Steroid saponin in traditional Chinese medicine radix physochlainae as well as extraction and purification method and application thereof

Abstract

The invention discloses steroid saponin in traditional Chinese medicine physochlaina macrophylla as well as an extraction and purification method and application thereof. The structural formula of the steroid saponin is as shown in formula I in the specification. According to the present invention, the chemical components in the physochlaina macrophylla 70% ethanol extract are separated and purified by using the traditional separation methods such as normal phase silica gel column chromatography, reverse phase ODS column chromatography, high performance liquid chromatography and the like, the steroid saponin component is found for the first time, and the new steroid saponin, namely the compound represented by the formula I, is obtained; the research on the activity of new steroid saponin components in radix physochlainae shows that the steroid saponin components have anti-inflammatory activity. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a steroidal saponin in a traditional Chinese medicine Panax notoginseng and an extraction and purification method and application thereof. Background Art

[0002] Chinese ginseng (Physochlainae Radix) is the dried root of Physochlainainfundibularis Kuang, a plant of the Solanaceae family. It first appeared in the "Supplement to Compendium of Materia Medica" written by Zhao Xuemin in the Qing Dynasty: "It is shaped like ginseng, with a dark blue skin and heart. This ginseng comes from Huashan Mountain in Shaanxi Province. Eating it causes vomiting." Hence the name Chinese ginseng, and it is distributed in Henan, Shanxi and other provinces. According to the 2020 edition of the "Chinese Pharmacopoeia", Chinese ginseng is warm in nature, sweet and slightly bitter in taste; it has the effects of warming the lungs and removing phlegm, relieving asthma and cough, and calming the nerves. It is mostly used clinically to treat symptoms such as cold phlegm, asthma and cough, palpitations and insomnia, and is also often used to treat cough, asthma, and tuberculosis among the people. In addition, studies have shown that Chinese ginseng also has certain therapeutic effects on diseases such as stomach and abdominal pain, neurasthenia, and schizophrenia. Panax notoginseng is a traditional Chinese medicine for resolving phlegm, relieving cough and relieving asthma. Although literature has reported that Panax notoginseng and its Chinese patent medicine have pharmacological effects in treating cough, asthma and other symptoms, previous studies have mainly focused on the analysis and determination of the tropane alkaloids it contains, and there has been no literature report on a comprehensive and systematic study of its ingredients, resulting in a weak material basis for its efficacy, and a systematic study of the related mechanism of action has not yet been carried out. Summary of the invention

[0003] The purpose of the present invention is to provide a steroidal saponin in Chinese medicinal material Panax notoginseng and a method for extracting and purifying the same.

[0004] The steroidal saponin in the Chinese medicinal herb Panax notoginseng provided by the present invention has a structural formula as shown in Formula I:

[0005]

[0006] The compound represented by formula I, and its pharmaceutically acceptable salts, esters, solvates, stereoisomers or tautomers also fall within the protection scope of the present invention.

[0007] The compound of formula I of the present invention can be used in the form of a pharmaceutically acceptable salt or solvate derived from an inorganic acid or an organic acid. The term "pharmaceutically acceptable salt" refers to a salt that is suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and is commensurate with a reasonable effect / risk ratio within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. The salt can be prepared by reacting the free base functionality of the compound of the present invention with a suitable organic acid.

[0008] The preparation method of the compound shown in formula I provided by the present invention comprises the following steps: extracting Panax notoginseng with an ethanol solution having a volume fraction of 50% to 95%, collecting the extract; and separating the compound shown in formula I from the extract.

[0009] Furthermore, the ethanol solution may be an ethanol solution with a volume fraction of 70%.

[0010] Furthermore, the extraction is performed at least once; during each extraction, the solid-liquid ratio of the Chinese ginseng root to the ethanol solution is 1:10-20, and the time for each extraction is 5-10 days.

[0011] According to an embodiment of the present invention, the extraction method is leaching, and the extraction is performed 3 times. During each extraction, the solid-liquid ratio of the Huashan ginseng ethanol solution can be 1:20, and the time of each leaching can be 7 days.

[0012] Furthermore, the method also includes the steps of treating the extract as follows: concentrating the extract under reduced pressure to obtain a crude extract; adding distilled water to disperse the crude extract evenly, then extracting with petroleum ether, ethyl acetate, and water-saturated n-butanol in sequence, collecting the water-saturated n-butanol extract and concentrating it under reduced pressure to obtain an n-butanol extraction portion; and separating the compound shown in Formula I from the n-butanol extraction portion.

[0013] According to an embodiment of the present invention, the ratio of the crude extract to distilled water is 1:10; the petroleum ether extraction is performed twice, the ethyl acetate extraction is performed three times, and the water-saturated n-butanol extraction is performed five times; the amount of each extraction solvent used in each extraction is the same as the amount of the distilled water used.

