticides has also led to serious resistance and resurgence of insect pests [1]. The current consensus asserts that the development of new pesticides should be based on sustainable development, environmental protection, and ecological balance. In order to achieve sustainable development, many scientists have undertaken the search for low toxicity, low residue and environmentally friendly biopesticides, among which botanical pesticides are an important part. Botanical insecticides are attracting global attention as new tools to kill or suppress insect pest populations. Generally, natural products are particularly attractive as templates because of their structural diversity. They can be used directly and have been used as models for the development of several successful insecticides that introduce new mechanisms of action, which are greatly needed Ring-seco limonoids are mainly divided into demolition of a single ring (ring A-seco group, ring B-seco group, ring C-seco group, and ring D-seco group), demolition of two rings (rings A,B-seco group, rings A,D-seco group, and rings B,D-seco group), and demolition of three rings (rings A,B,D-seco group). In particular, the ring C-seco group, which belongs to the group of demolition of a single ring, can be further divided into five classes (azadirachtin/melia-carpin-class, azadirachtinin/meliacarpinin-class, salanninclass, nimbolinin-class, nimbin-class, and nimbolidin-class) [9], while the rings of the A,Bseco group, belonging to the group of demolition of two rings, can be further divided into prieurianin-class and others. In the prieurianin-class, aphanamixoid-type belong to its Ring-seco limonoids are mainly divided into demolition of a single ring (ring Aseco group, ring B-seco group, ring C-seco group, and ring D-seco group), demolition of two rings (rings A,B-seco group, rings A,D-seco group, and rings B,D-seco group), and demolition of three rings (rings A,B,D-seco group). In particular, the ring C-seco group, which belongs to the group of demolition of a single ring, can be further divided into five classes (azadirachtin/melia-carpin-class, azadirachtinin/meliacarpinin-class, salanninclass, nimbolinin-class, nimbin-class, and nimbolidin-class) [9], while the rings of the A,B-seco group, belonging to the group of demolition of two rings, can be further divided     showed antifeedant activity against 16 insect species (E. paenulata, E. varivestis, H. armigera, L. decemlineata, L. migratoria, O. nubilalis, P. saucia, P. striolata, P. brassicae, P. rapae, P. xylostella, R. speratus, R. prolixus, S. gregaria, S. littoralis, and S. litura) ( Table 2) [9,17,[21][22][23]29,31]. In these chemicals, azadirachtin, namely azadirachtin A, was the most active and has been successfully used as a botanical insecticide. Azadirachtin B and L also showed significant activity. Normally, the widely used various neem-based insecticide preparations consisted of not only azadirachtin A but also other similar azadirachtins, such as azadirachtin B and L. Still, the activity of other azadirachtins and some other types of chemicals deserves more attention. For example, epoxyprieurianin showed an obvious antifeedant activity on H. armigera (EC 50 = 3.2 µg/mL, 7 d). Another chemical, 1-tigloyl-3-acetyl-azadirachtol, showed good activity on E. varivestis. These chemicals could be developed as antifeedant agents on some specific insects in the future [9,32,33].  Overall, 49 chemicals isolated from 14 plant species (Aglaia elaeagnoidea (A. Juss.), A. polystachya, A. excelsa, A. indica, C. canjerana, C. eichleriana, C. guianensis, C. dugessi, C. fissilis, C. salvadorensis, C. sinensis, Chisocheton ceramicus (Miq.) C.DC., Chisocheton erythrocarpus Hiern, and C. paniculatus) in Meliaceae exhibited poisonous activity on 10 insect species (A. aegypti, A. albopictus, A. gambiae, A. stephensi, A. sexdens rubropilosa, C. quinquefasciatus, D. balteata, P. xylostella, S. frugiperda, and S. littoralis) (Table 3) [9,19,20,25,26,28,43]. Normally, the poisonous activity was not the most important of many plant-derived chemicals. However, azadirachtin did show good poisonous activity against S. littoralis. Other chemicals such as azadirachtin O, azadirachtin P, azadirachtin Q, azadirachtin B, azadirachtin L, azadirachtin M, 11α-azadirachtin H, and azadirachtol also showed good poisonous activity on P. xylostella, with LD 50 (24 or 96 h) values ranging from 0.75 to 3.92 µg/g [9,33].
The following sections describe the insecticidal plant species, the corresponding insecticidal chemicals, and their activities in detail.

