An alkaloid, its preparation method and application

Abstract

The application relates to the field of natural medicines, and discloses an alkaloid, a preparation method and application thereof, the alkaloid having a structure shown in formula I and pharmaceutically acceptable salts thereof. The alkaloid is separated from a n-butanol extraction part of an ethanol extract of Dendrobium huoshanense stems through silica gel column chromatography and a preparation thin layer separation technology, and the structure is identified through nuclear magnetic resonance and high-resolution mass spectrometry. Through cell experiments, it is verified that the alkaloid prepared in the application can significantly relieve inflammation induced by LPS, inhibit human gastric cancer cell (AGS) proliferation and promote apoptosis; molecular docking analysis shows that the alkaloid prepared in the application can be significantly docked with active sites of induced iNOS, IL-6, TNF-alpha and MMP9, and the binding energy is-9.5~(-8.6) kcal / mol.

Description

Technical Field

[0001] This invention relates to the field of natural medicines, and more specifically, to an alkaloid, its preparation method, and its application. Background Technology

[0002] Diterpenoid alkaloids possess a wide range of biological activities, such as anti-inflammatory, analgesic, antiarrhythmic, and antitumor effects, and are considered to be the most promising natural compounds for cancer treatment. However, the complex ring systems and numerous chiralities in the structure of diterpenoid alkaloids make it impractical to prepare them in the laboratory or industrial setting using total synthesis or biomimetic synthesis. Therefore, finding naturally sourced diterpenoid alkaloids is of great significance for the development of innovative traditional Chinese medicine drugs. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides an alkaloid, its preparation method, and its application.

[0004] This invention provides an alkaloid with the molecular formula C 22 H 29 O 12 N3, and having the structure shown in Formula I and its pharmaceutically acceptable salt:

[0005]

[0006] Formula 1.

[0007] Preferred: Pharmaceutically acceptable salts include salts formed by compounds with inorganic or organic acids.

[0008] Furthermore: pharmaceutically acceptable salts include hydrochloride, sulfate, hydrogen sulfate, phosphate, acetate, propionate, butyrate, lactate, methanesulfonate, p-toluenesulfonate, maleate, benzoate, succinate, tartrate, citrate, fumarate, taurine, and gluconate.

[0009] A method for preparing an alkaloid, comprising the following steps:

[0010] S1: After crushing the dried stems of Dendrobium huoshanense, reflux extraction with ethanol was performed, and the obtained ethanol extract was concentrated under reduced pressure until no alcohol odor was detected, thus obtaining the ethanol extract;

[0011] S2: Dissolve the obtained ethanol extract in water at a ratio of 1:10, and then extract with petroleum ether, ethyl acetate and n-butanol in sequence to obtain four phases: petroleum ether phase, ethyl acetate phase, n-butanol phase and aqueous phase.

[0012] S3: The n-butanol phase was separated by silica gel column chromatography using a chloroform:methanol gradient elution from 80:1 to 0:100. Fractions of the same type were combined according to thin-layer chromatography, and twelve major fractions were obtained. These twelve fractions were designated as Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, Fr.H, Fr.I, Fr.J, Fr.K, and Fr.L.

[0013] Fraction Fr.G was separated into two sub-fractions, Fr.G1 and Fr.G2, by silica gel column chromatography using a solvent system of chloroform and methanol in a ratio of 30:1. Fr.G2 was then subjected to Sephadex LH-20 gel column chromatography and eluted with methanol to obtain a secondary fraction, Fr.G2a.

[0014] S4: The secondary fraction Fr.G2a was separated by preparative thin-layer chromatography using chloroform and methanol in a 10:1 ratio as the solvent system, ultimately yielding a product with the molecular formula C. 22 H 29 O 12 Compounds of N3.

[0015] Preferred method: In step S1, the dried stems of Dendrobium huoshanense are pulverized and then subjected to reflux extraction three times with 90% ethanol.

[0016] The application of an alkaloid in the preparation of in vitro anti-inflammatory and anti-gastric cancer active drugs.

