Insecticidal Triterpenes in Meliaceae: Plant Species, Molecules, and Activities: Part II (Cipadessa, Melia)

Plant-originated triterpenes are important insecticidal molecules. Research on the insecticidal activity of molecules from Meliaceae plants has always been a hotspot due to the molecules from this family showing a variety of insecticidal activities with diverse mechanisms of action. In this paper, we discussed 116 triterpenoid molecules with insecticidal activity from 22 plant species of five genera (Cipadessa, Entandrophragma, Guarea, Khaya, and Melia) in Meliaceae. In these genera, the insecticidal activities of plants from Entandrophragma and Melia have attracted substantial research attention in recent years. Specifically, the insecticidal activities of plants from Melia have been systemically studied for several decades. In total, the 116 insecticidal chemicals consisted of 34 ring-intact limonoids, 31 ring-seco limonoids, 48 rearranged limonoids, and 3 tetracyclic triterpenes. Furthermore, the 34 ring-intact limonoids included 29 trichilin-class chemicals, 3 azadirone-class chemicals, and 1 cedrelone-class and 1 havanensin-class limonoid. The 31 ring-seco limonoids consisted of 16 C-seco group chemicals, 8 B,D-seco group chemicals, 4 A,B-seco group chemicals, and 3 D-seco group chemicals. Furthermore, among the 48 rearranged limonoids, 46 were 2,30-linkage group chemicals and 2 were 10,11-linkage group chemicals. Specifically, the 46 chemicals belonging to the 2,30-linkage group could be subdivided into 24 mexicanolide-class chemicals and 22 phragmalin-class chemicals. Additionally, the three tetracyclic triterpenes were three protolimonoids. To sum up, 80 chemicals isolated from 19 plant species exhibited antifeedant activity toward 14 insect species; 18 chemicals isolated from 17 plant species exhibited poisonous activity toward 10 insect species; 16 chemicals isolated from 11 plant species possessed growth-regulatory activity toward 8 insect species. In particular, toosendanin was the most effective antifeedant and insect growth-regulatory agent. The antifeedant activity of toosendanin was significant. Owing to its high effect, toosendanin has been commercially applied. Three other molecules, 1,3-dicinnamoyl-11-hydroxymeliacarpin, 1-cinnamoyl-3-methacryl-11-hydroxymeliacarpin, and 1-cinnamoyl-3-acetyl-11-hydroxymeliacarpin, isolated from Melia azedarach, exhibited a highly poisonous effect on Spodoptera littoralis; thus, they deserve further attention.


Introduction
Currently, chemical insecticides are still undoubtedly the most useful method to control insect pests. However, it is also clear that the residue of certain insecticides could lead to some possible negative impacts on human health, food safety, and the ecological environment. Therefore, the agrochemical industry is continuously searching for new insecticides. Natural products are valuable resources due to the vast biodiversity of plants and microbes. Plant-derived insecticidal molecules are secondary metabolites in plants. Generally, these secondary metabolites cause less environment pollution and are safer to natural enemies. Due to their structural diversity and biological characteristics, plantderived natural products have received significant attention as lead compounds. Therefore, the application of these natural plant products as alternatives to synthetic insecticides has attracted more attention in recent years [1][2][3][4].
Triterpenes, as the main bioactive chemical compounds in Meliaceae plants, have attracted significant attention owing to their exclusive structural characteristics and remarkable biological activity. Due to their multiple bioactivities, the Meliaceae plants have been used as folk herbs in treating leprosy, eczema, asthma, malaria, fever, and pain. To date, diverse insecticidal molecules have been isolated from Meliaceae plants. A great many studies have revealed that, in these plants, triterpenoids were the active molecules [5,6]. Triterpenes are terpenoids derived from squalene, usually composed of 30 carbon atoms. The structural classification of triterpenoids is mainly grouped into six groups, including linear triterpenes, simple cyclic triterpenes (monocyclic triterpenes, bicyclic triterpenes, and tricyclic triterpenes), tetracyclic triterpenes, pentacyclic triterpenes, nortriterpenes, and triterpenoid saponins (Figure 1) [7]. This review, as a continuation of our first review ("insecticidal triterpenes in Meliaceae: plant species, molecules, and activities of eight genera (Aglaia, Aphanamixis, Azadirachta, Cabralea, Carapa, Cedrela, Chisocheton, and Chukrasia) in Meliaceae" [7]), covers naturally occurring insecticidal triterpenoids from five genera (Cipadessa, Entandrophragma, Guarea, Khaya, and Melia) in Meliaceae. Herein, we summarize the insecticidal plant species, insecticidal phytochemicals and their structures, various insecticidal activities, the structureactivity relationship (SAR), the insecticidal mechanism of action, and the environmental toxicity of the active insecticidal chemicals, hoping to offer some constructive information for the exploration of these chemicals as the lead compounds of novel insecticides. Furthermore, the future research perspectives are discussed.