Aglaia
In the Aglaia genus, two species, including A. elaeagnoidea and A. odorata, have been reported to show insecticidal activity. Previous phytochemical investigation and bioactivity studies on the Aglaia genus have shown the main chemical group of this genus to be rocaglamide derivatives (flavaglines) [53]. However, triterpenoids were also the main insecticidal active constituents in this genus.  IGR: insect growth inhibitory activity; LPE: larval phase extended; FI: fecundity inhibition; RF 50 : reduced feeding by 50%; PWI: pupal weight inhibition.
6α-acetoxygedunin, belonging to ring D-seco limonoids, was isolated from A. elaeagnoidea and could reduce the growth of the European corn borer O. nubilalis at 50 µg/mL [17,51]. A. odorata has been reported to show insecticidal activity on the cotton leafworm S. littoralis [54,55]. However, most of the reported compounds with insecticidal activity extracted from this species were rocaglaol derivatives. In addition, some triterpenoids, such as eleganoside A and odoratanone A, have also been reported to be extracted from A. odorata, but their insecticidal activities have not been described [56][57][58].
In these chemicals, prieurianin and epoxyprieurianin exhibited antifeedant activity against the cotton bollworm, H. armigera and the EC 50 values were 18.8 µg/mL and 3.2 µg/mL, respectively, after 7 d [34]. Further study has shown that prieurianin-type limonoids, zaphaprinin I, showed strong insecticidal activities against the aphid S. avenae, with a mortality score of 99, which was the same with the positive control thiamethoxam. Both Zaphaprinin I and Zaphaprinin R showed strong insecticidal activities against the diamondback moth/cabbage moth, P. xylostella and both mortalities were scored as 99, which was the same with the positive control thiamethoxam [63].
Aphapolynin A has been found to cause a mortality score of 66 against the diamondback moth P. xylostella in a leaf-disk assay at 500 µg/mL. Mortality was assessed relative to untreated control wells, with wells showing significant levels of mortality scored as 99, and wells without significant mortality scored as 0 [19,64]. Similarly, aphapolynin C, aphapolynin D, aphapolynin F, and dregenana-1 were found to possess obvious insecticidal activity against the banded cucumber beetle, D. balteata in a leaf-disk assay at 500 µg/mL [19,34,65].

Ring A-seco Limonoids
Aphanalide E, aphanalide F, aphanalide G, and aphanalide H were found to cause mortalities scored as 33-99 against the banded cucumber beetle D. balteata in a leaf-disk assay at 500 µg/mL at 5-9 days. Mortality was assessed relative to untreated control wells, with wells showing significant levels of mortality scored as 99, and wells without significant mortality scored as 0 [19,64].

Azadirachta
In this genus, three species, A. indica, A. excels, and A. siamensis were reported to show insecticidal activity with triterpenoids.

