Preparation method of vetiver spirane sesquiterpenoids extracted from belladonna roots and application of vetiver spirane sesquiterpenoids

Abstract

The invention relates to a preparation method and application of a vetiver spirane type sesquiterpenoid compound extracted from belladonna roots, effectively solves the problem of preparing a new vetiver spirane type sesquiterpenoid compound from the belladonna roots and realizes the application of the new vetiver spirane type sesquiterpenoid compound in development of anti-inflammatory drugs, and the compound is (3R, 5S, 6R)-solavetivone-13-O-beta-D-glucose-(1-> 6)-beta-D-glucopyranoside, and the molecular formula of the compound is C27H42O12. The method comprises the following steps: soaking and extracting belladonna roots with diluted hydrochloric acid, concentrating under reduced pressure, dispersing with water, adjusting the pH value, extracting with ethyl acetate and n-butyl alcohol in sequence, recovering a solvent from n-butyl alcohol extract under reduced pressure, carrying out alkaline silica gel column chromatography, gradient elution with a dichloromethane-methanol system, Toyopeal column chromatography and dichloromethane-methanol 1: 1 elution on the obtained extract, merging fractions with the same main spots, concentrating under reduced pressure, concentrating under reduced pressure, concentrating under reduced pressure, concentrating under reduced pressure, concentrating under reduced pressure, and concentrating under reduced pressure. And carrying out HPLC (High Performance Liquid Chromatography) separation by using an ODS-AQ column, and collecting a chromatographic peak with the retention time tR of 29.5-30.7 minutes to obtain a compound 1. The raw materials are rich, the preparation method is easy to operate, the medicine is effectively used for preparing the anti-inflammatory medicine, and the new application and the medicinal value of the belladonna roots are developed.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a vetiverane-type sesquiterpenoid compound, an extraction method and application thereof. Background Art

[0002] Atropa belladonna L. is a perennial herbaceous plant of the genus Atropa in the Solanaceae family, native to Europe. It has been introduced and cultivated in provinces and cities in my country, including Shandong, Hunan, Henan, and Zhejiang. Its roots, stems, and leaves are all used medicinally, with a global history of nearly 2,000 years. It is included in the pharmacopoeias of China, the United States, Japan, and other countries, primarily for the treatment of gastric spasms, gastric ulcers, duodenal ulcers, and biliary colic. Scopolamine alkaloids are its main active ingredients. Current research on Atropa belladonna, both domestically and internationally, has primarily focused on the determination, quality control, and pharmacological effects of these alkaloids, with limited research on characteristic components other than the alkaloids. Furthermore, there have been few reports on the chemical composition of Atropa belladonna roots in recent years, and to date, no published reports have been published on the preparation of vetiverane-type sesquiterpenes with anti-inflammatory activity from Atropa belladonna roots. Summary of the Invention

[0003] (1) Technical problems solved

[0004] In view of the deficiencies of the existing technology, the present invention provides a vetiverane-type sesquiterpenoid compound extracted from the root of belladonna, and an extraction method and application thereof, which solves the technical problem of how to extract vetiverane-type sesquiterpenoid compounds from the root of belladonna.

[0005] (2) Technical solution

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] In a first aspect, the present invention provides a vetiverane-type sesquiterpenoid compound extracted from the root of belladonna, wherein the compound is:

[0008] (3R,5S,6R)-solavetivone-13-O-β-D-glucose-(1→6)-β-D-glucopyranoside, whose molecular formula is C 27 H 42 O 12 , the compound structure is shown in Formula I,

[0009]

[0010] Preferably, the compound has a degree of unsaturation of 7.

[0011] Preferably, the vetiverane-type sesquiterpenoid compound has anti-inflammatory activity. As the concentration of the vetiverane-type sesquiterpenoid compound increases, the anti-inflammatory activity first increases and then decreases. When the compound concentration is 50 μM, it has a significant inhibitory effect on NO release.

[0012] In a second aspect, the present invention provides a method for extracting the vetiverane-type sesquiterpenoid compound as described in the first aspect, comprising the following steps:

[0013] (1) The dried roots of belladonna were crushed, and a 0.03 mol / L hydrochloric acid solution was added at a solid-liquid ratio of 1 g:5-10 mL. The mixture was immersed and extracted 2-3 times at room temperature, each time for 5-7 days. The filtrates were combined and concentrated under reduced pressure to obtain a total alkaloid extract.

