An anti-inflammatory active extract of lily, its preparation method and application

Abstract

The present invention provides a lily anti-inflammatory active extract, its preparation method and application. Take dry lily medicinal materials, extract with 60% ethanol, filter, combine the extracts, recover ethanol from the extracts and concentrate them into a thin extract. Pass this thin extract through an adsorption resin, first elute with water until colorless, discard the eluate, then elute with 80% ethanol, recover ethanol to obtain an extract. Add this extract to a silica gel column, elute with a dichloromethane-methanol gradient, and identify the eluate by thin layer chromatography. Then pass through a reversed-phase silica gel RP-18 column, elute with a methanol-water gradient, and identify the eluate by thin layer chromatography. Further purify by repeatedly passing through a Sephadex LH-20 gel column to obtain lilyin A. This active extract can effectively show obvious inhibitory activity against NO produced by LPS-induced RAW264.7 cells, with a significant anti-inflammatory effect, up to 95.09%, higher than the anti-inflammatory effect of the commonly used anti-inflammatory drug dexamethasone.

Description

Technical Field

[0001] The present invention relates to the extraction of active ingredients from plants, and particularly to an anti-inflammatory active extract of lily, a preparation method thereof and an application thereof. Background Art

[0002] Lily is a traditional Chinese medicinal material that can be used both as medicine and food in China. It has various varieties and is widely distributed, and has high nutritional and pharmacological effects, such as relieving cough and reducing phlegm, sedative and hypnotic, immune regulation, anti-tumor, antioxidant, anti-inflammatory, anti-stress injury, anti-depression, hypoglycemic and antibacterial, etc. Currently, the research mainly focuses on polysaccharides, steroidal saponins, alkaloids, phenols and steroidal glycosides. By studying the biochemical activities of the chemical components of lily plants, it is found that some active substances have anti-tumor, hypoglycemic, antioxidant, anti-inflammatory and other effects.

[0003] NO is a small molecule compound with a wide range of biological effects. It can directly participate in the regulation of cytokines related to inflammation and plays an important role in immune regulation. Research has confirmed that there is a close relationship between NO and inflammation. Its biochemical processes in the body mainly include three basic reactions: 1) binding to the heme group of soluble guanylate cyclase (sGC), activating sGC to produce and release cGMP; 2) being inactivated by oxyhemoglobin to generate methemoglobin and nitrite; 3) reacting with superoxide ions to generate toxic strong oxidants (ONOO). During the inflammatory reaction process, due to the action of injury factors, some tissues are damaged, resulting in vasodilation, increased local blood volume, and increased exudation of plasma and white blood cells from blood vessels. Then repair occurs, and the damaged tissues are healed. Thus, it can be seen that the inflammatory reaction is closely related to the repair process. The inflammatory reaction has potential harmfulness, and excessive inflammatory reaction will cause serious damage to the body. The vascular reaction is the central link but not the only link of the inflammatory reaction. The immune system, blood coagulation system, fibrinolytic system and cytokines are all necessary factors for the inflammatory reaction. And NO not only plays a major role in the vascular reaction, but also participates in the immune system reaction and the regulation reaction of platelets, and plays an important role as a cytokine in the inflammatory reaction. A large number of studies believe that the occurrence of acute and chronic inflammation is related to NO.

[0004] Currently, the components with anti-inflammatory activity in lily extracts include saponins, kaempferol glycosides, quercetin glycosides and chalcones. Anti-inflammatory drugs are the second largest category of drugs after anti-infective drugs, and people pay more and more attention to the research of anti-inflammatory drugs in natural drugs. Summary of the Invention

[0005] In order to provide a new anti-inflammatory active extract of lily, the present invention first provides a preparation method of an anti-inflammatory active extract of lily, comprising the following steps:

[0006] S1. Take the dried lily medicinal material and reflux extract it twice with 60% ethanol;

[0007] S2. Filter and combine the two extraction solutions in step S1, recover ethanol from the extraction solution and concentrate it into a thin extract;