Ring D-seco Chemicals
In this group, three chemicals were reported to show insecticidal activity and they were gedunin, 7-deacetylgedunin, and 6β-hydroxygedunin.
Azadirone showed antifeedant activity against the Colorado potato beetle L. decemlineata with an antifeedant index of 11.6 ± 6.3 (100 µg/mL) (starved for 6 h and feed for 20 h) [37]. Azadiradione and epoxyazadiradione were also reported to show antifeedant activities to some extent against the diamondback moth P. xylostella [24]. Further, azadiradione, 7-deacetylazadiradione, and 7-deacetyl-17β-hydroxyazadiradione were isolated from the seeds of A. indica and they showed growth inhibitory activity against the tobacco budworm H. virescens and the EC 50 values were 560, 1600, and 240 µg/mL, respectively. Similarly, 17β-hydroxyazadiradione also showed antifeedant activity and the PC 95 value at the lower subterranean termite R. speratus was 235.6 µg/disc after 30 days [23].
Additionally, the other ring intact limonoids, azadiraindin A and meliatetraolenone, were reported to show insecticidal activity. Azadiraindin A showed antifeedant activities against the diamondback moth P. xylostella. The antifeedant rate was 28% at 2000 µg/mL after 48 h [24]. Meliatetraolenone, isolated from the leaves of A. indica, showed insecticidal activities against the mosquito A. stephensi and the LC 50 value was 16 µg/mL after 24 h [44].

Pentanortriterpenoids
In this group, seven chemicals have been reported to show insecticidal activity and they were 11α-azadirachtin H, azadirachtin I, azadirachtin L, azadirachtin M, azadirachtin P, nimbinene, and nimbandiol. There were five kinds of azadirachtin analogs (11αazadirachtin H, I, L, M, and P) that belonged to pentanortriterpenoids. 11α-azadirachtin H, azadirachtin L, azadirachtin M, and azadirachtin P, which were reported to have insecticidal activities, were isolated from the seed kernels of A. excelsa. The LD 50 values (24 h) of these derivatives against the diamondback moth P. xylostella were 5.75, 10.27, 8.46, and 2.19 µg/g, respectively [33].
Nimbinene exhibited growth inhibitory activity on insects and the EC 50 values of nimbinene on the cotton bollworm H. armigera and the tobacco cutworm S. litura were 391.4 and 404.5 µg/mL, respectively after 7 days [35]. Further, nimbandiol were found to show antifeedant activity on the lower subterranean termite R. speratus and the PC 95 values was 254.4 µg/disc after 30 days [23].

Octanortriterpenoids
Desfurano-6α-hydroxyazadiradione, isolated from fresh leaves of A. indica, showed insecticidal activity on the mosquito A. stephensi and the LC 50 value was 43 µg/mL after 24 h [39]. Comparatively, desfuranoazadiradione showed relatively weak antifeedant activity on the diamondback moth P. xylostella to some extent as demonstrated by the low mortality rate (39.6% after 48 h) at a high concentration (2000 µg/mL) [24].

Protolimonoids
Odoratone, isolated from the leaves of A. indica, showed insecticidal activities against the mosquito A. stephensi and the LC 50 value was 154 µg/mL after 24 h [44]. Another protolimonoid isolated from this plant was meliantriol, found to be a feeding inhibitor preventing locust chewing [52].

Carapa
In this genus, until now, only C. guianensis has been reported to show insecticidal activity [95,96].

The Ring Intact Limonoid: Cedrelone
Cedrelone showed no antifeedant effect. However, cedrelone could affect the development and reproduction of the variegated cutworm P. saucia. After 9 days of feeding, the EC 50 value of growth inhibition of cedrelone on P. saucia was found to be 53.1 µg/mL. By injection to the 6th instar of P. saucia, cedrelone inhibited growth, delayed development, and resulted in considerable larval mortality [43,50,104,105].
As for the 10,11-linkage limonoid cipadesin B, it was reported to possess an effect on A. sexdens rubropilosa. At 100 µg/mL, the S 50 values of cipadesin B on A. sexdens rubropilosa was 9 d [103].

Pentacyclic Triterpenes
The two pentacyclic triterpenes, oleanolic acid and oleanonic acid, belong to oleanane triterpenes. They were reported to possess an effect on A. sexdens rubropilosa and the S 50 values of oleanolic acid and oleanonic acid at 100 µg/mL on this insect were 6 d and 8 d, respectively [103].
Azadiradione showed growth inhibitory activity on the tobacco budworm H. virescens. The EC 50 value (EC 50 value was the effective concentration of additive necessary to reduce larval growth to 50% of the control values) was 560 µg/mL. In addition, the EC 50 of its alkaline hydrolysis product, 7-deacetylazadiradione, was 1600 µg/mL [27,30,109]. Chisocheton compound F, isolated from C. paniculatus, showed antifeedant activity against the large white butterfly P. brassicae [38].

