Preparation and Application of Anti-inflammatory Active Phenylethanoid Glycosides in Qianjinzhui

Abstract

The present invention belongs to the field of pharmaceutical technology, and provides two phenylethanoid glycoside compounds with anti-inflammatory activity in Flemingia philippinensis Merr. ex Rolfe, and their preparation methods and applications. Specifically, it relates to the extraction and isolation of two novel phenylethanoid glycoside compounds from Flemingia philippinensis Merr. ex Rolfe, namely compound 1 (xylanchoside A) and compound 2 (xylanchoside B) as shown in the structural formula; compounds 1-2 have inhibitory effects on the cytokines IL-6 and TNF-α secreted by LPS-induced macrophages RAW264.7, and can form a pharmaceutical composition with a pharmaceutical carrier for the preparation of anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the fields of Chinese medicine chemistry and pharmaceutical technology, and particularly relates to two novel phenylpropanoid glycoside compounds, a preparation method thereof, and an application thereof in the preparation of anti-inflammatory drugs. Background Art

[0002] Qianjinzhui is the dried tuber of Xylanche himalaica Hook.f.et Thoms. of the Orobanchaceae family, which often parasitizes on the roots of plants of the Rhododendron genus. It is mainly distributed in Yunnan, Qinghai, Tibet, Sichuan, Hubei and other regions. It is dug in summer and autumn, and the spherical tuber is used as medicine after air-drying or sun-drying. It has the effects of dispelling wind and activating collaterals, regulating qi and strengthening the stomach, and is used for rheumatic arthralgia, irregular menstruation, stomachache, abdominal distension, hernia and cough. Some ethnic minorities in Yunnan also use Qianjinzhui to treat schistosomiasis, mumps and aconite poisoning and other diseases. As a unique medicinal plant in Yunnan, the research on its chemical constituents and biological activities is very weak at present. In order to explore the medicinal value of Qianjinzhui, the present invention takes the active part of Qianjinzhui as the research object, and uses the extraction and separation methods of natural medicine chemistry to conduct a systematic study on its material basis. Two novel phenylpropanoid glycoside compounds, compound 1 (xylanchoside A) and compound 2 (xylanchoside B), are isolated and purified therefrom, and it is screened that the two compounds have inhibitory effects on the cytokines IL-6 and TNF-α secreted by LPS-induced macrophages RAW264.7, and can be used for the preparation of anti-inflammatory drugs. So far, there is no report on compound 1 (xylanchoside A) and compound 2 (xylanchoside B) in the prior art, nor is there a report on a pharmaceutical composition with them as active ingredients, nor is there a report on the application of the pharmaceutical composition in the preparation of anti-inflammatory drugs. Summary of the Invention

[0003] The purpose of the present invention is to provide a preparation method, a pharmaceutical composition and an application of two new compounds 1 (xylanchoside A) and 2 (xylanchoside B) with medicinal value. In order to explore the medicinal value of Qianjinzhui, during the study on the material basis of the active part, two novel phenylpropanoid glycoside compounds 1 and 2 that have not been published are isolated and purified from Qianjinzhui; compounds 1 and 2 have inhibitory effects on the cytokines IL-6 and TNF-α secreted by LPS-induced macrophages RAW264.7, and can form a pharmaceutical composition with a pharmaceutical carrier for the preparation of anti-inflammatory drugs.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides two novel phenylethanoid glycoside compounds, compound 1 (xylanchoside A) and compound 2 (xylanchoside B), whose structures are shown as follows:

[0006]

[0007] The present invention also provides a preparation method of the novel phenylethanoid glycoside compounds 1-2 in the root tubers of Flemingia philippinensis Merr. ex Rolfe, comprising the following steps:

[0008] (1) The dried rhizomes of Flemingia philippinensis Merr. ex Rolfe are crushed into coarse powder, decocted twice with water, adding 10 times the amount of water each time, decocting for 1.5 hours, combining the decoctions, filtering, and concentrating the filtrate under reduced pressure to a relative density of 1.1 (60 °C) at 60-70 °C, cooling, adding 95% ethanol to make the alcohol content reach 70%, standing for 24 hours, filtering, and concentrating the filtrate under reduced pressure to an extract with a relative density of 1.1-1.2 (60 °C).

