Novel β-Macrocarpene-Type Sesquiterpenoids with Anti-Insect Activity from Corn Silks

A phytochemical study of corn silk resulted in the isolation of nine novel β -macrocarpene-type sesquiterpenoids ( 1 – 9 ) along with one known analogue ( 10 ). The structural elucidation of these compounds was accomplished using comprehensive spectral data, X-ray crystallography, and electronic circular dichroism (ECD) calculations. Bioassay revealed that at a concentration of 20 μg/g, compounds 2, 3, 6, and 7 significantly reduced the weight of Spodoptera frugiperda, 4 inhibited the growth of S. litura, whereas compound 10 inhibited weight of both insects. In vitro enzyme assays showed that compound 10 inhibited carboxylesterase (CarE) and glutathione S-transferase (GST) enzyme activities in S. litura but did not impact these enzymes in S. frugiperda . Furthermore, transcriptome analysis suggested that 10 can upregulate the expression of the cytochrome P-450 monooxygenase (CPY450) and GST genes in S. frugiperda, whereas increased expression of carcinine transporter-like gene and decreased expression of trypsin were observed in S. litura . Overall, this research reveals the potential of corn sesquiterpenoids for developing environmentally friendly insecticides. Cite this paper: Chin. J. Chem. 2024, 42, XXX—XXX. DOI: 10.1002/cjoc.202400XXX Novel β -Macrocarpene-Type Sesquiterpenoids with Anti-Insect Activity from Corn Silks Yu-Jia Wang, a,c,# Jun-Yu He, b,c,# Long Chen, d Zi-Wei Li, e Ming-Hua Qiu,* , a,c,f Jin-Feng Qi,* ,b,c and Xing-Rong Peng* ,a, c,f a Key Laboratory of Phytochemistry and Natural Medicines, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China b Department of Economic Plants and Biotechnology, Yunnan Key Laboratory for Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China c Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China d Tea Research Institute, Yunnan Academy of Agricultural Sciences, Kunming, 65020, China e Department of Agronomy and Biological Science, Dehong Teacher’s College, Mangshi 678400, China f State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, China

and Originality Content Corn ( Zea mays L.), one of the most important food crops in the world, is not only the main source of food for humans but also a key component of animal feed. [1] Beyond its nutritional value, corn has significant economic importance. It is used in the production of biofuels, such as ethanol, contributing to renewable energy sources and reducing reliance on fossil fuels. Additionally, corn is a vital raw material in the manufacture of numerous products, including corn syrup, starch, and biodegradable plastics. Overall, the significance of corn extends beyond agriculture; it plays a vital role in the economy, energy production, and food systems worldwide. However, the production of corn faces many threats, [2-3] among which pests such as Spodoptera frugiperda and Spodoptera litura pose serious threats. [4-5] These pests not only lead to a decrease in yield but also affect the quality of corn, causing considerable losses to farmers and the agricultural economy. Chemical pesticides are widely used for controlling these pests. [6] However, long-term dependence on chemical pesticides may lead to pest resistance issues and have negative impacts on the ecological environment and food safety, as residues can remain on harvested products and enter the food chain. [7] Therefore, exploring effective defense compounds to replace or assist the use of chemical pesticides has become an important issue for sustainable agricultural development and food safety. [7] Research has shown that the use of natural defense compounds from plant sources can effectively increase the resistance of plants to pests while reducing its dependence on chemical pesticides [8-11] These extracts, derived from various parts of plants such as leaves, flowers, and seeds, contain a variety of bioactive compounds that can effectively repel or kill insects. [12-14] Many plant extracts exhibit low toxicity to humans and non-target organisms, making them an environmentally friendly alternative to synthetic pesticides. For example, essential oils like pyrethrum, derived from chrysanthemums, are known for their rapid action against a broad spectrum of insects. [15] The increasing interest in sustainable agriculture has led researchers to explore and develop more applications of plant extracts, highlighting their potential as effective tools for integrated pest management and promoting ecological balance in agricultural systems. Figure 1 Structures of compounds 1 ‒ 10 (red: new compounds). Corn produces a range of terpenoid phytoalexins, such as diterpenoids (kauralexins and dolabralexins) and sesquiterpenoids (zealexins), in significant quantities as a defense mechanism against various fungal pathogens. These compounds act to directly inhibit the growth of the pathogens. [16-18] The biosynthetic pathways of maize terpenoid phytolexins (MTPs) have been clarified through the identification of over 30 MTP biosynthetic synthases, along with the discovery of various sesquiterpenes and diterpenes derived from corn silk and roots, which are modified by cytochrome P450 enzymes, underlying the inherent diversity of maize terpene defense systems. [17] Therefore, the aim of this study was to explore defense terpenoids from silk, the reproductive organ of corn, and analyze their potential for controlling S. frugiperda and S. litura, providing a theoretical foundation and practical guidance for the promotion of environmentally friendly pest control in corn, further ensuring food safety. Results and Discussion The phytochemical investigation