[0014] Furthermore, the specific method for isolating the compound represented by formula I from the n-butanol extraction part is as follows:

[0015] Take 400g of the n-butanol extraction part, add an appropriate amount of methanol to redissolve, then mix the sample in 500g of 80-120 mesh silica gel, wet-load the column, and load the sample into the silica gel chromatographic column (2.5kg of 200-300 mesh silica gel is loaded into the glass chromatographic column). The silica gel chromatographic column uses dichloromethane-methanol as the mobile phase for gradient elution, and the elution ratio is 50:0-0:50 (the ratios are: dichloromethane-methanol = 50:0, 50:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0:1, v / v). The amount of mobile phase used for each elution ratio is 2-4 column volumes, and each portion of eluent is collected as 1 / 4 of the column volume, and 120 portions of eluent are obtained. Each eluate was detected by thin layer chromatography under 254nm, 365nm ultraviolet light and sunlight (with 10% sulfuric acid-ethanol as the color developer), and the 1st to 4th, 5th to 9th, 10th to 14th, 15th to 22nd, 23rd to 31st, 32nd to 47th, 48th to 56th, 57th to 68th, 69th to 86th, 87th to 94th, 95th to 102nd, 103th to 108th, 109th to 113th, 114th to 118th, and 119th to 120th eluates with similar chromatographic behaviors were combined to obtain components Fr.1-15 in sequence.

[0016] Take component Fr.4 and perform gradient elution with methanol-water as mobile phase through reverse phase ODS column chromatography, with concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% methanol in sequence. The amount of mobile phase used for each elution concentration is 1 to 3 column volumes, and each portion of eluate is collected as 1 / 5 of the column volume, to obtain 153 portions of eluate; in the thin layer chromatography identification, each eluate is detected by thin layer chromatography under 254nm, 365nm ultraviolet light and sunlight (with 10% sulfuric acid-ethanol as a color developer), and the 1st to 8th, 9th to 19th, 20th to 31st portions with similar chromatographic behaviors are combined The 32nd to 45th, 46th to 68th, 69th to 82nd, 83rd to 96th, 97th to 104th, 105th to 111th, 112th to 118th, 119th to 125th, 126th to 130th, 131st to 137th, 138th to 145th, 146th to 153rd eluents were sequentially obtained to obtain 15 components in total, which were marked as components Fr. 4A to 4O;

[0017] Component Fr.4I was separated and purified by preparative HPLC using methanol-water as the mobile phase at a ratio of 61:39 (v / v) to obtain compound 1 (t R =23.6min).

[0018] The present invention also provides the use of the compound represented by the above formula I or its pharmaceutically acceptable salt, ester, solvate stereoisomer or tautomer in the preparation of at least one of the following products:

[0019] 1) Products for preventing and / or alleviating and / or treating inflammation;

[0020] 2) Products for the prevention and / or treatment of acute lung injury.

[0021] Furthermore, the acute lung injury is caused by inflammation.

[0022] Specifically, the prevention and / or treatment of acute lung injury is embodied in at least one of the following aspects:

[0023] a) Inhibit the increase in the number of inflammatory cells and the increase in total protein content in bronchoalveolar lavage fluid caused by acute lung injury;

[0024] b) Reduce lung tissue edema caused by acute lung injury;

[0025] c) Alleviate and / or improve lung pathological changes caused by acute lung injury;

[0026] d) inhibiting the increased expression levels of IL-6, IL-1β and TNF-α in bronchoalveolar lavage fluid caused by acute lung injury;

[0027] e) inhibiting the increased expression levels of COX-2, IL-6 and TNF-α in lung tissue caused by acute lung injury;

[0028] f) Inhibit the increased expression levels of TLR4, MyD88 and NF-κB proteins in lung tissue caused by acute lung injury.

[0029] Furthermore, the product may be a medicine, a composition, a health product or other biological product.

[0030] The invention also provides a product.

[0031] The product comprises the compound represented by formula I of the present invention or its pharmaceutically acceptable salt, ester or solvate, stereoisomer or tautomer.

[0032] The active ingredient of the above-mentioned product may be the compound represented by Formula I or a pharmaceutically acceptable salt, ester, solvate, stereoisomer or tautomer thereof. The active ingredient of the above-mentioned product may also contain other biological components or non-biological components. Those skilled in the art may determine the other active ingredients according to the therapeutic effect of the product.

[0033] The product has at least one of the following effects: 1) preventing and / or alleviating and / or treating inflammation; 2) preventing and / or treating acute lung injury.

[0034] The product may be a medicine, a composition, a health product or other biological product.

[0035] In the above, the product contains not only the compound of formula I or its pharmaceutically acceptable salt, ester, solvate, stereoisomer or tautomer, but also a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier may be 0.1-99.9% by weight of the total weight of the product.