[0009] (2) The extract is mixed with silica gel and separated by silica gel column chromatography, and gradient elution is carried out with chloroform-methanol at a ratio of 15:1 → 1:1 as the eluent, collecting each fraction respectively, and monitoring by TLC to combine the same fractions to obtain 11 component segments Fr1-Fr11;

[0010] (3) Take the component segment Fr8, carry out MCI column chromatography, and carry out gradient elution with methanol-water at a ratio of 10% → 40% as the eluent, collecting each fraction respectively, and monitoring by TLC to combine the same fractions to obtain 13 fraction groups Fr8-1-Fr8-13.

[0011] (4) Take the fraction group Fr8-13, separate it by Sephadex LH20 gel chromatography column, and elute with methanol to purify compound 1.

[0012] (5) Take the component segment Fr7, carry out MCI column chromatography, and carry out gradient elution with methanol-water at a ratio of 10% → 70% as the eluent, collecting each fraction respectively, and monitoring by TLC to combine the same fractions to obtain 10 fraction groups Fr7.1-Fr7.10.

[0013] (6) Take the fraction group Fr7.3, mix it with silica gel and separate it by silica gel column chromatography, and carry out gradient elution with dichloromethane-methanol-water at a ratio of 7:2.5:1 → 6:4:1 as the eluent, collecting each fraction respectively, and monitoring by TLC to combine the same fractions to obtain 8 fraction groups Fr7.3.1-Fr7.3.8.

[0014] (7) Take the fraction group Fr7.3.5 and separate it by RP 18 reverse-phase chromatography column, and carry out gradient elution with 15% → 70% methanol solution, and detect by TLC to combine the same fractions to obtain 9 sub-fraction groups Fr7.3.5.1-Fr7.3.5.9.

[0015] (8) The sub-fraction group Fr7.3.5.5 was separated by silica gel column chromatography, eluted with dichloromethane-methanol-water at a ratio of 7:2.5:1, and the same fractions were combined by TLC detection to obtain 4 sub-sub-fraction groups Fr7.3.5.5.1 to Fr7.3.5.4.

[0016] (9) The sub-sub-fraction group Fr7.3.5.5.4 was separated by a Sephadex LH20 gel chromatography column and eluted with methanol to obtain Compound 2 after purification.

[0017] In the preparation method, the Qianjinzhui used is Xylanche himalaica Hook.f.et Thoms. of the family Orobanchaceae.

[0018] The present invention provides a pharmaceutical composition and its preparation, which contain one or more of the anti-inflammatory active phenylethanoid glycosides in the above-mentioned Qianjinzhui or a pharmaceutically acceptable salt of the compound, as well as a pharmaceutically acceptable carrier and excipient; it also includes one or a combination of a pharmaceutically acceptable carrier, excipient, and diluent. The pharmaceutical composition preparation of the present invention is divided into an oral administration pharmaceutical composition or an injection administration pharmaceutical composition according to the administration route. The present invention has no special limitation on the dosage form, and the dosage forms well-known in the art can be selected, including but not limited to tablets, capsule preparations, granules, sprays, sustained-release tablets, oral liquids, powders, infusion granules, or injection preparations.

[0019] The present invention also provides the use of the anti-inflammatory active phenylethanoid glycosides in the above-mentioned Qianjinzhui or a pharmaceutically acceptable salt of the compound or the above-mentioned pharmaceutical composition in the preparation of anti-inflammatory drugs.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. The present invention provides 2 new compounds - novel phenylethanoid glycosides, Compound 1 (xylanchoside A) and Compound 2 (xylanchoside B).

[0022] 2. The present invention provides a preparation method for novel phenylethanoid glycosides 1-2, which has easily available raw materials and is easy to operate.