[0017] The beneficial effects of this invention are as follows: The novel alkaloid disclosed herein can potentially reduce nitric oxide (NO) production in LPS-stimulated RAW264.7 macrophages. ELISA experiments show that this alkaloid significantly reduces LPS-induced inflammation by inhibiting the expression of TNF and IL-6. Furthermore, this alkaloid effectively inhibits the survival and proliferation of AGS cells and promotes apoptosis in a concentration-dependent manner. Attached Figure Description

[0018] Figure 1 The chemical structural formula of compound DHS-DA-1 of this invention is shown below;

[0019] Figure 2 This is the mass spectrum of compound DHS-DA-1 of the present invention;

[0020] Figure 3 The infrared spectrum of compound DHS-DA-1 of this invention;

[0021] Figure 4 The ultraviolet spectrum of compound DHS-DA-1 of this invention;

[0022] Figure 5This is a thin-layer chromatography (TLC) image of the compound DHS-DA-1, potassium bismuth iodide, from this invention.

[0023] Figure 6 The compound DHS-DA-1 of this invention 1 HNMR spectrum (600MHz, CD3OD);

[0024] Figure 7 The compound DHS-DA-1 of this invention 13 CNMR spectrum (151MHz, CD3OD);

[0025] Figure 8 The HSQC spectrum of compound DHS-DA-1 of this invention is shown below.

[0026] Figure 9 The compound DHS-DA-1 of this invention 1 H- 1 HCOSY spectrum;

[0027] Figure 10 The HMBC spectrum of compound DHS-DA-1 of this invention is shown below.

[0028] Figure 11 The NOESY spectrum of compound DHS-DA-1 of this invention is shown below.

[0029] Figure 12 HMBCand of compound DHS-DA-1 of this invention 1 H- 1 HCOSY connection diagram;

[0030] Figure 13 This is a NOESY connection diagram of compound DHS-DA-1 of the present invention;

[0031] Figure 14 This is a graph showing the anti-inflammatory effects of the compound DHS-DA-1 from this invention.

[0032] Figure 15 This is a graph showing the AGS cytotoxicity results of the compound DHS-DA-1 of this invention;

[0033] Figure 16 This diagram shows the 2D and 3D binding sites of the compound DHS-DA-1 and the active site of protein nitric oxide synthase.

[0034] Figure 17 This diagram shows the 2D and 3D binding sites of the active sites of the compound DHS-DA-1 and interleukin-6 in this invention.

[0035] Figure 18This diagram shows the 2D and 3D binding sites of the compound DHS-DA-1 and the active site of the protein tumor necrosis factor-α.

[0036] Figure 19 This diagram shows the 2D and 3D binding sites of the active sites of the compound DHS-DA-1 and matrix metalloproteinase 9 in this invention. Detailed Implementation

[0037] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, features described in some examples may be combined in other examples.

[0038] Example 1

[0039] This embodiment presents a method for preparing alkaloids, comprising the following steps:

[0040] S1: After crushing the dried stems of Dendrobium huoshanense, reflux extraction with ethanol was performed, and the obtained ethanol extract was concentrated under reduced pressure until no alcohol odor was detected, thus obtaining the ethanol extract;

[0041] S2: Dissolve the obtained ethanol extract in water at a ratio of 1:10, and then extract with petroleum ether, ethyl acetate and n-butanol in sequence to obtain four phases: petroleum ether phase, ethyl acetate phase, n-butanol phase and aqueous phase.

[0042] S3: The n-butanol phase was separated by silica gel column chromatography using a chloroform:methanol gradient elution from 80:1 to 0:100. Fractions of the same type were combined according to thin-layer chromatography, and twelve major fractions were obtained. These twelve fractions were designated as Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, Fr.H, Fr.I, Fr.J, Fr.K, and Fr.L.

