A terpenoid compound and a preparation method thereof

Abstract

The application discloses a terpenoid compound and a preparation method thereof, and belongs to the technical field of biological medicines. A. costaricensis Scale fermentation is carried out, rich secondary metabolites are obtained, and two new terpenoid compounds are obtained through extraction, separation, purification and identification. Compound 1 and compound 2 are both isolated from aspergillus for the first time, so that it is known that under the condition of the culture medium containing halogen salt, the strain can produce secondary metabolites with rich and diverse structures. The application not only enriches the secondary metabolites of aspergillus, expands the structural diversity of aspericin terpenoid compounds, but also preliminarily reveals the anti-tumor activity potential of part of the compounds, so as to lay a material foundation and experimental basis for subsequent in-depth development of activity evaluation, mechanism and structure-activity relationship research.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to terpenoid compounds derived from secondary metabolites of endophytic fungi of Rauvolfia pulveratum and their preparation methods. Background Technology

[0002] Emetic Rauvolfia ( Rauvolfia vomitoria *Rauvolfia* (also known as Rauvolfia var. aurantiacus) belongs to the genus *Rauvolfia* in the family Apocynaceae. Widely distributed in tropical and subtropical regions, it is an important medicinal plant in traditional Chinese medicine used to treat hypertension, mental stress, and infectious diseases. *Rauvolfia* plants are an important natural source of monoterpenoid indole alkaloids; over 290 compounds of this class have been isolated, exhibiting various pharmacological activities including anti-Alzheimer's disease, antioxidant, anti-inflammatory, antibacterial, and vasodilatory effects. In recent years, new monoterpenoid alkaloid structures have been continuously discovered from *Rauvolfia var. aurantiacus*, and some compounds, when used in combination with cisplatin, have shown significant synergistic inhibitory effects on the proliferation of human ovarian cancer cells. Therefore, *Rauvolfia var. aurantiacus* remains a valuable plant resource for exploring bioactive natural products.

[0003] Plant endophytic fungi can produce structurally diverse and highly active secondary metabolites, making them a hot topic in natural product chemistry research. *Aspergillus* ( Aspergillus Fungi are a widely distributed group of ascomycetes, with over 260 known species. They have attracted considerable attention due to their abundant secondary metabolites and are an important source for discovering novel bioactive molecules. Studies have shown that Aspergillus fungi can produce compounds of various structural types, including polyketides, terpenes, alkaloids, and butenolides, many of which possess antitumor, antibacterial, anti-inflammatory, antiviral, and agricultural bactericidal activities. However, despite significant progress in the production of industrial enzyme preparations (such as Aspergillus niger and Aspergillus oryzae) and the study of secondary metabolites, the depth of chemical research varies considerably among different species. A. costaricensis Belonging to the Aspergillus niger group, it has attracted attention in recent years in the biotechnology field due to its strong carbohydrate-active enzyme secretion capacity, exhibiting high xylanase and pectinase activity, and showing significant application potential in plant biomass degradation and agricultural waste resource utilization. However, currently, regarding... A. Costa Rican Research on the chemical composition of the secondary metabolites remains very limited. Only a few studies have reported the isolation of a handful of compounds, such as butenolactone and naphthopyranone, from its fermentation products, and related activity studies have mainly focused on anti-inflammatory and anti-elastase activities. The chemical basis for other pharmacological activities of this strain, such as antitumor and antibacterial activity, is still unclear, and systematic and in-depth chemical composition research is urgently needed.

[0004] In the study of microbial secondary metabolites, many microbial biosynthetic gene clusters are silenced or underexpressed under conventional culture conditions, resulting in high repetition rates of known compounds and reduced efficiency in discovering new compounds. To overcome this bottleneck, the OSMAC (One Strain Many Compounds) strategy has been widely used to regulate microbial metabolic pathways. This strategy can effectively activate silent gene clusters and significantly increase the chemical diversity of secondary metabolites produced by a single strain by altering the culture medium composition (e.g., species, halide addition), culture conditions (e.g., solid / liquid state, pH, temperature), or by using co-culture. However, the systematic application of the OSMAC strategy is currently lacking. A. Costa Rican Research reports on the mining of secondary metabolites indicate that the metabolic potential of this strain under different culture conditions and the chemical nature of its active components still need to be revealed.

[0005] In summary, existing technologies regarding endophytic fungi derived from Rauvolfia oleracea are limited. A. costaricensis Studies on its chemical composition are not yet systematic, especially lacking a systematic review of its metabolite diversity and antitumor activity based on the OSMAC strategy. Therefore, it is necessary to provide a method for studying the endophytic fungi of *Rauvolfia pulveratula*. A. costaricensis The method for preparing secondary metabolites was developed to enrich the chemical composition library of this strain and provide a material basis and experimental evidence for the development of novel antitumor lead compounds. Summary of the Invention

[0006] The technical problem to be solved by this invention is the treatment of plant endophytic fungi. A. costaricensis The compounds prepared from the secondary metabolites of the terpenoid were used for preliminary screening of their in vitro antitumor activity, providing a terpenoid compound and its preparation method.