[0023] 3. The present invention provides that novel phenylethanoid glycosides 1-2 have inhibitory effects on the cytokines IL-6 and TNF-α secreted by LPS-induced macrophages RAW264.7, and can form a pharmaceutical composition with a pharmaceutical carrier for the preparation of anti-inflammatory drugs.

[0024] The present invention further enriches the chemical substance basis of the active part of Qianjinzhui, lays a foundation for the relevant bioactivity tests of the monomer compounds obtained subsequently, provides active leading compounds for new drug development, and also provides a theoretical basis for the research and development of the drug property of Qianjinzhui. Description of the Drawings

[0025] Figure 1 Structural formulas of Compound 1 and Compound 2;

[0026] Figure 2 For Compound 1 1 H- 1 H COSY and HMBC correlations;

[0027] Figure 3 For Compound 2 1 H- 1 H COSY and HMBC correlations;

[0028] Figure 4 Effects of Compound 1 and Compound 2 on cell viability;

[0029] Figure 5 Effects of Compound 1 and 2 on cytokines IL-1β, IL-6 and TNF-α in LPS-induced macrophages RAW264.7. Detailed Embodiments

[0030] To better understand the essence of the present invention, the following further illustrates the substantial content of the present invention in conjunction with embodiments, but does not limit the present invention thereto.

[0031] In combination with the embodiments of the present invention, the technical solutions in the present invention are clearly and completely described. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0032] Example 1: Preparation of Compound 1 (xylanchoside A) and Compound 2 (xylanchoside B)

[0033] (1) Crushing of medicinal materials

[0034] 17 kg of Qianjinzhui medicinal materials are crushed into coarse powder by a CSJ-60 type coarse crusher.

[0035] (2) Preparation of active part

[0036] The crude powder of Qianjinzhui is decocted twice with water. Each time, 10 times the amount of water (170 L) is added, and it is decocted for 1.5 hours. The decoction is filtered through a 200-mesh filter screen. The aqueous decoctions are combined, and the filtrate is concentrated under reduced pressure at 60 - 70 °C to a relative density of 1.1 (60 °C). After cooling, 95% ethanol is added to make the ethanol content reach 70%. It is left standing for 24 hours and then filtered. The filtrate is concentrated under reduced pressure to an extract of relative density 1.1 - 1.2 (60 °C), weighing 6.5 kg, and the yield is 38.2%.

[0037] (3)Segmentation by silica gel chromatography column

[0038] Take 2 kg of the Qianjinzhui extract obtained in step 2, mix it with silica gel (100 - 200 mesh), and separate it by silica gel (100 - 300 mesh) column chromatography. Dichloromethane - methanol or chloroform - methanol with a ratio of 15:1 → 1:1 is used as the eluent for gradient elution. Each fraction is collected separately, and the same fractions are combined by TLC monitoring. A total of 11 component segments are obtained, named Fr1 - Fr11 respectively.

[0039] (4)Isolation and purification of compound 1

[0040] Take 200 g of the extract of component segment Fr8 obtained in step 3, and separate it by MCI column chromatography. Methanol - water with a ratio of 10% → 40% (v / v) is used as the eluent for gradient elution. Each fraction is collected separately, and the same fractions are combined by TLC monitoring. 13 fraction groups are obtained, named Fr8 - 1 - Fr8 - 13 respectively; Take 8.23 g of the extract of fraction group Fr8 - 13, separate it by Sephadex LH20 gel chromatography column, and elute with methanol to purify compound 1 (50 mg).