[0043] Fraction Fr.G was separated into two sub-fractions, Fr.G1 and Fr.G2, by silica gel column chromatography using a solvent system of chloroform and methanol in a ratio of 30:1. Fr.G2 was then subjected to Sephadex LH-20 gel column chromatography and eluted with methanol to obtain a secondary fraction, Fr.G2a.

[0044] S4: The secondary fraction Fr.G2a was separated by preparative thin-layer chromatography using chloroform and methanol in a 10:1 ratio as the developing solvent, ultimately yielding a fraction with the molecular formula C. 22 H 29 O 12Compounds of N3.

[0045] In step S1, the dried stems of Dendrobium huoshanense are pulverized and then extracted three times by reflux with 90% ethanol.

[0046] Example 2

[0047] In this embodiment, an alkaloid is prepared, and the specific process is as follows:

[0048] 80 kg of dried stems of *Dendrobium huoshanense* were pulverized and extracted three times by reflux with 90% ethanol (2 × 800 L). The combined ethanol extracts were concentrated under reduced pressure to obtain a total ethanol extract (10.5 kg). The obtained extract was dissolved in water and then extracted sequentially with petroleum ether, ethyl acetate, and n-butanol to obtain petroleum ether fraction (0.65 kg), ethyl acetate fraction (0.95 kg), n-butanol fraction (1.2 kg), and water fraction (7.7 kg), respectively. The n-butanol fraction was separated by silica gel column chromatography using a chloroform:methanol gradient elution from 80:1 to 0:100. Twelve major fractions (A–L) were obtained by combining fractions of the same type using thin-layer chromatography. Fr.G (5 g) was separated into two fractions, Fr.G1 and Fr.G2, by silica gel column chromatography using a chloroform:methanol solvent system in a 30:1 ratio. Fr.G2 (200 mg) was then subjected to Sephadex LH-20 gel column chromatography, eluted with methanol, to obtain a secondary fraction Fr.G2a. Subsequently, Fr.G2a was separated by preparative thin-layer chromatography using a 10:1 ratio of chloroform and methanol as a solvent system, finally yielding the compound with a total mass of 55.0 mg.

[0049] The compound is a white powder with the molecular formula C. 22 H 29 O 12 N3, its structural formula is as follows Figure 1 As shown. This is demonstrated by HRESIMS spectroscopy at m / z 566.4288 [M+K]. +（ The calculated value of 566.5745 was confirmed. Figure 2 The IR spectrum showed the characteristic functional groups of the benzene ring at 1595 cm⁻¹. -1 and hydroxyl groups at 3380cm -1 The existence of ( Figure 3 The UV spectrum at 240 nm and 270 nm shows typical characteristics of benzene rings and hydroxyl groups. Figure 4 The Dragendorff reagent test was positive. Figure 5 ).

[0050] compound 1 HNMR spectra (600MHz, CD3OD) (Table 1, Figure 6The characteristic signals of two aromatic protons [δ] were displayed. H 6.73 (1H, s, H-8), δ H 6.66 (1H, s, H-9)], two methoxy groups [δ H 3.87 (3H, s, H-20OCH3)] and [δ H 3.85 (3H, s, H-19OCH3)], three methylene groups [δ H 4.29 (1H, td, J=10.4, 6.7Hz, H-15), δ H 3.92 (1H, m, H-15), δ H 3.92 (1H, m, H-16), δ H 4.29 (1H, td, J=10.4, 6.7Hz, H-16), δ H 3.78 (1H, dd, J=12.0, 2.4Hz, H-18), δ H 3.67 (1H, dd, J = 12.0, 5.2 Hz, H-18)], three hydroxyl groups [δ H 7.33 (1H, s), δ H 6.73 (1H, s), δ H 6.66 (1H, s)], a primary amino group [δ H 3.85 (2H, s)], a secondary amino group [δ H 1.31 (1H, m)], the remaining signal is methine. 13 CNMR (151 MHz, CD3OD) and DEPT-135 spectra (Table 1, Figure 7 8) A total of 22 carbon atoms were identified, including six quaternary carbons (δ). C 154.39, C-1;δ C 149.32, C-2;δ C 139.51, C-3;δ C 136.18, C-4;δ C 135.54, C-5; δ C 133.03, C-6), two methoxy groups (δ C 57.02, C-19;δ C 56.76, C-20), three methylene groups (δ C 72.83, C-15;δ C 72.88, C-16;δ C 62.53 (C-18), 11 methines. The above spectroscopic data indicate that this compound can be considered a diterpenoid alkaloid with 22 skeletal carbons, which is quite unique. This inference was further confirmed by subsequent analysis of 2DNMR data.

