Compound extracted from belladonna roots and having anti-inflammatory activity as well as preparation method and application of compound

Abstract

The invention relates to a compound which is extracted from belladonna roots and has anti-inflammatory activity as well as a preparation method and application of the compound, the compound with a novel structure is found from the belladonna roots, the application problem of the novel compound in preparation of anti-inflammatory drugs is realized, and the technical scheme for solving the problem is that the compound is (2S)-1, 2-trigoyl-3-beta-D-glauronide-glycinol, the molecular formula is C19H27O11, the unsaturation degree is 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17 According to the present invention, the raw materials are rich, the preparation method is easy to operate, the guidance quality is strong, the product purity is high, and the compound has significant inhibition effect on the generation of RAW264.7 cell nitric oxide with LPS-induced inflammatory reaction, has anti-inflammatory activity, can be effectively used for preparing anti-inflammatory drugs, develops the medicinal value of the belladonna root, and has significant economic and social benefits.

Description

1. Technical Field

[0001] The invention relates to the field of medicine, in particular to a compound with anti-inflammatory activity extracted from belladonna roots and a preparation method and application thereof. 2. Background Technology

[0002] Belladonna root is the dried root of Atropa belladonna L., a plant of the Solanaceae family. It is a traditional medicinal plant and an important source plant for tropane alkaloids as specified in the Chinese Pharmacopoeia. It has the effects of antispasmodic, analgesic, antisecretory, and mydriasis. It is mainly used to stop night sweats, gastric spasm, gastric ulcer, duodenal ulcer, bronchial hypersecretion, relieve cardia and pyloric spasm, and stop spasmodic cough. Belladonna root has rich pharmacological effects and is currently mainly used to make clinical preparations such as belladonna extract, belladonna fluid extract, belladonna tablets, belladonna tincture, and vitamin U belladonna aluminum capsules. However, there is little research on its chemical components, and most of the chemical components are known. Whether new compounds can be extracted from belladonna roots and used in the preparation of anti-inflammatory drugs has not been reported yet. III. Summary of the invention

[0003] In view of the above situation, in order to solve the defects of the prior art, the purpose of the present invention is to provide a compound with anti-inflammatory activity extracted from belladonna roots and its preparation method and application, which can effectively solve the problem of discovering novel structural compounds from belladonna roots and realizing the application of new compounds in the preparation of anti-inflammatory drugs.

[0004] The technical solution provided by the present invention is to provide a compound with anti-inflammatory activity extracted from the root of belladonna, the compound is (2S)-1,2-tigloyl-3-β-D-glucuronide-glycerol (compound 1), the molecular formula is C 19 H 27 O 11 , the unsaturation degree is 6, and the structural formula is:

[0005]

[0006] The preparation method of the compound comprises the following steps:

[0007] 1) Crush the dried roots of Belladonna, extract them twice in a 0.03 mol / L dilute hydrochloric acid solution at room temperature, let the extract stand, filter to remove the residue, and concentrate the filtrate to obtain a total alkaloid extract;

[0008] 2) dissolving and dispersing the total alkaloid extract obtained in step 1) in distilled water, adjusting the pH to 3 with a 5% hydrochloric acid solution, extracting with ethyl acetate to remove acidic and extremely small amounts of low-polar components; adjusting the pH to 7 with a 25% concentrated ammonia solution, extracting with ethyl acetate again to remove neutral components; adjusting the pH to 10 with a 25% concentrated ammonia solution, extracting with water-saturated n-butanol, and recovering the solvent under reduced pressure to obtain an n-butanol fraction;

[0009] 3) The n-butanol fraction obtained in step 2) was subjected to alkaline silica gel column chromatography, gradient elution was performed using a dichloromethane-methanol system, and detection was performed using anisaldehyde-concentrated sulfuric acid spray, and the same fractions of the main spot were combined to obtain components Fr.A to Fr.E;

[0010] 4) The component Fr.D obtained in step 3) was subjected to alkaline silica gel column chromatography, gradient elution was performed using a dichloromethane-methanol system, and anisaldehyde-concentrated sulfuric acid spray was used for identification, and the same fractions of the main spot were combined to obtain components Fr.D-1 to Fr.D-4;

[0011] 5) The component Fr.D-1 obtained in step 4) was subjected to Toyopeal column chromatography, eluted with dichloromethane-methanol 1:1, and sprayed with anisaldehyde-concentrated sulfuric acid for identification. 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;

[0012] 6) The component Fr.D-1-5 obtained in step 5) was separated on a semi-preparative HPLC using an ODS-AQ column, and the residues with retention time t R = =18.2~19.7min chromatographic peak, and recover the solvent under reduced pressure to obtain the compound 1 of the present invention.