of the corn silks resulted in the isolation of ten sesquiterpenoids ( 1 ‒ 10 ) (Figure 1), among which nine new compounds were identified as stigmayic acids A‒I ( 1 ‒ 9 ), as detailed below. Known compound 10 (stigmene C) was confirmed by comparing its NMR spectroscopic data with previously reported data. [19] Stigmayic acid A ( 1 ) was obtained as a colorless single crystal, and its molecular formula was established as C 15 H 20 O 4 based on the HRESIMS ion observed at m/z 287.1256 [M + Na] + (calculated 287.1254). The 1 H NMR spectrum of 1 displayed two singlet methyl proton signals at δ H 1.04 and δ H 1.06, along with two sp 2 methine proton signals at δ H 6.90 (s) and δ H 6.14 (s). The 13 C NMR-DEPT spectra of 1 revealed fifteen distinct carbon resonances, which were categorized as two methyl groups, five methylene groups, two olefinic/aromatic methine carbons ( δ C 123.1; δ C 136.7), and six quaternary carbons, including one ketone carbonyl ( δ C 203.2), one carboxyl group ( δ C 171.0), two olefinic/aromatic quaternary carbons ( δ C 131.5; δ C 171.2), and one oxygenated quaternary carbon ( δ C 72.5). These spectral data indicate that compound 1 is a macrocarpene-type sesquiterpene, structurally similar to stigmene B, [19] with the notable exception that the chemical shift of C-15 in compound 1 is shifted downfield compared to that in stigmene B. [19] moreover, the molecular weight of 1 was one more than that of stigmene B. Thus, we deduced that C-15 in 1 was a carboxyl group. The HMBC correlations (Figure 2) of H-3 ( δ H 6.90, s) and H 2 -5 ( δ H 2.39, m) with C-15 further confirmed the above deduction. Moreover, X-ray crystallographic analysis (Figure 3) was used to determine the planar structure and revealed that compound 1 was a pair of enantiomers (1 R - 1 :1 S - 1 = 9:1). Furthermore, 1a and 1b were obtained through separation via a chiral column (Figure S86). Their opposite rotation optical values ( 1a : [α] 20 D 4.67; 1b : [α] 20 D ‒12.44) and CD curves (Figure S87) demonstrated the presence of 1 R - 1 and 1 S - 1 . Moreover, 1a and 1b were assigned as 1 S - 1 and 1 R - 1, respectively by comparing their calculated ECD curves with the experimental curves (Figure 4). Finally, the structure of 1 was determined. Figure 2 Selected HMBC (H→C) and 1 H- 1 H COSY (HH) correlations of 1 ‒ 3, 5, 6, and 8 . Figure 3 ORTEP diagrams of 1, 2, and 9 . Figure 4 Experimental and calculated ECD curves of compounds 1, 4 ‒ 7, and 10 . The molecular formula of stigmayic acid B ( 2 ) was determined to be C 15 H 22 O 3 based on HRESIMS data, which displayed a peak at m/z 251.1644 [M + H] + (calculated as 251.1642). Analysis of its 1D NMR spectra showed similarities to those of stigmene C [19] with the primary distinction being the replacement of the double bonds at C-3 and C-4 in stigmene C with a methylene group ( δ C 29.9) and a methine group ( δ C 44.2) in compound 2 . The HMBC correlations (Figure 2) of H-1 with C-2, C-3, C-5, and C-6, as well as the correlations of H 2 -3 and H 2 -5 with C-15 ( δ C 180.9), along with the 1 H- 1 H COSY correlations of H-1/H 2 -2/H 2 -3/H-4/H 2 -5/H 2 -6, supported this structural assignment. Additionally, X-ray crystallographic analysis confirmed that compound 2 exists as a mesomer. (Figure 3), which was consistent with the deduction based on its low optical rotation value ([α] 21.2 D ‒4.67) and baseline CD curve. In addition, the relative configurations of H-1 and H-4 were β- and α -based on X-ray crystallographic analysis (Figure 3). Thus, the structure of 2 was determined. The molecular formula of compound 3 was determined to be C 15 H 20 O 4 based on its HRESIMS data, which showed a peak at m/z 287.1257 [M + Na] + (calculated as 287.1254). The 1D NMR spectra of 3 exhibited similarities to those of stigmene C, [19] with the notable difference being the presence of an oxygenated methine ( δ C 75.0) in 3, instead of a methylene group in stigmene C. Detailed analysis of the HMBC spectrum (Figure 2) of 3 revealed key correlations: H-1, H-10, H-12, H 3 -13, and H 3 -14 with the oxygenated methine ( δ C 75.0), as well as the oxygenated methine proton ( δ H 3.99, s) with C-1 ( δ C 38.3), C-9 ( δ C 38.8), C-10 ( δ C 48.1), and C-12 ( δ C 124.2). These correlations confirmed that the hydroxyl group is attached to C-8. Furthermore, a colorless crystal was obtained through the evaporation of MeOH, and X-ray crystallographic analysis (Figure 5) demonstrated that compound 3 exists as a pair of epimers, and the ratio of 1S,8 S - 3 ( 3a ) to 1 R,8 S - 3 ( 3b ) was 9:1. However, they were not separated, although we have tried many methods. Theoretically, the CD curve of the mixture corresponds to that of 1 S,8 S - 3 ( 3a, major epimer). The calculated ECD method further confirmed the above deduction (Figure 5). Thus, the structure of mixture 3 was established and named stigmayic acid C ( 3 ). Stigmayic acid D ( 4 ) has a molecular formula of C 15 H 22 O 4, determined by the HRESIMS ion at m/z 289.1418 [M + Na] + (calcd. 289.1410), showing two more hydrogens than 3 . Additionally, the 1D NMR spectroscopic data of 4 reseamble those of 3, however, the double bond signals at C-3 and C-4 or at C-5 and C-4 in 4 were replaced by one methylene and one methine. The HMBC spectrum of compound 4 displayed correlations of H-1 with C-2, C-3, C-5, and C-6 and of H 2 -3 and H 2 -5 with C-4 and C-15. Moreover, the 1 H- 1 H COSY correlations of H-1/H 2 -2/H 2 -3/H-4/H 2 -5/H 2 -6 were observed, which indicated that compound 4 had a cyclohexanocarboxylic acid fraction. Considering the plane symmetry of the cyclohexanocarboxylic acid fraction, compound 4 had only one chiral carbon, C-8. Furthermore, the absolute configuration of C-8 was established to be S by comparing the calculated ECD curve of 8 S - 4 with the experimental CD curve (Figure 4). Therefore, the structure, including the stereo structure, of 4 was established. Figure 5 (A) ORTEP diagram of mixture 3 ; (B) experimental and calculated ECD curves of compound 3 . Figure 6 Regression analysis of experimental versus calculated NMR chemical shifts of 1 S,8 R,11 S - 5 (red dots), 1 S,8 S,11 S - 5 (blue dots), 1 S,8 S,5 R - 5 (green dots), and 1 S,8 R,5 S - 5 (purple dots), with linear fitting shown as a line. Table 1. 