Monoterpene indole alkaloids extracted from crinum asiaticum and preparation method and application thereof

Abstract

This invention belongs to the field of natural product chemistry and relates to 12 monoterpenoid indole alkaloids isolated from *Cynodon dactylon*, a plant belonging to the genus *Cynodon* in the family Apocynaceae. The crude extract of *Cynodon dactylon* roots was sequentially separated using silica gel column chromatography, HP20 column chromatography, ODS column chromatography, and HPLC to ultimately obtain 12 oxidized monoterpenoid indole alkaloids. This invention also relates to the evaluation of the antifeedant activity of these compounds against diamondback moths. Activity screening results showed that compounds 2 and 3 exhibited significant activity, with compound 2 having the lowest half-maximum inhibitory concentration (IC50), making it the most promising antifeedant in this series. Furthermore, prediction results from the ApisTox bee toxicity dataset confirmed the good safety profile of these compounds for bees. The preparation method of this invention is simple, reproducible, and yields high-purity compounds. The obtained compounds exhibit significant antifeedant activity against diamondback moths and good environmental safety, possessing the potential to be developed into novel green biological pesticides or antifeedants.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, specifically relating to 12 monoterpenoid indole alkaloids isolated from Cynodon dactylon, their preparation methods, and their application in inhibiting feeding activity against diamondback moth. Background Technology

[0002] Diamondback moth ( Plutella xylostella Diamondback moth (Linnaeus) is one of the world's most destructive invasive pests, posing a serious threat to cruciferous crops. With its exceptional migratory and dispersal abilities, it has successfully established itself in multiple climate zones and rapidly built up colonies in various regions. The global cost of controlling the diamondback moth is enormous annually, while its rapidly evolving resistance to multiple chemical pesticides, coupled with limited alternative management strategies, has led to a continuous decline in the effectiveness of chemical control. Against this backdrop, the development of environmentally friendly pesticides with novel mechanisms of action has become a pressing priority.

[0003] The importance of natural products in insecticide development has been systematically validated. Analysis of a dataset containing 800 pesticide compounds shows that natural products have a far greater impact on the insecticide field than on fungicides and herbicides. Natural products and their derivatives directly account for 31% of all insecticide compounds; if synthetic compounds containing natural product pharmacophores are included, this proportion rises to 74%, strongly indicating that natural products are one of the richest sources of insecticidal lead compounds. Indole alkaloids, such as evodiamine, have been clearly identified as important insecticidal lead compounds acting on insect reniform receptors, and the systematic elucidation of their structure-activity relationships and structural modification strategies has laid a theoretical foundation for the development of novel indole insecticides. However, most current research on natural product insecticides still focuses on the activity screening of crude extracts or the derivatization of known active compounds, while research that truly elucidates the targets and signaling pathways in insects from natural products with entirely new skeletons remains quite limited. *Cynodon dactylon* (… Tabernaemontana corymbosa *Roxb. ex Wall.*, belonging to the genus *Roxb.* in the family Apocynaceae, is widely distributed in Yunnan Province, southern Guangxi, and Southeast Asia, and is a representative source of indole alkaloids. However, the in vivo targets and molecular pathways of monoterpenoid indole alkaloids (MIAs) derived from *Roxb. ex Wall.* in insect systems have not yet been systematically characterized, constituting an important research gap. Therefore, conducting systematic target and pathway studies on novel MIAs from *Roxb. ex Wall.*, integrating chemometrics-guided methods, omics analysis, and molecular biology validation, will help fill this research gap and provide key lead compounds and theoretical foundations for the development of natural product-derived insecticides with novel mechanisms of action. Summary of the Invention

[0004] The purpose of this invention is to provide a solution from *Cymbidium goeringii* (a type of shrub). Tabernaemontana corymbosaTwelve monoterpenoid indole alkaloids were isolated from Roxb. exWall. Methods for extraction and preparation of these monoterpenoid indole alkaloids are provided, along with their applications in inhibiting feeding on diamondback moth. The structures of the monoterpenoid indole alkaloids are shown below: .