[0014] (2) suspending the total alkaloid extract in distilled water, adjusting the suspension to pH 3 with a 5% hydrochloric acid solution, and extracting with ethyl acetate to obtain an ethyl acetate extract with a pH of 3 and an acid solution;

[0015] (3) adjusting the acid aqueous solution to pH = 7 with 25% concentrated ammonia solution, and extracting with ethyl acetate to obtain an ethyl acetate extract with a pH = 7 and a neutral solution;

[0016] (4) adjusting the neutral solution to pH = 10 with a 25% concentrated ammonia solution, extracting with water-saturated n-butanol to obtain an n-butanol extract with a pH of 10, and recovering the solvent under reduced pressure to obtain an n-butanol extract;

[0017] (5) The n-butanol extract was dissolved in methanol, and dry-mixed with alkaline silica gel at a mass ratio of 1:1. The sample was loaded onto an alkaline silica gel column with a diameter-to-height ratio of 1:3. Gradient elution was performed using a dichloromethane-methanol system with volume ratios of 1:0, 50:1, 25:1, 10:1, and 0:1, with 5 column volumes of elution at each ratio. Anisaldehyde-concentrated sulfuric acid was used for spray detection, and fractions with the same main spot were combined. The solvent was recovered under reduced pressure to obtain components Fr.A, Fr.B, Fr.C, Fr.D, and Fr.E.

[0018] (6) The component Fr.D was dissolved in methanol and dry-mixed with alkaline silica gel at a mass ratio of 1:1. The sample was loaded onto an alkaline silica gel column with a diameter-to-height ratio of 1:6. Gradient elution was performed using a dichloromethane-methanol system with volume ratios of 50:1, 25:1, 10:1, and 0:1, with 8 column volumes eluted at each ratio. Anisaldehyde-concentrated sulfuric acid spray detection was used, and the same fractions of the main spot were combined. The solvent was recovered under reduced pressure to obtain components Fr.D-1 to Fr.D-4.

[0019] (7) The component Fr.D-1 was dissolved in dichloromethane-methanol in a volume ratio of 1:1 and separated by Toyopeal column chromatography. The elution was isocratic with dichloromethane-methanol in a volume ratio of 1:1 at a flow rate of 1.5 mL / min. Anisaldehyde-concentrated sulfuric acid spray detection was performed, and detection was performed once every 15 mL. The fractions with the same main spot were combined and the solvent was recovered under reduced pressure to obtain components Fr.D-1-1 to Fr.D-1-6.

[0020] (8) The component Fr.D-1-3 was dissolved in methanol and further separated on a semi-preparative HPLC with an ODS-AQ column. The mobile phase was acetonitrile: water in a volume ratio of 25:75 at a flow rate of 3 mL / min. The retention time t was collected. R = =29.5~30.7min chromatographic peak, the solvent was recovered under reduced pressure to obtain vetiverane-type sesquiterpenoid compounds.

[0021] Preferably, step (1) satisfies at least one of the following conditions:

[0022] The dosage ratio of belladonna root to dilute hydrochloric acid is 1g:7mL;

[0023] The number of maceration extractions was 2;

[0024] The immersion and extraction time for each time was 7 days.

[0025] In a third aspect, the present invention provides a use of the vetiverane-type sesquiterpenoid compound as described in the first aspect in the preparation of anti-inflammatory drugs.

[0026] Preferably, the anti-inflammatory drug is prepared using the vetiverane-type sesquiterpenoid compound as an active ingredient.

[0027] (3) Beneficial effects

[0028] The present invention provides a vetiverane-type sesquiterpenoid compound, its extraction method, and its application. Compared with the existing technology, it has the following beneficial effects:

[0029] The extraction method disclosed in the present invention is easy to operate, has strong guidance, and produces high product purity. The extracted vetiverane-type sesquiterpenoid compounds have a significant inhibitory effect on the production of nitric oxide in RAW264.7 cells induced by LPS. As the concentration of the compound increases, the inhibitory effect first increases and then weakens. Therefore, the compound can be used as an active ingredient in the preparation of anti-inflammatory drugs, expanding the medicinal value of belladonna roots and having huge economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 is the compound 1 of this example 1 H-NMR (CD3OD, 500 MHz) spectrum.

[0032] Figure 2 is the compound 1 of this example 13 C-NMR (CD3OD, 500 MHz) spectrum.