[0036] Among them, the pharmaceutically acceptable carrier includes microcrystalline cellulose, sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, mannitol, sorbitol, sorbic acid or potassium salt, sodium pyrosulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin A, vitamin C, vitamin E, vitamin D, azone, disodium EDTA, calcium sodium EDTA, carbonate, acetate, phosphate of monovalent alkali metal or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, Fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinyl pyrrolidone, glycerol, propylene glycol, ethanol, Tween 60-80, Span-80, beeswax, lanolin, liquid paraffin, cetyl alcohol, gallic acid esters, agar, triethanolamine, basic amino acids, urea, allantoin, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate and microcrystals.

[0037] The drug described in the present invention can be prepared into any pharmaceutical dosage form such as tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, pills, powders, pills, suspensions, solutions, injections, ointments, plasters, and sprays.

[0038] The present invention uses conventional separation methods such as normal phase silica gel column chromatography, reverse phase ODS column chromatography and high performance liquid chromatography to separate and purify the chemical components in 70% ethanol extract of Panax notoginseng, and discovers steroidal saponins for the first time, and obtains a new steroidal saponin. Through the activity study of the new steroidal saponin component in Panax notoginseng, it is found that it has anti-inflammatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the HR-ESI-MS spectrum of Infundilide A;

[0040] Figure 2 For Infundilide A 1H NMR spectrum (600MHz, pyridine-d 5 );

[0041] Figure 3 For Infundilide A 13 C NMR spectrum (150MHz, pyridine-d 5 );

[0042] Figure 4 DEPT 135 spectrum of Infundilide A;

[0043] Figure 5 is the HSQC spectrum of Infundilide A;

[0044] Figure 6 For Infundilide A 1 H- 1 H COSY spectrum;

[0045] Figure 7 is the HMBC spectrum of Infundilide A;

[0046] Figure 8 is the NOESY spectrum of Infundilide A;

[0047] Fig. 9 GC-MS spectrum of Infundilide A; AD-galactose; BD-glucose; C.InfundilideA;

[0048] Fig.10 The key COSY (bold line) and HMBC (arrow) related signals of Infundilide A.

[0049] Fig.11 This is the X-ray ORTEP image of Infundilide A.

[0050] Fig.12 Infundilide A significantly reduced lung tissue damage in ALI mice. (AB) The number of inflammatory cells in BALF; (C) Quantification of total protein content in BALF; (D) Wet / dry weight ratio of lung tissue.

[0051] Fig.13 This is the effect of Infundilide A on the pathological changes of lung tissue in ALI mice.

[0052] Fig.14Infundilide A regulates the expression levels of inflammatory factors in the lung tissue of ALI mice; (AC) The effect of Infundilide A on the expression levels of IL-1β, TNF-α and IL-6 in the BALF of ALI mice; (DG) Immunofluorescence detection of the effect of Infundilide A on the expression levels of COX-2, IL-6 and TNF-α in the lung tissue of ALI mice.

[0053] Fig.15 Immunohistochemical staining was used to detect the effect of Infundilide A on the expression of TLR4, MyD88, and NF-κB proteins in the lung tissue of ALI mice. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0055] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0056] Example 1. Preparation of a new steroidal saponin Infundilide A from Panax notoginseng

[0057] 1. Experimental instruments and materials

[0058] P850 fully automatic polarimeter (China Haineng Future Technology Group Co., Ltd.); Bruker Ascend-600MHz nuclear magnetic resonance spectrometer (Bruker, Germany); Q Exactive Focus LC-MS / MS liquid chromatography-mass spectrometry (Thermo Fisher, USA); Büchi pump Manager C-610 medium pressure chromatograph (Büchi, Switzerland); Waters 2695-2998-2414 analytical high performance liquid chromatograph (Waters, USA); Shimadzu Essentia LC-16P preparative high performance liquid chromatograph (SHIMADZU, Japan); Sharpsil-U C18 analytical chromatographic column (4.6 mm × 250 mm, 5 μm, Kelanmu Experimental Technology (Shanghai) Co., Ltd.); Sharpsil-U C18 preparative chromatographic column (21.2 mm × 250 mm, 5 μm, Kelanmu Experimental Technology (Shanghai) Co., Ltd.); column chromatography silica gel (200-300 mesh, 80-120 mesh, Qingdao Ocean Chemical Plant); column chromatography ODS (YMC Company, Japan); chromatographic grade methanol (OCEANPAK Company, Sweden); other reagents were of analytical grade (Tianjin Zhiyuan Chemical Reagent Co., Ltd.).

[0059] The Chinese ginseng was purchased in Queshan County, Henan Province in September 2021 and was identified as the dried root of Physochlaina infundibularisKuang, a plant of the Solanaceae family, by Professor Pei Xiangping of the Chinese Medicine Identification Teaching and Research Section, School of Chinese Medicine and Food Engineering, Shanxi University of Traditional Chinese Medicine. The original specimen (20210915) is preserved in the Shanxi Modern Chinese Medicine Engineering Laboratory of Shanxi University of Traditional Chinese Medicine.

