Urtica cannabina neoglycoside and preparation and application methods thereof
High-purity urticaria glycosides were extracted from the roots of *Urtica macrantha* using macroporous resin, silica gel column chromatography, and preparative HPLC separation techniques. This method solved the problems of low purity and cumbersome operation in existing technologies, and achieved significant anti-inflammatory, antioxidant, and 5α-reductase inhibition effects, while reducing preparation costs.
Patent Information
- Application Number
- CN202410713818.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-04
- Publication Date
- 2025-12-05
AI Technical Summary
Existing technologies for extracting new glycosides from *Urtica fuciformis* have low purity, low yield, and are cumbersome to operate with a long separation cycle.
Using macroporous resin, silica gel column chromatography, and preparative HPLC, urticaria glycoside I and urticaria glycoside II with a purity greater than 95% were separated from the aqueous decoction of nettle root. The chromatographic peaks with specific absorption wavelengths were collected by centrifugation of the aqueous decoction, macroporous resin column chromatography, silica gel column chromatography, and preparative HPLC.
It improved the purity and yield of urticaria glycosides, simplified the operation process, reduced the preparation cost, and achieved significant anti-inflammatory, antioxidant and 5α-reductase inhibition effects.
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Figure CN121064263A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, and particularly relates to a nettleside and a preparation and application method thereof. BACKGROUND
[0002] Urtica cannabina L. is a plant of Urticaceae Urtica, has the effects of expelling wind and removing dampness, activating blood and relieving spasm, and detoxification, and is commonly used for treating BPH, urolithiasis, eczema, rheumatism and other diseases in folk. SUMMARY
[0003] The present application aims at the problems of low purity, low yield, complicated operation and long separation period of the components obtained by the existing extraction technology, and provides a nettleside and a preparation and application method thereof. Two kinds of nettleside I and nettleside II with purity greater than 95% and significant anti-inflammatory, antioxidant and 5alpha-reductase inhibitory activities are prepared from the water decoction of the roots of Urtica cannabina L. by macroporous resin column chromatography, silica gel column chromatography and preparative HPLC separation.
[0004] The present application is realized by the following technical scheme:
[0005] The present application relates to a kind of nettleside compounds, including: nettleside I and nettleside II, and the chemical structure is as shown in the following:
[0006] The present application relates to the application of the above-mentioned nettleside compounds, which is used for preparing anti-prostate hyperplasia drugs or for identifying Urtica cannabina L.
[0007] The present application relates to the extraction method of the above-mentioned nettleside compounds. The dried roots of Urtica cannabina are decocted with water, the water decoction is centrifuged, and then subjected to macroporous resin column chromatography, silica gel column chromatography and preparative HPLC separation. Two kinds of nettleside I and nettleside II with purity greater than 95% are obtained by collecting the main chromatographic peaks with maximum absorption wavelengths of 314 nm and 326 nm, respectively.
[0008] The Urtica cannabina L. is the dried roots of Urtica cannabina L.
[0009] The macroporous resin column chromatography uses AB-8 type macroporous resin and ethanol solution as the eluent.
[0010] The silica gel column chromatography is detected by TLC, and the fractions with Rf of 0.2-0.5 are combined.
[0011] The extraction method specifically comprises the following steps: crushing the roots of U. hirta, decocting the roots in water, subjecting the decocted liquid to AB-8 macroporous resin column chromatography, sequentially eluting impurities with water and 40% ethanol, then eluting with 80% ethanol, collecting the 80% ethanol eluent, and recovering the solvent under reduced pressure to obtain U. hirta neoside extract; subjecting the U. hirta neoside extract to silica gel column chromatography, eluting with dichloromethane-methanol (9:1, V / V), recovering the solvent, and discarding the extract; then eluting with dichloromethane-methanol (17:3, 4:1, 3:1, V / V), collecting the eluent, and detecting by TLC; combining the fractions with the same main spot (Rf 0.2-0.5), recovering the solvent under reduced pressure, and obtaining U. hirta neoside crude product. The U. hirta neoside crude product is subjected to preparative HPLC separation (acetonitrile-water 1:4-1:1, 30 min; DAD detector), and the main peaks with maximum absorption wavelengths of 314 nm and 326 nm are collected respectively to obtain U. hirta neoside I and U. hirta neoside II.
[0012] The decoction is performed for 0.5-1 h, and the decoction is performed for 1-2 times.
[0013] The macroporous resin column chromatography is performed with AB-8 macroporous resin, and the amount of the macroporous resin is 1-2 times the amount of the medicinal material. The eluent for removing impurities is water and 40% ethanol, and then 80% ethanol is used for elution, and the amount of the eluent is 3-5 times the column volume (BV).