[0033] Figure 3 HSQC correlation spectrum of compound 1 in this example.

[0034] Figure 4 This is the HMBC correlation spectrum of compound 1 in this example.

[0035] Figure 5 This is the key NOESY correlation spectrum of compound 1 in this example.

[0036] Figure 6 The experimental and calculated ECD spectra of compound 1 in this example.

[0037] Figure 7 This is the IR spectrum of compound 1 in this example.

[0038] Figure 8 is the structural formula of compound 1 in this example. DETAILED DESCRIPTION

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0040] The embodiments of the present application provide a method for extracting vetiverane-type sesquiterpenoid compounds from belladonna roots, and an extraction method and application thereof, thereby solving the technical problem of how to extract vetiverane-type sesquiterpenoid compounds from belladonna roots.

[0041] The technical solution in the embodiments of the present application is to solve the above technical problems, and the overall idea is as follows:

[0042] Compound 1 with anti-inflammatory activity was extracted from the roots of belladonna, specifically (3R,5S,6R)-solavetivone-13-O-β-D-glucose-(1→6)-β-D-glucopyranoside, whose molecular formula is C 27 H 42 O 12 , the unsaturation is 7.

[0043] The preparation method thereof comprises the following steps:

[0044] (1) The dried roots of belladonna were crushed, and a 0.03 mol / L hydrochloric acid solution was added at a solid-liquid ratio of 1 g:5-10 mL. The mixture was immersed and extracted 2-3 times at room temperature, each time for 5-7 days. The filtrates were combined and concentrated under reduced pressure to obtain a total alkaloid extract.

[0045] (2) suspending the total alkaloid extract in distilled water, adjusting the suspension to pH 3 with a 5% hydrochloric acid solution, and extracting with ethyl acetate to obtain an ethyl acetate extract with a pH of 3 and an acid solution;

[0046] (3) adjusting the acid aqueous solution to pH = 7 with 25% concentrated ammonia solution, and extracting with ethyl acetate to obtain an ethyl acetate extract with a pH = 7 and a neutral solution;

[0047] (4) adjusting the neutral solution to pH = 10 with a 25% concentrated ammonia solution, extracting with water-saturated n-butanol to obtain an n-butanol extract with a pH of 10, and recovering the solvent under reduced pressure to obtain an n-butanol extract;

[0048] (5) The n-butanol extract was dissolved in methanol, and dry-mixed with alkaline silica gel at a mass ratio of 1:1. The sample was loaded onto an alkaline silica gel column with a diameter-to-height ratio of 1:3. Gradient elution was performed using a dichloromethane-methanol system with volume ratios of 1:0, 50:1, 25:1, 10:1, and 0:1, with 5 column volumes of elution at each ratio. Anisaldehyde-concentrated sulfuric acid was used for spray detection, and fractions with the same main spot were combined. The solvent was recovered under reduced pressure to obtain components Fr.A, Fr.B, Fr.C, Fr.D, and Fr.E.

[0049] (6) The component Fr.D was dissolved in methanol and dry-mixed with alkaline silica gel at a mass ratio of 1:1. The sample was loaded onto an alkaline silica gel column with a diameter-to-height ratio of 1:6. Gradient elution was performed using a dichloromethane-methanol system with volume ratios of 50:1, 25:1, 10:1, and 0:1, with 8 column volumes eluted at each ratio. Anisaldehyde-concentrated sulfuric acid spray detection was used, and the same fractions of the main spot were combined. The solvent was recovered under reduced pressure to obtain components Fr.D-1 to Fr.D-4.

[0050] (7) The component Fr.D-1 was dissolved in dichloromethane-methanol in a volume ratio of 1:1 and separated by Toyopeal column chromatography. The elution was isocratic with dichloromethane-methanol in a volume ratio of 1:1 at a flow rate of 1.5 mL / min. Anisaldehyde-concentrated sulfuric acid spray detection was performed, and detection was performed once every 15 mL. The fractions with the same main spot were combined and the solvent was recovered under reduced pressure to obtain components Fr.D-1-1 to Fr.D-1-6.

[0051] (8) The component Fr.D-1-3 was dissolved in methanol and further separated on a semi-preparative HPLC with an ODS-AQ column. The mobile phase was acetonitrile: water in a volume ratio of 25:75 at a flow rate of 3 mL / min. The retention time t was collected. R = =29.5~30.7min chromatographic peak, the solvent was recovered under reduced pressure to obtain vetiverane-type sesquiterpenoid compounds.