[0060] 2. Extraction and separation

[0061] Take 10kg of dried Chinese ginseng, crush it into uniform small pieces, and extract it with 20 times 70% ethanol at room temperature for 3 times, each time for 7 days. Combine the extracts, use a rotary evaporator to reduce pressure and concentrate (40°C) to obtain 2.05kg of crude extract. Add 20L of distilled water to the crude extract and disperse it evenly, then extract it with 20L of petroleum ether twice, 20L of ethyl acetate three times, and 20L of water-saturated n-butanol five times, combine the same extracts, and use a rotary evaporator to reduce pressure and concentrate to obtain 112.6g of petroleum ether extraction, 347.2g of ethyl acetate extraction, and 545.2g of n-butanol extraction.

[0062] Take 400g of the n-butanol extraction part, add an appropriate amount of methanol to redissolve, then mix the sample in 500g of 80-120 mesh silica gel, wet-load the column, and load the sample into the silica gel chromatographic column (2.5kg of 200-300 mesh silica gel is loaded into the glass chromatographic column). The silica gel chromatographic column uses dichloromethane-methanol as the mobile phase for gradient elution, and the elution ratio is 50:0-0:50 (the ratios are: dichloromethane-methanol = 50:0, 50:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0:1, v / v). The amount of mobile phase used for each elution ratio is 2-4 column volumes, and each portion of eluent is collected as 1 / 4 of the column volume, and 120 portions of eluent are obtained. Using 10% sulfuric acid-ethanol as a color developer, each eluate was detected by thin layer chromatography under 254nm, 365nm ultraviolet light and sunlight, and the 1st to 4th, 5th to 9th, 10th to 14th, 15th to 22nd, 23rd to 31st, 32nd to 47th, 48th to 56th, 57th to 68th, 69th to 86th, 87th to 94th, 95th to 102nd, 103rd to 108th, 109th to 113th, 114th to 118th, and 119th to 120th eluates with similar chromatographic behaviors were combined to obtain component Fr.1-15 in sequence.

[0063] Take component Fr.4 and perform gradient elution through reverse phase ODS column chromatography with methanol-water as mobile phase, the concentrations of which are 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% methanol. The amount of mobile phase used for each elution concentration is 1 to 3 column volumes, and each portion of eluent is collected as 1 / 5 of the column volume, to obtain 153 portions of eluent; in the thin layer chromatography detection, 10% sulfuric acid-ethanol is used as a color developer, and each eluent is detected by thin layer chromatography under 254nm, 365nm ultraviolet light and sunlight, and the 1st to 8th portions, 9th to 19th portions, and 20th to 31st portions with similar chromatographic behaviors are combined. , the 32nd to 45th, 46th to 68th, 69th to 82nd, 83rd to 96th, 97th to 104th, 105th to 111th, 112th to 118th, 119th to 125th, 126th to 130th, 131st to 137th, 138th to 145th, 146th to 153rd eluents, a total of 15 components were obtained in order, marked as components Fr. 4A to 4O;

[0064] Component Fr.4I was separated and purified by preparative HPLC using methanol-water as the mobile phase at a ratio of 61:39 (v / v) to obtain compound 1 (t R =23.6min, 27.6mg).

[0065] 3. Structural Elucidation

[0066] 3.1 Hydrolysis of compound 1 and GC analysis of sugar residues

[0067] Compound 1 (2.0 mg) dissolved in MeOH (2.0 mL) was treated with 2.0 N HCl and dried at 90 °C for 3 h. The dried residue was heated in H 2 O and CH 2 Cl 2 Extraction was performed in . The aqueous phase was filtered and the solvent was evaporated, then the residue, D-glucose, D-galactose (standard, 0.5 mg each) were dissolved in 1 mL of anhydrous pyridine, 1.5 mg of L-cysteine ​​methyl ester hydrochloride was added, and the reaction was shaken at 60°C for 1 hour. Then 112.5 μL of hexamethyldisilazane and 37.5 μL of trimethylchlorosilane were added, and the reaction was shaken at 60°C for 30 minutes. The resulting silicon ether derivatives were extracted with n-hexane and water, respectively, and the n-hexane layer was filtered with a 0.22 μm microporous filter membrane for detection. GC-MS program temperature rise: initial temperature 50°C, increased to 190°C at 40°C / min, then increased to 200°C at 0.4°C / min, and finally increased to 280°C at 20°C / min; injection temperature 280°C; carrier gas: He; injector temperature 250°C; split ratio: 20:1; injection volume: 1 μL.