Below, we review the insecticidal plant species, the corresponding insecticidal chemicals, and their activities in detail.

Cipadessa
In the genus Cipadessa, two species, C. baccifera and C. cinerascens, have been reported to show insecticidal activities. Additionally, limonoids isolated from the leaves of C. baccifera showed moderate antimalarial activity [8,9].
The acetone extract of C. baccifera inhibited the freshly laid eggs of the mosquito C. quinquefasciatus. The acetone extract of the leaf of C. baccifera showed smoking toxicity toward mosquitoes A. stephensi, A. aegypti, and C. quinquefasciatus [59,60]. The hexane and dichloromethane extracts from the fruits of C. baccifera showed toxicity toward the leaf-cutting ant, A. sexdens rubropilosa [61]. Likewise, the hexane extract from the leaves of C. baccifera showed insecticidal activity toward the cotton bollworm, H. armigera. Further studies revealed that the petroleum ether extract reduced the pupation rate and pupal weight and caused a higher percentage of malformed adults. However, the hexane extract reduced the fecundity and egg hatchability in the first-generation adults [62].
Febrifugin A, khayasin T, cipadesin, febrifugin, ruageanin A, and cipadesin A showed poisonous activity toward the fall armyworm, S. frugiperda. At 50 mg/kg, the total cycle mortalities of febrifugin A and khayasin T toward the fall armyworm were 73.3% and 50%, respectively. However, the total cycle mortalities of the other four chemicals toward the fall armyworm were less than 40%. Febrifugin, khayasin T, cipadesin, and cipadesin A also showed growth-inhibitory activity toward the fall armyworm. At 50 mg/kg, febrifugin and khayasin T shortened larval phases by 1.8 and 1.2 days, respectively. At 100 mg/kg, cipadesin A and cipadesin shortened the larval phases by 2.1 and 0.8 days, respectively. Meanwhile, febrifugin also showed antifeedant activity toward the fall armyworm at 100 mg/kg [43,47,51,61,63,70,71].
Additionally, khayasin exhibited marked insecticidal activity toward the fifth larvae of coconut leaf beetle, B. longissimi, with an LC 50 value of 7.28 µg/mL at 24 h [53].

Rings B,D-Seco Limonoids
In this group, two andirobin-type chemicals, cipadonoid B and cineracipadesin G, were reported to show insecticidal activity.
Cineracipadesin G showed antifeedant activity toward the fruit fly, D. melanogaster. The antifeedant index was 32.8% at 1 mM after 17 h [42]. An in vitro assay at the insect nicotinic acetylcholine receptor (nAChR) was performed for cipadonoid B, and the pI 50 value was found to be 4.2, showing that this chemical was a weak antagonist of the insect nAChR [47,72].
Ring D-seco chemical: gedunin possessed various activities toward insects. It showed antifeedant activity toward the lower subterranean termite, R. speratus, with a PC 95 value of 218.4 µg/disc after 30 days [77]. Gedunin also showed poisonous activity toward the fall armyworm, S. frugiperda, and growth-inhibitory activity toward the cotton bollworm, H. armigera [77,78]. In our previous review, we summarized its activity. Therefore, further information can be obtained from the review by Lin (2021) [7]. Additionally, secomahoganin showed antifeedant activity toward the African cotton leafworm, S. littoralis, at 1000 µg/mL [31].