Chukrasia
C. tabularis has been reported to show insecticidal activity. From this species, five rearranged limonoids, belonging to tetranortriterpenoids, were isolated. Specifically, they were phragmalins, which belonged to the 2,30-linkage group of the rearranged limonoids. The five chemicals were tabulalin, tabulalide A, tabulalide B, tabulalide D, and tabulalide E. They all showed antifeedant activity against the third instar larvae of the cotton leafworm S. littoralis. Among them, tabulalin and tabulalide D were active at 500 µg/mL. Tabulalides A, B, and E were active at 1000 µg/mL at 2-12 h after the treatment [42,[110][111][112][113].

Structures of the Insecticidal Chemicals
In total, 102 insecticidal chemicals have been summarized, including 96 nortriterpenes, 4 tetracyclic triterpenes, and 2 pentacyclic triterpenes. The structures of the chemicals are shown in Figures 3-21.

Structure-Activity Relationship (SAR) of the Insecticidal Chemicals
Traditional insecticide discovery effectively contributes to the development of new insecticides but is limited by high costs and long cycles. Structure-activity relationship (SAR) methods were introduced to evaluate the activity of compounds virtually, which saves significant costs for determining the activities of the compounds experimentally [114].
An SAR study on the antifeedant effects and developmental delays of three different azadirachtin A derivatives against E. varivestis showed that the hydroxy group at C-11 is important for high mortality rates and a single bond between C-22 and C-23 increases the degree of efficiency. An exchange of the large ester group ligands at C-1 and C-3 with hydroxy groups in combination with a single bond between C-22 and C-23 and a hydroxy group at C-11 leads to high feeding activity and a degree of efficiency of about 100% [115]. Interestingly, another study aiming to understand the structure-related bioactivities of the limonoids based on the insect antifeedant and growth-regulating activities of 22 limonoids (both natural and their derivatives) against the tobacco cutworm, S. litura, indicated that the C-seco limonoids (azadirachtins A, B, D, H, and I) were the most effective compounds as a group, while the intact limonoids (cedrelone and its derivatives) were the least effective. The cyclohexenone A ring and the α-hydroxy enone group in the B ring appear to be important for antifeedant activity. The presence of a cyclohexenone or 1,2-epoxide in the A ring coupled with an α-hydroxy enone in the B ring correlated well with growth regulatory activity. An acetoxy at C-7 instead of α-hydroxy enone, and perhaps the carbonyl at C-16, increase growth regulatory activity. The absence of 14-15 epoxide may not drastically reduce antifeedant activity and growth regulatory activity [41].

Structure-Activity Relationship (SAR) of the Insecticidal Chemicals
Traditional insecticide discovery effectively contributes to the development of new insecticides but is limited by high costs and long cycles. Structure-activity relationship (SAR) methods were introduced to evaluate the activity of compounds virtually, which saves significant costs for determining the activities of the compounds experimentally [114]. Oleanolic acid Oleanonic acid