[0014] The silica gel column chromatography is performed with silica gel with a particle size of 200-300 mesh, and the sample amount is 1:30-1:50 (sample amount / silica gel, The elution is performed with dichloromethane-methanol (17:3, 4:1, 3:1, V / V), and each gradient elution is performed for 3 BV at a flow rate of 1 BV / h and 0.25 BV / fraction. The eluent is collected, detected by TLC, and the fractions with Rf of 0.2-0.5 are combined, and the solvent is recovered under reduced pressure to obtain U. hirta neoside crude product.
[0015] The preparative HPLC separation is performed with a C 18Column (250*20mm, 5μm), mobile phase acetonitrile-water, gradient elution for 30min (acetonitrile-water 1:4~1:1, V / V); flow rate 10ml / min; DAD detector, collect two main peaks at 314, 326nm, respectively, and recover the solvent to obtain refined Urticularin. BRIEF DESCRIPTION OF DRAWINGS
[0016] Figure 1 Urticularin I is a new glycoside from Urtica fissa 13 C-NMR chart (DMSO-d6, 150MHz);
[0017] Figure 2 Urticularin II is a new glycoside from Urtica fissa 13 C-NMR chart (DMSO-d6, 150MHz). DETAILED DESCRIPTION Example 1
[0018] This example includes the following steps:
[0019] Step 1. Urtica fissa root 1kg, add 12L water and decoct for 1h, centrifuge the extract, and perform column chromatography on macroporous resin (1kg), elute with water and 40% ethanol (3BV each) in sequence to remove impurities; elute with 80% ethanol (3BV) to recover ethanol to obtain Urticularin extract (25g).
[0020] Step 2. Take the Urticularin extract above and perform column chromatography on silica gel (750g of silica gel), elute with dichloromethane-methanol (9:1, V / V) for 6BV, recover the solvent, and discard the extract. Elute with dichloromethane-methanol (17:3, 4:1, 3:1, V / V), 3BV for each gradient, and collect the eluate. TLC detection, combine the fractions with main spots Rf at 0.2-0.5, and recover the solvent to obtain Urticularin crude product (427mg).
[0021] Step 3. Perform preparative HPLC separation on the Urticularin crude product, and collect two main chromatographic peaks at 314, 326nm, respectively, to obtain refined Urticularin I product 25mg (purity 97.0%, HPLC method) and refined Urticularin II product 17mg (purity 96.6%, HPLC method).
[0022] The physical properties and spectral data of the obtained components are as follows:
[0023] Urticularin I: white powder; UV (MeOH) λ max : 314nm; IR (KBr) v max : 3442, 2922, 1634, 1026cm-1 HR-ESI-MS: 481.1398 ([M-H] - C 22 H 25 O 12 ; calculated 481.1396); 1 H、 13 C-NMR: see Table 1.
[0024] Urticae Novel Glycoside II: white powder; UVλ max : 326 nm; IR v max : 3442, 2920, 1633, 1219, 720 cm -1 ; HR-ESI-MS: 511.1459 [M-H] - (C 23 H 27 O 13 , calculated 511.1452); 1 H and 13 CNMR: see Table 1.
[0025] Table 1.1H-NMR (600 MHz), 13C-NMR (150 MHz) data of Urticae Novel Glycoside I, II (DMSO-d6) 1 H-NMR (600 MHz), 13 C-NMR (150 MHz) data (DMSO-d6) Example 2
[0026] This example includes the following steps:
[0027] Step 1. Urticae radix 1 kg was decocted twice with 8 L water for 0.5 h each time, and the two extracts were combined. The extract was centrifuged and subjected to column chromatography on a macroporous resin (2 kg), eluted with water, 40% ethanol (5 BV each) to remove impurities, and 80% ethanol (5 BV) to recover the ethanol to obtain a Urticae novel glycoside extract (32 g).
[0028] Step 2. The Urticae novel glycoside extract was subjected to column chromatography on silica gel (1200 g of silica gel), eluted with dichloromethane-methanol (9:1, V / V) for 5 BV, and the solvent was recovered and the extract was discarded. Elution was continued with dichloromethane-methanol (17:3, 4:1, 3:1, V / V) for 3 BV each gradient, and the eluate was collected. TLC detection was performed, and the fractions with main spots Rf at 0.2-0.5 were combined, and the solvent was recovered to obtain a Urticae novel glycoside crude product (506 mg).
[0029] Step 3. The crude Urticularin was separated by preparative HPLC, and two main chromatographic peaks with maximum absorption wavelength of 314 nm and 326 nm were collected, respectively, to obtain refined Urticularin I 33 mg (purity 96.0%, HPLC method) and refined Urticularin II 24 mg (purity 95.4%, HPLC method). Example 3
[0030] This example includes the following steps:
[0031] Step 1. Urtica fissa root 1 kg was added with 15 L of water and decocted for 0.75 h, and the extract was centrifuged and subjected to column chromatography with macroporous resin (1.5 kg), and eluted with water and 40% ethanol (4 BV each) in sequence to remove impurities; and eluted with 95% ethanol for 4 BV, and the ethanol was recovered to obtain Urticularin extract (29 g).