[0013] The invention discloses an application of a compound with anti-inflammatory activity extracted from the root of belladonna in the preparation of an anti-inflammatory drug.

[0014] The invention has abundant raw materials, an easy-to-operate preparation method, strong guidance, and high product purity. The compound has a significant inhibitory effect on the production of nitric oxide in RAW264.7 cells with LPS-induced inflammatory response, has anti-inflammatory activity, can be effectively used to prepare anti-inflammatory drugs, expands the medicinal value of belladonna roots, and has significant economic and social benefits. IV. Description of the drawings

[0015] Figure 1 It is the molecular structure formula diagram of compound 1 of the present invention.

[0016] Figure 2 The compound 1 of the present invention 1 H-NMR (500 MHz, CD3OD) spectrum.

[0017] Figure 3 The compound 1 of the present invention 13 C-NMR (125MHz, CD3OD) spectrum.

[0018] Figure 4 It is the HSQC correlation spectrum of compound 1 of the present invention.

[0019] Figure 5 It is the HMBC correlation spectrum of compound 1 of the present invention.

[0020] Figure 6 This is the key NOESY correlation spectrum of compound 1 of the present invention.

[0021] Figure 7 It is the ECD spectrum of compound 1 of the present invention. V. Specific implementation methods

[0022] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0023] Example 1

[0024] The present invention, when implemented, specifically comprises the following steps:

[0025] 1) 30 kg of dried belladonna root was crushed, 0.03 mol / L hydrochloric acid solution was added according to the solid-liquid ratio of 1 g:7 mL, and the mixture was extracted twice at room temperature, each time for 7 days, the filtrate was combined, and the relative density was concentrated under reduced pressure to 1.2-1.4 to obtain 1.2 kg of total alkaloid extract;

[0026] 2) The total alkaloid extract obtained in step 1) is suspended in distilled water, and the suspension is adjusted to pH=3 with a 5% hydrochloric acid solution, and extracted with ethyl acetate 5 times to remove acidic and extremely small amounts of low-polar components; the acid aqueous solution is adjusted to pH=7 with a 25% concentrated ammonia solution, and extracted with ethyl acetate 5 times again to remove neutral components; the acid aqueous solution is adjusted to pH=10 with a 25% concentrated ammonia solution again, extracted 5 times with water-saturated n-butanol, and the solvent is recovered under reduced pressure to obtain 182.2 g of n-butanol fraction;

[0027] 3) dissolving the n-butanol fraction obtained in step 2) in methanol, dry-mixing with alkaline silica gel at a mass ratio of 1:1, loading onto an alkaline silica gel column with a diameter-to-height ratio of 1:3, gradient eluting with dichloromethane-methanol volume ratios of 1:0, 50:1, 25:1, 10:1, 0:1, eluting for 5 column volumes at each ratio, spraying with anisaldehyde-concentrated sulfuric acid for detection, detecting once every 500 mL, combining fractions with the same main spot, recovering the solvent under reduced pressure, and obtaining components Fr.A, Fr.B, Fr.C, Fr.D, and Fr.E;

[0028] 4) The component Fr.D (45.3 g) obtained in step 3) was dissolved in methanol, and mixed with alkaline silica gel by dry method at a mass ratio of 1:1, and loaded on an alkaline silica gel column with a diameter-to-height ratio of 1:6. Gradient elution was performed with dichloromethane-methanol volume ratios of 50:1, 25:1, 10:1, and 0:1, and 8 column volumes were eluted at each ratio. Anisaldehyde-concentrated sulfuric acid was used for spray detection, and the detection was performed once every 300 mL. The same fractions of the main spot were combined, and the solvent was recovered under reduced pressure to obtain components Fr.D-1, Fr.D-2, Fr.D-3, and Fr.D-4;

[0029] 5) The component Fr.D-1 (4.2 g) obtained in step 4) was dissolved in dichloromethane-methanol in a volume ratio of 1:1, and separated by Toyopeal column chromatography. The product was isocratically eluted with dichloromethane-methanol in a volume ratio of 1:1 at a flow rate of 1.5 mL / min. Anisaldehyde-concentrated sulfuric acid was used for spray detection. The detection was performed 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;

[0030] 6) The component Fr.D-1-5 obtained in step 5) was dissolved in methanol and further separated on a semi-preparative HPLC with an ODS-AQ column. The mobile phase was a volume ratio of methanol: water = 23:77, the flow rate was 3 mL / min, and the retention time t was collected. R =18.2~19.7min chromatographic peak, concentrated and dried to obtain compound 1 of the present invention.