Protolimonoid melianone showed poisonous and antifeedant activities toward the lower subterranean termite, R. speratus, at 100 µg/disc after 30 days. The mortality of R. speratus at 30 days was 95% [41]. Melianodiol showed poisonous activity toward the larvae of the mosquito A. aegypti. The LC 50 value was 14.44 mg/mL and the LC 90 value was 17.54 mg/mL after 24 h. According to the results, melianodiol could be regarded as a potential candidate for use as an ecologically sound biocontrol agent for reducing the larval population of this vector [20]. The other chemical, 3β-O-tigloylmelianol, was effective against the oogenesis and ecdysis of R. (Boophilus) microplus at concentrations of 0.01%, 0.005%, 0.0025%, and 0.00125%. After 48 h, the sexual gland index (GSI) decreased by 50% at all three concentrations [21].
According to these reports, the ethanol extract of the stem bark of K. ivorensis had termiticidal activity [84]. The ethanol extracts of K. grandifoliola and K. senegalensis had ovicidal properties and larvicidal properties against the first-instar larvae of C. maculatus [85]. Moreover, the seed oil of K. senegalensis showed high potential for the control of the cowpea beetle, C maculatus [86]. Further research revealed that the acetone, ethanol, hexane, and methanol extracts of K. senegalensis also showed insecticidal activity toward the mosquito C. annulirostris [87]. In addition, K. senegalensis gum could be employed as an emulsifying agent in the formulation industry [88].

Ring-Intact Limonoids: Anthothecol and Azadirone
The two chemicals of this group, anthothecol and azadirone, can be further classified into two subgroups. Anthothecol is a cedrelone-type limonoid, while azadirone is an azadirone-class chemical.

Ring-Intact Limonoids
The most-studied chemical in this group was toosendanin. Toosendanin is a trichilinclass limonoid isolated from M. toosendan and M. azedarach [37,49]. The principle bioactive chemicals in M. toosendan are toosendanin-type limonoids known as tetranortriterpenoids and intact-ring protolimonoids, which are euphane-or tirucallane-type triterpenoids. It is believed that toosendanin and its derivatives are formed by the loss of four carbons from the side chain of the euphane (20R) or tirucallane (20S) skeleton, which then cyclize to form the 17β-furan ring [5].
Toosendanin and its derivatives demonstrate high insecticidal activity and are important insecticidal molecules derived from plants. In China, toosendanin is used as an important Chinese traditional insecticide and has been registered and commercialized. Studies on the insecticidal activity of various formulations of toosendanin and residue analysis using IC-ELISA can be easily found. The studied formulations include 2% toosendanin EW, 2% toosendanin ME, 2% toosendanin SL, and 2% toosendanin WP [190,191].
Specially, toosendanin has marked systemic properties. It could control the newly hatched larvae of the rice yellow stem borer, T. incertulas, inside of the rice stem [195]. Toosendanin showed strong antifeedant activity toward insects such as P. rapae, O. furnacalis, P. saucia, S. incertulas, O. furnacalis, L. compta, P. xylostrella, S. litura, A. citricidis, and T. aurantia [5,32,37,44,57,58]. Toosendanin at the concentration of 0.01% could result in a 100% antifeedant rate against the tobacco cutworm, S. litura, while toosendanin at the concentration of 0.1% could result in an antifeedant rate of 76.5% against the lawn caterpillar, S. abyssinia [196]. Using the leaf disc choice test, the DC 50 value (concentration deterring feeding by 50%) was 8.04 µg/cm 2 against the fourth-instar larvae of the variegated cutworm, P. saucia [57]. On the cotton bollworm, H. armigera, the EC 50 value (concentration inhibiting larval growth by 50% relative to controls) of toosendanin was 26.8 µg/mL 7 days after the treatment. The FI 50 (dietary concentration showing 50% feeding inhibition) value for toosendanin on the third-instar larvae of the cotton bollworm, H. armigera, was 56.6 µg/mL [32]. As for the ladybird beetle, E. paenulata, the ED 50 value of toosendanin was 3.69 µg/cm 2 after 24 h [100]. It was also effective against the third-instar larvae of the African cotton leafworm, S. littoralis, at 200 µg/mL [5]. Other studies showed that the MIC value of toosendanin was 300 µg/mL against the southern armyworm, S. eridania, in 2-24 h [37]. Meanwhile, toosendanin also exhibited poisonous activity and insect growth-inhibitory activity. For example, toosendanin showed growth-inhibitory effects on the variegated cutworm, P. saucia, with an EC 50 value of 42.3 µg/mL 7 days after treatment [55]. When tested at a concentration of 0.05%, toosendanin could inhibit the body weight of the Asian corn borer, O. furnacalis, by 61.52% after 2 days of treatment [195]. Toosendanin possessed obvious poisonous activity toward the fall armyworm, S. frugiperda, with an LC 50 value of 7.0 µg/mL [5]. It also showed poisonous activity toward the rice weevil, S. oryzae, and the rusty grain beetle, C. ferrugineus. After 6 weeks, the LC 50 values of toosendanin on them were 675 and 1875 µg/mL, respectively [49]. Another study showed that, when tested using the method of topical application, 0.4 µg of toosendanin resulted in a mortality of 58.33% at an average of 3.1 days on the Asian corn borer, O. furnacalis [194]. Furthermore, toosendanin, at 10 µg/mL, could deter the oviposition activity of the cabbage looper, T. ni, and the diamondback moth [139].