[0051] For HSQC and 1 H- 1 Analysis of the HCOSY spectral data provided a clear allocation of hydrogen and carbon resonances associated with hydrogen atoms in DHS-DA-1 (Table 1, Figure 9 and 10 First, the chemical shifts of C1, C2, C3, C4, C5, and C6, between 130 and 155, were identified as six quaternary carbon atoms, presumably forming a hexasubstituted benzene ring, designated as segment A. Second, COSY spectral analysis revealed cross-peaks between H7 and H10 / H14; between H14 and H13; and between H10 and H21, and H21 and H11. 1 H- 1 The HCOSY correlation profile depicts fragment B, containing H13-H14-H7-H10-H21-H11. Furthermore, the cross peaks between H12 and H17 / H18, and H22 and H15 / H16, suggest the presence of two fragments, designated as fragments C and D, respectively. Based on the HMBC spectrum of DHS-DA-1 (Table 1), Figure 11 According to C5 and H16 (C-16, δ) C 72.88); C4 and H8 (C-8, δ C A heterocyclic atom was identified based on the HMBC correlation of 104.78. C 87.15) and C-18 (δ C 62.53) to H-11 (δ) H 4.78; C-8, δ C The HMBC cross peak at 87.15 indicates that the connection between C-11, C-14, and C-18 may be through a nitrogen atom, thus forming a six-membered nitrogen heterocycle. Similarly, based on the HMBC cross peak at C-10 (δ... C 87.57) to H-9 (δ) H The HMBC correlation of 4.73) forms a pentacyclic heterocycle linked to an amino group; from C-12 (δ C 78.31) to H-13 (δ) H 3.42) and from C-13 (δ C 77.79) to H-17 (δ) H The HMBC correlation of 3.41) suggests that DHS-DA-1 may contain a five-membered nitrogen-oxygen heterocycle. Furthermore, C-11 (δ... C 87.15) and H-8 (δ H 6.73) and C-21 (δ C 55.69) and H22 (δ HThe HMBC cross peaks between 3.14 and C-11 suggest the relationship between C-11 / H-8 and C21 (δ). C An oxygen bridge exists between C-6 and H-19, and between C-17 and H-20. HMBC cross-peaks were also observed between C-6 and H-19, and between C-17 and H-20. Based on the above analysis, a possible planar structure of an unusual C22-diterpene alkaloid is deduced. Figure 12 ).

[0052] By interpreting NOESY data ( Figure 13 Table 1 illustrates the relative configurations of the compounds.

[0053] The NOESY interactions observed between H-8 / H-9, H-11, H-19, H-20, H-21, and H-22, and the correlations between H-9 / H-10 and H-19, H-20, H-21, and H-22, indicate the coplanar orientation of C-8 and N-9. Furthermore, the correlation between H-12 and H-18, and the cross-peaks connecting H-15 and H-16 with H-21 and H-22, indicate the β-direction of H-12, H-18, H-15, H-16, H-21, and H-22. Finally, the chemical structure of the compound was determined and named DHS-DA-1, a novel diterpenoid alkaloid with 22 skeletal carbons derived from *D. huoshanense*. Additionally, a search of the SciFinder database confirmed it as a novel compound.

[0054] Table 1. 1H NMR (600MHz) and 13CNMR (151MHz) data for DHS-DA-1

[0055]

[0056]

[0057]

[0058] Example 3

[0059] This example is used to evaluate the in vitro anti-inflammatory activity of compound DHS-DA-1.