[0007] To address the aforementioned technical problems, the objective of this invention is to provide a compound with the following structural formula: Formula 1 or Formula 2.

[0008] Specifically, it is prepared according to the following steps: including the following steps: Step 1, Preparation Stage: Take healthy, disease-free Rauvolfia pulveratum stem segments, remove surface soil and impurities, and in a clean bench, soak the stem segments sequentially in 75% ethanol for 30 seconds to 1 minute, then soak them in 2.5% sodium hypochlorite solution for 2 to 5 minutes, followed by rinsing with sterile water 3 to 4 times. Blot dry the surface moisture, and spread the sterile water from the final rinse onto a PDA plate. Incubate at 28°C for 3 days. Then, cut the surface-sterilized stem segments into small pieces of approximately 0.5 cm, place them on PDA plates, and incubate at 28°C for 3 to 7 days. Once fungal hyphae have grown along the edges of the tissue blocks, pick the newly formed hyphae from the edges and transfer them to fresh PDA plates. After multiple purifications, obtain single colonies. A. costaricensis ; Step 2 Aspergillus costaricensis Endophytic fungal fermentation: The activated fungi are fermented in a clean bench... A. Costa Rican Inoculate into PDAs and incubate for 3-5 days. After sterilizing the rice culture medium in an autoclave, [the following steps are taken]. A. costaricensis The strain was inoculated into rice culture medium and cultured at 28°C and 60% RH for 30-40 days. Step 3 Aspergillus costaricensis Extraction and extraction of fermentation products: The fermented rice culture medium was extracted three times with methanol at room temperature for 7 days each time. The extracts were combined, filtered, and concentrated under reduced pressure to obtain crude extract. The crude extract was suspended in warm water and then fractionally extracted with petroleum ether, ethyl acetate, and n-butanol. After standing, the layers were separated, and each extract phase was filtered and concentrated under reduced pressure to obtain extracts of the corresponding polar fractions, which were then weighed. Step 4 Aspergillus costaricensis Isolation and purification of fermentation products: A. costaricensisThe ethyl acetate fraction was separated using a normal-phase, atmospheric-pressure column chromatography with silica gel stirred in a 100-200 mesh column. Gradient elution was performed using petroleum ether-ethyl acetate (8:1–1:5 v / v) and dichloromethane-methanol (20:1–6:1 v / v). The resulting fractions were combined using thin-layer chromatography to obtain nine fractions, Fr.1–Fr.9. Fraction Fr.9 was further separated using reversed-phase silica gel column chromatography with gradient elution using methanol-water (20%–90% v / v) to obtain nine fractions, Fr.9-1–Fr.9. -9; Fr.9-4 was further separated by normal-phase silica gel column chromatography with a mesh size of 200-300, using dichloromethane-methanol at a volume ratio of 100:1→15:1 to elute, yielding five fractions Fr.9-4-1 to Fr.9-4-5. Fr.9-4-4 was further purified by normal-phase silica gel column chromatography and LH-20 gel column chromatography to obtain fraction Fr.9-4-4-1. Fr.9-4-4-1 was further purified by semi-preparative HPLC with a mobile phase of MeOH-H2O, 50:50, 2 mL / min, v / v, yielding compound 1; Furthermore, step 4 can also be: Aspergillus costaricensis Isolation and purification of fermentation products: A. costaricensis The ethyl acetate fraction was separated by mixing the sample with silica gel in a 100-200 mesh column and separating it on a normal-phase atmospheric pressure column. The sample was eluted with a gradient of petroleum ether-ethyl acetate (8:1 to 1:5 v / v) and dichloromethane-methanol (20:1 to 6:1 v / v). The obtained fractions were combined by thin-layer chromatography to obtain 9 fractions Fr.1 to Fr.9. Fr.7 was further separated by reversed-phase silica gel column chromatography, eluting with a methanol-water gradient of 20% to 90% (v / v) to obtain eight fractions Fr.7-1 to Fr.7-8. Fr.7-5 was then further separated by normal-phase silica gel column chromatography (200-300 mesh), eluting with dichloromethane-methanol at a v / v ratio of 100:1 to 30:1 to obtain five fractions Fr.7-5-1 to Fr.7-5-5. Fr.7-5-3 was separated by normal-phase silica gel column chromatography and further purified by LH-20 gel column chromatography to obtain fraction Fr.7-5-3-1. Fr.7-5-3-1 was further purified by semi-preparative HPLC under the following mobile phase conditions: MeCN-H2O, 25:75, 2 mL / min, v / v, to obtain compound 2.

