Alkaloid as well as preparation method and application thereof

Abstract

The invention relates to the field of natural medicines, and discloses an alkaloid and a preparation method and application thereof, and the alkaloid has a structure as shown in a formula I and a pharmaceutically acceptable salt thereof. The alkaloid is separated from an n-butyl alcohol extraction part of a dendrobium huoshanense stem ethanol extract through silica gel column chromatography and a preparative thin-layer separation technology, and the structure of the alkaloid is identified through nuclear magnetic resonance and high-resolution mass spectrometry. Cell experiments prove that the alkaloid prepared by the invention can remarkably relieve inflammation induced by LPS, inhibit proliferation of human gastric cancer cells (AGS) and promote apoptosis of the AGS; molecular docking analysis shows that the alkaloid prepared by the invention can be remarkably docked with active sites of inducible iNOS, IL-6, TNF-alpha and MMP9, and the binding energy is 9.5-8.6 kcal / mol.

Description

Technical Field

[0001] The present invention relates to the field of natural medicines, and more specifically, it relates to an alkaloid, its preparation method and application. Background Art

[0002] Diterpenoid alkaloids have a wide range of biological activities, such as anti-inflammatory, analgesic, anti-arrhythmic, anti-tumor, etc. They are considered to be the most promising natural compounds for the treatment of cancer at present. However, the ring system in the structure of diterpenoid alkaloids is complex and there are many chiral centers. It is not practical to prepare them by total synthesis or biomimetic synthesis in the laboratory or on an industrial scale. Finding diterpenoid alkaloids from natural sources is of great significance for the development of innovative traditional Chinese medicines. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides an alkaloid, its preparation method and application.

[0004] The present 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 salts:

[0005]

[0006] Formula 1.

[0007] Preferably, the pharmaceutically acceptable salts include salts formed by the compound and inorganic acids or organic acids.

[0008] Furthermore, the pharmaceutically acceptable salts include hydrochloride, sulfate, bisulfate, phosphate, acetate, propionate, butyrate, lactate, mesylate, tosylate, maleate, benzoate, succinate, tartrate, citrate, fumarate, taurate, gluconate.

[0009] A preparation method of an alkaloid, comprising the following steps:

[0010] S1: After crushing the dry stems of Dendrobium huoshanense, reflux extraction is carried out with ethanol, and the obtained ethanol extract is concentrated under reduced pressure until there is no alcohol smell to obtain an ethanol extract;

[0011] S2: Dissolve the obtained ethanol extract in water according to a ratio of 1:10, and then extract successively with petroleum ether, ethyl acetate and n-butanol to obtain four parts: petroleum ether phase, ethyl acetate phase, n-butanol phase and water phase;

[0012] S3: The n-butanol phase was separated by silica gel column chromatography, eluted with chloroform: methanol in a gradient from 80:1 to 0:100, and the same fractions were combined according to thin layer chromatography detection. Then, twelve main fractions were obtained, which were denoted 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, respectively;

[0013] Fraction Fr.G was eluted through silica gel column chromatography using a solvent system of chloroform and methanol in a ratio of 30:1 to separate two sub-fractions, Fr.G1 and Fr.G2. Fr.G2 was further separated by 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 a solvent system of chloroform and methanol in a ratio of 10:1, and finally a compound with the molecular formula C 22 H 29 O 12 N3 was obtained.

[0015] Preferably: In step S1, after the dried stems of Dendrobium huoshanense were crushed, they were refluxed and extracted three times with 90% ethanol.