[0068] 3.2 Structural analysis

[0069] Compound 1 was obtained as colorless crystals (methanol). HR-ESI-MS in negative ion mode gave [MH] at m / z 931.4539 - Molecular ion peak (calcd. for 931.4539); given at m / z 977.4599 [M+HCOO] - Ion peak (calcd.for977.4593), combined 1 H-NMR, 13 C-NMR suggests that its molecular formula is C 45 H 72 O 20 , the calculated unsaturation degree is 10. In addition, HR-ESI-MS in positive ion mode of compound 1 gave [MC 6 H 10 O 5 +H] + Ion peak (calcd. for 771.4167); at m / z 609.3630 gives [MC 12 H 20 O 10 +H] + Ion peak (calcd.for609.3639); at m / z 447.3110 gives [MC 18H 30 O 15 +H] + Ion peak (calcd.for447.3101). Based on the above information, it is speculated that compound 1 is a glycoside compound containing three hexoses.

[0070] In compound 1 1 H-NMR (pyridine-d 5 , 600MHz), one olefin proton signal δ H 5.30 (1H, d, J = 4.1 Hz), 3 sugar terminal proton signals δ H 5.23 (1H, d, J = 7.7 Hz), 5.13 (1H, d, J = 7.7 Hz), 4.91 (1H, d, J = 7.7 Hz); 3 groups of methyl proton signals can be seen in the high field region δ H 1.08(3H,d,J=6.5Hz), 0.90(3H,s), 0.86(3H,s).

[0071] Compound 1 13 C-NMR (pyridine-d 5 , 150MHz), 45 carbon signals can be clearly observed, and combined with the hydrogen spectrum, it can be judged that there is a group of characteristic carbon signals of trisaccharide β-D-Gal-(1→4)-β-D-Glc-(1→2)-β-D-Glcδ C 102.6, 73.2, 75.5, 81.0, 75.0, 60.3, 105.2, 86.1, 77.6, 70.2, 78.9, 61.5, 106.9, 76.7, 78.4, 71.8, 78.0, 63.1; excluding the 18 carbons of the sugar part, there are 27 carbon signals remaining. Combining the hydrogen spectrum and mass spectrum, it is speculated that the compound is C 27 Steroidal saponins.

[0072] Combined with the DEPT spectrum, HSQC was used to assign hydrogen atoms to the carbon atoms directly connected to them (Table 1). 1 H- 1 In the H COSY spectrum, three directly connected fragments can be observed, see Fig.10 ; In the HMBC spectrum of compound 1 ( Fig.10 ), δ H 0.86(H-19) and δ C 140.9 (C-5), 50.2 (C-9), 37.4 (C-10), 36.9 (C-1) are related, so we can judge δ H 0.86 is the proton signal at C-19, and 1 H-1 The two fragments constituting the A and B rings are connected in the H COSY spectrum; δ H 0.90(H-18) and δ C 59.2 (C-17), 56.8 (C-14), 40.7 (C-13), and 39.5 (C-12) are related, so δ H 0.90 is the proton signal at C-18, and the C and D rings can also be connected to the former; in addition, δ H 2.50 (H-20), 4.26 (H-21a), 4.06 (H-21b), 4.05 (H-24), 3.69 (H-26a), 3.65 (H-26b) are all consistent with δ C There is a HMBC related signal between 111.7 (C-22), and the F ring is further connected to the E ring to form the C 27 Steroidal aglycones.

[0073] In the HMBC spectrum of compound 1, the galactose terminal proton δ H 4.90 and aglycone 3 (δ C 78.0) There are related signals, the first glucose terminal proton δ H 5.13 and galactose 4 (δ C 81.0) There are related signals, the second glucose sugar terminal proton δ H 5.20 and the first glucose 2 position (δ C 86.1), and thus the connection mode between the trisaccharides and the aglycone was determined.

[0074] Compound 1 was hydrolyzed with HCl, and the hydrolyzate was treated and analyzed by GC, and identified as D-galactose (t R =9.181min) and D-glucose (t R =12.219min).

[0075] In the NOESY spectrum of compound 1, the related signals of H-18 and H-20, H-18 and H-27 were observed, and the relative configurations of C-21 and C-27 were determined to be α and β, respectively. Since qualified crystals of compound 1 were obtained in methanol solvent, Cu Kα X-ray crystallography was used for analysis ( Fig.11) determined that the absolute configurations of all chiral positions were: 3S, 8S, 9S, 10R, 13S, 14S, 16S, 17R, 20R, 22R, 24S, 25S, and further confirmed that the trisaccharide group was β-D-Gal-(1→4)-β-D-Glc-(1→2)-β-D-Glc. The Scifinder database search showed that compound 1 was a new steroidal saponin named Infundilide A. The structural formula of compound 1 is shown in Formula I.