[0009] Furthermore, the rice culture medium in step 2 includes: 200 g rice, 1 g peptone, 1 g yeast extract, 1 g monosodium glutamate, 4% sea salt, and 250 mL water, and is sterilized in an autoclave at 121°C for 20 min.

[0010] This invention has the following beneficial effects: This invention targets the endophytic fungi of the genus Aspergillus in Rauvolfia var. mongolica that induce vomiting. A. Costa Rican Large-scale fermentation yielded abundant secondary metabolites, which were then extracted, purified, and identified as two novel terpenoid compounds. Both terpenoid compound 1 and compound 2 were isolated from Aspergillus species for the first time, demonstrating that the strain can produce structurally rich and diverse secondary metabolites under halide-containing culture conditions.

[0011] From a chemotactic perspective, compounds 1 and 2 isolated in this invention are rare aspericin-type terpenoid derivatives found in Aspergillus fungi. Compound 2, in particular, exhibits structural features including deoxymethylation at C-15 (replacing a hydroxymethyl group with a methyl group linked to a double bond) and hydroxylation at C-12 (replacing a methyl group with a hydroxymethyl group). Such novel modifications are rarely reported in Aspergillus terpenoid secondary metabolites. These findings further expand the structural diversity of Aspergillus terpenoid compounds.

[0012] This invention not only enriches the secondary metabolites of Aspergillus fungi and expands the structural diversity of aspericin terpenoids, but also preliminarily reveals the antitumor activity potential of some compounds, laying a material basis and experimental evidence for further in-depth research on activity evaluation, mechanism of action and structure-activity relationship. Attached Figure Description

[0013] Figure 1 The proton NMR spectrum (CD3OD) of compound 1 , 600MHz); Figure 2 The carbon NMR spectrum (CD3OD) of compound 1 , 150MHz); Figure 3 The DEPT spectrum (CD3OD) of compound 1 is shown. Figure 4 For compound 1 1 H- 1 H COSY spectrum (CD3OD); Figure 5 The HSQC spectrum (CD3OD) of compound 1 is shown. Figure 6 The HMBC spectrum (CD3OD) of compound 1 is shown. Figure 7 The NOSEY spectrum (CD3OD) of compound 1 is shown. Figure 8 The infrared spectrum of compound 1 is shown below. Figure 9 This is the high-resolution mass spectrum of compound 1; Figure 10 For compound 1 1 H- 1 Correlation plot of HCOSY (thick blue line), HMBC (red arrow), and NOESY (dashed red line); Figure 11 The 1H NMR spectrum of compound 2 (CD3OD, 600MHz); Figure 12 The carbon NMR spectrum of compound 2 (CD3OD, 150MHz); Figure 13 The DEPT spectrum (CD3OD) of compound 2 is shown. Figure 14 For compound 2 1 H- 1 H COSY spectrum (CD3OD); Figure 15 The HSQC spectrum (CD3OD) of compound 2 is shown. Figure 16 The HMBC spectrum (CD3OD) of compound 2 is shown. Figure 17 The NOSEY spectrum (CD3OD) of compound 2 is shown. Figure 18 The infrared spectrum of compound 2 is shown below. Figure 19 This is the high-resolution mass spectrum of compound 2; Figure 20 For compound 2 1 H- 1 Correlation plot of HCOSY (thick blue line), HMBC (red arrow), and NOESY (dashed red line); Figure 21 For compounds 1 and 2 at 40 μ Figure 1. In vitro cell proliferation inhibition activity and survival rate of A549 and MCF-7 cells at M concentration. Detailed Implementation

[0014] The present invention will be described in detail below with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but should not be considered as limiting the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0015] Example 1: The structures of the compounds in this example are shown in Formulas 1 and 2: Formula 1 or Formula 2.

[0016] Preparation of compounds 1 and 2 1.1 Experimental Instruments and Materials 1.1.1 Experimental Materials This experiment selected five endophytic fungi, *Basilella* (…). Talaromyces purpureogenus ), Penicillium ( Penicillium kongii Aspergillus ( ) A. costaricensis Alternaria ( ) Alternaria burnsii ) and genus *Nyctaginus* ( Periconia pseudobyssoides The aforementioned endophytic fungi were obtained from the laboratory.