[0032] Step 2. The Urticularin extract was subjected to column chromatography with silica gel (1450 g of silica gel), and eluted with dichloromethane-methanol (9:1, V / V) for 4 BV, and the extract was discarded after recovery of the solvent. Elution was performed with dichloromethane-methanol (17:3, 4:1, 3:1, V / V) for 3 BV each gradient, and the eluate was collected. TLC detection was performed, and the main spots were combined in the fractions of 0.2-0.5, and the solvent was recovered to obtain crude Urticularin (462 mg).
[0033] Step 3. The crude Urticularin was separated by preparative HPLC, and two main chromatographic peaks with maximum absorption wavelength of 314 nm and 326 nm were collected, respectively, to obtain refined Urticularin I 33 mg (purity 96.0%, HPLC method) and refined Urticularin II 24 mg (purity 95.4%, HPLC method). Example 4
[0034] This example relates to the application of the above-mentioned Urticularin to the treatment of prostatic hyperplasia, which includes:
[0035] a) Inhibition of 5α-reductase activity: The sample was dissolved in 2% methanol by ultrasonic, and was prepared into 0.2, 1.0, 5.0, 20.0, 100.0 μM solution for use. The 5α-reductase solution, testosterone solution, NADPH-Na4, sample / finasteride and Tris buffer were reacted at 37°C for 60 min, centrifuged at 10,000 rpm for 20 min at 4°C, the supernatant was filtered (0.22 μm), 20 μL was injected, and the peak area of testosterone in the reaction solution at 0 min and 60 min was analyzed by HPLC to determine the inhibition of the sample (S) on 5α-reductase by the change of the peak area of the substrate testosterone before and after reaction. A blank control (B, Tris buffer instead of finasteride, to observe the activity of 5α-reductase in the reaction system without adding enzyme inhibitor) was also set. The inhibition rate of the sample at different concentrations (inhibition rate (%) = (S 60min -B 60min ) / (B 0min -B 60min ) x 100%) and the half-inhibitory concentration (IC 50 ) were calculated.
[0036] b) Inhibition of prostate cell proliferation: The human prostate hyperplasia cell line BPH-1 in logarithmic growth phase was taken, inoculated in a 96-well culture plate, 5 x 10 4 cells were added in each well, and the culture solution was replaced after the cells were attached for 24 h. The culture solution containing the sample (concentration: 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 μM) was added in each well, and a blank group, finasteride control group and sample group were set, each group had 3 replicate wells, and the culture was carried out for 48 h. The concentration of the sample was 100 μM. After the culture, 5 mg / mL MTT 20 μL was added in each well, and the culture was carried out for another 4 h. The culture supernatant in the well was aspirated, DMSO 150 μL was added in each well, and the well was shaken on a shaker for 10 min. After the purple crystals were completely dissolved, the absorbance was measured at 570 nm, and the cell growth inhibition rate [inhibition rate (%) = (absorbance of the control well / absorbance of the sample well) x 100%] and the half-inhibitory concentration (IC 50 )
[0037] c) Anti-inflammatory activity: (1) Cytotoxicity investigation: the RAW 264.7 cells in logarithmic growth phase were taken, digested with 0.25% trypsin, diluted with the culture solution to a final concentration of 1 x 10 5 cells / mL, 100 μL was inoculated in each well of a 96-well culture plate, and was placed in a 37°C, 5% CO2 incubator overnight. After the cells were attached, 100 μL of each concentration of the sample (concentration: 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64, 128 μM) was added, and the cytotoxicity of the sample was investigated by MTT method. (2) Inflammatory factor determination: the RAW 264.7 cell suspension was inoculated into 96-well plate, 1 mL per well, and the cell number was 1 x 10 5 After 24 h of culture, the culture medium was carefully aspirated, and LPS was added to make the final concentration of LPS in each group 5 μg / mL. 100 μL of each concentration of drug solution (drug solution concentration: 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 μM; 100 μL of culture medium was added to the blank control group) was added, and each group had 5 replicate wells. The culture was continued for 24 h, and the release amount of TNF-α, IL-1β, and IL-10 in the supernatant was detected by the kit, and the inhibition rate and half inhibition concentration (IC 50 ) of the sample were calculated.
[0038] The above experiment was repeated 3 times in parallel, and the average value and standard deviation (SD) were calculated. The specific results are shown in Table 2.