[0005] This invention includes the following preparation techniques: A method for preparing monoterpenoid indole alkaloids from Cynodon umbellatus includes the following steps: The dried leaves of *Cynodon dactylon* were extracted three times by refluxing with 70% ethanol. The extracts were combined, concentrated under reduced pressure to obtain a total crude extract, which was then extracted with n-butanol and separated by silica gel column chromatography using a gradient elution of dichloromethane:methanol from 100:1 to 1:1 to obtain Fr.A-Fr.D. Fr.C was separated by HP20 and ODS using a gradient elution of 10%-90% methanol-water to obtain Fr.C1-Fr.C6. After identifying Fr.C5 by bismuth potassium iodide color development, further analysis was performed using the retention time information provided by RP-HPLC-MS. The results showed that it contained a large number of oxidized monoterpenoid indole alkaloids (MIAs) within the retention time window of 15-34 min. Therefore, Fr.C5 was subjected to silica gel column chromatography with a gradient elution of dichloromethane:methanol system at a volume ratio of 100:0 to 3:1, followed by RP-HPLC with an acetonitrile-water mobile phase at a volume ratio of 40:60-10:90 to obtain compounds 1-9 and 1S-3S.

[0006] The extraction and preparation method described in this invention uses *Cymbidium ensifolium*, a plant belonging to the genus *Cymbidium* of the family Apocynaceae (family Apocynaceae). Tabernaemontana corymbosa Roxb. ex Wall.).

[0007] Preferably, in the preparation method, dried leaves of *Cynodon dactylon* are extracted three times by reflux with 70% industrial ethanol, each time for 3 hours.

[0008] The structural identification results of the compounds obtained in this invention are as follows: The structures of compounds 1-9 and 1S-3S were identified using high-resolution mass spectrometry, one-dimensional NMR, two-dimensional NMR, calculated NMR, calculated ECD, and X-ray single-crystal diffraction. The corresponding spectra are shown below. Figures 1 to 37 As shown, the corresponding main structural characterization data are shown in Tables 1 and 2.

[0009] Compound 1: Pale yellow blocky crystals; [ α ]20 D -30.00 ( c0.1, CH3OH); HRESIMS gives a quasi-molecular ion peak at 357.21725 [M + H] + (calcd for C 21 H 29 O3N2 (357.21727), the molecular weight of this compound was determined to be 356, and the molecular formula is C. 21 H 28 O3N2; UV (CH3OH) λ max 206, 282 nm; By analyzing its 1 H NMR, 13 CNMR, HSQC spectrum, HMBC spectrum 1 H 1 The structure of compound 1 was determined by HCl COSY spectroscopy, NOESY spectroscopy, quantum chemical calculations, and X-ray single-crystal diffraction, identifying it as a novel skeletal compound.

[0010] Compound 1S: The structure of compound 1S was determined by quantum chemical calculations through comparison with the NMR data of compound 1, and it is the C7 epimer of compound 1.

[0011] Compound 2: Pale yellow blocky crystals; α ]20 D -27.50 ( c 0.1, CH3OH); HRESIMS gives a quasi-molecular ion peak at 355.20129 [M + H] + (calcd for C 21 H 27 O3N2 (355.20162), the molecular weight of this compound was determined to be 354, and the molecular formula is C. 21 H 26 O3N2;UV (CH3OH)λ max 207, 252 nm; By analyzing its 1 H NMR, 13 C NMR, HSQC spectrum, HMBC spectrum 1 H 1 The structure of compound 2 was determined by HCl COSY spectroscopy, NOESY spectroscopy, quantum chemical calculations, and X-ray single-crystal diffraction, identifying it as a novel skeletal compound.

[0012] Compound 2S: The structure of compound 2S was determined by quantum chemical calculations through comparison with the NMR data of compound 2, and it is the C7 epimer of compound 2.

[0013] Compound 3: White amorphous powder; α ]20 D -22.50 ( c0.1, CH3OH); HRESIMS gives a quasi-molecular ion peak at 341.18634 [M + H] + (calcd for C 20 H 25 O3N2 (341.18597), the molecular weight of this compound was determined to be 340, and the molecular formula is C. 20 H 24 O3N2;UV (CH3OH)λ max 206, 282 nm; By analyzing its 1 H NMR, 13 C NMR, HSQC spectrum, HMBC spectrum 1 H 1 The structure of compound 3 was determined by HCl COSY spectroscopy, NOESY spectroscopy, and quantum chemical calculations, identifying it as a novel skeletal compound.

[0014] Compound 3S: The structure of compound 3S was determined by quantum chemical calculations through comparison with the NMR data of compound 3, and it is the C7 epimer of compound 3.

[0015] Compound 4: pale yellow powder; α ]20 D -35.00 ( c 0.1, CH3OH); HRESIMS gives a quasi-molecular ion peak at 371.19720 [M + H] + (calcd for C 21 H 27 O4N2 (371.19653), the molecular weight of this compound was determined to be 370, and the molecular formula is C. 21 H 26 O4N2;UV (CH3OH)λ max 209, 251, 281 nm; By analyzing its 1 H NMR, 13 CNMR, HSQC spectrum, HMBC spectrum 1 H 1 The structure of compound 4 was determined by HCl COSY spectroscopy, NOESY spectroscopy, and quantum chemical calculations, identifying it as a novel skeletal compound.