[0017] 1.1.2 Experimental Apparatus Table 1 Experimental Apparatus

[0018] 1.1.3 Experimental reagents and consumables Table 2 Experimental Reagents and Consumables

[0019] 1.2 Experimental Methods 1.2.1 Fermentation of 5 endophytic fungi based on the OSMAC strategy In the early stages of the laboratory, Rauvolfia oleracea (… Rauvolfia vomitoria Twenty endophytic fungi were obtained from the stems after surface disinfection and aseptic isolation. Healthy, disease-free Rauvolfia pulveratula stem segments were taken and rinsed with running water to remove surface dirt and impurities. In a clean bench, the stem segments were successively immersed in 75% ethanol for 30 seconds to 1 minute, then in 2.5% sodium hypochlorite solution for 2 to 5 minutes, and then rinsed 3 to 4 times with sterile water to thoroughly remove residual disinfectant. Finally, the surface moisture was blotted dry with sterile filter paper. The sterile water from the last rinse was spread on PDA plates and incubated at 28 °C for 3 days to verify the thoroughness of surface disinfection. The surface-disinfected stem segments were cut into small pieces of about 0.5 cm and placed on PDA plates. They were incubated at 28 °C for 3 to 7 days. After fungal hyphae grew from the edges of the tissue blocks, the newly grown hyphae were picked and transferred to fresh PDA plates. After multiple purifications, single colonies were obtained.

[0020] Based on colony morphology, color, growth rate, and spore structure, 20 endophytic fungi were initially classified and obtained. Five strains (*Basilella*, *Penicillium*, *Aspergillus*, *Alternaria*, and *Nyctalococcus*) were selected for subsequent OSMAC culture experiments. The strains were first activated on PDA plates for 3–5 days, and then aseptically inoculated into autoclaved rice culture medium (formulation shown in Table 3) in a clean bench. The five endophytic fungi were cultured for 7 days at 28°C and 60% humidity under two different fermentation conditions to investigate the effect of different culture media on the production of secondary metabolites.

[0021] Table 3 Composition of the culture medium

[0022] 1.2.2 Extraction of secondary metabolites from 5 endophytic fungi After fermentation, the rice culture medium was extracted twice at room temperature with 85% methanol for 7 days each time. The extracts were combined and concentrated under reduced pressure to obtain a crude extract. Further extraction with ethyl acetate (repeated three times) was performed, and the organic phase was collected and concentrated under reduced pressure to obtain an ethyl acetate extract.

[0023] 2.1 Experimental Instruments and Materials 2.1.1 Instruments Table 4 Experimental Instruments

[0024] 2.1.2 Reagents Table 5 Experimental Reagents and Consumables

[0025] 2.2 Experimental Methods 2.2.1 Aspergillus costaricensis Endophytic fungal fermentation In the clean bench, the already activated A. costaricensis Inoculate into PDAs and incubate for 3-5 days. After sterilizing the rice culture medium in an autoclave, ... A. costaricensis The strain was inoculated into rice culture medium with the second formulation and cultured at 28°C and 60% RH for 30-40 days.

[0026] 2.2.2 Aspergillus costaricensis Extraction and extraction of fermentation products The fermented rice culture medium was extracted three times with methanol at room temperature, each time for 7 days. The extracts were combined, filtered, and concentrated under reduced pressure to obtain a crude extract. The crude extract was suspended in warm water and then fractionally extracted with petroleum ether, ethyl acetate, and n-butanol. After standing, the extracts were separated into layers, filtered, and concentrated under reduced pressure to obtain extracts of the corresponding polar fractions, which were then weighed.

[0027] 2.2.3 Aspergillus costaricensis Isolation and purification of fermentation products right A. costaricensisThe ethyl acetate fraction was separated using a normal-phase, atmospheric-pressure column chromatography with 100-200 mesh silica gel mixed with the sample. Gradient elutions were performed using petroleum ether-ethyl acetate (8:1, 6:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:5, v / v) and dichloromethane-methanol (20:1, 15:1, 10:1, 6:1, v / v) as eluents. The resulting fractions were spotted onto a thin-layer silica gel plate and observed under 254 nm UV light. The colorimetric results, combined with 10% sulfuric acid-ethanol staining, led to the combination of identical fractions, yielding nine fractions (Fr.1–Fr.9). Fraction Fr.9 was further separated using reversed-phase silica gel column chromatography (ODS) with a methanol-water gradient elution (20%, 35%, 50%, 75%, 80%, 90%), yielding nine fractions (Fr.9-1–Fr.9-9).

[0028] Fraction Fr.9-4 was further separated by normal-phase silica gel column chromatography with a mesh size of 200-300, using dichloromethane-methanol (100:1, 50:1, 30:1, 20:1, 15:1) as the eluent to obtain five fractions (Fr.9-4-1 to Fr.9-4-5). Fr.9-4-4 was further purified by normal-phase silica gel column chromatography and LH-20 gel column chromatography (MeOH) to obtain fraction (Fr.9-4-4-1). Fr.9-4-4-1 was further purified by semi-preparative HPLC with a mobile phase of (MeOH-H2O, 50:50, 2 mL / min, v / v) to obtain compound 1 (2.03 mg, t R =21.5 min).