[0041] (5)Isolation and purification of compound 2

[0042] Take 324.51 g of the fraction Fr7 extract obtained in step 3, perform MCI column chromatography, and use methanol-water with a gradient of 10%→70% as the eluent for gradient elution. Collect each fraction separately, monitor by TLC and combine the same fractions to obtain 10 fraction groups, named Fr7.1~Fr7.10 respectively; take 200 g of the Fr7.3 fraction group extract, mix it with silica gel (100~200 mesh), perform silica gel (100~300 mesh) column chromatography separation, and use dichloromethane-methanol-water with a gradient of 7∶2.5∶1→6∶4∶1 (v / v / v) as the eluent for gradient elution. Collect each fraction separately, monitor by TLC and combine the same fractions to obtain 8 fraction groups, named Fr7.3.1~Fr7.3.8 respectively; take 40 g of the Fr7.3.5 fraction group extract, perform RP18 reverse phase chromatography column separation, and use a methanol solution with a gradient of 15%→70% for gradient elution. Detect by TLC and combine the same fractions to obtain 9 sub-fraction groups, named Fr7.3.5.1~Fr7.3.5.9 respectively; take 11 g of the Fr7.3.5.5 sub-fraction group extract, mix it with silica gel (100~200 mesh), perform silica gel (100~300 mesh) column chromatography separation, and use dichloromethane-methanol-water with a ratio of 7∶2.5∶1 (v / v / v) as the eluent for elution. Detect by TLC and combine the same fractions to obtain 4 sub-sub-fraction groups, named Fr7.3.5.5.1~Fr7.3.5.4 respectively; take the Fr7.3.5.5.4 sub-sub-fraction group (210 mg), perform Sephadex LH20 gel chromatography column separation, and elute with methanol to purify and obtain compound 2 (24.1 mg).

[0043] Example 2 Structure Identification of New Compounds

[0044] (1)Structure Identification of Compound 1

[0045]

[0046] xylanchoside A

[0047] Molecular formula: C 37 H 48 O 21

[0048] Appearance: Pale yellow oily substance

[0049] HRESI-MS m / z: 851.2585 [M+Na] + (Calculated value: C 37 H 48 O 21 Na, 851.2580)

[0050] Compound 1, a pale yellow oily substance, with a molecular formula of C + ) and 13 determined by positive-ion HRESI-MS m / z 851.2585 (calcd. 851.2580 [M+Na] 37 H 48 O 21 . Combining with HSQC, the 1 H and 13 C NMR (Table 2) of compound 1 showed three typical anomeric signals [4.47 (d, J = 8.1 Hz, H-1″), 4.80 (dd, J = 7.8, 6.5 Hz, H-1″′), 4.57 (d, J = 7.8 Hz, H-1″′′); δ C 101.83 (C-1″), 102.50 (C-1″′), 105.58 (C-1″′′)] and the characteristic signal of rhamnose at C-6 [1.31 (d, J = 6.2 Hz, H-6″′, δ C 18.16 (C-6″′)], one acetyl group [δ H 1.97 (d, J = 4.2 Hz); δ C 20.86, 171.25], two sets of ABX spin system aromatic signals [δ H 6.62 (d, J = 1.9 Hz, H-2), 6.65 (d, J = 8.0 Hz, H-5), 6.50 (dd, J = 8.0, 1.9 Hz, H-6), δ H 7.05 (d, J = 1.9 Hz, H-2′), 6.79 (t, J = 8.3 Hz, H-5′), 6.92 (dd, J = 8.3, 1.9 Hz, H-6′); δ C 117.19 (C-2), 116.27 (C-5), 121.16 (C-6), 115.03 (C-2′), 116.53 (C-5′), 123.14 (C-6′)], one set of methylene signals [δ H 2.68 (dd, J = 10.9, 6.1 Hz, H-7; δ C 36.29 (C-7)], one set of oxygenated methylene signals [δ H 3.99 (dd, J = 6.1, 3.5 Hz, H-8a), 3.61 (m, H-8b), δ C71.84 (C-8)], a set of signals of trans-olefins [6.30 (d, J = 15.9 Hz, H-7′), 7.57 (d, J = 15.9 Hz, H-8′); δ C 114.73 (C-7′), δ C 147.72 (C-8′)] and one ketone carbonyl [δ C 169.05 (C-9′)]. Based on the above information speculation, compound 1 is a typical phenyl ethanol glycoside compound. For the comparison of the 1 H and 13 C NMR data of this compound and Monoacetylrossicaside A, the only difference is that in Monoacetylrossicaside A, 80.21 (C-3″) and 62.14 (C-6″) shift to lower field to 82.14 (C-3″), 64.25 (C-6″), and it is speculated that the position of the caffeoyl group may have changed. In the HMBC spectrum, H-6″ (δ H 4.36, 4.50) is correlated with C-9′ (169.05), indicating that the caffeoyl group is connected to glucose C-6″, which confirms the above speculation. To sum up, the structure of compound 1 is a new compound not reported in the literature and is named xylanchoside A. 1 1H NMR and 13 13C NMR (DEPT) data are shown in Table 1.