[0031] It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of protection of the present invention. Any changes and modifications that do not depart from the overall concept of the present invention should fall within the scope of protection of the present invention.

[0032] The compound obtained by the present invention was determined to be a new compound extracted from the root of belladonna: (2S)-1,2-tigloyl-3-β-D-glucuronide-glycerol, with a molecular structure shown in Figure 1 As shown, compound 1 was subjected to an activity experiment. The results showed that it had a significant inhibitory effect on the production of NO in RAW264.7 cells induced by LPS inflammation and had anti-inflammatory activity. The specific determination and experimental data are as follows:

[0033] 1. Structural Identification

[0034] Compound 1: brown yellow powder, HR-ESI-MS m / z 431.1554[MH] - (called for C 19 H 27 O 11,431.1548), the molecular formula of the compound is determined to be C 19 H 28 O 11 . 1 H-NMR spectrum shows 28 proton signals, among which δ H 6.84 (2H, m, H-3', H-3") is the proton signal connected to the double bond, δ H 1.79 (12H, brs, H3-4', H3-5', H3-4", H3-5") are the four methyl proton signals connected to the olefin. 13 C NMR spectrum of δ C 169.1 (C-1'), 168.7 (C-1") are carbonyl carbon signals, δ C 129.3 (C-2'), 129.4 (C-2"), 139.3 (C-3'), 139.5 (C-3") are two groups of olefin carbon signals, δ C 12.1 (C-5'), 12.1 (C-5"), 14.4 (C-4'), and 14.5 (C-4") are four groups of methyl carbon signals, and based on the chemical shifts of their olefinic proton signals, it is inferred that there are two tigloyl fragments in the structure.

[0035] 1 The high-field region of the H-NMR spectrum also shows a signal δ attributable to the protons at the end of the sugar H 4.84 (1H, d, J = 8.0 Hz, H-1"'), at δ H Multiple overlapping oxygen-linked methine proton signals were given between 3.43 and 3.95, suggesting that there may be a sugar fragment in the structure. 1 H- 1 There is a correlation between H2-1 / H-2 and H-2 / H2-3 in the H COSY spectrum, confirming the existence of a C1(H2)-C2(H)-C3(H2) fragment in the structure. In addition, 1 H- 1 There is a correlation between H-1"' / H-2"', H-2"' / H-3"', H-3"' / H-4"', and H-4"' / H-5"' in the H COSY spectrum. C 176.2 (C-6”'), 106.7 (C-1”'), 74.6 (C-3”'), 73.8 (C-5”'), 73.1 (C-2”'), 72.1 (C-4”') carbon signals, H-5”' and H-4”' in the HMBC spectrum are respectively related to C-6”', confirming that the sugar is β-D-glucuronic acid. Combined with the HMBC spectrum, H-1”' and δ C67.3 (C-3), indicating that the β-D-glucuronic acid is connected to the 3-position of the propanol group. In the HMBC spectrum, H-1 was found to be correlated with C-1', and H-2 was found to be correlated with C-1", indicating that two groups of tigloyl groups were connected to the 1 and 2 positions of the propanol group, respectively. In the NOESY spectrum, related signals were observed at H-1"' / H-2"', H-4"', and H-3"' / H-5"', confirming that the sugar was glucuronic acid. The structure of the compound was identified as 1,2-tigloyl-3-β-D-glucuronide-glycerol.

[0036] The stereo configuration of the compound was determined by comparing the experimental and theoretical ECD data and combining the acid hydrolysis method. The compound was subjected to acid hydrolysis, and the optical rotation value of the sugar was positive, so the configuration of glucuronic acid in the compound was D-type. The calculated ECD spectrum of (2S)-AB-2 was basically consistent with the experimental ECD spectrum of the compound, so the structure of the compound was determined to be (2S)-1,2-tigloyl-3-β-D-glucuronide-glycerol.