1D NMR spectroscopic data (600/150 MHz, CD 3 OD) of compounds 1 ‒ 7 ( δ in ppm, J in Hz). | 1 H | 13 C | 1 H | 13 C | 1 H | 13 C | 1 H | 13 C | 1 H | 13 C | 1 H | 13 C | 1 H | 13 C | | | 1 | 72.5 C | 2.27, m | 46.6 CH | 2.67, m | 38.3 CH | 2.36, t (11.8) | 42.9 CH | 2.36, m | 37.9 CH | 2.29, m | 51.0 CH | 2.49, m | 37.0 CH | | | 2 | 2.21, d (18.7); 2.55, d (19.2) | 37.0 CH 2 | 1.33, m; 1.88, m | 30.8 CH 2 | 2.25, m; 2.40, m | 31.0 CH 2 | 1.19, m; 2.02, m | 32.3 CH 2 | 2.27, m; 2.34, m | 31.9 CH 2 | 4.35, d (9.4) | 69.6 CH | 2.28, m; 2.40, m | 31.3 CH 2 | | 3 | 6.90, s | 136.7 CH | 1.50, m; 2.08, m | 29.9 CH 2 | 6.99, s | 139.3 CH | 1.50, m; 2.08, m | 30.1 CH 2 | 7.04, s | 140.5 CH | 6.81, s | 142.4 CH | 7.04, s | 141.1 CH | | 4 | 131.5 C | 2.27, m | 44.2 CH | 131.5 C | 2.28, t (12.2) | 44.5 CH | 131.1 C | 132.5 C | 131.4 C | ||||| | 5 | 2.39, m | 22.2 CH 2 | 1.50, m; 2.08, m | 29.9 CH 2 | 2.28, m; 2.37, m | 25.2 CH 2 | 1.50, m; 2.08, m | 30.1 CH 2 | 2.22, m; 2.32, m | 25.3 CH 2 | 2.27, m; 2.21, m | 25.5 CH 2 | 2.27, m | 25.4 CH 2 | | 6 | 1.75, m; 1.89, m | 31.7 CH 2 | 30.8 CH 2 | 1.53, m; 2.02, m | 27.9 CH 2 | 1.19, m; 2.02, m | 32.3 CH 2 | 1.44, m; 1.92, m | 28.7 CH 2 | 1.71, m; 1.86, m | 26.9 CH 2 | 1.30, m; 1.90, m | 29.6 CH 2 | | | 7 | 171.2 C | 171.2 C | 169.8 C | 171.1 C | 144.5 C | 168.7 C | 171.1 C | ||||||| | 8 | 2.36, s | 40.6 CH 2 | 2.30, s | 43.2 CH 2 | 3.99, s | 75.0 CH | 3.95, s | 75.0 CH | 3.42, s | 74.2 CH | 2.35, s | 42.8 CH 2 | 3.83, s | 81.9 CH | | 9 | 34.7 C | 34.5 C | 38.8 C | 38.8 C | 36.1 C | 34.5 C | 34.5 C | ||||||| | 10 | 2.24, s | 51.9 CH 2 | 2.22, s | 51.9 CH | 2.13, d (16.5); 2.47, d (16.5) | 48.1 CH 2 | 2.12, d (16.5); 2.40, d (16.5) | 48.0 CH 2 | 1.54, d (8.4) | 39.5 CH 2 | 2.25, s | 52.0 CH | 1.16, m; 1.73, m | 31.1 CH 2 | | 11 | 203.2 C | 203.3 C | 202.3 C | 202.5 C | 4.13, m | 67.1 CH | 202.9 C | 2.07, m | 24.2 CH 2 | ||||| | 12 | 6.14, s | 123.1 CH | 5.85, s | 123.5 CH | 5.79, s | 124.2 CH | 5.77, s | 123.7 CH | 7.04, m | 126.9 CH | 5.94, s | 125.9 CH | 5.54, t (3.4) | 124.5 CH | | 13 | 1.04, s | 27.8 CH 3 | 1.03, s | 28.2 CH 3 | 1.01, s | 26.6 CH 3 | 0.98, s | 26.5 CH 3 | 0.84, s | 24.6 CH 3 | 1.05, s | 28.4 CH 3 | 0.88, s | 25.1 CH 3 | | 14 | 1.06, s | 28.5 CH 3 | 1.03, s | 28.2 CH 3 | 1.03, s | 23.7 CH 3 | 1.02, s | 23.8 CH 3 | 1.00, s | 27.7 CH 3 | 1.05, s | 28.4 CH 3 | 1.04, s | 27.1 CH 3 | | 15 | 171.0 C | 180.9 C | 171.1 C | 180.3 C | 170.8 C | 170.3 C | 171.1 C | ||||||| | 1’ | 4.38, d (7.7) | 103.7 CH | |||||||||||| | 2’ | 3.15, m | 75.6 CH | |||||||||||| | 3’ | 3.17, m | 77.8 CH | |||||||||||| | 4’ | 3.25, t (9.2) | 71.8 CH | |||||||||||| | 5’ | 3.31, m | 78.2 CH | |||||||||||| | 6’ | 3.65, dd (11.7, 5.8); 3.84, m | 62.9 CH 2 | Structural analysis of compounds 1 ‒ 4 revealed that when a double bond was present in the cyclohexanocarboxylic acid moiety, two isomers were detected, and their ratio was approximately 1:9 (1 R : 1 S ). In addition, we used a chiral column to analyze a known compound, stigmene C ( 10 ), which revealed that compound 10 was still a pair of enantiomers (Figure S95). Moreover, we collected 10a and 10b . The CD and optical rotation values of only 10a are sufficient to be determined ([α] 21.2 D ‒70.5). The calculated ECD curve confirmed that the absolute configuration of 10a was 1 S (Figure 4). Thus, the aforementioned information prompted us to deduce that their biogenetic precursors could be a pair of isomers. Research has reported that enantiomeric analysis of β -macrocarpene and the β -bisabolene product, two known precursors of sesquiterpenes in maize, revealed the presence of major ( S )-enantiomers and trace ( R )-enantiomers. [20] Thus, we still consider that compounds 5 ‒ 7 are epimers and that the 1 S -isomer is the majority. Given that they are not easily separated, the stereochemical structures of 5 ‒ 7 was elucidated as their major isomers. HRESIMS and 13 C NMR data of stigmayic acid E ( 5 ) gave a mo Table 2. 1D NMR spectroscopic data (600/150 MHz, CD 3 OD) of compounds 8 and 9 ( δ in ppm, J in Hz). | 1 H | 13 C | 1 H | 13 C | | | 1 | 143.7 C | 142.8 C | || | 2 | 7.69, d (8.3) | 127.4 CH | 7.76, d (8.2) | 128.1 CH | | 3 | 8.06, d (8.3) | 131.1 CH | 8.07, d (8.2) | 130.6 CH | | 4 | 134.7 C | 134.8 C | || | 5 | 7.69, d (8.3) | 127.4 CH | 7.76, d (8.2) | 128.1 CH | | 6 | 8.06, d (8.3) | 131.1 CH | 8.07, d (8.2) | 130.6 CH | | 7 | 159.7 C | 159.2 C | || | 8 | 2.76, s | 43.0 CH 2 | 4.40, s | 73.9 CH | | 9 | 34.6 C | 38.5 C | || | 10 | 2.37, s | 51.8 CH 2 | 2.37, s | 47.1 CH 2 | | 11 | 202.7 C | 202.1 C | || | 12 | 6.43, s | 125.8 CH | 6.30, s | 126.4 CH | | 13 | 1.14, s | 28.3 CH 3 | 1.05, s | 26.1 CH 3 | | 14 | 1.14, s | 28.3 CH 3 | 1.16, s | 25.1 CH 3 | | 15 | 170.5 C | 170.9 C | -lecular formula C 15 H 20 O 4, suggesting that it has two more hydrogen atoms than 1 . A careful examination of the 1D NMR spectral data for compounds 5 and 1 revealed the absence of a ketone group at C-11 in 5, along with the presence of an oxygenated methine. This finding was corroborated by the HMBC correlations (Figure 2), which showed connections between