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

[0017] The beneficial effects of the present invention are as follows: The novel alkaloid disclosed in the present invention can potentially reduce the production of nitric oxide (NO) in LPS-stimulated RAW264.7 macrophages. ELISA experiments showed that the alkaloid significantly reduced LPS-induced inflammation by inhibiting the expression of TNF and IL-6. In addition, the alkaloid was able to effectively inhibit the survival and proliferation of AGS cells and promote apoptosis in a concentration-dependent manner. Description of the Drawings

[0018] Figure 1 It is the chemical structural formula of the compound DHS-DA-1 of the present invention;

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

[0020] Figure 3 It is the infrared spectrum of the compound DHS-DA-1 of the present invention;

[0021] Figure 4 It is the ultraviolet spectrum of the compound DHS-DA-1 of the present invention;

[0022] Figure 5Thin-layer detection diagram of potassium bismuth iodide for the compound DHS-DA-1 of the present invention;

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

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

[0025] Figure 8 HSQC spectrum of the compound DHS-DA-1 of the present invention;

[0026] Figure 9 For the compound DHS-DA-1 of the present invention 1 H- 1 HCOSY spectrum;

[0027] Figure 10 HMBC spectrum of the compound DHS-DA-1 of the present invention;

[0028] Figure 11 NOESY spectrum of the compound DHS-DA-1 of the present invention;

[0029] Figure 12 HMBC and 1 H- 1 HCOSY connection diagram;

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

[0031] Figure 14 Anti-inflammatory result diagram of the compound DHS-DA-1 of the present invention;

[0032] Figure 15 AGS cytotoxicity result diagram of the compound DHS-DA-1 of the present invention;

[0033] Figure 16 2D and 3D binding site diagrams of the compound DHS-DA-1 of the present invention and the active center of protein nitric oxide synthase;

[0034] Figure 17 2D and 3D binding site diagrams of the compound DHS-DA-1 of the present invention and the active center of protein interleukin-6;

[0035] Figure 182D and 3D binding site diagrams of compound DHS-DA-1 of the present invention and the active center of protein tumor necrosis factor-α;

[0036] Figure 19 2D and 3D binding site diagrams of compound DHS-DA-1 of the present invention and the active center of protein matrix metalloproteinase 9; Detailed implementation manners

[0037] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that discussing these embodiments is only to enable those skilled in the art to better understand and thus implement the subject matter described herein. Without departing from the scope of protection of the content of this specification, changes can be made to the functions and arrangements of the elements discussed. Each example can omit, substitute, or add various processes or components as needed. Additionally, the features described relative to some examples can also be combined in other examples.

[0038] Example 1

[0039] In this example, a preparation method of an alkaloid is proposed, including the following steps:

[0040] S1: After crushing the dried stems of Dendrobium huoshanense, reflux extraction is carried out with ethanol, and the obtained ethanol extract is concentrated under reduced pressure until there is no alcohol smell to obtain an ethanol extract;

[0041] S2: The obtained ethanol extract is dissolved in water at a ratio of 1:10, and then extracted successively with petroleum ether, ethyl acetate, and n-butanol to obtain four parts: petroleum ether phase, ethyl acetate phase, n-butanol phase, and water phase;

[0042] S3: The n-butanol phase is separated by silica gel column chromatography, eluted with chloroform:methanol from 80:1 to 0:100 in a gradient manner, and the same fractions are combined according to thin-layer chromatography detection, and then twelve main fractions are obtained. These twelve fractions 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;

[0043] Fraction Fr.G is eluted via silica gel column chromatography using a solvent system of chloroform and methanol at a ratio of 30:1 to separate two sub-fractions Fr.G1 and Fr.G2. Fr.G2 is further separated by Sephadex LH-20 gel column chromatography and eluted with methanol to obtain a secondary fraction Fr.G2a.

[0044] S4: The secondary fraction Fr.G2a is separated by preparative thin-layer chromatography using chloroform and methanol at a ratio of 10:1 as the developing agent, and finally a compound with the molecular formula C 22 H 29 O 12Compound of N3.

[0045] Among them: In step S1, after the dried stems of Dendrobium huoshanense are crushed, they are subjected to three times of reflux extraction with 90% ethanol.