[0008] S3. Load the thin extract obtained in step S2 onto D101 macroporous adsorption resin, elute with water, and discard the water eluate; then elute with 80% ethanol, collect the eluate, and concentrate it under reduced pressure to obtain an extract;

[0009] S4. Load the extract obtained in step S3 onto a silica gel column for chromatography, use dichloromethane - methanol as the eluent, elute with a gradient, collect the eluate, and identify it by thin - layer chromatography, then combine;

[0010] S5. Load the product obtained in step S4 onto a reverse - phase silica gel RP - 18 column, use methanol - water as the eluent, elute with a gradient, collect the eluate, identify it by thin - layer chromatography, and combine;

[0011] S6. Further purify the product obtained in S5 by passing it through a Sephadex LH - 20 gel column repeatedly to obtain the lily anti - inflammatory active extract.

[0012] Preferably, the lily medicinal material in step S1 is the fleshy bulb of lily.

[0013] Preferably, the mass ratio of the lily medicinal material to the ethanol used for the two extractions in step S1 is 1:11:10.

[0014] Preferably, the extraction time for each extraction in step S1 is 1 h.

[0015] Preferably, the relative density of the thin extract in step S2 is 1.05.

[0016] Preferably, the volume ratio of dichloromethane to methanol for gradient elution of the eluate in step S4 is 9:1 - 0:10.

[0017] Preferably, the volume ratio of methanol to water for gradient elution of the eluate in step S5 is 4:6 - 10:0.

[0018] Preferably, the lily anti - inflammatory active extract is lilyin A, as shown in formula 1:

[0019]

[0020] Based on a general inventive concept, the present invention also provides the application of the lily anti - inflammatory active extract in the preparation of a drug for treating inflammation.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) An anti-inflammatory active extract of lilium brownii in the present invention can effectively show obvious inhibitory activity against NO produced by LPS-induced RAW264.7 cells, with a significant anti-inflammatory effect of up to 95.09%, higher than that of the commonly used anti-inflammatory drug dexamethasone.

[0023] (2) A preparation method of an anti-inflammatory active extract of lilium brownii in the present invention, through purification and concentration steps, the extracted extract can be used as the main pharmacodynamic component for anti-inflammatory and anti-inflammatory, further interpreting the role of the anti-inflammatory active extract of lilium brownii. The extraction and concentration methods are efficient, the extraction method is relatively stable and the quality is controllable, providing a reference for mass production and wide promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 It is crucial for liliin A of the extract in Example 1 of the present invention 1 H- 1 H COSY (bold), HMBC (arrows) and ROESY related, where A is 1 H- 1 H COSY (bold) and ROESY related diagram, B is HMBC (arrows) and ROESY related diagram;

[0026] Figure 2 It is the COSY spectrum diagram of liliin A of the extract in Example 1;

[0027] Figure 3 It is the DEPY spectrum diagram of liliin A of the extract in Example 1;

[0028] Figure 4 It is the HMBC spectrum diagram of liliin A of the extract in Example 1;

[0029] Figure 5 It is the HSQC spectrum diagram of liliin A of the extract in Example 1;

[0030] Figure 6 It is the ROESY spectrum diagram of liliin A of the extract in Example 1;

[0031] Figure 7 It is the H spectrum diagram of liliin A of the extract in Example 1;

[0032] Figure 8 It is the C spectrum of liliumin A, the extract of Example 1. Detailed implementation mode

[0033] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0034] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, the modifications or replacements made to the methods, steps or conditions of the present invention all belong to the scope of the present invention.

[0035] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are all commercially available.