[0076]

[0077] Table 1 Infundilide A 1 H-NMR (600MHz,pyridine-d 5 )and 13 C-NMR (150MHz,pyridine-d 5 )data

[0078]

[0079]

[0080] 3.3 Information about Infundilide A

[0081] Molecular formula: C 45 H 72 O 20

[0082] HR-ESI-MS m / z:931.4539[MH] -

[0083] State: Colorless crystal

[0084] Specific rotation:

[0085] Example 2: Study on the anti-inflammatory activity of a new steroidal saponin Infundilide A from Panax notoginseng

[0086] The LPS-induced RAW264.7 inflammatory cell model was used to investigate the anti-inflammatory activity of Infundilide A. The specific method was as follows: RAW264.7 cells were resuscitated by conventional methods and placed in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO 2 The Griess reagent method was used to detect the effect of compound Infundilide A on the NO release of RAW264.7 cells induced by LPS. 6RAW 264.7 cells at 1 μg / mL were seeded in 96-well plates with 3 replicates per group. After pretreatment with Infundilide A for 1 h, cells were stimulated with LPS at a concentration of 1 μg / mL. After incubation, the culture supernatant was separated and mixed with Griess reagent. NaNO 2 A standard curve was generated and the absorbance of the mixture was measured at 540 nm. L-NG-monomethylarginine monoacetate (L-NMMA) was used as a positive control.

[0087] Table 2 Inhibitory effect of Infundilide A on LPS-induced NO release in RAW264.7

[0088]

[0089] Infundilide A inhibits NO release induced by LPS in RAW264.7 inflammatory cells. 50 The value is 6.27 μM. Therefore, Infundilide A has certain potential in developing into an anti-inflammatory drug.

[0090] Example 3: Infundilide A inhibits inflammatory response in mice with LPS-induced acute lung injury

[0091] Acute lung injury (ALI) is a serious clinical pathological syndrome characterized by acute onset, diffuse lung parenchymal damage and the resulting acute respiratory failure. Lipopolysaccharide (LPS), as the main component of the cell wall of Gram-negative bacteria, is one of the important pathogenic factors that induce ALI. LPS activates the body's immune response, triggering a series of complex inflammatory reactions and leading to lung tissue damage. Therefore, finding effective anti-inflammatory drugs to alleviate ALI has important clinical significance and research value.

[0092] In recent years, the extraction of natural compounds with anti-inflammatory activity from traditional Chinese medicine has become a hot topic in the field of drug research and development. We discovered a new steroidal saponin component from Panax notoginseng, Infundilide A, which showed good anti-inflammatory potential in in vitro anti-inflammatory activity studies. Therefore, by constructing an LPS-induced ALI mouse model, we further explored the role of Infundilide A in alleviating inflammatory response and lung injury.

[0093] 1 Materials and Methods

[0094] 1.1 Materials

[0095] 48 6-8 week old C57BL / 6J male mice, weighing (20±2) g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., with a certificate number of 110324241105595753 and a license number of SCXK (Beijing) 2024-0001, and were kept in the Experimental Animal Center of Shanxi University of Traditional Chinese Medicine. Before the experiment, all animals were kept in the experimental animal room for one week to fully adapt to the living and breeding environment. The animal room controlled the ambient temperature at 23-25°C, the humidity at 50%-60%, the light-dark cycle at 12h, and the animals were free to eat and drink during the experiment.

[0096] This experiment was approved by the Medical Ethics Committee of Shanxi University of Traditional Chinese Medicine (approval number: AWE202410427), and all experimental operations were performed in strict accordance with the guiding principles for animal research.

[0097] 1.2 Reagents and instruments

[0098] Infundilide A (prepared in Example 1), LPS (SIGMA-ALDRICH, Co. L2630-10MG), dexamethasone acetate tablets (Zhejiang Xianjun Pharmaceutical Co., Ltd., LB24106), IL-6 (Shanghai Fankewei Biotechnology Co., Ltd., F3066-A), IL-1β (Shanghai Fankewei Biotechnology Co., Ltd., F2923-B), TNF-α (Shanghai Fankewei Biotechnology Co., Ltd., F3056-A), rabbit primary antibody IL-6 (Beijing Bioson Biotechnology Co., Ltd., bs-0782R), rabbit primary antibody T NF-α (Beijing Bio-Tech Biotechnology Co., Ltd., bs-10802R), rabbit primary antibody TLR4 (Aibotek Biotechnology Co., Ltd., A5258), rabbit primary antibody MyD88 (Beijing Bio-Tech Biotechnology Co., Ltd., bs-1047R), rabbit primary antibody NF-κB (Aibotek Biotechnology Co., Ltd., A19653), rabbit primary antibody COX-2 (Beijing Bio-Tech Biotechnology Co., Ltd., bs-0732R), goat anti-rabbit immunoglobulin (Ig) G-horseradish peroxidase (HRP) secondary antibody ... Co., Ltd., BC10268988), sodium citrate (Tianjin Kaitong Chemical Reagent Co., Ltd., 20220610), citric acid (Tianjin Fengchuan Chemical Reagent Technology Co., Ltd., 20190226), sodium dihydrogen phosphate (Tianjin Tianli Chemical Reagent Co., Ltd., 20230528), disodium hydrogen phosphate (Tianjin Tianli Chemical Reagent Co., Ltd., 20240102), PBS (Wuhan Sewell Technology Co., Ltd., G4202-500ML), peroxidase blocker (Wuhan Elerite Biotechnology Co., Ltd., DU04Z4FR 6025), goat serum (Wuhan Sewell Technology Co., Ltd., MPC2302068), DAB stain (Jiangsu KeyGen Biotech Co., Ltd., 20240820), hematoxylin stain (Feijing Biotechnology Co., Ltd., 20240622), DAPI stain (Beijing Solebold Technology Co., Ltd., C0065), BCA protein concentration kit (Beijing Solebold Technology Co., Ltd., 240009024), digital pathology slide scanner (Ningbo Jiangfeng Bioinformatics Co., Ltd., KF-PRO-005-EX), Zeiss Axio Imager.Z2 (Carl Zeiss (Shanghai) Management Co., Ltd.), electronic balance (Shanghai Zhuojing Electronic Technology Co., Ltd., 0347622049), HC-3016R centrifuge (Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.).