[0046] Example 2

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

[0048] 80 kg of dried stems of Dendrobium huoshanense are crushed and subjected to three times of reflux extraction with 90% ethanol (2×800 L). The combined ethanol extracts are concentrated under reduced pressure to obtain the total alcohol extract (10.5 kg). The obtained extract is dissolved in water and then successively extracted with petroleum ether, ethyl acetate, and n-butanol to obtain the 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 is separated by silica gel column chromatography and eluted with chloroform: methanol from 80:1 to 0:100 gradient, and the same fractions are combined according to thin-layer chromatography detection to obtain twelve main fractions (A - L). Fr.G (5 g) is separated into two sub-fractions, Fr.G1 and Fr.G2, via silica gel column chromatography using a solvent system of chloroform and methanol in a ratio of 30:1. Fr.G2 (200 mg) is further separated by Sephadex LH-20 gel column chromatography and eluted with methanol to obtain a secondary fraction Fr.G2a. Subsequently, Fr.G2a is separated by preparative thin-layer chromatography using a solvent system of chloroform and methanol in a ratio of 10:1, and finally a compound is obtained with a total mass of 55.0 mg.

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

[0050] The 1 1H NMR spectrum of the compound (600 MHz, CD3OD) (Table 1, Figure 6showed the characteristic signals of two aromatic protons [δ H 6.73 (1H, s, H-8), δ H 6.66 (1H, s, H-9)], two methoxyl groups [δ H 3.87 (3H, s, H-20OCH3)] and [δ H 3.85 (3H, s, H-19OCH3)], three methylenes [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, 5.2Hz, H-18)], three hydroxyl groups [δ H 7.33 (1H, s), δ H 6.73 (1H, s), δ H 6.66 (1H, s)], one primary amino group [δ H 3.85 (2H, s)], one secondary amino group [δ H 1.31 (1H, m)]. The remaining signals were for methines. 13 13C NMR (151MHz, CD3OD) and DEPT-135 spectra (Table 1, Figure 7 , 8) in total identified 22 carbon atoms, 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 methoxyl groups (δ C 57.02, C-19; δ C 56.76, C-20), three methylenes (δ C 72.83, C-15; δ C 72.88, C-16; δ C 62.53, C-18), 11 methines. The above spectral data indicated that the compound could be considered a diterpenoid alkaloid with 22 skeletal carbons. This alkaloid was very unique, and this inference was further confirmed by subsequent analysis of the 2D NMR data.

[0051] Analysis of the HSQC and 1 H- 1 HCOSY spectral data provided unambiguous assignments of the hydrogen and carbon resonances associated with the hydrogen atoms in DHS-DA-1 (Table 1, Figure 9 and 10 ). First, the chemical shift values of C1, C2, C3, C4, C5, and C6, which were between 130 and 155, were identified as six quaternary carbon atoms, and it was speculated that they formed a hexasubstituted benzene ring, which was designated as fragment A. Second, cross-peaks between H7 and H10 / H14; H14 and H13; H10 and H21; and H21 and H11 were observed through COSY spectral analysis. These 1 H- 1 HCOSY correlations delineated fragment B containing H13-H14-H7-H10-H21-H11. In addition, cross-peaks between H12 and H17 / H18; H22 and H15 / H16 suggested the presence of two fragments, which were respectively designated as fragment C and fragment D. According to the HMBC spectrum of DHS-DA-1 (Table 1, Figure 11 ), based on the HMBC correlations of C5 with H16 (C-16, δ C 72.88); C4 with H8 (C-8, δ C 104.78), a heteroatom was determined. According to the HMBC cross-peaks from C-14 (δ C 87.15) and C-18 (δ C 62.53) to H-11 (δ H 4.78; C-8, δ C 87.15), it was indicated that the connection between C-11, C-14, and C-18 might be through a nitrogen atom, thus forming a six-membered nitrogen heterocycle. Similarly, according to the HMBC correlation from C-10 (δ C 87.57) to H-9 (δ H 4.73), a five-membered heteroatom connected to an amino group was formed; the HMBC correlations 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 3.41) indicated that a five-membered nitrogen-oxygen heterocycle might exist in DHS-DA-1. In addition, C-11 (δ C 87.15) and H-8 (δ H 6.73), as well as C-21 (δ C 55.69) and H22 (δ HHMBC cross-peaks between 3.14) suggest an oxygen bridge between C-11 / H-8 and C21 (δ C 55.69) / H22. HMBC cross-peaks between C-6 and H-19, and between C-17 and H-20 were also observed. Based on the above analysis, a possible planar structure of an unusual C22-diterpenoid alkaloid was speculated ( Figure 12 ).