[0051]

[0052] (2) Structure data of compound 2.

[0053]

[0054] xylanchoside B

[0055] Molecular formula: C 29 H 44 O 18

[0056] Appearance: white solid

[0057] HRESI-MS m / z: 703.2414 [M+Na] + , (calculated value: C 20 H 44 O 18 Na, 703.2420)

[0058] Compound 2, white solid, according to the positive ion HR-ESI-MS (m / z 703.2414 [M+Na]+ , Calculated value: 703.2420) determined the molecular formula to be C 29 H 44 O 18 , with an unsaturation degree of 8. 1 H and 13 The H C NMR (Table 2) spectrum shows a typical ABX spin system [δ H 6.67 (d, J = 2.1 Hz, H-3), 6.63 (dd, J = 2.1, 8.2 Hz, H-5), 6.80(d, J = 8.2 Hz, H-6); δ C 117.1 (C-3), 121.2 (C-5), 112.8(C-6) ], one H 3.81(s); δ H C methoxy group [δ H 1.97 (s); δ C 20.9, 171.6], one H 2.70 (m, H-7; δ H C methylene group signal [δ H 3.63 (m, H-8a), 4.05 (m,H-8b), δ C 71.5 (C-8)], three H 4.44 (d, J = 8.1 Hz, H-1'), 4.78 (d, J =1.6 Hz, H-1"), 4.56 (d, J = 7.8 Hz, H-1‴); δ H C 101.7 (C-1'), 102.6(C-1"), 105.6(C-1‴)]. From the above information, it can be seen that compound 2 is a typical Figure 3 ), in the HMBC spectrum, H-1' is correlated with C-8 (δ Figure 3 C 71.5), H-1" is correlated with C-3' (δ C 82.7), H-1‴ is correlated with C-4" (δ C 83.3), indicating that the sugar chain is linked at the C-8 position, rhamnose is linked at C-3', and glucose is linked at C-4". In addition, H-1' and H-2' [δ H 4.75 (m)] are correlated with δ CIt was related to 171.6, indicating that the acetyl group was connected to the C-2' position. By the coupling constant J > 7.0 Hz, glucose was determined to be in the β configuration; in addition, rhamnose was determined to be in the α configuration by comparing the chemical shifts of C-3 and C-5 with those of α-L-Rha and β-L-Rha. To sum up, the structure of compound 2 was determined to be a new phenylethanoid glycoside compound and named xylanchoside B. 1 The H NMR and 13 C NMR (DEPT) data are shown in Table 2.

[0059]

[0060] Example 3: In vitro anti-inflammatory activities of compounds 1-2 in macrophage RAW264.7

[0061] (1) Detection of the effects of compounds 1-2 on cell viability by CCK8 method

[0062] Using DMEM medium, RAW264.7 cells were cultured routinely. When the cell confluence reached 80%, the cell morphology was observed to be normal under the microscope. The cells were seeded in 96-well plates at a density of 2×10 5 cells / mL, 100 μL / well. After culturing overnight in a carbon dioxide incubator, the supernatant in the wells was discarded. Sample solutions of different concentrations of compound 1 and compound 2 (200, 100, 50, 25, 12.5 μg / mL) were added to treat the cells, 100 μL per well, with 6 wells for each concentration. The blank control group was added with the same volume of DMEM and cultured in a carbon dioxide incubator. After 24 hours, 10 μL of CCK-8 reagent solution was added to each well. After culturing for 4 hours in a carbon dioxide incubator, the absorbance was measured at a wavelength of 450 nm with an enzyme-linked immunosorbent assay reader, and the cell viability was statistically analyzed. The results are as Figure 4 shown: The maximum non-toxic doses of compound 1 and compound 2 on RAW264.7 cells were 50 μg / mL.