[0016] Compound 5: a yellow oily substance; α ]20 D +7.50 ( c 0.1, CH3OH); HRESIMS gives a quasi-molecular ion peak at 415.22321 [M + H] + (calcd for C 23 H 31O5N2 (415.22275), the molecular weight of this compound was determined to be 414, and the molecular formula is C. 23 H 30 O5N2;UV (CH3OH)λ max 207, 281 nm; By analyzing its 1 H NMR, 13 C NMR, HSQC spectrum, HMBC spectrum 1 H 1 The structure of compound 5 was determined by HCl COSY spectroscopy, NOESY spectroscopy, and quantum chemical calculations, identifying it as a novel skeletal compound.

[0017] Compound 6: Yellow oily substance; α ]20 D -77.50 ( c 0.1, CH3OH); HRESIMS gives a quasi-molecular ion peak at 369.17981 [M + H] + (calcd for C 21 H 25 O4N2 (369.18088), the molecular weight of this compound was determined to be 368, and the molecular formula is C. 21 H 24 O4N2;UV (CH3OH)λ max 205, 282 nm; By analyzing its 1 H NMR, 13 C NMR, HSQC spectrum, HMBC spectrum 1 H 1 The structure of compound 6 was determined by HCl COSY spectroscopy, NOESY spectroscopy, and quantum chemical calculations, identifying it as a novel skeletal compound.

[0018] Compound 7: a pale yellow oily substance; α ]20 D -31.50 ( c 0.1, CH3OH); HRESIMS gives a quasi-molecular ion peak at 429.23837 [M + H] + (calcd for C 24 H 33 O5N2 (429.23840), the molecular weight of this compound was determined to be 428, and the molecular formula is C. 24 H 32 O5N2; UV (CH3OH) λ max 208, 252, 282 nm; By analyzing its 1 H NMR, 13 CNMR, HSQC spectrum, HMBC spectrum 1H 1 The structure of compound 7 was determined by HCl COSY spectroscopy, NOESY spectroscopy, and quantum chemical calculations, identifying it as a novel skeletal compound.

[0019] Compound 8: a yellow oily substance; α ]20 D +17.50 ( c 0.1, CH3OH); HRESIMS gives a quasi-molecular ion peak at 401.20541 [M + H] + (calcd for C 22 H 29 O5N2 (401.20710), the molecular weight of this compound was determined to be 400, and the molecular formula is C. 22 H 28 O5N2;UV (CH3OH)λ max 206, 282 nm; By analyzing its 1 H NMR, 13 C NMR, HSQC spectrum, HMBC spectrum 1 H 1 The structure of compound 8 was determined by HCl COSY spectroscopy, NOESY spectroscopy, and quantum chemical calculations, identifying it as a novel skeletal compound.

[0020] Compound 9: a colorless oily substance; α ]20 D +10.00 ( c 0.1, CH3OH); HRESIMS gives a quasi-molecular ion peak at 357.21750 [M + H] + (calcd for C 21 H 29 O3N2 (357.21727), the molecular weight of this compound was determined to be 356, and the molecular formula is C. 21 H 28 O3N2;UV (CH3OH)λ max 206, 282 nm; By analyzing its 1 H NMR, 13 C NMR, HSQC spectrum, HMBC spectrum 1 H 1 The structure of compound 9 was determined by HCl COSY spectroscopy, NOESY spectroscopy, and quantum chemical calculations, identifying it as a novel skeletal compound.

[0021] Table 1. Compounds 1-5 1 H (600 MHz) and 13 C NMR (150 MHz) data Compounds 6-9 in Table 2 1 H (600 MHz) and 13 C NMR (150 MHz) data This invention provides the application of oxidized monoterpenoid indole alkaloids or their pharmaceutically acceptable salts from *Cynodon dactylon* in the preparation of antifeedant for diamondback moth, and the application of compositions of such compounds with pesticide-acceptable carriers or adjuvants in the control of diamondback moth.

[0022] Activity screening results showed that compounds 2 and 3 exhibited significant antifeedant activity, as indicated by AFC. 50 The AFC of compound 2 was determined. 50 It has the lowest value and is the most promising antifeeding active compound in this series.