[0099] 2. Solution Preparation

[0100] 2.1 Infundilide A solution

[0101] 20.0 mg of the Infundilide A monomer compound prepared from Panax notoginseng in Example 1 was weighed, and an appropriate amount of physiological saline was added to prepare an Infundilide A mother solution with a mass concentration of 1 mg / mL.

[0102] 2.2 Dexamethasone acetate solution

[0103] Dexamethasone acetate tablets were taken, ground into powder in a mortar, and physiological saline was added to prepare a solution with a mass concentration of 0.25 mg / mL.

[0104] 3 Modeling and grouping

[0105] 48 mice were randomly divided into 4 groups, 12 mice in each group, namely control group, model group, dexamethasone group (5 mg / kg) and Infundilide A group (20 mg / kg). The dexamethasone group and Infundilide A group were intraperitoneally injected with the corresponding drugs once a day, and the control group and model group were intraperitoneally injected with an equal volume of saline for 3 consecutive days. Except for the control group, the mice in the other groups were gently pinched by the left hand after the last administration of the mouse neck, so that the mouse was vertically tilted back, the left thumb pressed the mouth, and the right hand held a microinjector to absorb LPS saline solution (containing LPS 0.5 mg / mL) and dripped it through the left nostril of the mouse. The modeling dose was 5 mg / kg to establish the ALI model. The control group was dripped with an equal amount of saline.

[0106] 4. Collection of bronchoalveolar lavage fluid (BALF)

[0107] 12 hours after LPS induction, the eyeballs of mice were removed for blood collection. Six mice were randomly selected from each group and fixed on the operating table with their limbs facing upward. The chest and neck skin was disinfected with 75% alcohol, and then the neck muscles were separated with sterilized surgical scissors to fully expose the trachea. A syringe equipped with a soft needle was used to perform tracheal intubation in the main bronchi. 1 mL of pre-cooled PBS was slowly pushed from the trachea through the bronchus into the right lung. At this time, the chest was obviously swollen. After staying for 1-2 minutes, it was slowly withdrawn. Be careful not to use excessive force to cause rupture and bleeding of the lung tissue. Without changing the lavage fluid, the lavage fluid was injected into the lungs again and withdrawn again. This process was repeated 3 times to collect all BALF. The collected BALF was then centrifuged at 3000 rpm for 10 minutes (4°C) in a refrigerated centrifuge. The obtained precipitate was placed at 4°C for standby use, and the supernatant was frozen at -80°C for subsequent testing.

[0108] 5 indicators detection

[0109] 5.1 Wright-Giemsa staining to detect and count inflammatory cells in BALF

[0110] Resuspend the cell pellet in BALF to prepare a cell suspension, take 20 μL of the suspension for Wright-Giemsa staining, and then count the cells. Place the cell smear on the staining rack and evenly cover it with an appropriate amount of staining solution. After incubation at room temperature for 2 minutes, add an equal volume of PBS and gently shake to ensure that PBS and Wright-Giemsa staining solution are evenly mixed. After standing at room temperature for 10 minutes, gently rinse the smear from one end with distilled water, wait for it to dry completely, and then observe and analyze it under a microscope.

[0111] 5.2 Detection of total protein concentration in BALF by BCA method

[0112] The total protein concentration in the collected BALF supernatant was detected by the BCA method, and the operation was carried out strictly in accordance with the instructions of the kit. After the reaction was terminated, the optical density (OD) value of each sample was detected at 562nm by means of an enzyme reader, and then the protein concentration was calculated according to the standard curve.

[0113] 5.3ELISA kit to detect the levels of IL-6, IL-1β and TNF-α in BALF

[0114] The levels of IL-6, IL-1β and TNF-α in BALF supernatant were determined according to the ELISA kit instructions.