[0052] The relative configuration of the compound was elucidated by interpreting the NOESY data ( Figure 13 , Table 1).

[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, H-22 indicate the coplanar orientation of C-8 and N-9. In addition, the correlation between H-12 and H-18, and the cross-peaks connecting H-15 and H-16 with H-21, H-22 of an alkaloid and its preparation method and application indicate the β-orientation 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, it was confirmed to be a new compound by querying the SciFinder database.

[0054] Table 1 1HNMR(600MHz) and 13CNMR(151MHz) data of DHS-DA-1

[0055]

[0056]

[0057]

[0058] Example 3

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

[0060] Cell viability was evaluated by the MTT method. Macrophages were cultured in DMEM containing 10% fetal bovine serum. After incubation for 24 h, 100 µL of the test compound was added. After 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 a wavelength of 570 nm using a microplate reader, and the 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, the cells were cultured in 96-well plates at a density of 9×10 5 cells per well to promote NO production. In addition, 10 μM dexamethasone was used as a positive control, while cell medium without drugs was used as a blank control. The culture plates were pre-treated with different concentrations of DHS-DA-1 for 1 hour and then incubated for 24 hours with or without 1 μg / mL lipopolysaccharide (LPS). RAW264.7 macrophages were treated with 1 μg / mL lipopolysaccharide and DHS-DA-1 for 24 hours. Subsequently, the culture medium was collected to evaluate the expression levels of certain pro-inflammatory cytokines, such as TNF-α and IL-6, and dexamethasone was used as a positive control. The experiments were performed using ELISA kits and were carried out according to the instructions provided by the manufacturer.

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

[0063] In the ELISA assay, the inhibitory effect of DHS-DA-1 on the expression of pro-inflammatory cytokines such as TNF-α and IL-6 was evaluated. The results showed that LPS stimulation led to a significant increase in the levels of these cytokines compared to the control group. In contrast, treatment with DHS-DA-1 significantly reduced the LPS-induced cytokine production, showing a dose-dependent relationship (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 used as a positive control ( Figure 14 C and 14D). These data indicate that DHS-DA-1 exerts an anti-inflammatory effect by regulating the expression of pro-inflammatory cytokines.

[0064] Example 4

[0065] This example was 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 humid environment at 37 °C and 5% CO2. Cell viability was evaluated using the MTT method with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Briefly, cells were seeded into 96-well plates at an initial density of 2×10 4 cells per well per milliliter. After 24 hours of culture, the cells were exposed to different concentrations of the compound for 48 hours, with 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 incubated for 4 hours under humid conditions. Then, the supernatant was removed, and the resulting crystals were dissolved with 150 μL of DMSO. 5-Fluorouracil (Sigma Aldrich Shanghai Trading Co., Ltd.) was used as a positive control. The absorbance was then measured using a microplate reader at a wavelength of 570 nm.

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

[0068] Example 5

[0069] In this example, to further understand the main binding interactions, molecular docking simulations were performed for DHS-DA-1, focusing on key proteins related to inflammation: 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 could effectively bind within the active site of iNOS, as shown in ( Figure 16 A, Table 2), with a calculated binding energy of -9.0 kcal / mol. DHS-DA-1 formed five conventional hydrogen bond interactions with the His101 and Cys115 residues in the active center of iNOS (Figure 16 B, Table 2). In addition, DHS-DA-1 also established hydrophobic interactions with the Ala104 residue ( Figure 16 C), and formed π-cation interactions with the Cys115 residue ( Figure 16 C).

[0071] 5FUC is a complex formed by interleukin-6 (IL-6) and the gp80 receptor. The results of docking studies showed that DHS-DA-1 successfully embedded into the IL-6 binding site, as shown in ( Figure 17 A, Table 2), and the obtained binding energy was -9.5 kcal / mol. In addition, DHS-DA-1 interacted with Arg40 through hydrogen bonds ( Figure 17 B). It is worth noting that the key intermolecular forces inhibiting IL-6 activity seem to involve specific types of hydrogen bonds, including two carbon-hydrogen bond interactions and two attractive charge interactions with the Glu172 residue ( Figure 17 C).