[0036] Example 1

[0037] Extract liliumin A

[0038] S1. Take 20 kg of dry lily bulbs, reflux and extract with 60% ethanol twice, 1 hour each time. Add 220 kg of 60% ethanol for the first time and 200 kg of 60% ethanol for the second time;

[0039] S2. Filter and combine the two extraction solutions in step S1. Recover ethanol from the extraction solution and concentrate it to a thin extract with a relative density of 1.05;

[0040] S3. Load the thin extract obtained in step S2 onto D101 macroporous adsorption resin, elute with water, and discard the water eluate; then elute with 80% ethanol, collect the eluate, recover ethanol, and concentrate under reduced pressure to obtain an extract;

[0041] S4. Load the extract obtained in step S3 onto a silica gel column for chromatography, use dichloromethane - methanol with a volume ratio of 9:1 to 0:10 as the eluent, perform gradient elution, collect the eluate, and identify it by thin layer chromatography, then combine;

[0042] S5. Load the product obtained in step S4 onto a reverse phase silica gel RP - 18 column, use methanol - water with a volume ratio of 4:6 to 10:0 as the eluent, perform gradient elution, collect the eluate, identify it by thin layer chromatography, combine, and recover methanol;

[0043] S6. Further purify the product obtained in S5 by repeatedly passing it through a Sephadex LH - 20 gel column to obtain liliumin A.

[0044] Key of liliumin A 1 H - 1 H COSY (bold), HMBC (arrows) and ROESY are related as Figure 1 shown; 1 H- 1 H COSY is a hydrogen-hydrogen correlation spectroscopy ( 1 H- 1 H correlation spectroscopy); HMBC is a heteronuclear multiple bond correlation spectroscopy (Heteronuclear Multiple Bond Correlation); ROESY is the abbreviation of Rotating frame Overhauser Enhancement Spectroscopy, which is the NOESY spectrum in the rotating coordinate system (i.e., 1 H- 1 H nuclear Overhauser effect two-dimensional spectrum).

[0045] The COSY spectrum, DEPT spectrum, HMBC spectrum, HSQC spectrum and ROESY spectrum of lilacin A are as Figures 2 - 6 shown; The COSY spectrum is a hydrogen-hydrogen correlation spectrum; The DEPT spectrum is a type of carbon nuclear magnetic resonance spectrum (abbreviation of Distortionless Enhancement by Polarization Transfer, also known as the non-distorted polarization transfer technique); The HMBC spectrum is a heteronuclear multiple bond correlation spectrum, which is a multiple bond carbon-hydrogen correlation spectrum; The HSQC spectrum is a directly connected carbon-hydrogen correlation spectrum (abbreviation of heteronuclear singular quantum correlation); The ROESY spectrum is the NOESY spectrum in the rotating coordinate system (i.e., 1 H- 1 H nuclear Overhauser effect two-dimensional spectrum).

[0046] The H spectrum of lilacin A is as Figure 7 shown, and the spectral data are 11H-NMR (CD3OD, 600 MHz) δ: 3.87 (2H, m, H-3, H-3′), 3.59 (2H, m, H-6a, H-6a′), 3.51 (2H, m, H-6b, H-6b′), 2.35 (2H, m, H-7a, H-7a′), 2.03 (2H, m, H-8a, H-8a′), 1.95 (4H, overlap, H-7b, H-7b′, H-8b, H-8b′), 1.79 (2H, m, H-11, H-11′), 1.69 (2H, m, H-10a, H-10a′), 1.59 (2H, m, H-10b, H-10b′), 1.02 (6H, d, J = 6.6 Hz, 12-CH3, 12′-CH3), 0.99 (6H, d, J = 6.6 Hz, 13-CH3, 13′-CH3);

[0047] The carbon spectrum of lilacin A is as shown in Figure 8 and the spectral data are 13 13C-NMR (CD3OD, 150 MHz) δ: 170.2 (C-1, C-1′), 167.7 (C-4, C-4′), 57.6 (C-9, C-9′), 55.7 (C-3, C-3′), 45.3 (C-6, C-6′), 42.3 (C-10, C-10′), 28.4 (C-7, C-7′), 24.1 (C-11, C-11′), 21.9 (C-12, C-12′), 21.7 (C-8, C-8′), 20.5 (C-13, C-13′).