H-11 and C-7, C-9, C-10, and C-12; between H-1 and C-7, C-8, and C-12; and between H-8 and C-7, C-9, C-10, and C-12. Additionally, the 1 H- 1 H COSY correlations (Figure 2) indicated a relationship involving H-12, H-11, and H 2 -10. The relative configurations at C-8 and C-11 were assigned as S * and R *, respectively, based on the calculated NMR data analyzed using the DP4+ method (Figure 6). To further establish the absolute configuration of compound 5, an electronic circular dichroism (ECD) calculation was performed, and the computed ECD spectrum for 1 S,8 S,11 R -5 ( 5a ) aligned well with the experimental circular dichroism curve. Consequently, the structure of stigmayic acid E ( 5 ) was defined unequivocally. Stigmayic acid F ( 6 ) was confirmed to have the molecular formula C 15 H 20 O 4 based on HRESIMS and 1D NMR spectroscopic analyses. While the 1D NMR data of compound 6 exhibited similarities to those of compound 3, a notable difference was observed in the chemical shifts of the oxygenated methine: 6 ( δ C 69.6) compared to 3 ( δ C 75.0). This significant difference indicates that the oxygenated methine in compound 6 is not located at C-8, as it is in compound 3 . Further analysis through HMBC correlations (Figure 2) revealed connections between H-1 and H 2 -6 with the oxygenated methine, as well as the proton on the oxygenated methine showing correlation with C-1, C-7, C-3, C-4, and C-6. Additionally, the 1 H- 1 H COSY correlations involving H-5, H-6, H-1, H-2, and H-3 supported the conclusion that the hydroxyl group is attached to C-2. The absolute configuration at C-1 in the major isomer was assigned as R based on its biosynthetic pathway. Thus, the potential absolute configurations of compound 6 could be either 1 R,2 R or 1 R,2 S . Upon comparison of the calculated ECD curves for 1 R,2 R -6 and 1 R,2 S -6, the stereostructure of compound 6a was determined to be 1 R,2 S (Figure 4). Consequently, the structure of stigmayic acid F ( 6 ) was established. The molecular formula of stigmayic acid G ( 7 ) was assigned as C 21 H 32 O 8 from its HRESIMS m/z 435.1989 [M + Na] + (calcd. 435.1989). Its 1D NMR spectra revealed a sesquiterpenoid fraction like that of 3 and a glucose moiety. Combination of the downfield shift of C-8 ( δ C 75.0 for 3 ; δ C 81.9 for 7 ) with a series of HMBC correlations from H-8 to C-1’ and from H-1 and H-12 to C-8 illustrated that the glucose moiety was linked to 8-OH. 1 S,8 S of 7a was established by comparing the calculated and experimental CD curves (Figure 4). The presence of β -D-glucose was verified by analyzing the HPLC results of the hydrolysate and comparing the retention time with that of β -D-glucose documented in the literature. [21] Therefore, the structure of 7 was determined. The 1D NMR spectrum of 8 (stigmayic acid H) was similar to that of stigmene A, [19] except that the molecular weight of 8 had one more hydrogen than the latter one and the downfield shift of C-15 in 8 . This information illustrated that C-15 in 8 could be a carboxyl, which corresponds to its molecular formula of C 15 H 16 O 3 . Further evidence was established from the HMBC correlations (Figure 2) of H-3 and H-5 with C-15. The structure of 8 was finally elucidated. The 1D NMR spectroscopic data of compounds 8 and 9 indicated that they possess the same skeleton, with the sole distinction being the substitution of the oxygenated methine at C-8 in compound 8 with a methylene group in compound 9 . The HMBC correlations of H 2 -8 with C-1, C-7, C-9, C-10, C-12, C-13, and C-14 further confirmed the above deduction. X-ray diffraction analysis of 9 confirmed that C-8 was S -configuration (Figure 3). Therefore, the structure of 9 was established and named as stigmayic acid I. Compounds 1 ‒ 10 are classified as β -macrocarpene-type sesquiterpenoids, and their plausible biosynthetic pathway was deduced starting from the precursor β -bisabolene (Scheme S1). Research has shown that β -bisabolene is initially cyclized to β -macrocarpene via the catalysis of TPS6 and TPS11. [22] The methyl group at C-15 of β -macrocarpene is subsequently oxidized to a carboxyl group (zealexin A1) by the P450 enzyme CYP71Z18. [23] Additionally, the C-1, C-2, C-8, and C-11 carbons are further oxidized to hydroxyl or carbonyl groups, as observed in compounds 3, 5, 6, and 10 . In compound 7, the 8-OH group is glycosylated with glucose under the action of a glycosyltransferase. Furthermore, ring A in compounds 3 and 10 is oxidized to phenyl in compounds 9 and 8, respectively, whereas the double bond between C-2 and C-3 in the A ring of compounds 3 and 10 is reduced, resulting in the formation of compounds 4 and 2, respectively. The discovery of these diverse structures indicates that numerous post-modification enzymes, which play crucial roles in the construction of the chemical defense system, are present in maize. To evaluate the anti-insect activity of the isolates against S. frugiperda and S. litura, a feeding bioassay was conducted with neonate larvae, which is a commonly used method in evaluating the insecticidal activity of natural compounds. [24] The results revealed that compounds 2, 3, 6, 7, and 10 significantly reduced the weight of S. frugiperda, whereas compounds 4 and 10 inhibited the growth of S. litura at a concentration of 20 μg/g compared with the control group (Figure 7A, B, D). Among them, only compound 10 inhibited two insect herbivores. Considering that 10 was a pair of enantiomers, 10a and 10b were separated for further anti-insect activity assay. Notably, both 10a and 10b significantly suppressed the growth of S. litura ; however, their inhibitory effects were weaker than that of 