[0029] Fr.7 was further separated by reversed-phase silica gel column chromatography (ODS) with a methanol-water gradient (20%, 35%, 50%, 75%, 80%, 90%) to obtain 8 fractions (Fr.7-1 to Fr.7-8). Fr.7-5 was further separated by normal-phase silica gel column chromatography (200-300 mesh) with dichloromethane-methanol (100:1, 80:1, 50:1, 30:1) as the eluent to obtain 5 fractions (Fr.7-4-1 to Fr.7-4-5). Fr.7-5-3 was separated by normal-phase silica gel column chromatography and further purified by LH-20 gel column chromatography (MeOH) to obtain fraction (Fr.7-5-3-1). Fr.7-5-3-1 was further purified by semi-preparative HPLC under the following mobile phase conditions: (MeCN-H2O, 25:75, 2 mL / min, v / v) to obtain compound 2 (40.88 mg). t R =28.6 min).

[0030] The chemical structure of compound 1 (yellow waxy substance) obtained in Example 1 was identified. Compound 1 is readily soluble in methanol, and its color development in 10% sulfuric acid ethanol is brown. High-resolution mass spectrometry (HR-ESIMS) indicates that the quasi-molecular ion peak of compound 1 is [missing value]. m / z 337.19743[M + Na] + (Calculated value 337.41197), the molecular formula of compound 1 is inferred to be C. 17 H 30 O5, calculated unsaturation degree is 3. In the IR spectrum, the values ​​at 3427, 1733, and 1670 cm⁻¹ are... -1 The presence of an absorption peak at this location indicates the presence of a hydroxyl group in the compound. 1 The HNMR spectrum shows that the compound has one olefin proton: 5.35 (1H, d, J = 10.1 Hz, H-5); one methoxy proton: 3.65 (3H, s, OCH3-17); and signals for two methine protons: 2.62 (1H, m, H-4) and 2.45 (1H, q). J =7.9 Hz, H-2); signals for 3 pairs of methylene protons: 2.11 (1H, m, H-9a), 2.05 (1H, m, H-8a), 1.91 (1H, m, H-8b), 1.64 (1H, m, H-9b), 1.63 (1H, m, H-3a), 1.38 (1H, m, H-3b); signals for 4 methyl protons: 1.17 (3H, s, Me-16), 1.12 (6H, d, J =6.6 Hz, Me-12, 13), 0.99 (3H, d, J =6.6 Hz, Me-14); via 13 The C NMR (CD3OD, 150 MHz) and DEPT spectra revealed 17 carbon signals, including one carbonyl carbon signal. δ C 179.2 (C-1); Group 1 olefin carbon signal δ C 139.9 (C-6) δ C 136.6 (C-5); 3 sps 3 Hybridized carbon-oxygenated signals δ C 87.2 (C-10) δ C 85.0 (C-7) δ C 73.7 (C-11); 1 hydroxymethylene group δ C58.0 (C-15); 1 methoxy carbon signal δ C 52.1 (C-17); 4 methyl carbon signals: δ C 23.6 (C-16) δ C 21.7 (C-14) δ C 18.2 (C-12) δ C 17.7 (C-13); its structure is preliminarily inferred to be a terpene. (The sentence appears to be incomplete and requires further context.) 1 H- 1 The H COSY and HMBC spectra show correlations between H-2 / H-3 and H-4 / H-5, confirming a continuous carbon chain from C-2 to C-5. H-7 / H-8 / H-9 and H-8 are correlated with C-10, and the chemical shifts observed at (C-10) and (C-7) confirm the presence of a 2,5-disubstituted tetrahydrofuran ring. The HMBC spectrum shows that (H-17) is associated with (C-1), indicating the presence of a methyl ester structure in the molecule; H-15 is associated with C-5, C-6, and C-7, confirming that the hydroxymethyl group is linked to C-6; H-7 is associated with C-5, C-6, C-9, and C-15, proving that the aliphatic chain is linked to the tetrahydrofuran ring at C-7; H-16 is associated with C-9, C-10, and C-11, and H-11 is associated with C-9, C-10, C-12, and C-16, confirming the C-10 quaternary carbon and the 1-hydroxyethyl substituent on the tetrahydrofuran ring.

[0031] The NOESY spectrum shows that H-7 is related to H-15a. Figure 10 The correlation between H-16 and H-11 indicates that the hydroxymethyl group on the tetrahydrofuran ring is on the same side as the aliphatic chain. Furthermore, the correlation between H-2 and H-13 supports the stereoconfiguration of C-2 and C-4 being consistent with the known configuration of asperic acid A. Compound 1 has the same configuration as the known compound asperic acid; therefore, the relative configuration of compound 1 is determined.