In addition, when compared with toosendanin, the nimbolinin-type chemical nimbolinin B showed a relatively weaker antifeedant activity toward S. eridania with an MIC of 1000 µg/mL in 2-24 h (compared to 300 µg/mL for toosendanin) [37]. Furthermore, studies on nimbolinin-type chemicals volkensin and hydroxylactone (isolated from M. volkensii) revealed that they were antifeedant agents against the third-instar larvae of the fall armyworm, S. frugiperda. In the choice assays using corn leaf discs, the ED 50 values of the two molecules were 3.5 and 6 µg/cm 2 , respectively (15 h) [29].

Structures of the Insecticidal Chemicals
A total of 116 insecticidal chemicals were summarized, including 34 ring-intact limonoids, 31 ring-seco limonoids, 48 rearranged limonoids, and 3 tetracyclic triterpenes. The structures of the chemicals are shown in Figures 3-19.
In continuation of the program aimed at the discovery and development of natural product-based insecticidal agents, according to the insecticidal activity of 12 semi-synthesized 28-acyloxy derivatives of toosendanin (2a-l) against the pre-third-instar larvae of the rice ear-cutting caterpillar, M. separata, in vivo at the concentration of 1 mg/mL, Xu et al. (2011) concluded that the butanoyloxy or phenylacryloyloxy moiety at the 28-position of toosendanin was essential for insecticidal activity [200]. Furthermore, Zhang et al. (2013) synthesized 18 alkyl/alkenylacyloxy derivatives at the C-28 position adopting the exo-configuration of toosendanin (3a-r) via the reaction of toosendanin with fatty acids in the presence of N,Ndiisopropylcarbodiimide and 4-dimethylaminopyridine. The activity of these 18 molecules tested on the pre-third-instar larvae of the rice ear-cutting caterpillar, M. separata, revealed that compounds 3e and 3o displayed more promising insecticidal activity than their natural precursor, toosendanin. It was revealed that, for the n-alkyloyloxy series derivatives, the proper length of the side chain R at the C-28 position of toosendanin was very important for insecticidal activity [201]. Another structure-activity relationship study of toosendanin derivatives indicated that the sites around R4 and R5 also contributed to the activity [202].                     Khayanolide B and 1-O-acetylkhayanolide B with a C2-C14 ether linkage and hydroxyl group at C8 were more potent antifeedants than khayanolide A with a C8-C14 epoxide group and a keto-carbonyl group at C-2. The presence of a hydroxyl group at C-1 slightly enhanced the antifeedant activity of khayanolide B compared with the acetoxy group in 1-O-acetylkhayanolide B, indicating that the substituents at C-1 in this type of molecule had no marked effect on antifeedant activity [54].
Analysis revealed that febrifugin A had a furan-ring oxygenated group at C-21 and a hydroxy group at C-23, which contributed to the insecticidal activity. The high insecticidal activity of febrifugin A further confirmed that the hydroxyl group on C-23 and the carbonyl group on C-21 had a great influence on the activity. Compared with the furan ring oxygenated at C-21 and C-23, intact and seco-rings and an intact furan ring in the limonoids showed more significant antifeedant activity [43,47].