[0060] Cell viability was assessed using the MTT assay. Macrophages were cultured in DMEM containing 10% fetal bovine serum. After 24 h of incubation, 100 µL of the test compound was added. After another 24 h, 20 µL of MTT was added to each well. After 4 h, the supernatant was discarded, and 150 µL of DMSO was added to each well. After shaking for 1 min, the absorbance of each well was measured at 570 nm using a microplate reader, and cell viability was calculated.

[0061] The inhibitory activity of the isolated compounds against nitric oxide (NO) production in RAW264.7 cells was evaluated using Griess reagent. Specifically, cells were stocked at 9 × 10⁶ cells per well. 5 Cells were cultured at a density of [number] cells in 96-well plates to promote NO production. Additionally, 10 µM dexamethasone was used as a positive control, while drug-free cell culture medium served as a blank control. Culture plates were pretreated with different concentrations of DHS-DA-1 for 1 hour, followed by incubation for 24 hours with or without 1 µg / mL lipopolysaccharide (LPS). RAW264.7 macrophages were treated with 1 µg / mL LPS and DHS-DA-1 for 24 hours. Subsequently, culture medium was collected to assess the expression levels of certain pro-inflammatory cytokines, such as TNF-α and IL-6, with dexamethasone used as a positive control. Experiments were performed using ELISA kits and according to the manufacturer's instructions.

[0062] like( Figure 14 As shown in A), DHS-DA-1 at concentrations of 6.25-100 μM had no cytotoxic effect on RAW264.7 cells. Therefore, DHS-DA-1 at concentrations of 6.25-100 μM was selected for further investigation. The NO content in the control group cells was relatively low, but it increased 12-fold compared to the control group in the LPS-treated group (model group, 1 μg / ml) (P<0.01). Figure 14 B). Pretreatment with dexamethasone (positive control, 10 μM) and DHS-DA-1 (50 or 100 μM) significantly downregulated NO levels (P<0.05).

[0063] The inhibitory effect of DHS-DA-1 on the expression of pro-inflammatory cytokines such as TNF-α and IL-6 was evaluated in an ELISA assay. The results showed that LPS stimulation led to a significant increase in the levels of these cytokines compared to the control group. Conversely, DHS-DA-1 treatment significantly reduced LPS-induced cytokine production in a dose-dependent manner (P<0.05). Notably, at a concentration of 100 μM, DHS-DA-1 showed a significant inhibitory effect on cytokine production, almost approaching the effect of dexamethasone as a positive control. Figure 14 (C and 14D). These data suggest that DHS-DA-1 exerts its anti-inflammatory effect by regulating the expression of pro-inflammatory cytokines.

[0064] Example 4

[0065] This embodiment is used to evaluate the in vitro anti-gastric cancer cell activity of compound DHS-DA-1.

[0066] AGS cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics. The culture environment was a humidified 37°C with 5% CO2. Cell viability was assessed using the MTT assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. In short, cells were grown at a rate of 2 × 10⁶ cells / well per mL. 4 Cells were initially distributed at a density in 96-well plates. After 24 hours of culture, cells were exposed to different concentrations of compounds for 48 hours, followed by a 24-hour delay before treatment. Dimethyl sulfoxide (DMSO) was used as a negative control at a final concentration of 0.1% (v / v). At specified time intervals, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated under humid conditions for 4 hours. The supernatant was then removed, and the resulting crystals were dissolved in 150 μL of DMSO. 5-Fluorouracil (Sigma-Aldrich Shanghai Trading Co., Ltd.) was used as a positive control. The absorbance was then measured at 570 nm using a microplate reader.

[0067] like( Figure 15 As shown in A), DHS-DA-1 exhibited a dose-dependent inhibitory effect on the viability of AGS cells. As a positive control, 5-FU showed a significant effect in inhibiting the proliferation of gastric cancer cells. Figure 15 B). These results indicate that DHS-DA-1 effectively reduces AGS cell proliferation while promoting apoptosis in a concentration-dependent manner.