[0032] Table 6. Compound 1 1 H NMR, 13 C NMR, HMBC, 1 H- 1 H COSY Data Origin Table

[0033] Compound 2 (yellow waxy substance) obtained in Example 1 was used to identify its chemical structure. Compound 2 is readily soluble in methanol, turns brown in 10% sulfuric acid ethanol, and high-resolution mass spectrometry (HR-ESIMS) shows a quasi-molecular ion peak for compound 2. m / z 323.19743[M + Na] + (Calculated value 323.38509), the molecular formula of compound 2 is inferred to be C. 16 H 28 O5, with a calculated degree of unsaturation of 3. Its molecular formula is greater than that of compound 1 (C...). 18 H 28 O5 has one less CH2 unit, suggesting a high degree of structural similarity between the two. The ¹H and ¹³C NMR spectra of compound 2, compared to compound 1, exhibit highly consistent skeletal signals, including: a tetrasubstituted double bond (…). δ C 135.8, C-6; 132.6, C-5), a carbon atomized quaternary carbon ( δ C 85.9, C-10), two hydroxymethyl groups ( δ C 86.7, C-7; δ H 4.32, dd, J = 9.5, 5.8 Hz; δ C 78.6, C-11; δ H 3.56, dd, J = 8.0, 3.0 Hz), and the same methyl substitution pattern (four methyl signals). The key difference is: (1) the chemical shift of H-15 in compound 2 is significantly high-field shifted to δ H 1.62 (s, 3H), the corresponding carbon signal is located at δ C 12.0 indicates that C-15 changes from a methylene group (-CH2O-) in compound 1 to a methyl group (=C-CH3) linked to a double bond. This change is confirmed by the HMBC-related signal: H-15 ( δ H 1.62) and C-5 ( δ C 132.6) C-6 ( δ C 135.8) and C-7 ( δ C 86.7) There is a correlation. (2) The methyl group (C-12) attached to C-11 in compound 1 is replaced by a hydroxymethyl group (-CH2OH) in compound 2. δ H 3.74 (dd,J = 11.2, 3.1 Hz) and 3.46 (dd, J = 11.3, 8.0 Hz) and its corresponding carbon signal ( δ C 64.3), in the HMBC spectrum, this group of protons is associated with C-9, C-10, and C-11. Based on the above analysis, and combined with detailed 2D NMR spectra (¹H-¹H COSY, HSQC, HMBC), the planar structure of compound 2 was determined as follows: Figure 20 As shown, it is a C-15 deoxymethylated and C-12 hydroxylated analog of compound 1.

[0034] Compound 2 shares the same skeleton and most of the chiral centers as compound 1. By comparing the ¹H-¹H coupling constants between the key protons and the NOESY correlation signals, its relative configuration was determined to be consistent with compound 1. In the NOESY spectrum, H-2 correlates with H-13, indicating that the methyl group at C-2 is on the same side as C-13; H-5 correlates with H-14, indicating that the methyl group at the end of the double bond (C-14) is on the same side as H-5; and H-7 correlates with H-15, further confirming that the methyl group at C-15 is on the same side as C-7. The key H-11 proton and its adjacent H-12a proton (… δ H There is a strong correlation between H-12b (3.74) and H-12b (the inverse of H-12b). δ H The correlation is relatively weak (3.46), combined with its coupling constant (H-12a, J = 3.1 Hz, H-12b J = 8.0 Hz), the relative configuration at C-11 was determined. NOESY and coupling constant were in perfect agreement with compound 1. Considering that compound 2 was obtained from the same fungal fermentation product as compound 1 and their biosynthetic pathways are highly related, it is inferred that compound 2 and compound 1 have the same absolute relative configuration.

[0035] Table 7 Compound 2 1 H NMR, 13 C NMR, HMBC, 1 H- 1 H COSY Data Origin Table

[0036] Experimental Example: Screening for in vitro antitumor activity of compounds 1 and 2 3.1 Experimental Instruments and Materials 3.1.1 Experimental Materials Table 8 Experimental Reagents and Consumables

[0037]

[0038] 3.1.2 Experimental Apparatus Table 9 Experimental Instruments

[0039]

[0040] 3.2 Experimental Method (MTT Method) 3.2.1 Preparation of cell culture medium

[0041] Take 45 mL of DMEM, add 5 mL of fetal bovine serum (final concentration 10%) and store at 4℃.

[0042] 3.2.2 A549 and MCF-7 cell culture methods

[0043] Cell thawing was performed by removing cryovials containing MCF-7 and A549 cell lines stored at -80 °C and immediately placing them in a 37 °C water bath. Gently agitate (the water level should not exceed the cap) to rapidly and completely thaw the cryopreservation solution. Then, remove the cryovials, wipe the surface dry, and disinfect with 75% ethanol spray. Place them in a clean bench, add an appropriate amount of DMEM, transfer to a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 2 mL of culture medium, and repeatedly pipette until the cells are completely resuspended. Transfer to a 25 cm⁻¹ container of DMEM containing 10% bovine serum. 2 Culture flasks. The culture flasks are placed in a cell culture incubator at 37°C and 5% CO2 for incubation.