[0052] Furthermore, in step (1), the ratio of belladonna root to dilute hydrochloric acid is 1 g:7 mL, the number of immersion extractions is 2 times, and the immersion extraction time for each time is 7 days.

[0053] The new compound extracted from the root of belladonna was identified as a sesquiterpenoid compound of the vetiverane type. The extraction method is easy to operate, has strong guidance, and the product purity is high. The compound can be effectively used to prepare anti-inflammatory drugs, which has expanded the medicinal value of belladonna root and has huge economic and social benefits.

[0054] In order to better understand the above technical solution, the above technical solution will be described in detail below with reference to the accompanying drawings and specific implementation methods.

[0055] Example 1

[0056] This embodiment provides a method for extracting vetiverane-type sesquiterpenoid compounds from the roots of belladonna, comprising the following steps:

[0057] (1) 30 kg of dried belladonna root was crushed, and 0.03 mol / L hydrochloric acid solution was added at a solid-liquid ratio of 1 g:7 mL. The mixture was immersion-extracted twice at room temperature, each time for 7 days. The filtrates were combined and concentrated under reduced pressure to obtain 1.2 kg of total alkaloid extract.

[0058] (2) suspending the total alkaloid extract of step (1) in distilled water, adjusting the suspension to pH = 3 with 5% hydrochloric acid solution, and extracting with ethyl acetate five times to obtain an ethyl acetate extract with a pH = 3 and an acid solution;

[0059] (3) The acid solution was adjusted to pH = 7 with 25% concentrated ammonia solution, and extracted again with ethyl acetate 5 times, and the pH = 7 ethyl acetate extract and neutral solution were obtained;

[0060] (4) The neutral solution was adjusted to pH = 10 with 25% concentrated ammonia solution, extracted 5 times with water-saturated n-butanol to obtain an n-butanol extract with a pH of 10, and the solvent was recovered under reduced pressure to obtain 182.2 g of n-butanol extract;

[0061] (5) The n-butanol extract was dissolved in methanol and dry-mixed with alkaline silica gel at a mass ratio of 1:1. The sample was loaded onto an alkaline silica gel column with a diameter-to-height ratio of 1:3. Gradient elution was performed using a dichloromethane-methanol system with volume ratios of 1:0, 50:1, 25:1, 10:1, and 0:1, with 5 column volumes of elution at each ratio. Anisaldehyde-concentrated sulfuric acid spray detection was used, and the fractions with the same main spot were combined. The solvent was recovered under reduced pressure to obtain components Fr.A, Fr.B, Fr.C, Fr.D, and Fr.E.

[0062] (6) Component Fr.D (45.3 g) was dissolved in methanol and dry-mixed with alkaline silica gel at a mass ratio of 1:1. The sample was loaded onto an alkaline silica gel column with a diameter-to-height ratio of 1:6. Gradient elution was performed using a dichloromethane-methanol system with volume ratios of 50:1, 25:1, 10:1, and 0:1, with 8 column volumes eluted at each ratio. Anisaldehyde-concentrated sulfuric acid spray detection was used, and the same fractions of the main spot were combined. The solvent was recovered under reduced pressure to obtain components Fr.D-1, Fr.D-2, Fr.D-3, and Fr.D-4.

[0063] (7) Component Fr.D-1 (4.2 g) was dissolved in dichloromethane-methanol in a volume ratio of 1:1 and separated by Toyopeal column chromatography. Elution was performed isocratically with dichloromethane-methanol in a volume ratio of 1:1 at a flow rate of 1.5 mL / min. Spray detection was performed using anisaldehyde-concentrated sulfuric acid, with detection occurring every 15 mL. Fractions with the same main spot were combined and the solvent was recovered under reduced pressure to obtain components Fr.D-1-1 to Fr.D-1-6.

[0064] (8) Component Fr.D-1-3 was dissolved in methanol and further separated on a semi-preparative HPLC with an ODS-AQ column. The mobile phase was acetonitrile: water in a volume ratio of 25:75 at a flow rate of 3 mL / min. The retention time t was collected. R = =29.5~30.7min chromatographic peak, the solvent was recovered under reduced pressure to obtain compound 1.