[0068] Example 5

[0069] To further understand the key binding interactions, molecular docking simulations were performed on DHS-DA-1, focusing on key inflammation-related proteins: inducible nitric oxide synthase (iNOS, PDBID: 4CX7), interleukin-6 (IL-6, PDBID: 5FUC), and tumor necrosis factor-α (TNF-α, PDBID: 7JRA). This assessment aimed to determine whether the results of the docking analysis supported the results of the anti-inflammatory experiments.

[0070] 4CX7 is a complex formed by human inducible nitric oxide synthase (iNOS) and the compound (R)-6-(3-amino-2-5-2-6-amino-4-methylpyridin-2-yl)ethyl)pyridin-3-yl)propyl)-4-methylpyridin-2-amine. Docking analysis showed that DHS-DA-1 can effectively bind to the active site of iNOS, such as ( Figure 16 As shown in Table 2 (A), the calculated binding energy is -9.0 kcal / mol. DHS-DA-1 forms five conventional hydrogen bond interactions with the His101 and Cys115 residues of the iNOS active center ( Figure 16 B, Table 2). Furthermore, DHS-DA-1 also established hydrophobic interactions with Ala104 residues (B, Table 2). Figure 16 C), and formed a π-cation interaction with Cys115 residues ( Figure 16 C).

[0071] 5FUC is a complex formed by interleukin-6 (IL-6) and the gp80 receptor. Docking studies showed that DHS-DA-1 successfully intercalated into the IL-6 binding site, such as ( Figure 17 As shown in Table 2 (A), the obtained binding energy is -9.5 kcal / mol. Furthermore, DHS-DA-1 interacts with Arg40 via hydrogen bonds (…). Figure 17 B). Notably, the key intermolecular forces inhibiting IL-6 activity appear to involve specific types of hydrogen bonds, including two carbon-hydrogen bond interactions and two attractive charge interactions with Glu172 residues (B). Figure 17 C).

[0072] The 7JRA complex consists of human tumor necrosis factor (TNF-α) bound to 2-[5-(3-chloro-4-{[1R-1-2-fluorophenyl)ethyl]amino}quinolin-6-yl)pyridin-2-yl]propanol. Docking studies indicate that DHS-DA-1 effectively interacts with the interleukin-6 (IL-6) pocket, exhibiting a binding energy of -8.6 kcal / mol. Figure 18 A, Table 2). The stability of this ligand within the binding pocket is primarily achieved through four hydrogen bonds formed with Lys174, Pro176, Glu192, and Pro176 residues (A, Table 2). Figure 18 B, Table 2). It was also noted that the key intermolecular interactions promoting the inhibition of TNF-α activity include a unique class of hydrogen bond compositions, comprising two carbon-hydrogen bond interactions and five charge-attracting interactions involving Pro189, Lys188, and Glu192 residues (B, Table 2). Figure 18 C).

[0073] Table 2. Binding energy of DHS-DA-1 with nitric oxide synthase, tumor necrosis factor-α, and interleukin-6 proteins.

[0074]

[0075] The natural compound DHS-DA-1, isolated from *D. huoshanense*, showed potential cytotoxic activity against gastric cancer cells (AGS). Therefore, molecular docking analysis was performed to elucidate the binding mode of DHS-DA-1 within the hydrophobic pocket of matrix metalloproteinase (PDB:4H1Q) using AutoDockVina software. (See 3D diagram). Figure 7As shown in the figure, DHS-DA-1 binds well to the amino acid residues Ala191, Tyr245, Pro246, Ala189, Ser237, Ser238, His236, and His230 in MMP9. The binding energy between DHS-DA-1 and MMP9 is relatively strong, at -8.6 kcal / mol. Figure 19 A). DHS-DA-1 forms three conventional hydrogen bond interactions with residues Gly217 and Tyr248 at the active site of MMP9 ( Figure 19 C). Furthermore, it forms a π-donor hydrogen bond interaction and a π-cation interaction with the active center Tyr218 (C). Figure 19 B).