[0023] The beneficial effects of this invention are as follows: The oxidized monoterpenoid indole alkaloids described herein have well-defined structures, with compounds 1-9 and 1S-3S being novel skeletal compounds. These compounds are easy to isolate and purify, exhibit significant antifeedant activity against diamondback moth, and demonstrate good environmental safety, showing potential for development into novel green biological pesticides or antifeedants. Furthermore, the preparation method described in this invention is simple, low-cost, and easily scalable for industrial production. Attached Figure Description

[0024] Figure 1 HSQC spectrum of compound 1.

[0025] Figure 2 HMBC spectrum of compound 1.

[0026] Figure 3 Compound 1 1 H- 1 H COSY spectrum.

[0027] Figure 4 NOESY spectrum of compound 1.

[0028] Figure 5 HSQC spectrum of compound 2.

[0029] Figure 6 HMBC spectrum of compound 2.

[0030] Figure 7 Compound 2 1 H- 1 H COSY spectrum.

[0031] Figure 8NOESY spectrum of compound 2.

[0032] Figure 9 HSQC spectrum of compound 3.

[0033] Figure 10 HMBC spectrum of compound 3.

[0034] Figure 11 Compound 3 1 H- 1 H COSY spectrum.

[0035] Figure 12 NOESY spectrum of compound 3.

[0036] Figure 13 HSQC spectrum of compound 4.

[0037] Figure 14 HMBC spectrum of compound 4.

[0038] Figure 15 Compound 4 1 H- 1 H COSY spectrum.

[0039] Figure 16 NOESY spectrum of compound 4.

[0040] Figure 17 HSQC spectrum of compound 5.

[0041] Figure 18 HMBC spectrum of compound 5.

[0042] Figure 19 Compound 5 1 H- 1 H COSY spectrum.

[0043] Figure 20 NOESY spectrum of compound 5.

[0044] Figure 21 HSQC spectrum of compound 6.

[0045] Figure 22 HMBC spectrum of compound 6.

[0046] Figure 23 Compound 6 1 H- 1 H COSY spectrum.

[0047] Figure 24 NOESY spectrum of compound 236.

[0048] Figure 25HSQC spectrum of compound 7.

[0049] Figure 26 HMBC spectrum of compound 7.

[0050] Figure 27 Compound 7 1 H- 1 H COSY spectrum.

[0051] Figure 28 NOESY spectrum of compound 7.

[0052] Figure 29 HSQC spectrum of compound 8.

[0053] Figure 30 HMBC spectrum of compound 8.

[0054] Figure 31 Compound 8 1 H- 1 H COSY spectrum.

[0055] Figure 32 NOESY spectrum of compound 8.

[0056] Figure 33 HSQC spectrum of compound 9.

[0057] Figure 34 HMBC spectrum of compound 9.

[0058] Figure 35 Compound 9 1 H- 1 H COSY spectrum.

[0059] Figure 36 NOESY spectrum of compound 9.

[0060] Figure 37 (A) Heatmap of selective and non-selective feeding rejection rates for compounds 1-9; (B) Prediction results from the ApisTox bee toxicity dataset; (C) AFC of compounds 2 and 3. 50 . Detailed Implementation

[0061] Example 1 Umbrella-shaped dogtooth flower ( Tabernaemontana corymbosaThe dried leaves of *Roxb. ex Wall* were extracted three times by reflux with 70% ethanol. The extracts were combined and concentrated under reduced pressure to obtain a total crude extract, which was then extracted with n-butanol and separated by silica gel column chromatography using a gradient elution of dichloromethane:methanol from 100:1 to 1:1 to obtain Fr.A-Fr.D. Fr.C was separated by HP20 and ODS using a gradient elution of 10%-90% methanol-water to obtain Fr.C1-Fr.C6. After identifying Fr.C5 by bismuth potassium iodide color development, further analysis was performed using the retention time information provided by RP-HPLC-MS. The results showed that it contained a large number of oxidized monoterpenoid indole alkaloids (MIAs) within the retention time window of 15-34 min. Therefore, Fr.C5 was subjected to silica gel column chromatography with a gradient elution of dichloromethane:methanol system at a volume ratio of 100:0 to 3:1, followed by RP-HPLC with an acetonitrile-water mobile phase at a volume ratio of 40:60-10:90 to obtain compounds 1-9 and 1S-3S.

[0062] In the method described above, the *Cymbidium goeringii* species used is *Cymbidium goeringii*, a plant belonging to the genus *Cymbidium* in the family Apocynaceae. Tabernaemontana corymbosa Roxb. ex Wall).

[0063] In the method described above, *Cymbidium goeringii* (a type of shrub) is taken. Tabernaemontana corymbosa The dried leaves of Roxb. ex Wall were extracted three times by reflux with 70% industrial ethanol for 3 hours each time.