Structure-Activity Relationship (SAR) of the Insecticidal Chemicals
Traditional insecticide discovery effectively contributes to the development of new insecticides but is limited by high costs and long cycles. Structure-activity relationship (SAR) methods were introduced to evaluate the activity of compounds virtually, which saves significant costs for determining the activities of the compounds experimentally [114]. Based on 25 limonoids isolated from the fruits of A. polystachya, including seven new prieurianin-type limonoids, aphapolynins C-I, and one new C3-C6 connected aphanamolidetype limonoid aphanamolide B, along with 17 known compounds, a structure-activity analysis revealed that the α,β-unsaturated lactone and 14,15-epoxy moieties were essential for insecticidal activity [19]. Further structure-activity relationship analysis of the aphanamixoids indicated that the olefinic bond, the ∆ 2,30 configuration, and the substituent at C-12 significantly affected the antifeedant potency [18]. Antifeedant effect comparison of prieurianin, prieurianin acetate, epoxyprieurianin, and epoxyprieurianin acetate revealed that, first, epoxy compounds are more efficacious and, second, that acetylation enhances the activity of these rings A,B-seco-type limonoids [34].
A structure-activity study based on 11 molecules (nimbandiol, 17-hydroxyazadiradione, deacetylnimbin, 17-epiazadiradione, deacetylsalannin, azadiradione, nimbin, and deacetylgedunin), gedunin, salannin, and epoxyazadiradione) revealed that the furan ring, αβunsaturated ketone, and hydroxyl group each played an important role in determining the antifeedant activity. Specifically, a hydroxyl group at C-7 increased the antifeedant activity of gedunin [23]. Later, a further structure-activity study revealed that a hydroxyl group at C-7 reduced the insect growth inhibitory activity and the antifeedant activity of azadiradione, while a hydroxyl group at C-17 increased the activity of azadiradione and 7deacetylazadiradione. Compared with 7-deacetylazadiradione, the parent natural product contained hydroxyl groups at both the C-7 and C-17 positions, which might contribute to the activity [27,30,109]. Hydroxyl groups in other groups of limonoids were also found to influence biological activity. For example, acetylation or ketonization of the C-7 or C-l 2 hydroxyl groups in the trichilins rendered them inactive as antifeedants against larvae of the southern armyworm, S. eridania (Cramer). On the other hand, deacetylation of the C-1 acetate group in nomilin rendered it inactive as a growth inhibitor against larvae of the fall armyworm and the corn earworm [23,30]. Additionally, comparison of the activities of β-photogedunin and gedunin indicated that oxidation of the furan ring led to a decrease in insecticidal activity [48].