[0063] (2) Detection of the effects of compounds 1-2 on LPS-induced inflammatory-related indicators in macrophage RAW264.7 by ELISA assay

[0064] Using DMEM medium, RAW264.7 cells were cultured routinely. When the cell confluence reached 80%, the cell morphology was observed to be normal under the microscope. The cells were seeded in 96-well plates at a density of 2×10 5Inoculate at a density of Figure 5 per mL into a 6-well plate, 2 mL per well. After culturing overnight in a carbon dioxide incubator, discard the supernatant in the wells. For both Compound 1 and Compound 2, sample solutions with concentrations of 50 and 25 μg / mL are added as the high and low dose groups (diluted with DMEM, and the concentrations are confirmed by the results of the cytotoxicity test). At the same time, Lipopolysaccharide (LPS) is added for model establishment, with a concentration of 200 ng / ml, 1 mL per well, and 3 wells for each concentration. The blank control group is added with the same volume of DMEM, and the model control group is added with a DMEM solution containing the same concentration of LPS, and then cultured in a carbon dioxide incubator. After 24 hours, aspirate the cell supernatant and use a kit to detect IL-1β, IL-6, and TNF-α. The results are as

[0065] Example 4: New compound pharmaceutical preparations

[0066] (1) Take 5.0 g of Compound 1 or / and Compound 2, 5.0 g of microcrystalline cellulose, and 14.7 g of compressible starch, mix them evenly, add an appropriate amount of 2.5% polyethylene glycol, mix them evenly, and prepare spherical pellets by the extrusion-spheronization method. Load the pellets into capsules to make capsules;

[0067] (2) Grind Compound 1 or / and Compound 2 into fine powder, pass through a 100-mesh sieve. Take 5.0 g and mix it evenly with 16.0 g of polyoxyethylene and 3.0 g of sodium chloride that have passed through a 100-mesh sieve. Add a 2% polyvinylpyrrolidone (95% ethanol) binder to make soft materials, granulate through a 20-mesh sieve, dry at 40°C, screen through an 18-mesh sieve for sizing, tableting, and perform semi-permeable membrane coating with cellulose acetate as the matrix material and polyethylene glycol as the pore-forming agent (10% of the coating solid content) to make sustained-release tablets.

[0068] (3) Take Compound 1 or / and Compound 2 and prepare oral liquid according to the conventional oral liquid preparation method.

[0069] As can be seen from the above examples, the present invention provides two novel phenylethanoid glycoside compounds, Compound 1 (xylanchoside A) and Compound 2 (xylanchoside B), and their preparation methods; Compounds 1-2 have inhibitory effects on the cytokines IL-6 and TNF-α secreted by LPS-induced macrophages RAW264.7, and can form a pharmaceutical composition with a pharmaceutical carrier for the preparation of anti-inflammatory drugs.

[0070] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a compound or a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising one or more of the compound or its pharmaceutically acceptable salt, or a pharmaceutical preparation comprising a therapeutically effective amount of the pharmaceutical composition and a pharmaceutically acceptable carrier in the preparation of an anti-inflammatory drug; Among them, The compound is Compound 2, and its chemical structural formula is as follows:

2. The application according to claim 1, wherein The pharmaceutically acceptable carrier is selected from one or more of fillers, disintegrants, binders, and lubricants.

3. The application according to claim 1, characterized in that, The pharmaceutical preparation is a tablet, a capsule preparation, a granule, a spray, an oral liquid, a powder, or an injection preparation.

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