[0037] Table 1 NMR data of compound 1 (in CD3OD)

[0038]

[0039] 2. Activity Detection

[0040] 2.1 Cytotoxicity assay of compound 1

[0041] RAW264.7 cells were cultured in DMEM complete medium (containing 10% fetal bovine serum) in an incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were selected for the experiment.

[0042] Take RAW264.7 cells in the logarithmic growth phase and dilute them to 1×10 5 / mL of cell suspension was inoculated in a 96-well plate, and 100μL of cell suspension was added to each well. After culturing in a 37℃, 5% CO2 incubator for 24 hours, the supernatant was discarded, and 100μL of culture medium containing different concentrations of the above-mentioned compounds was added respectively. The compound concentrations were set as: 0, 5, 10, 25, 50, 100μM, i.e., blank group and drug-treated group, and 3 replicate wells were set for each group. Incubate in a 37℃, 5% CO2 incubator for 24 hours, add 10μL of MTT to each well, continue culturing for 3 hours, discard the supernatant, add 100μL of DMSO to each well, shake on a shaker for 10 minutes, and the purple crystals will be fully dissolved. The OD value of each well is measured at 490nm using an enzyme marker. And calculate the cell survival rate, 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), and the experiment was repeated three times. Compared with the blank group, compound 1 had no cytotoxic activity against RAW264.7 cells at a concentration of 5-100 μM, and the cell viability was above 98%. Therefore, 5, 10, 25, 50, and 100 μM were selected as the subsequent dosing concentrations.

[0043] 2.2 Anti-inflammatory activity detection of compound 1

[0044] Cells in the logarithmic growth phase were seeded in 24-well plates (2×10 5 Cells / mL), cultured in an incubator at 37°C and 5% CO2 environment, and divided into a blank group, a model group, and a drug group after 24 hours of culture. Compound 1 of different concentrations (5, 10, 25, 50, 100 μM) was added to the drug group, and after 2 hours of culture, except for the blank group, an appropriate amount of LPS was added to the remaining groups to make the final concentration of 1 μg / mL. After continuing to culture for 24 hours, 100 μL of the culture supernatant was transferred to an ELISA plate, and the OD value was measured at 540 nm with an ELISA instrument according to the instructions of the NO kit, and the NO release inhibition rate was calculated. Each group repeated 3 independent experiments. As shown in Table 2, the results show that different concentrations of compound 1 have a significant inhibitory effect on the NO release of RAW264.7 cells induced by LPS inflammatory response, indicating that compound 1 has good anti-inflammatory activity.

[0045] 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%.

[0046] Table 2 Effect of compound 1 on NO release induced by LPS in RAW264.7 cells

[0047]

[0048]

[0049] Conclusion

[0050] In summary, it can be seen that the raw materials of the present invention are rich and the preparation method is easy to operate. The compound 1 extracted from the roots of belladonna has anti-inflammatory activity, no cytotoxic activity, and the cell viability is above 98%. It has a significant inhibitory effect on the production of nitric oxide in RAW264.7 cells with LPS-induced inflammatory response, and can be effectively used to prepare anti-inflammatory drugs, which has opened up new medicinal value of belladonna roots, has huge development and application prospects, and has huge economic and social benefits.

[0051] It should be pointed out that the above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession can make changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A compound with anti-inflammatory activity extracted from the root of Belladonna, characterized in that The compound is (2S)-1,2-tigloyl-3-β-D-glucuronide-glycerol, with a molecular formula of C 19 H 27 O 11 , the unsaturation is 6, and the structural formula is:

2. The method for preparing the compound with anti-inflammatory activity extracted from the root of Belladonna as claimed in claim 1, characterized in that: The following steps are involved: 1) Crush the dried roots of Belladonna, soak and extract twice with 0.03 mol / L dilute hydrochloric acid solution at room temperature, let the soaking solution stand, filter to remove the residue, and concentrate the filtrate to obtain the total alkaloid extract; 2) dissolving and dispersing the total alkaloid extract obtained in step 1) in distilled water, adjusting the pH to 3 with a 5% hydrochloric acid solution, extracting with ethyl acetate to remove acidic and extremely small amounts of low-polar components; adjusting the pH to 7 with a 25% concentrated ammonia solution, extracting with ethyl acetate again to remove neutral components; adjusting the pH to 10 with a 25% concentrated ammonia solution, extracting with water-saturated n-butanol, and recovering the solvent under reduced pressure to obtain an n-butanol fraction; 3) The n-butanol fraction obtained in step 2) was subjected to alkaline silica gel column chromatography, gradient elution was performed using a dichloromethane-methanol system, and detection was performed using anisaldehyde-concentrated sulfuric acid spray, and the same fractions of the main spot were combined to obtain components Fr.A to Fr.E; 4) The component Fr.D obtained in step 3) was subjected to alkaline silica gel column chromatography, gradient elution was performed using a dichloromethane-methanol system, and anisaldehyde-concentrated sulfuric acid spray was used for identification, and the same fractions of the main spot were combined to obtain components Fr.D-1 to Fr.D-4; 5) The component Fr.D-1 obtained in step 4) was subjected to Toyopeal column chromatography, eluted with dichloromethane-methanol 1:1, and sprayed with anisaldehyde-concentrated sulfuric acid for identification. 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; 6) The component Fr.D-1-5 obtained in step 5) was separated on a semi-preparative HPLC using an ODS-AQ column, and the residues with retention time t R =The chromatographic peak at 18.2-19.7min was obtained by recovering the solvent under reduced pressure.