[0044] Cell passage is performed by observing cell fusion density and viability under a microscope. Cells are ready for passage when fusion reaches 85% or higher in the culture flask. Transfer the cells to a clean bench from the incubator, discard the culture medium, rinse twice with 1 mL of PBS and discard the PBS. Add 1 mL of trypsin to the culture flask and place it in the incubator for 2-3 minutes to digest. Observe under a microscope that the cells have become rounded and floating. Add 2 mL of culture medium to stop digestion and resuspend the cells. Transfer the liquid from the culture flask to a centrifuge tube and centrifuge. Repeat the centrifugation process. Collect the lower layer and add 10% fetal bovine serum (FBS) DEME medium using a pipette, resuspending the cells until a homogeneous suspension is formed. Re-seed the cells into new cell culture flasks at a ratio of 1:2 to 1:3 for continued culture.

[0045] For cell cryopreservation, remove the cells from the incubator when the cell density reaches approximately 80%, discard the old culture medium, rinse with sterile PBS solution, discard the PBS solution, and repeat the washing once to thoroughly remove residual serum; add trypsin to the culture flask, place it flat in the incubator, and allow it to digest statically; after digestion, add complete culture medium, repeat the above centrifugation operation, discard the supernatant, add 1-2 mL of serum-free cell cryopreservation solution, mix well, transfer to cryovials for labeling and sealing, and after gradient cooling of the cryovials, transfer them to a liquid nitrogen tank or a -80°C freezer for storage.

[0046] 3.2.3 Initial screening for anti-tumor cell proliferation activity

[0047] Cells in good growth condition and in the logarithmic growth cycle were selected from the cell culture flasks, digested with trypsin, centrifuged, mixed, and diluted, and then counted using a hemocytometer. For seeding the test cells into plates, the density of the two cell types was first adjusted to obtain a density of 1 × 10⁻⁶ cells / mL. 5 Two cell suspensions were prepared at a concentration of 100 cells / mL. The two cell suspensions were then evenly seeded into two 96-well plates using a pipette, with 100 mL added to each well. μ L cell suspension was cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h.

[0048] Cells cultured in 96-well plates for 24 hours were replaced with a medium containing different monomeric compounds at a concentration of 40%. μ M (this concentration is a commonly used threshold for screening the in vitro antitumor activity of natural products) DEME medium, each plate has 2 positive control groups (doxorubicin and cisplatin) and 1 blank control group, and each compound has 3 parallel wells. The 96-well plates were cultured in a cell culture incubator at 37°C and 5% CO2 for 48 h. The drug-containing medium in the plate was discarded, and 20 mg of the medium was added to each well. μ After incubating the culture medium in L of MTT solution for 4 hours, remove the culture medium with a pipette and add 100 μL of the solution to each well. μ L-DMSO was used to dissolve formazan produced intracellularly. The absorbance was measured using a microplate reader, with the detection wavelength set at 490 nm.

[0049] Formula for calculating cell viability: , All of the above experiments need to be repeated independently at least 3 times, and the data obtained are expressed as mean ± SD.

[0050] 3.2.4 Rescreening for anti-tumor cell proliferation activity

[0051] Based on 40 μThe results of the compound screening at concentration M identified compounds with an inhibition rate of more than 50% against tumor cell growth. Five different concentration gradients were then used for secondary screening, using the same method as the initial screening.

[0052] Experimental results 3.3.1 Initial screening results of anti-tumor cell proliferation activity To evaluate the effects of endophytic fungi A. costaricensis The in vitro antitumor potential of the two compounds isolated from [the study] was investigated using human breast cancer cells (MCF-7) and lung cancer cells (A549) as models. The MTT assay was employed to evaluate the antitumor activity of all compounds at 40 [units unspecified]. μ Preliminary proliferation inhibition activity was tested at M concentration, and the results are expressed as cell viability (%). The results are as follows: Figure 21 As shown in Table 10. The initial screening results show that at 40... μ At the M concentration, the compounds showed weak inhibitory activity against both types of tumor cells, with compound 1 showing a certain inhibitory effect on the proliferation of the MCF-7 cell line.

[0053] Table 10 Effects of compounds 1-2 and positive control agents on the survival rate of MCF-7 and A549 cells. .

Claims

1. A terpenoid compound, characterized in that, The structural formula of the compound is as follows: Formula 1 or Formula 2.