[0065] The structure of the compound 1 is as follows:

[0066] (3R,5S,6R)-solavetivone-13-O-β-D-glucose-(1→6)-β-D-glucopyranoside), the molecular structure is as follows Figure 8 Activity testing of compound 1 showed that it had anti-inflammatory activity, and as the dosage of compound 1 increased, its anti-inflammatory activity first increased and then weakened.

[0067] The specific structure determination and activity detection methods are as follows:

[0068] (1) Structure determination

[0069] Compound 1: yellow powder, easily soluble in methanol. IR spectrum (see Figure 7 ) showed that the compound contained hydroxyl groups (3217 cm -1 ), carbonyl (1707cm -1 ) and double bond (1597cm -1 ) and other structural fragments. HR-ESI-MS m / z581.2555[M+Na] + (called for C 27 H 42 O 12 Na, 581.2568), the molecular formula of the compound is determined to be C 27 H 42 O 12 .

[0070] 1 H-NMR spectrum (see Figure 1 ) shows two groups of olefinic hydrogen proton signals [δ H 5.73 (1H, s, H-9), 5.16 (1H, s, H-12α), 5.01 (1H, s, H-12β)], 2 groups of methyl hydrogen signals [δ H 2.00 (3H, s, H-10), 0.99 (3H, d, J = 6.7 Hz, H-6)], a series of aliphatic hydrogen signals δ H 1.58-2.78, the remaining signals of the hydrogen spectrum can be attributed to two glucose groups, and according to the end group hydrogen coupling constant, they are all β-configuration [δ H 4.64(1H,d,J=7.8Hz,H-1”), 4.44(1H,d,J=8.0Hz,H-1′). 13 The C-NMR spectrum showed 27 carbon signals, including 4 olefinic carbon signals [δ C 111.4 (C-12), 125.8 (C-9), 148.9 (C-11), 170.6 (C-10)], a conjugated carbonyl carbon signal [δ C 202.0(C-8)](see Figure 2 ). Combined with the HSQC spectrum, in addition to the sugar signal, the carbon spectrum also shows a quaternary carbon signal [δ C 51.6(C-5)], 2 methyl carbon signals [δ C 16.1(C-14),21.2(C-15)], 4 methylene carbon signals [δ C42.1(C-1),34.1(C-3),35.5(C-4),43.8(C-7)], 1 methine carbon signal [δ C 40.4(C-6)](see Figure 3 ), the above NMR data suggest that the compound is a sesquiterpene derivative with α,β-unsaturated ketone.

[0071] HMBC spectrum (see Figure 4 ) showed that H-7 was related to C-6 and C-8, H-14 was related to C-5, C-6 and C-7, and H-15 was related to C-5, C-10 and C-9, suggesting the presence of cyclohexenone fragment in the compound. 1 H- 1 The H COSY spectrum revealed correlations between H2-1 / H-2, H-2 / H2-3, and H2-3 / H2-4, confirming the presence of a C1(H2)-C2(H)-C3(H2)-C4(H2) fragment in the structure, along with the formation of a cyclopentane fragment. The HMBC spectrum revealed correlations between H-1 and H-4 with C-6 and C-10, respectively, while H-2 with C5 and C-4, respectively. This confirmed that the cyclohexadienone fragment was linked to the cyclopentane via a spirocyclic ring at the C-5 position, suggesting that the compound is a sesquiterpene glycoside of the vetiverane type. The HMBC spectrum revealed correlations between H2-13 and C-2, C-11, and C-12, and between H2-12 and C-11 and C-2, suggesting the presence of a propenyl fragment at C-2, with a glucosyl group at C-13. Therefore, the planar structure of compound 1 was determined to be: solavetivone-13-O-β-D-glucose-(1→6)-β-D-glucopyranoside.

[0072] The NOESY spectrum showed the presence of related signals at H-2 / H-1β, H-3β, H12β, H-3β / H-4β, H-6 / H-1β, H-2, H-4β, H-1α / H-4α / H2-13, and H3-14 / H-4β, which confirmed the relative configuration of compound 1 (see Figure 5 The stereo configuration of the compound was determined by comparing the experimental and theoretical ECD data and combining CAD technology. The compound was hydrolyzed to obtain aglycone and glucose. The glucose residue was analyzed by CAD detector and determined to be D configuration by comparing the retention time with the reference substance. The calculated ECD spectrum of compound 1 is basically consistent with the experimental ECD spectrum of the compound (see Figure 6 ), thus determining its absolute configuration to be 3R,5S,6R.