3. The method for preparing a compound having anti-inflammatory activity extracted from the root of Belladonna according to claim 2, characterized in that: The following steps are involved: 1) 30 kg of dried belladonna root was crushed, 0.03 mol / L hydrochloric acid solution was added according to the solid-liquid ratio of 1 g:7 mL, and the mixture was extracted twice at room temperature, each time for 7 days, the filtrates were combined, and the relative density was concentrated under reduced pressure to 1.2-1.4 to obtain a total alkaloid extract; 2) The total alkaloid extract obtained in step 1) is suspended in distilled water, and the suspension is adjusted to pH=3 with a 5% hydrochloric acid solution, and extracted with ethyl acetate for 5 times to remove acidic and extremely small amounts of low-polar components; the acid aqueous solution is adjusted to pH=7 with a 25% concentrated ammonia solution, and extracted with ethyl acetate for 5 times to remove neutral components; the acid aqueous solution is adjusted to pH=10 with a 25% concentrated ammonia solution again, extracted with water-saturated n-butanol for 5 times, and the solvent is recovered under reduced pressure to obtain an n-butanol fraction; 3) dissolving the n-butanol fraction obtained in step 2) in methanol, dry-mixing with alkaline silica gel at a mass ratio of 1:1, loading onto an alkaline silica gel column with a diameter-to-height ratio of 1:3, gradient eluting with dichloromethane-methanol volume ratios of 1:0, 50:1, 25:1, 10:1, 0:1, eluting for 5 column volumes at each ratio, spraying with anisaldehyde-concentrated sulfuric acid for detection, detecting once every 500 mL, combining fractions with the same main spot, recovering the solvent under reduced pressure, and obtaining components Fr.A, Fr.B, Fr.C, Fr.D, and Fr.E; 4) Dissolve the component Fr.D obtained in step 3) in methanol, dry-mix with alkaline silica gel at a mass ratio of 1:1, load on an alkaline silica gel column with a diameter-to-height ratio of 1:6, and perform gradient elution with dichloromethane-methanol volume ratios of 50:1, 25:1, 10:1, and 0:1, eluting for 8 column volumes at each ratio, using anisaldehyde-concentrated sulfuric acid spray detection, detecting once every 300 mL, combining the same fractions of the main spot, and recovering the solvent under reduced pressure to obtain components Fr.D-1, Fr.D-2, Fr.D-3, and Fr.D-4; 5) The component Fr.D-1 obtained in step 4) was dissolved in dichloromethane-methanol in a volume ratio of 1:1, and separated by Toyopeal column chromatography. The product was isocratically eluted with dichloromethane-methanol in a volume ratio of 1:1 at a flow rate of 1.5 mL / min. Anisaldehyde-concentrated sulfuric acid was used for spray detection. The 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; 6) The component Fr.D-1-5 obtained in step 5) was dissolved in methanol and further separated on a semi-preparative HPLC with an ODS-AQ column. The mobile phase was a volume ratio of methanol: water = 23:77, the flow rate was 3 mL / min, and the retention time t was collected. R =18.2~19.7min chromatographic peak, concentrate and dry to obtain.

4. Use of the compound with anti-inflammatory activity extracted from the root of Belladonna as claimed in claim 1 in the preparation of anti-inflammatory drugs.

5. Use of the compound with anti-inflammatory activity extracted from the root of belladonna prepared by the preparation method according to claim 2 or 3 in the preparation of anti-inflammatory drugs.

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