10 (Figure 7C). In addition, the quantification analysis of 10 in different parts of maize indicated that the content of 10 was more than 350 μg/g (dry weight) in the roots and silk, which is greater than that in the leaves and tassel (Figure 7E). Thus, considering the high content and significant inhibition of 10, we chose it for subsequent in vitro enzyme activity and transcriptome analyses to reveal the underling mechanism of the herbivore resistance activity. The midgut that secreted detoxifying enzymes is also a key player in insecticide resistance. Insects undergo a detoxification process comprising three phases: phase I, phase II, and phase III. Cytochrome P450 monooxygenases (CYPs), glutathione S-transferases (GSTs), and esterases (ESTs) are involved in the first two phases and serve as effective targets for plant secondary compounds used in insecticides. [25-27] Therefore, we assessed the activities of these detoxification enzymes in the guts of S. frugiperda and S. litura at 0, 24, and 48 hours after treatment with an artificial diet containing 20 μg/g of compound 10 via in vitro enzyme activity assays. The results revealed that there was no significant difference in the activity of the P450 enzyme in the midgut of S. litura at 24 and 48 hours (Figure 8A). However, the Figure 7 (A, B) Resistance of compounds 2 - 4, 6, 7, and 10 against S. frugiperda and S. litura at a concentration of 20 μg/g; (C) Resistance of compounds 10, 10a, and 10b against S. litura ; (D) Inhibition of the growth of two insects by compound 10 ; (E) Content of 10 in different parts of maize. Figure 8 Effects of compound 10 on the detoxification enzymes in the guts of S. litura (A-C) and S. frugiperda (D-F) (CK: control; T24: treatment 24 hour; T48: treatment 48 hour). activity of CarE was significantly reduced at both 24 and 48 hours (Figure 8B). The activity of GST was inhibited at 48 hours (Figure 8C). In contrast, the activities of the three detoxification enzymes in the midgut of S. frugiperda did not noticeably change (Figure 8D-F). These findings suggest that compound 10 affects the weight of both insect species through different targets and signaling pathways. To further investigate the mechanism by which stigmene C ( 10 ) weakens insect adaptability, we performed RNA-seq and PCA on the midgut of insects fed with compounds at 0 (Control), 24, and 48 h, distinguishing transcriptome samples (Figure S96A and S97A) at different time points and indicating that the samples can be used for subsequent data analysis. After 24 h of treatment (control vs T24 h), the midgut of S. frugiperda larvae presented a total of 45 DEGs, including 14 upregulated DEGs and 31 downregulated DEGs. After 48 hours of feeding (Con vs T48h), there were a total of 101 DEGs in the midgut, including 32 upregulated DEGs and 69 downregulated DEGs. There was a total of unique 87 DEGs in the transcriptome of plants fed for 48 h compared with those fed for 24 h (T24h vs T48h). Among these, 23 DEGs were upregulated, while 64 DEGs were downregulated (Figure S96B, see Table S131). Further UpSet analysis revealed that there were 5 upregulated DEGs in Con vs T24h and Con vs T48h, mainly P450 and GST, whereas 1 downregulated DEG was an eye-specific diacylglycerol kinase (Figure S96B, see Table S131). GO enrichment analysis revealed that the DEGs of Con vs T48h were enriched in the greatest number of entries, and the upregulated genes were enriched mainly in molecular functions such as metal ion binding and the physiological processes of sulfur-containing compounds, organic acids, and small molecule metabolism. The downregulated genes were enriched in several cellular components, such as ribosomes; molecular functions, such as structural activity; and physiological processes, such as the biosynthesis of organic nitrogen compounds. The upregulated and downregulated DEGs in Con vs T24h were enriched in only one and two entries, and the DEGs in T24h vs T48h were enriched in only a few entries (Figure S96C-D). After being fed the compound for 24 h (Con vs T24h), the midgut of S. litura larvae presented a total of 54 DEGs, including 43 upregulated DEGs and 9 downregulated DEGs. After 48 h of feeding (Con vs T48h), 50 DEGs were detected in the midgut, including 3 upregulated DEGs and 47 downregulated DEGs. A total of 122 DEGs were present in the transcriptome of plants fed for 48 h compared with those fed for 24 h (T24h vs T48h), including 9 upregulated DEGs and 113 downregulated DEGs (Figure S97B, see Table S132). Further UpSet analysis revealed that in the Con vs T24h and Con vs T48h comparisons, one upregulated DEG was a carnitine transporter-like gene, whereas two downregulated DEGs were trypsin, alkaline C-like, and E3 ubiquitin protein ligases (Figure S97B, see Table S132). GO enrichment analysis revealed that the DEGs of Con vs T48h were enriched in the highest number of entries, with upregulated genes enriched mainly in transport-related entries and downregulated genes enriched in molecular function entries related to oxidoreductases. The upregulated DEGs of Con vs T24h and T24h vs T48h were not enriched in the entries, whereas the downregulated DEGs were enriched in 4 entries (Figure S97C-D). There are notable differences in the midgut transcriptome profiles of S. frugiperda and S. litura, and the changes in gene expression related to detoxifying enzymes and other physiological processes are inconsistent. Therefore, stigmene C ( 10 ) may influence the adaptability of these two insects by affecting the upstream regulatory nodes of detoxifying enzymes and other physiological processes, thereby altering various downstream physiological processes. To