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

[0073] Table 2 Binding energies 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 effects on 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. As shown in the 3D figure ( Figure 7As shown in (), 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 center of MMP9 ( Figure 19 C). In addition, it also forms a π-donor hydrogen bond interaction and a π-cation interaction with Tyr218 at the active center ( Figure 19 B).

[0076] The present invention provides an alkaloid DHS-DA-1, which is isolated from the n-butanol fraction of the stem of D. huoshanense by silica gel column chromatography and preparative thin layer chromatography, and its structure is elucidated by nuclear magnetic resonance and high-resolution mass spectrometry analysis. The results of anti-inflammatory studies show that DHS-DA-1 can significantly alleviate LPS-induced inflammation, which is demonstrated by the reduction of nitric oxide (NO) production and the decrease in the expression levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Molecular docking analysis also shows that the binding interactions of DHS-DA-1 with proteins such as iNOS, TNF-α, and IL-6 are significant. In addition, DHS-DA-1 can effectively inhibit the proliferation of AGS cells in a concentration-dependent manner and promote their apoptosis. Molecular docking further reveals the possible mechanisms of the anti-inflammatory and anti-gastric cancer activities of DHS-DA-1. As a potential resource for discovering novel anti-inflammatory alkaloids, DHS-DA-1 provides theoretical insights into the derivation of functional components from natural products for gastric cancer prevention and treatment. Therefore, this study is of great significance for further exploring the medicinal properties of this rare and endangered traditional Chinese medicine, D. huoshanense.

[0077] The above describes the embodiments of this example, but this example is not limited to the above specific implementation manners. The above specific implementation manners are merely illustrative and not restrictive. Under the inspiration of this example, those of ordinary skill in the art can also make many forms, all of which fall within the protection scope of this example.

Claims

1. An alkaloid, characterized in that, Its molecular formula is C 22 H 29 O 12 N3, and it 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 salts include salts formed by the compound with inorganic acids and organic acids.

3. An alkaloid according to claim 2, characterized in that, The pharmaceutically acceptable salts include hydrochloride, sulfate, bisulfate, phosphate, acetate, propionate, butyrate, lactate, mesylate, tosylate, maleate, benzoate, succinate, tartrate, citrate, fumarate, taurate, gluconate.

4. A method for preparing an alkaloid, characterized in that, It includes the following steps: S1: After pulverizing the dried stems of Dendrobium huoshanense, reflux extraction is carried out with ethanol, and the obtained ethanol extract is concentrated under reduced pressure until there is no alcohol smell to obtain an ethanol extract; S2: The obtained ethanol extract is dissolved in water at a ratio of 1:10, and then successively extracted with petroleum ether, ethyl acetate and n-butanol to obtain four parts: petroleum ether phase, ethyl acetate phase, n-butanol phase and water phase; S3: The n-butanol phase is separated by silica gel column chromatography, eluted with chloroform: methanol from 80:1 to 0:100 in a gradient manner, and the same fractions are combined according to thin layer chromatography detection, and then twelve main fractions are obtained. These twelve fractions 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; 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. Fr.G2 is further subjected to Sephadex LH-20 gel column chromatography and eluted with methanol to obtain a secondary fraction Fr.G2a; S4: The secondary fraction Fr.G2a was separated by preparative thin layer chromatography using chloroform and methanol in a ratio of 10:1 as the developing agent, and finally a compound with the molecular formula C 22 H 29 O 12 N3 was obtained.

5. The preparation method of an alkaloid according to claim 4, characterized in that, In step S1, after pulverizing the dried stems of Dendrobium huoshanense, reflux extraction is carried out with 90% ethanol.

6. Use of an alkaloid, characterized in that, Use in the preparation of drugs with in vitro anti-inflammatory and anti-gastric cancer activities.

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