[0076] This invention presents an alkaloid, DHS-DA-1, isolated from the n-butanol fraction of *D. huoshanense* stems by silica gel column chromatography and preparative thin-layer chromatography, and its structure was elucidated by NMR and high-resolution mass spectrometry. Anti-inflammatory studies showed that DHS-DA-1 significantly alleviated LPS-induced inflammation, as evidenced by reduced nitric oxide (NO) production and decreased expression levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Molecular docking analysis also revealed significant binding interactions between DHS-DA-1 and proteins such as iNOS, TNF-α, and IL-6. Furthermore, DHS-DA-1 effectively inhibited the proliferation of AGS cells and promoted apoptosis in a concentration-dependent manner. Molecular docking further elucidated the possible mechanisms of DHS-DA-1's anti-inflammatory and anti-gastric cancer activities. DHS-DA-1, as a potential resource for discovering novel anti-inflammatory alkaloids, provides theoretical insights into the derivation of functional components from natural products for the prevention and treatment of gastric cancer. Therefore, this study is of great significance for further research on the medicinal properties of this rare and endangered traditional Chinese medicine, D. huoshanense.

[0077] The embodiments of this example have been described above. However, this example is not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms based on the guidance of this example, and all of them are within the protection scope of this example.

Claims

1. An alkaloid characterized in that, The molecular formula is C 22 H 29 O 12 N3, and has the structure shown in formula I and its pharmaceutically acceptable salts: ； Formula I.

2. The alkaloid according to claim 1, characterized in that, The pharmaceutically acceptable salt includes the salt of the compound with inorganic acid, organic acid.

3. The alkaloid according to claim 2, characterized in that, The pharmaceutically acceptable salt includes hydrochloride, sulfate, bisulfate, phosphate, acetate, propionate, butyrate, lactate, methanesulfonate, p-toluenesulfonate, maleate, benzoate, succinate, tartrate, citrate, fumarate, taurinate, gluconate.

4. A process for the preparation of the alkaloid as claimed in claim 1, characterized in that, The method comprises the following steps: S1: crushing the dry stems of Dendrobium huoshanense, and extracting the crushed stems by reflux extraction with ethanol, and concentrating the obtained ethanol extract under reduced pressure to remove the alcohol taste to obtain an ethanol extract; S2: dissolving the obtained ethanol extract in water according to a ratio of 1:10, and sequentially extracting the ethanol extract with petroleum ether, ethyl acetate and n-butanol to obtain four parts of petroleum ether phase, ethyl acetate phase, n-butanol phase and water phase; S3: separating the n-butanol phase by silica gel column chromatography, eluting with chloroform:methanol from 80:1 to 0:100 gradient, and combining the same fractions according to thin layer chromatography detection, and then obtaining twelve main fractions, which are respectively denoted as Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, Fr.H, Fr.I, Fr.J, Fr.K, Fr.L; The fraction Fr.G is eluted by silica gel column chromatography with a solvent system of chloroform and methanol at a ratio of 30:1 to separate two sub-fractions Fr.G1 and Fr.G2, and the Fr.G2 is further eluted by Sephadex LH-20 gel column chromatography with methanol to obtain a sub-fraction Fr.G2a; S4: The sub-fraction Fr.G2a was separated by preparative thin layer chromatography using chloroform and methanol in a ratio of 10:1 as developing agent, resulting in the compound of formula C 22 H 29 O 12 N3.

5. A process for the preparation of an alkaloid as claimed in claim 1 according to claim 4, wherein, In step S1, the dry stems of Dendrobium huoshanense are crushed, and then extracted by reflux extraction with 90% ethanol.

6. Use of an alkaloid as claimed in claim 1, wherein, The application in the preparation of an in-vitro anti-inflammatory and anti-gastric cancer active drug.

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