2. The method for preparing a terpene compound as described in claim 1, characterized in that, Includes the following steps: Step 1, Preparation Stage: Take healthy, disease-free Rauvolfia pulveratum stem segments, remove surface soil and impurities, and in a clean bench, soak the stem segments sequentially in 75% ethanol for 30 seconds to 1 minute, then soak them in 2.5% sodium hypochlorite solution for 2 to 5 minutes, followed by rinsing with sterile water 3 to 4 times. Blot dry the surface moisture, and spread the sterile water from the final rinse onto a PDA plate. Incubate at 28°C for 3 days. Then, cut the surface-sterilized stem segments into small pieces of approximately 0.5 cm, place them on PDA plates, and incubate at 28°C for 3 to 7 days. Once fungal hyphae have grown along the edges of the tissue blocks, pick the newly formed hyphae from the edges and transfer them to fresh PDA plates. After multiple purifications, obtain single colonies. A. costaricensis ; Step 2 Aspergillus costaricensis Endophytic fungal fermentation: The activated fungi are fermented in a clean bench... A. costaricensis Inoculate into PDAs and incubate for 3-5 days. After sterilizing the rice culture medium in an autoclave, [the following steps are taken]. A. costaricensis The strain was inoculated into rice culture medium and cultured at 28°C and 60% RH for 30-40 days. Step 3 Aspergillus costaricensis Extraction and extraction of fermentation products: The fermented rice culture medium was extracted three times with methanol at room temperature for 7 days each time. The extracts were combined, filtered, and concentrated under reduced pressure to obtain crude extract. The crude extract was suspended in warm water and then fractionally extracted with petroleum ether, ethyl acetate, and n-butanol. After standing, the layers were separated, and each extract phase was filtered and concentrated under reduced pressure to obtain extracts of the corresponding polar fractions, which were then weighed. Step 4 Aspergillus costaricensis Isolation and purification of fermentation products: A. costaricensis The ethyl acetate fraction was separated by mixing the sample with silica gel in a 100-200 mesh column and separating it on a normal-phase atmospheric pressure column. The sample was eluted with a gradient of petroleum ether-ethyl acetate (8:1 to 1:5 v / v) and dichloromethane-methanol (20:1 to 6:1 v / v). The obtained fractions were combined by thin-layer chromatography to obtain 9 fractions Fr.1 to Fr.

9. Fr.9 was further separated by reversed-phase silica gel column chromatography with a gradient elution of 20%–90% methanol-water (v / v) to obtain nine fractions Fr.9-1–Fr.9-9. Fr.9-4 was further separated by normal-phase silica gel column chromatography (200–300 mesh) with a dichloromethane-methanol (v / v) of 100:1–15:1 to obtain five fractions Fr.9-4-1–Fr.9-4-5. Fr.9-4-4 was further purified by normal-phase silica gel column chromatography and then by LH-20 gel column chromatography to obtain fraction Fr.9-4-4-1. Fr.9-4-4-1 was further purified by semi-preparative HPLC under the following mobile phase conditions: MeOH-H2O, 50:50, 2 mL / min, v / v, to obtain compound 1. The method for preparing a terpene compound according to claim 1, characterized in that step 4 is: Aspergillus costaricensis Isolation and purification of fermentation products: A. costaricensis The ethyl acetate fraction was separated by mixing the sample with silica gel in a 100-200 mesh column and separating it on a normal-phase atmospheric pressure column. The sample was eluted with a gradient of petroleum ether-ethyl acetate (8:1 to 1:5 v / v) and dichloromethane-methanol (20:1 to 6:1 v / v). The obtained fractions were combined by thin-layer chromatography to obtain 9 fractions Fr.1 to Fr.

9. Fr.7 was further separated by reversed-phase silica gel column chromatography, eluted with a methanol-water gradient of 20% to 90% (v / v), to obtain 8 fractions Fr.7-1 to Fr.7-8; Fr.7-5 was further separated by normal-phase silica gel column chromatography with a mesh size of 200-300, using dichloromethane-methanol at a volume ratio of 100:1 to 30:1 to elute the fraction, yielding five fractions Fr.7-5-1 to Fr7-5-5. Fr.7-5-3 was separated by normal-phase silica gel column chromatography and further purified by LH-20 gel column chromatography to obtain fraction Fr.7-5-3-1. Fr.7-5-3-1 was further purified by semi-preparative HPLC with the following mobile phase conditions: MeCN-H2O, 25:75, 2 mL / min, v / v, to obtain compound 2.

3. The method for preparing a terpene compound as described in claim 1, characterized in that, Includes the following steps: The rice culture medium in step 2 consists of: 200 g rice, 1 g peptone, 1 g yeast extract, 1 g monosodium glutamate, 4% sea salt, and 250 mL water. The mixture is then sterilized in an autoclave at 121°C for 20 min.

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