Insecticidal Mechanism of Action
A study of the insecticidal mechanism of action (MOA) of triterpenoids mainly focused on the MOA of azadirachtin with few MOA studies on other molecules. For example, it was demonstrated that both rings A,B-seco-type limonoids aphapolynin C and aphanalide H inhibited a nicotine response with IC 50 at 3.13 µg/mL (aphapolynin C) and 1.59 µg/mL (aphanalide H), respectively, and aphanalides H also inhibited a GABA response with IC 50 at 8.00 µg/mL [19]. Currently, azadirachtin is widely recognized as one of the most promising plant compounds for pest control in organic agriculture and one of the best alternatives to conventional insecticides in IPM programs [71,116]. The MOA study of azadirachtin has been a hot topic. However, even after many years of study, the exact molecular mechanism of action of azadirachtin has yet to be fully understood [117,118]. So far, the principal azadirachtin action on insects could be categorized into four groups: effects on neuro-endocrine activity, effects on reproduction, anti-feedancy, and cellular and molecular effects [116].
The primary antifeeding effect of azadirachtin seems to be mediated by gustatory chemosensillas and linked to inhibition on the rate of firing of sugar-sensitive cells of the gustatory chemoreceptors by activating bitter sensitive gustatory cells [119][120][121]. An internal feedback mechanism called secondary antifeedancy, including a long-term reduction in food intake, and deleterious effects on different insect tissues (muscles, fat body, gut epithelial cells), has also been reported [122][123][124]. In addition, azadirachtin showed an agonistic effect on dopaminergic neurons and can induce aversive taste memory in Drosophila melanogaster, and such memory is regulated by dopaminergic signals in the brain resulting in inhibition of the proboscis extension response (PER) [125].
Azadirachtin is an antagonist of 20-hydroxyecdysone (20E) and juvenile hormone (JH), two principal hormones in insects. The major action of azadirachtin has been its effect on hemolymph ecdysteroid and JH titers by inhibition of the secretion of morphogenetic peptide hormone (PTTH) and allatotropins from the corpus cardiacum complex, resulting in the IGD effects such as a failure of adult emergence, reduced pupation, or malformation. Moreover, azadirachtin could influence the activity of ecdysone 20-monooxygenase, which is a cytochrome P450-dependant hydroxylase responsible for the conversion of the steroid hormone ecdysone to its more active metabolite, and 20E. Furthermore, azadirachtin can cause degenerative structural changes in the nuclei in all endocrine glands (prothoracic gland, corpus allatum, and corpus cardiacum) responsible for controlling molting and ecdysis in insects, which would contribute to a generalized disruption of neuroendocrine function [117,122]. It was reported that the inhibition of growth and development in the fruit fly, D. melanogaster, after azadirachtin treatment was similar to those caused by disruption of the IIS pathway. In addition, azadirachtin can inhibit the excitatory cholinergic transmission and partly block the calcium channel, and this might interfere with different endocrinological and physiological actions in insects [126].
Owing to the interference of azadirachtin with yolk protein synthesis and or its uptake into oocytes, azadirachtin reduced the fecundity and fertility of several insects [127]. Sterility effects in females due to interference with vitellogenin synthesis and uptake into oocytes were also reported. In males, azadirachtin significantly decreases the number of cysts and the apical nuclei within the cysts in D. melanogaster, thereby inhibiting spermiogenesis [128][129][130]. In addition, azadirachtin was found to alter reproductive behavior, mating behavior, and oviposition behavior [128,131].
Additionally, the molecular insecticidal mechanisms of azadirachtin have been investigated and several explanations have been presented. For instance, it was found that azadirachtin could induce apoptosis through caspase-dependent pathways and could also inhibit protein synthesis and release by binding to specific proteins (such as heatshock protein, hsp 60), affected genes encoding key enzymes such as the gene encoding cytochrome oxidase-related proteins CYP307A1 and CYP314A1, which catalyze the 20hydroxyecdysone [132], and the gene encoding JH epoxide hydrolase, responsible for JH degradation by hydrolyzing the epoxide of JH [133][134][135].
In sum, recent work has demonstrated the MOA of azadirachtin to be complex and is not yet fully understood. Therefore, continued research is needed to reveal the ultimate MOA.

Future Outlook
Research on the insecticidal activity of Meliaceae plants has always received considerable attention. Investigations of Meliaceae plants over the past decades have led to some significant achievements.
Azadirachtin is the most successful botanical insecticide among the active compounds extracted from Meliaceae. Accordingly, the progress of the worldwide application of azadirachtin in controlling insect pests is inspiring. The application of azadirachtin can control insects, and at the same time, be safe for non-target arthropods. Such work demonstrates the effectiveness of a phytochemical for sustainable pest control in contrast to any negative effects of synthetic insecticide use.
In addition to azadirachtin, some azadirachtin analogs have also demonstrated strong insecticidal activities. Moreover, some compounds in Meliaceae possess more than one type of favorable activity, such as 7-deacetylgedunin, salannin, gedunin, azadirone, salannol, azadiradione, and methyl angolensate; some of which have multiple activities (poisoning, antifeeding, or growth inhibition). Among them, 7-deacetylgedunin and gedunin can be extracted from many Meliaceae plants. However, they are still in the primary stages of research and further studies on these compounds are needed. Their activities on insects should be systemically evaluated as well as their effects on non-target organisms and the environment. It is expected that 7-deacetylgedunin, gedunin, and so on, could be important molecules for managing insect pests in the near future.
Most of the compounds with obvious activity are only in the primary stages of research, and their mechanism of action and structure-activity relationship warrant further study. Generally, tetranortriterpenoids have complex structures and are difficult to synthesize. Therefore, it is of considerable significance to study the synthesis of tetranortriterpenoids with outstanding activity in Meliaceae.