[0073] Table 1 NMR data assignment of compound 1 (in CD3OD)

[0074]

[0075] (2) Activity detection

[0076] (1) Cytotoxicity assay of compound 1

[0077] RAW264.7 cells in the logarithmic growth phase were taken and diluted to 1×10 5 Cell suspension of 100 μL / mL was inoculated into a 96-well plate, 100 μL of cell suspension was added to each well, and cultured with DMEM medium containing 10% fetal bovine serum. After culturing in a 37°C, 5% CO2 incubator for 24 hours, the supernatant was discarded. 100 μL of complete culture medium containing compound 1 was added respectively, and the compound concentration was set to 0, 5, 10, 25, 50, and 100 μM, i.e., blank group and drug-treated group. Three replicate wells were set for each group, and cultured in a 37°C, 5% CO2 incubator for 24 hours. 10 μL of MTT was added to each well, and culture was continued for 3 hours. The supernatant was discarded, 100 μL of DMSO was added to each well, and after shaking on a shaker for 10 minutes, the purple crystals were fully dissolved, and the OD value of each well was measured at 490 nm using an enzyme marker. The cell survival rate was calculated, and the cell survival rate = (A 测 / A 空 ) × 100% (Note: A is the OD value of the drug group minus the OD value of the blank group; A is the OD value of the control group minus the OD value of the blank group). The experiment was repeated three times independently. Compound 1 showed no cytotoxic activity against RAW264.7 cells at concentrations of 5-100 μM compared to the blank group, with cell viability exceeding 95%. Therefore, 5, 10, 25, 50, and 100 μM were selected as subsequent dosing concentrations.

[0078] (2) Anti-inflammatory activity detection of compound 1

[0079] Cells in the logarithmic growth phase were seeded in 24-well plates (2×10 5 Cells were cultured in a 37°C, 5% CO2 incubator for 24 hours and divided into blank, model, and treatment groups. The treatment groups were treated with compound 1 at varying concentrations (5, 10, 25, 50, and 100 μM). After 2 hours of incubation, LPS was added to all groups except the blank group to a final concentration of 1 μg / mL. After another 24 hours of incubation, 100 μL of the culture supernatant was transferred to a microplate reader. The OD value was measured at 540 nm using a microplate reader according to the NO kit instructions, and the NO release inhibition rate was calculated. Three independent experiments were performed for each group. The results are shown in Table 2.

[0080] Calculation formula: NO inhibition rate = (NO content in model group - NO content in drug-treated group) / (NO content in model group - NO content in blank group) × 100%.

[0081] Table 2 Effect of compound 1 on LPS-induced NO release in RAW264.7 cells

[0082]

[0083] As shown in Table 2, compound 1 at various concentrations inhibited LPS-induced NO release from RAW264.7 cells, indicating that compound 1 possesses strong anti-inflammatory activity. As shown in Table 2, with increasing concentrations of compound 1, NO inhibition initially increased and then decreased. Therefore, the anti-inflammatory activity of compound 1 initially increased and then decreased with increasing dosage. When the dosage reached 50 μM, the NO inhibition rate reached a peak of 86.82 ± 8.91, indicating the strongest anti-inflammatory activity.

[0084] The present invention has been identified as a vetiverane-type sesquiterpenoid compound extracted from the root of belladonna. The preparation method is easy to operate and highly directional. The vetiverane-type sesquiterpenoid compound 1 of the present invention can effectively inhibit the production of the inflammatory factor NO and is effectively used in the preparation of anti-inflammatory drugs. This realizes the use of compound 1 in the preparation of anti-inflammatory drugs, opens up new drug value of belladonna root, and has great development and application prospects, with huge economic and social benefits.

[0085] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or device comprising the element.

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

[0087] The present invention uses the above-described embodiments to illustrate the detailed process flow of the present invention. However, the present invention is not limited to the above-described detailed process flow, that is, it does not mean that the present invention must rely on the above-described detailed process flow to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements of various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A vetivan-type sesquiterpenoid compound, characterized in that: The compound molecular formula is C 27 H 42 O 12 , the compound structure is shown in Formula 1, 2. The vetiverane-type sesquiterpenoid compound according to claim 1, characterized in that: The compound is: (3R,5S,6R)-solavetivone-13-O-β-D-glucose-(1→6)-β-D-glucopyranoside, wherein the compound has an unsaturation degree of 7.