further explore the potential targets of stigmene C ( 10 ), we found two and three facilitated trehalose transporter genes, namely SfTret1, SfTret1-like and SlTret1, SlTret1-like1, SlTret1-like2 (Table S132), respectively in DEGs of S. frugiperda and S. litura . Moreover, SfTret1 and SlTret1 are homologous genes (Figure S96). Therefore, it is possible that stigmene C ( 10 ) has certain common potential targets in inhibiting pests’ growth of the Noctuidae family in Lepidoptera. Previous research has indicated that enantiomeric β -bisabolene serves as the biosynthetic precursor for the formation of enantiomeric β -macrocarpene through the catalysis of TPS6 and TPS11. [22] Additionally, several β -macrocarpene-type sesquiterpenoids have been identified in roots and corn silk. [19, 28] However, there have been no reports addressing the enantiomeric nature of these sesquiterpenoids. In our current study, we not only isolated nine new highly oxygenated sesquiterpenoids from corn silk but also confirmed that β -macrocarpene-type sesquiterpenoids are enantiomers using NMR spectroscopy, X-ray crystallography analysis, and calculated ECD. Additionally, these polyoxygenated structures suggest the involvement of various post-modification enzymes, such as P450 enzymes. G. konell et al. confirmed that roots are the primary tissue responsible to produce β -bisabolene and β -macrocarpene, [22] indicating that post-modification enzymes may be present in the roots or/and the corn silks. Therefore, further investigation is needed to understand how corn silk to obtain these sesquiterpenoids. Furthermore, the key terpenoid synthases TPS6 and TPS11, along with their enzyme products β -bisabolene and β -macrocarpene, can be induced following leaf damage caused by S. littoralis . [22] This suggests that there may be a signaling mechanism that transmits information from herbivore-damaged leaves to the roots, potentially involving plant defense responses. [22] Our research has revealed for the first time that the active sesquiterpenoid compound 10 significantly inhibits the growth of two major lepidoptera insect herbivores. Furthermore, the enantiomers of 10 demonstrated more significant inhibition than the optically pure compounds, highlighting the importance of β -macrocarpene-type sesquiterpenoids in corn defense and the necessity of producing enantiomers. Quantitative analysis proved that the concentration of compound 10 in the root and stigma is notably higher than in other tissues, which indicates that corn silk may be vital in safeguarding corn seeds. Besides, our research suggests that exploring the biosynthetic pathway of compound 10 remains valuable for the development of insect-resistant varieties.

In summary, ten β -macrocarpene-type sesquiterpenoids ( 1 ‒ 10 ), comprising nine new compounds ( 1 ‒ 9 ) and one known analog ( 10 ) were isolated and identified from corn silk. Meanwhile, a plausible biosynthetic pathway for compounds 1 ‒ 10 was proposed. The discovery of structurally diverse sesquiterpenoids indicates the presence of post-modification enzymes such as P450 and glucosyl transferase in maize. Evaluation of their anti-insect activities revealed that stigmene C ( 10 ) significantly reduced the weights of S. frugiperda and S. litura . Furthermore, in vitro enzyme activity assays and transcriptome analysis demonstrated that stigmene C ( 10 ) inhibited two insects through different mechanisms. This research emphasizes the diversity of corn sesquiterpenoids and their effects against insects, demonstrating their significant role in corn defense and the potential for developing environmentally friendly and safe insecticides. Experimental Experimental procedure Silica gel (200–300 mesh, Qingdao Marine Chemical, Inc.). Lichroprep RP-18 (40–63 μm, Qingdao Marine Chemical, Inc.) and Sephadex LH-20 (20−150 μm, Pharmacia) were utilized for column chromatography. Methanol, chloroform, ethyl acetate, acetone and petroleum ether were acquired from Tianjing Chemical Reagents Co. (Tianjing, China). A Jasco P-1020 polarimeter (Jasco, Tokyo, Japan) was employed to measure optical rotation. CD spectra were obtained using a Chirascan instrument. Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker AV-600 MHz spectrometer (Bruker, Zurich, Switzerland), using tetramethyl chlorosilane (TMS) as an internal standard for chemical shifts. Electrospray ionization mass spectrometry (ESIMS) and high-resolution time-of-flight electrospray ionization mass spectrometry (HRTOF-ESIMS) spectra were recorded on an API QSTAR Pulsar spectrometer. Infrared (IR) spectra were obtained using a Bruker Tensor-27 instrument with KBr pellets. High-performance liquid chromatography (HPLC) separation was carried out on a Shimadzu series instrument equipped with a ZORBAX SB-C18 column (5 μm, 4.9 mm × 250 mm) and a ZORBAX SB-C8 column (5 μm, 4.9 mm × 250 mm). Plant materials Corn silk from Zea mays was collected in December 2021 from Dehong, Yunnan Province, China and a voucher (no. 20211201) was deposited in Key Laboratory of Phytochemistry and Natural Medicines, Kunming Institute of Botany, Chinese Academy of Sciences. Extraction and isolation Dry corn silk from Z. mays (10 kg) was extracted under reflux three times with 95% methanol (MeOH) to obtain the extract (600 g). The extract was suspended in water and then partitioned with ethyl acetate (EtOAc) to afford the EtOAc fraction, which was subsequently dissolved in MeOH and extracted with petroleum ether (PE) to obtain the MeOH fraction (60 g) and the PE fraction (127.6 g). The methanol fraction was separated by MeOH–H 2 O (30%→100%) on microporous absorption resin: Fr. I–V. Fraction III (70% fraction) was eluted by silica gel column chromatography (CH 2 Cl 2 -MeOH, 80: 1→0: 1) to obtain six fractions (Fr.III-1→ Fr.III-6). Each fraction was subsequently treated with Rp-18 (MeOH-H 2 O, 30%→100%), LH-20 (MeOH), and semipreparative HPLC to obtain thirty compounds. The specific separation procedure is shown in the Supplementary material. The crystallographic data (excluding structure factor tables) for the reported structures have been deposited at the Cambridge Crystallographic Data Center (CCDC) as supplementary publication no. CCDC 2342507 for 1 and CCDC 2342509 for 2, CCDC 2342496 for 3, and CCDC 2342508 for 9 . A copy of the data can be obtained free of charge from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: + 44(0)1223 336 033; e-mail: [email protected]). Stigmayic acid A (1) . colorless crystal (MeOH); [α] 21.3 D ‒12.67 ( c 0.21, MeOH); UV (MeOH); λ max (log ε): 235 (4.11), and 207 (4.00); 1 H NMR and 13 C NMR data: see Table 1; HRMS (ESI-TOF) m/z : 287.1256 [M + Na] + (calcd for C 15 H 20 O 4 Na, 287.1254). Crystal data for 1 . C 15 H 20 O 4, M = 264.31, a = 12.4919(4) Å, b = 5.7177(2) Å, c = 19.0192(6) Å, α = 90°, β = 96.1070(10)°, γ = 90°, V = 1350.74(8) Å 3, T = 150.0(2) K, space group P 121 /c 1, Z = 4, μ (Cu Kα) = 0.763 mm -1, 20934 reflections measured, 2468 independent reflections ( R int = 0.1000). The final R 1 value was 0.0612 ( I > 2 σ ( I )). The final wR ( F 2 ) value was 0.1602 ( I > 2 σ ( I )). The final R 1 value was 0.0883 (all data). The final wR ( F 2 ) value was 0.1623 (all data). The goodness of fit for F 2 was 1.225. S tigmayic acid B (2). colorless crystal (MeOH); [α] 21.2 D ‒4.67 ( c 0.12, MeOH); UV (MeOH); λ max (log ε): 239 (4.23), and 197 (3.91); 1 H NMR and 13 C NMR data: see Table 1; HRMS (ESI-TOF) m/z : 251.1644 [M + H] + (calcd for C 15 H 23 O 3, 251.1642). Crystal data for 2 . C 15 H 22 O 3, M = 250.32, a = 5.9161(6) Å, b = 6.2198(6) Å, c = 19.429(2) Å, α = 83.175(5)°, β = 83.458(5)°, γ = 75.332(4)°, V = 684.11(12) Å 3, T = 150.0(2) K, space group P -1, Z = 2, μ (Cu Kα) = 0.665 mm -1, 7963 reflections measured, 2499 independent reflections ( R int = 0.0790). The final R 1 value was 0.0607 ( I > 2 σ ( I )). The final wR ( F 2 ) value was 0.1625 ( I > 2 σ ( I )). The final R 1 value was 0.0877 (all data). The final wR ( F 2 ) value was 0.1835 (all data). The goodness of fit for F 2 was 1.070. S tigmayic acid C (3). colorless crystal (MeOH); [α] 21.3 D ‒69.42 ( c 0.27, MeOH); UV (MeOH); λ max (log ε): 226 (4.15), and 204 (4.01); 1 H NMR and 13 C NMR data: see Table 1; HRMS (ESI-TOF) m/z : 287.1257 [M + Na] + (calcd for C 15 H 20 O 4 Na, 287.1254). Crystal data for 3 . 2(C 15 H 20 O 4 )•H 2 O, M = 546.63, a = 11.4164(4) Å, b = 11.7911(4) Å, c = 11.9239(4) Å, α = 100.4030(10)°, β = 94.9030(10)°, γ = 114.0430(10)°, V = 1418.97(8) Å 3, T = 150. (2) K, space group P 1, Z = 2, μ (Cu Kα) = 0.770 mm -1, 39299 reflections measured, 10008 independent reflections ( R int = 0.0577). The final R 1 value was 0.0499 ( I > 2 σ ( I )). The final wR ( F 2 ) value was 0.1379 ( I > 2 σ ( I )). The final R 1 value was 0.0509 (all data). The final wR ( F 2 ) value was 0.1395 (all data). The goodness of fit for F 2 was 1.034. Flack parameter = 0.12(7). Stigmayic acid D (4). white powder (MeOH); [α] 21.3 D 1.64 ( c 0.17, MeOH); UV (MeOH); λ max (log ε): 196 (3.81), and 234 (4.06); 1 H NMR and 13 C NMR data: see Table 1; HRMS (ESI-TOF) m/z : 289.1418 [M + Na] + (calcd for C 15 H 22 O 4 Na, 289.1410). Stigmayic acid E (5). white powder (MeOH); [α] 21.3 D ‒24.59 ( c 0.27, MeOH); UV (MeOH); λ max (log ε): 206 (3.77), and 212 (3.75); 1 H NMR and 13 C NMR data: see Table 1; HRMS (ESI-TOF) m/z : 289.1414 [M + Na] + (calcd for C 15 H 22 O 4 Na, 289.1410). Stigmayic acid F (6). white powder (MeOH); [α] 21.3 D 11.14 ( c 0.14, MeOH); UV (MeOH); λ max (log ε): 207 (3.81), and 239 (3.95); 1 H NMR and 13 C NMR data: see Table 1; HRMS (ESI-TOF) m/z : 265.1437 [M + H] + (calcd for C 15 H 21 O 4, 265.1434). S tigmayic acid G (7). white powder (MeOH); [α] 21.3 D ‒45.27 ( c 0.50, MeOH); UV (MeOH); λ max (log ε): 204 (3.86), and 212 (3.84); 1 H NMR and 13 C NMR data: see Table 1; HRMS (ESI-TOF) m/z : 435.1989 [M + Na] + (calcd for C 21 H 32 O 8 Na, 435.1989). S tigmayic acid H (8). white powder (MeOH); [α] 21.3 D ‒1.00 ( c 0.10, MeOH); UV (MeOH); λ max (log ε):202 (3.90), 218 (3.82), and 290 (4.07); 1 H NMR and 13 C NMR data: see Table 2; HRMS (ESI-TOF) m/z : 245.1172 [M + H] + (calcd for C 15 H 17 O 3, 245.1172). Stigmayic acid I (9). colorless crystal (MeOH); [α] 21.3 D ‒6.09 ( c 0.23, MeOH); UV (MeOH); λ max (log ε):202 (4.07), 215 (3.97), and 291 (4.26); 1 H NMR and 13 C NMR data: see Table 2; HRMS (ESI-TOF) m/z : 261.1122 [M + H] + (calcd for C 15 H 17 O 4, 261.1121). Crystal data for 9 . C 15 H 16 O 4 •H 2 O, M = 278.29, a = 6.5149(6) Å, b = 6.9895(6) Å, c = 31.023(3) Å, α = 90°, β = 90°, γ = 90°, V = 1412.7(2) Å 3, T = 150.0(2) K, space group P 212121, Z = 4, μ (Cu Kα) = 0.816 mm -1, 10881 reflections measured, and 2586 independent reflections ( R int = 0.0911). The final R 1 value was 0.0495 ( I > 2 σ ( I )). The final wR ( F 2 ) value was 0.1301 ( I > 2 σ ( I )). The final R 1 value was 0.0609 (all data). The final wR ( F 2 ) value was 0.1367 (all data). The goodness of fit for F 2 was 1.062. Flack parameter = 0.02(15). Quantum chemical calculation The theoretical calculations of compounds 1 and 3 ‒ 7 was performed via Gaussian 16. [29] Conformational analysis was carried out. The optimized conformation geometries and thermodynamic parameters of all the conformations are provided. The conformers were optimized at the B3LYP/6-311G (d,p) level. The theoretical