Insecticidal Mechanism of Action
The study of the insecticidal mechanism of action (MOA) of triterpenoids from these five genera mainly focused on the MOA of toosendanin. Several MOA studies of other molecules reported on the inhibition of certain enzymes. For example, khayanolide B was reported to show weak inhibitory activities toward the enzymes acetylcholinesterase (AChE), butyrycholinesterase (BuChE), and lipoxygenase (LOX) in a concentration-dependent manner [203]. The inhibition of AChE by the mexicanolide limonoids 3-O-detigloyl-3-O-isobutyrylfebrifugin A, granatumin E, khaysin T, and 2'S-cipadesin A have also been reported, and they showed moderate inhibitory activities against AChE at 50 mM [37]. In addition, prieurianin was reported as an antagonist of 20-hydroxyecdysone. When tested with the D. melanogaster B-II cell line, the ED 50 value of prieurianin was 10 −5 M with a 20-hydroxyecdysone concentration of 5 × 10 −8 M [204].
The MOA of toosendanin has been systemically studied. Using the electrophysiological technique, the mechanism study of toosendanin as a feeding deterrent for the larvae of the cabbage butterfly, P. brassicae, demonstrated that toosendanin stimulated a deterrent receptor cell located in the medial maxillary sensillum styloconicum. Toosendanin, even at the low concentration of 10 −9 M, also inhibited the responses of both the sugar and the glucosinolate receptor cell localized in the lateral sensillum styloconicum, in a dose-dependent manner. However, the taste neurons responding to amino acids or deterrents in the lateral sensillum were not affected by toosendanin. Therefore, it could be concluded that the sensory code underlying feeding behavior was modulated by toosendanin via several different peripheral sensory mechanisms [205]. Further studies showed that toosendanin seemed to specifically induce feeding deterrence in the larvae of the cotton bollworm, H. armigera, and apparently stimulated deterrent receptor cells and reduced neural input from taste cells specialized to detect feeding stimulants [30].
The possible mechanism underlying the poisonous activity of toosendanin has also been analyzed. It was found that the activities of protease and microsome multifunctional oxidase (MFO) in the midgut tissue of the larvae of the cabbage worm, P. rapae, fed with toosendanin were inhibited. However, the activities of lipase, amylase, and acetylcholinesterase were not significantly affected. The physiological metabolism of the larvae was disturbed, and abnormal biological oxidation was carried out in the body, while the metabolic level decreased. Histological observation revealed degradation in the microvilli, hyperplasia of the smooth endoplasmic reticulum, and condensation of chromatin. Moreover, immunohistochemical analysis revealed that gold particles existed on the microvilli of columnar cells and goblet cells, and they gradually accumulated with the exacerbation of poisoning symptoms, showing that toosendanin targeted the microvilli of midgut cells. In addition, it inhibited the central nervous system of the larvae [206][207][208][209].
Furthermore, studies on the larvae and female adults of the mosquito, A. aegypti, revealed that topical application or ingested toosendanin dose-dependently disrupted yolk deposition in oocytes, blood ingestion and digestion, and ovary ecdysteroid production in blood-fed females [210]. It is noteworthy that medicinal studies demonstrated that toosendanin selectively affected neurotransmitter release, effectively antagonized botulism, induced cell differentiation and apoptosis, inhibited proliferation of various human cancer cells, and inhibited K + -channel and facilitated L-type Ca 2+ -channel activity [211]. These results are good starting points for further research on the MOA of toosendanin as an insecticidal molecule.

Environmental Toxicity
In fact, various extracts of plants or some pure chemicals in Meliaceae have been used as traditional medicines. The ethnomedical uses of the plant are as varied as the different cultures and geographical people that make use of the plant. For example, the stem bark of K. senegalensis has been used in the treatment of several conditions, including stomach pain, malaria, fever, and blennorrhagia, in Africa [212]. The pure chemical, toosendanin, isolated from M. toosendan, has been used to treat abdominal pain and as a digestive tract parasiticide in ancient China for about 1500 years [213].
Normally, naturally derived plant extracts or chemicals are easily degraded and thus cause less residue to remain in the environment. For example, three days after the field application at five times the dose recommended by the manufacturer, the residue of salannin on strawberry (LOQ 0.01 mg/kg) was not detectable [217]. Another study demonstrated that toosendanin was easily degradable. At the recommended dose, the final residues of toosendanin detected by IC-ELISA were 0.009 mg/kg in cabbage and 0.043 mg/kg in tobacco. In soil, toosendanin residue was not detectable [218].