3. The vetiverane-type sesquiterpenoid compound according to claim 1, wherein: The vetiverane-type sesquiterpenoid compound has anti-inflammatory activity, and as the concentration of the vetiverane-type sesquiterpenoid compound increases, the anti-inflammatory activity first increases and then decreases.

4. A method for extracting vetiverane-type sesquiterpenoid compounds according to any one of claims 1 to 3, characterized in that: The steps include: (1) The dried roots of belladonna were crushed, and a 0.03 mol / L hydrochloric acid solution was added at a solid-liquid ratio of 1 g:5-10 mL. The mixture was immersed and extracted 2-3 times at room temperature, each time for 5-7 days. The filtrates were combined and concentrated under reduced pressure to obtain a total alkaloid extract. (2) suspending the total alkaloid extract in distilled water, adjusting the suspension to pH 3 with a 5% hydrochloric acid solution, and extracting with ethyl acetate to obtain an ethyl acetate extract with a pH of 3 and an acid solution; (3) adjusting the acid aqueous solution to pH = 7 with 25% concentrated ammonia solution, and extracting with ethyl acetate to obtain an ethyl acetate extract with a pH = 7 and a neutral solution; (4) adjusting the neutral solution to pH = 10 with a 25% concentrated ammonia solution, extracting with water-saturated n-butanol to obtain an n-butanol extract with a pH of 10, and recovering the solvent under reduced pressure to obtain an n-butanol extract; (5) The n-butanol extract was dissolved in methanol, and dry-mixed with alkaline silica gel at a mass ratio of 1:

1. The sample was loaded onto an alkaline silica gel column with a diameter-to-height ratio of 1:

3. Gradient elution was performed using a dichloromethane-methanol system with volume ratios of 1:0, 50:1, 25:1, 10:1, and 0:1, with 5 column volumes of elution at each ratio. Anisaldehyde-concentrated sulfuric acid was used for spray detection, and fractions with the same main spot were combined. The solvent was recovered under reduced pressure to obtain components Fr.A, Fr.B, Fr.C, Fr.D, and Fr.E. (6) The component Fr.D was dissolved in methanol and dry-mixed with alkaline silica gel at a mass ratio of 1:

1. The sample was loaded onto an alkaline silica gel column with a diameter-to-height ratio of 1:

6. Gradient elution was performed using a dichloromethane-methanol system with volume ratios of 50:1, 25:1, 10:1, and 0:1, with 8 column volumes eluted at each ratio. Anisaldehyde-concentrated sulfuric acid spray detection was used, and the same fractions of the main spot were combined. The solvent was recovered under reduced pressure to obtain components Fr.D-1 to Fr.D-4. (7) The component Fr.D-1 was dissolved in dichloromethane-methanol in a volume ratio of 1:1 and separated by Toyopeal column chromatography. The elution was isocratic with dichloromethane-methanol in a volume ratio of 1:1 at a flow rate of 1.5 mL / min. Anisaldehyde-concentrated sulfuric acid spray detection was performed, and detection was performed once every 15 mL. The fractions with the same main spot were combined and the solvent was recovered under reduced pressure to obtain components Fr.D-1-1 to Fr.D-1-6. (8) The component Fr.D-1-3 was dissolved in methanol and further separated on a semi-preparative HPLC with an ODS-AQ column. The mobile phase was acetonitrile: water in a volume ratio of 25:75 at a flow rate of 3 mL / min. The retention time t was collected. R = =29.5~30.7min chromatographic peak, the solvent was recovered under reduced pressure to obtain vetiverane-type sesquiterpenoid compounds.

5. The preparation method according to claim 4, characterized in that The step (1) satisfies at least one of the following conditions: The dosage ratio of belladonna root to dilute hydrochloric acid is 1g:7mL; The number of maceration extractions was 2; The immersion and extraction time for each time was 7 days.

6. A use of the vetiverane-type sesquiterpenoid compound according to any one of claims 1 to 3, characterized in that: The vetiverane-type sesquiterpenoid compound is used for preparing anti-inflammatory drugs.

7. The use according to claim 6, characterized in that The anti-inflammatory drug is prepared by taking the vetiverane-type sesquiterpenoid compound as an active ingredient.

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