[0048] Experimental Example 1

[0049] Investigate the anti-inflammatory activity of lilacin A

[0050] Cell culture and grouping: RAW264.7 cells were cultured in DMEM medium containing 10% FBS + 1% double antibody; the above cells were changed with fresh medium every 2 - 3 days. When the cell confluence rate reached about 80%, the cells were digested with trypsin and subcultured by splitting into two parts. Logarithmically growing cells were seeded in 96-well plates and allowed to adhere. Except for the blank group, other groups were treated with lipopolysaccharide (LPS) for 24 hours, and then diluted lilacin A was added and incubated for 24 h. Each group was set with 3 replicates. Each drug was grouped as follows: (1) blank control group; (2) model control group (LPS group); (3) LPS + lilacin A 2.5 μM; (4) LPS + lilacin A 5 μM; (5) LPS + lilacin A 25 μM; (6) LPS + lilacin A 50 μM; (7) LPS + lilacin A 250 μM; (8) positive control group (LPS + dexamethasone).

[0051] IC50 Detection of NO Inhibition: (1) Digest and count the cells in the above groups, and inoculate them into a 96-well plate at a density of 1*10 4 cells / well, 100 μL per well. Each group is set with 3 replicate wells. (2) After the cells adhere and are pre-treated with low-serum medium for 24 h, then after adding drugs according to the above method for the corresponding time, add 10 μL / well of CCK8 to each well. Prepare the CCK8 solution with complete medium, remove the drug-containing medium, and add 100 μL of medium containing CCK8 to each well. (3) Continue to incubate at 37 °C and 5% CO2 for 1 hour, then analyze the absorbance (OD) value at 450 nm with a Bio-Tek microplate reader and take the average value. (4) Calculate the IC 50 value.

[0052] The results are shown in Table 1. As the content of lilacin A increases, the anti-inflammatory effect increases significantly. When the content of lilacin A is 100 μM, the anti-inflammatory effect is 95.09%, which is significantly higher than that of the commonly used anti-inflammatory drug dexamethasone.

[0053] After calculation, the IC 50 of lilacin A inhibiting NO release is 15.41 μM, indicating good anti-inflammatory activity.

[0054] Table 1 Anti-inflammatory Rates of Different Groups on LPS-Induced RAW264.7 Cells

[0055]

Claims

1. A preparation method of an anti-inflammatory active extract of lily, characterized in that, The following steps are involved: S1. Take dried lily medicinal material and extract it twice with 60% ethanol under reflux; S2, filtering and combining the two extracts in step S1, recovering ethanol from the extracts and concentrating them into a dilute extract; S3, the dilute extract obtained in step S2 is subjected to D101 macroporous adsorption resin, eluted with water, and the water eluate is discarded; then eluted with 80% ethanol, the eluate is collected, and concentrated under reduced pressure to obtain the extract; S4, subjecting the extract obtained in step S3 to silica gel column chromatography, using dichloromethane-methanol as the eluent, gradient elution, collecting the eluate, identifying it by thin layer chromatography, and combining them; S5, applying the product obtained in step S4 to a reverse phase silica gel RP-18 column, using methanol-water as the eluent, gradient elution, collecting the eluate, performing thin layer chromatography for identification, and combining; S6, further purifying the product obtained in S5 repeatedly through a Sephadex LH-20 gel column to obtain a lily extract with anti-inflammatory activity; In the step S4, the volume ratio of the eluent dichloromethane and methanol for gradient elution is 9:1 to 0:10; In the step S5, the volume ratio of the eluent methanol and water for gradient elution is 4:6 to 10:0; The structural formula of the lily anti-inflammatory active extract is shown in Formula 1 below:

2. The preparation method of the lily anti-inflammatory active extract according to claim 1, wherein, In step S1, the lily medicinal material is lily fleshy bulb.

3. The preparation method of the lily anti-inflammatory active extract according to claim 1, characterized in that, In step S1, the mass ratio of the lily medicinal material to the ethanol used for the two extractions is 1:11:

10.

4. The preparation method of the lily anti-inflammatory active extract according to claim 1, characterized in that, Each extraction time in step S1 is 1 hour.

5. The preparation method of the lily anti-inflammatory active extract according to claim 1, characterized in that, The relative density of the dilute extract in step S2 is 1.

05.

6. Use of an anti-inflammatory active extract of lily as shown in Formula 1 in the preparation of a drug for treating inflammation:

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