[0039] Table 2 Inhibition of prostate hyperplasia by Urticae Folium neoside n = 3
[0040] a positive control drug; / not tested
[0041] As can be seen from Table 2, both Urticae Folium neosides have significant inhibitory activities on 5α-reductase, human prostate cell proliferation, and inflammation, and can be used for the preparation of prostate hyperplasia drugs. Example 5
[0042] This embodiment relates to a rapid and qualitative identification method of Urticae Folium based on the above-mentioned Urticae Folium neoside. Specifically, the Urticae Folium roots are crushed, 2 g is taken, and 40 ml of ethanol is added for reflux extraction for 1 h, and the ethanol is recovered under reduced pressure to obtain an alcohol extract. The alcohol extract is dissolved in 5 ml of water, extracted with ethyl acetate (5 ml), and the solvent is recovered to obtain an extract. The extract is dissolved in 1 ml of methanol as a test solution. Another 1 mg of Urticae Folium neoside I and II is taken, dissolved in 1 ml of methanol, and used as a mixed control solution. The test solution and the control solution are spotted on the same silica gel G, developed with chloroform-methanol (4:1, V / V) as the developing agent, colored with 10% sulfuric acid-ethanol, and observed under daylight. The sample shows the same color spots as Urticae Folium neoside I and II at the same position.
[0043] Compared with the prior art, the method can simultaneously prepare two new compounds, removes interference of most water-soluble components by using macroporous resin column chromatography, effectively enriches the new urushibotanydendron glycosides in the urushibotanydendron, and improves the yield. When the silica gel column chromatography is performed, dichloromethane-methanol (9:1) is used for elution, and fatty acids, flavones, lignans and fat-soluble pigments are further removed, so that the urushibotanydendron glycosides can be simply and quickly prepared. The urushibotanydendron glycosides obtained are detected by HPLC, and the purity is more than 95%. The organic solvents used in the whole preparation process and the fillers used in the separation can be repeatedly used. On the other hand, two urushibotanydendron glycosides are simultaneously prepared, the raw materials and chemical reagents are fully utilized, and the preparation cost is greatly reduced.
[0044] The above specific embodiments can be adjusted in different ways by those skilled in the art without departing from the principles and purposes of the present application, the protection scope of the present application is subject to the claims and is not limited by the above specific embodiments, and each implementation scheme within the scope is subject to the present application.
Claims
1. A class of new urushibasides of the genus Urtica, characterized in that, Urticae Folium, and the chemical structure is as follows:
2. The use of the urushibotanyin compound of claim 1, characterized in that, The application relates to a preparation method of an anti-prostate hyperplasia medicine.
3. Use according to claim 2, characterized in that, The prostate hyperplasia includes inhibiting 5alpha-reductase activity, inhibiting human prostate cell proliferation and anti-inflammatory activity.
4. The use of the urushibotanyin compound of claim 1, characterized in that, The application relates to a method for identifying Urticae Folium.
5. The extraction method of the urushibotanyin compound according to claim 1, characterized in that, The refined Urticae Folium new glycoside I and the refined Urticae Folium new glycoside II are obtained by collecting two main chromatographic peaks with maximum absorption wavelengths of 314nm and 326nm respectively after the drying root of Urticae Folium is decocted with water, the obtained water decoction is centrifuged and subjected to macroporous resin column chromatography, and then the water decoction is subjected to silica gel column chromatography and preparation HPLC separation.
6. The extraction method according to claim 5, characterized in that, in particular The application relates to a preparation method of an anti-prostate hyperplasia medicine. The application relates to a preparation method of an anti-prostate hyperplasia medicine. The application relates to a preparation method of an anti-prostate hyperplasia medicine.
7. The extraction method according to claim 6, characterized in that, The application relates to a preparation method of an anti-prostate hyperplasia medicine.
8. The extraction method according to claim 6, characterized in that, The application relates to a preparation method of an anti-prostate hyperplasia medicine.
9. The extraction method of claim 6, wherein, The application relates to a preparation method of an anti-prostate hyperplasia medicine. The application relates to a preparation method of an anti-prostate hyperplasia medicine. The application relates to a preparation method of an anti-prostate hyperplasia medicine. The application relates to a preparation method of an anti-prostate hyperplasia medicine. The application relates to a preparation method of an anti-prostate hyperplasia medicine. The application relates to a preparation method of an anti-prostate hyperplasia medicine. The application relates to a preparation method of an anti-prostate hyperplasia medicine. The application relates to a preparation method of an anti-prostate hyperplasia medicine. The application relates to a preparation method of an anti-prostate hyperplasia medicine. The application relates to a preparation method of an anti-prostate hyperplasia medicine. 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The extraction method of claim 6, wherein, The HPLC separation was prepared by using a C 18 column (250*20mm, 5μm), mobile phase was acetonitrile-water, gradient elution for 30min (acetonitrile-water 1:4~1:1, V / V); the flow rate was 10ml / min; DAD detector detection, respectively, to collect two main peaks of 314, 326nm absorption wavelength, recovery solvent to obtain refined product of Urticae-Urticae Glycoside.