[0064] Example 2: Investigation of the antifeedant activity of compounds 1-9 Non-selective feeding rejection activity assay: Using a 1.5 L Wahaha water bottle cap as a punch, fresh cabbage leaves were punched into leaf dishes. The leaf dishes were immersed in 200 μg / mL of the test solution and the control solution for 15 s, respectively. After drying, four treated leaf dishes were placed in each dish, and five third-instar larvae of the diamondback moth, starved for 4 h, were introduced. Each group was repeated three times. The dishes were incubated at 25℃ and 65% humidity, and the feeding area was measured after 48 h.

[0065] Selective antifeeding activity assay: Leaf discs of equal area (200 μg / mL) and control leaf discs were alternately and symmetrically placed in the same petri dish. Five test insects starved for 4 h were inoculated into each dish. Each group was repeated 3 times under the same culture conditions. After 48 h, the feeding area of ​​the treatment group and the control group was measured respectively, and the antifeeding activity of each compound was evaluated by the antifeeding rate.

[0066] Initial screening revealed that, for example Figure 37 As shown, compounds 2 and 3 exhibited significant activity; subsequent predictions based on the ApisTox bee toxicity dataset confirmed their safety. For quantitative comparison, the half-maximal rejection concentration (AFC) was determined.50 ), of which compound 2 has AFC 50 The lowest value indicates that it is the most active antifeedant in this series.

[0067] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A monoterpenoid indole alkaloid compound from *Cynodon dactylon*, characterized in that, It can be any of the following structures: 。 2. The monoterpenoid indole alkaloid compound in *Cynodon dactylon* according to claim 1, characterized in that, The monoterpenoid indole alkaloid compound is derived from *Cynodon dactylon*, a plant belonging to the genus *Cynodon* in the family Apocynaceae. Tabernaemontana corymbosa It was isolated from Roxb. ex Wall.

3. A method for preparing a monoterpenoid indole alkaloid compound from *Cynodon dactylon* as described in claim 1 or claim 2, characterized in that, Includes the following steps: Umbrella-shaped dogtooth flower ( Tabernaemontana corymbosa The dried leaves of *Roxb. ex Wall* were extracted three times by refluxing with 70% ethanol. The extracts were combined and concentrated under reduced pressure to obtain the total crude extract, which was then extracted with n-butanol. Separation was performed by silica gel column chromatography with a gradient elution of dichloromethane:methanol from 100:1 to 1:1 to obtain Fr.A-Fr.D. Fr.C was then separated by HP20 and ODS, followed by a gradient elution of 10%-90% methanol-water to obtain Fr.C1-Fr.C6. Fr.C5 was identified by bismuth potassium iodide staining, and further analysis was performed using retention time information provided by RP-HPLC-MS. The results showed that it contained a large amount of oxidized monoterpenoid indole alkaloids within a retention time window of 15-34 min. Therefore, Fr.C5 was subjected to silica gel column chromatography with gradient elution of dichloromethane:methanol system at a volume ratio of 100:0 to 3:1, followed by RP-HPLC with acetonitrile-water mobile phase at a volume ratio of 40:60-10:90 to obtain compounds 1-9 and 1S-3S.

4. The method for preparing monoterpenoid indole alkaloids from *Cynodon dactylon* according to claim 3, characterized in that, The *Cymbidium goeringii* species used is *Cymbidium goeringii*, a plant belonging to the genus *Cymbidium* in the family Apocynaceae. Tabernaemontana corymbosa Roxb. ex Wall.).

5. The method for preparing monoterpenoid indole alkaloids from *Cynodon dactylon* according to claim 3, characterized in that, The dried leaves of Cynodon dactylon were extracted three times by reflux with 70% industrial ethanol, each time for 3 hours.

6. A food repellent composition, characterized in that, The composition comprises a monoterpenoid indole alkaloid compound or a pesticide-acceptable salt thereof from *Cynodon dactylon* as described in claim 1 or claim 2, and a pesticide-acceptable carrier or adjuvant.

7. The use of monoterpenoid indole alkaloids or their pesticide-acceptable salts from *Cynodon dactylon* as described in claim 1 or claim 2 in the preparation of a diamondback moth antifeedant.

8. The use of the antifeedant composition according to claim 6 in the preparation of an antifeedant for diamondback moth.

9. The use of the monoterpenoid indole alkaloid compound or its phytochemically acceptable salt from *Cynodon dactylon* as described in claim 1 or claim 2, or the antifeedant composition as described in claim 6, in the control of diamondback moth damage.

Images