calculation of the NMR and ECD was performed via time-dependent density functional theory (TDDFT) at the B3LYP/6-311G(d,p) level in MeOH with the PCM model. The ECD spectra of compounds 1 and 3 ‒ 7 was obtained by weighing the Boltzmann distribution rate of each geometric conformation. Acid hydrolysis of compound 7 The acid hydrolysis and structural identification including configuration of compound 7 was performed according to the previous protocol. [21] Comparison with an authentic sugar revealed that the sugar obtained from compound 7 exhibited a peak for D-(+)-glucose at 20.7 min. Anti-insect activity assay An artificial diet feeding experiment was used to determine the toxicity of compounds 2-4, 6, 7, and 10 to larvae of S. frugiperda and S. litura . [30] Neonate larvae of S. frugiperda and S. litura were randomly assigned to seven groups with (20 μg/g compounds, treatment group) or without compounds (control group) supplemented in the artificial diet. Each group contained more than 30 larvae, and the weights of the larvae were determined after 7 days. The toxicity of compounds 10a, 10b, and 10 to S. litura was evaluated via the same method. Quantification analysis of compound 10 in different tissue of corn Dry tissues (leaf, stigma, tassel, and root) were ground into powder and each tissue had three replicates. Approximately 20 mg (DW) of each tissue sample was collected, followed by extraction with 1 ml solution (50% methanol containing 0.5% formic acid). The extracts were analyzed on a HPLC-MS/MS system (LCMS-8040, Shimadzu) and quantified via standard curve. Measurements were conducted on an LC-20AD liquid chromatography system (Shimadzu). At a flow rate of 0.3 mL/min, 1 μL of each sample was injected onto a ODS column (1.6 μm, 75 × 2 mm) (Shim-pack XR-ODS III). A mobile phase composed of solvent A (0.05% formic acid) and solvent B (acetonitrile) was used in a gradient mode for the separation. The ion source was ESI and eluted compound 10 were ionized by electrospray (positive mode) and detected on an LCMS-8040 mass spectrometer (Shimadzu, Japan) using selected ion monitoring (SIM). SIM parameter was optimized using standard compound 10 (249.0, [M + H] + ). The source interface voltage was kept at 4.5 kv, with the dry gas at 15 L/min and the drying temperature at 300 ⁰C. Determination of detoxification enzyme activity Determination of P450, EST, and GST enzyme activities was based on the methods of Hou et al. [31] The midguts (The main digestive organ) of 5 th instar larvae treated for 0, 24, and 48 h were used as samples. Each treatment included 5 biological replicates, each of which contained 5 midguts. A homogenization buffer (0.1 M phosphate buffer, pH 7.6, containing 1 mM EDTA, 1 mM DTT, 1 mM PTU, 1 mM PMSF, and 20% glycerol) was used to extract the samples. The samples were subsequently centrifuged for 10 minutes at 4°C and 12000 g. The resulting supernatant was used as the enzyme source. If the supernatant was not clear enough, the centrifugation process was repeated. The protein content in the enzyme source was determined via Bradford’s (Bradford, 1976) method. [32] The absorbance (OD value) was measured at a wavelength of 595 nm, and a standard curve was plotted using bovine serum albumin (BSA). The detailed experimental methods are shown in Supplementary material. Transcriptome analysis 5 th instar (binge eating stage) S. litura and S. frugiperda larvae were fed an artificial diet supplemented with compound 10, whereas the control group was fed an artificial diet supplemented with solvent. After 24 and 48 h, the midgut of each larva was dissected, frozen and crushed in liquid nitrogen for RNA-seq. Each treatment included 3 biological replicates, and every replicate contained 5 midgut larvae (Details are shown in Supplementary material). [33] Statistical analysis Each treatment was repeated three times, and the data are presented as the mean value and standard error of the mean (SEM) of three independent replicates. The statistical significance of the data in different comparisons was analyzed via SPSS software (version 23.0, SPSS Inc., Chicago, IL, USA), with Student’s t test ( p < 0.05) and one-way ANOVA. Experimental Section should be given in sufficient detail to enable others to repeat your work. In theoretical papers, some technical details such as computational methods should be confined to an appropriately named section. Supporting Information The supporting information for this article is available on the WWW under https://doi.org/10.1002/cjoc.202400xxx.

This study was financially supported by the Yunnan Revitalization Talent Support Program “Young Talent” Project (XDYC-QNRC-2022-0480), the Basic Research Program of Yunnan Province (202301AT070332), and the General and Key Project of Applied Basic Research Program of Yunnan (202201AS070053). The authors are grateful to the sample collection of Ziwei Li.

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Gene 2024, 893, 147928-147938. | Manuscript received: XXXX, 2024 Manuscript revised: XXXX, 2024 Manuscript accepted: XXXX, 2024 Version of record online: XXXX, 2024 | | Left to Right: Yu-Jia Wang, Jun-Yu He, Long Chen, Zi-Wei Li, Ming-Hua Qiu, Jin-Feng Qi, Xing-Rong Peng | Entry for the Table of Contents Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 179views 95downloads Citations Download citation Yu-Jia Wang, Jun-Yu He, Long Chen, et al. Novel β-Macrocarpene-Type Sesquiterpenoids with Anti-Insect Activity from Corn Silks. Authorea. 08 February 2025. DOI: https://doi.org/10.22541/au.173898408.86519629/v1 DOI: https://doi.org/10.22541/au.173898408.86519629/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.