However, there were also some negative effects of these plant extracts or chemicals. Some extracts were reported to show a negative effect on rats. In detail, high doses of the crude water extract of K. grandifoliola reduced the Ca, P, Mg, and Zn levels of the bones and may have had an adverse effect on bone minerals in growing rats [219]. The methanol extracts of K. ivorensis were found to be relatively toxic, with an LD 50 value of 549 mg/kg per body weight of the mice [220]. The ethanolic extract of K. senegalensis adversely affected the function of the liver and kidneys of rats [221][222][223]. M. azedarach was also reported to possess a potent pregnancy interceptive property on the rat [224]. Isolated chemicals, such as methyl angolensate and toosendanin, were also reported to possess some negative effects. For example, methyl angolensate could cause the inhibition of smooth muscle and reduce the propulsive action of the gastrointestinal tract in mice [225]. The traditional medicinal chemical toosendanin had serious hepatotoxicity [226]. Severe cytoplasmic vacuolation and nuclear shrinkage were found in the liver of toosendanin-treated zebrafish [227]. Further studies revealed that toosendanin was pregnancy-toxic to animals (Table 5) [228].
Briefly, several studies have been conducted on the environmental toxicity of the extracts of plants or isolated chemicals from the five genera (Cipadessa, Entandrophragma, Guarea, Khaya, and Melia) in Meliaceae. Further studies are needed to elucidate the environmental toxicity of some important insecticidal chemicals for their future application in the field.

Future Outlook
The unique insecticidal properties of insecticidal plants, particularly Meliaceae, which are safe both for the environment and natural enemies, and their compatibility with the agroecosystem emphasize their potential value in the integrated control of insect pests [195].
The use of toosendanin as an agricultural insecticide, with marked systemic properties, showing various activities including antifeeding, deterring, growth-inhibitory, contact poisoning, and stomach poisoning activities, was recorded about 2000 years ago in ancient China. There are still various commercial formulations of toosendanin on the market. In addition, there are other molecules with obvious insecticidal activity that deserve further attention. Like toosendanin, gedunin possesses various activities toward insects and exhibits good potential to be used as a lead compound for the development of novel insecticides. Other chemicals, such as khayasin and 12-deacetyltoosendanin, also deserve further attention. Their activities toward insects should be systemically evaluated, and their effects on nontarget organisms and the environment should also be further studied.
Recently, knowledge of the biosynthesis of important bioactive molecules in plants has become increasingly important. Understanding the biosynthetic pathways and their regulation has led to attempts to metabolically engineer bioactive molecules more successfully and more easily in economically important plants.
However, despite the intensive investigation of limonoids over several decades, the biosynthetic pathway of these triterpenoids is less understood. Enzymes involved in the biosynthesis of limonoids have been partially identified and characterized in some plant species. For example, AiOSC1 from the neem tree produces a single triterpene, tirucalla-7,24-dien-3β-ol, indicating the importance of its role in azadirachtin biosynthesis [229]. Toosendanin from M. toosendan was proposed to be synthesized in a manner similar to azadirachtin, by cyclizing the precursor 2,3-oxidosqualene into a tirucalla-7,24-dien-3βol as the scaffold, followed by scaffold rearrangements and the formation of the furan ring [5]. Lian et al. (2020) elucidated that MtOSC1 was a key enzyme in the production of triterpene tirucalla-7,24-dien-3β-ol, while MtOSC6 (a lupeol synthase) was a key enzyme in the production of lupeol in M. toosendan. The product of MtOSC1 was the precursor for the biosynthesis of toosendanin. This research provided a foundation for toosendanin biosynthesis and presented an important building block for the synthesis of insecticidal triterpenoids using the synthetic biology approach [230].
Using synthetic biology methods, the identified enzymes could be used to model a biosynthetic pathway to produce large quantities of insecticidal molecules, such as azadirachtin and toosendanin. Therefore, further research needs to be carried out for the clear elucidation of the biosynthetic pathway of certain highly effective plant-derived insecticidal molecules. The biosynthesis of important plant-derived insecticidal molecules in Meliaceae will be a significant research topic of interest in the coming years.