[0115] 5.4 Determination of lung wet / dry weight (W / D) ratio

[0116] In each group, except for 6 mice from which BALF was collected, the fresh right lung tissues of the remaining 6 mice were weighed, the surrounding residual tissues were removed, and excess water and blood stains were cleaned with absorbent paper. The tissue weight was accurately weighed with a precision balance and recorded as the wet weight of the lung (W); the tissue was placed in an 80°C oven for dehydration for 24 hours to constant weight, and the tissue weight was accurately weighed and recorded as the dry weight of the lung (D). The degree of lung tissue edema was evaluated by calculating the lung W / D weight ratio.

[0117] 5.5 Observation of pathological changes in mouse lung tissue by HE staining

[0118] The left lung of the mouse was fixed with 4% paraformaldehyde for 24 hours, and then washed, gradient dehydrated, embedded, sliced, dewaxed, stained with H&E, dehydrated, and sealed. After the treatment, the pathological changes of the lung tissue were observed under an optical microscope and photographed.

[0119] 5.6 Immunofluorescence staining to detect the expression of COX-2, IL-6, and TNF-α in lung tissue

[0120] Paraffin sections were dewaxed in xylene I and II for 10 min each, and then dehydrated in anhydrous ethanol, 95%, 85% ethanol and distilled water for 5 min each. After marking with a histochemical pen, the sections were rinsed with PBS three times. The sections were then placed in sodium citrate buffer (pH 6.0) and microwaved for antigen repair (high temperature for 5 min, warm for 5 min, low temperature for 3 min), cooled naturally and rinsed with PBS. Endogenous peroxide blocker (3% H 2 O 2 ) and then rinsed with PBS for 10 minutes. Blocked with goat serum, COX-2, IL-6, and TNF-α primary antibodies (1:200) were added and incubated overnight (4°C). The next day, after rinsing with PBS, the cells were incubated with secondary antibodies for 3 hours, stained with DAPI for 10 minutes, rinsed with PBS, dehydrated, treated with autofluorescence quencher for 5 minutes, and observed under a fluorescence microscope.

[0121] 5.7 Immunohistochemical detection of TLR4, MyD88, and NF-κB protein expression in lung tissue

[0122] After paraffin sections were dewaxed and put into water, antigens were repaired by microwave in citric acid buffer (pH 6.0). After rinsing with PBS, primary antibodies of TLR4 (1:100), MyD88 (1:100), and NF-κB (1:200) were added and incubated overnight at 4°C. After rinsing with PBS the next day, the sections were incubated with secondary antibodies for 3 hours. Diaminobenzidine staining (DAB) solution was added for color development and the sections were rinsed with distilled water. Hematoxylin was used for counterstaining, and the sections were differentiated with 1% hydrochloric acid alcohol and rinsed with distilled water. Dehydration was performed with anhydrous ethanol, xylene was used for transparent treatment, and the sections were sealed with neutral gum. The staining results were then observed under a microscope.

[0123] 6 Statistical analysis

[0124] GraphPad Prism V9.5 software was used for data analysis, and the quantitative data that met the normal distribution were expressed as mean ± standard deviation. When the variance was equal, one-way analysis of variance (ANOVA) was used for comparison among multiple groups. P<0.05 indicated that the difference was statistically significant.

[0125] 7 Results

[0126] 7.1 Effect of Infundilide A on the Number of Inflammatory Cells and Total Protein Concentration in BALF of ALI Mice

[0127] The total number of inflammatory cells in BALF was counted by Wright-Giemsa staining. The results showed that compared with the control group, the total number of inflammatory cells in BALF of mice in the model group was significantly increased; compared with the model group, the total number of inflammatory cells in BALF of ALI mice treated with Infundilide A was significantly reduced ( Fig.12 AB, P<0.05).

[0128] The total protein concentration in BALF was determined using a BCA kit. The results showed that compared with the control group, the total protein concentration in BALF of the model group mice was significantly increased, reflecting the increased permeability of alveolar capillaries and the appearance of pulmonary tissue edema; compared with the model group, the total protein concentration in BALF of ALI mice treated with Infundilide A was significantly reduced ( Fig.12 C, P<0.01).

[0129] 7.2 Effect of Infundilide A on W / D of Lung Tissue in ALI Mice

[0130] Pulmonary edema is one of the main manifestations of lung injury. By comparing the wet weight and dry weight of lung tissue, the relative water content of lung tissue is evaluated, thereby indirectly reflecting the severity of pulmonary edema. Fig.12 As shown in D, compared with the control group, the W / D weight ratio of the model group was significantly increased (P<0.01); compared with the model group, after intervention with Infundilide A, the W / D weight ratio of the mouse lung tissue was significantly decreased (P<0.01).

[0131] 7.3 Effects of Infundilide A on Lung Morphology and Pulmonary Edema in ALI Mice

[0132] Through hematoxylin-eosin (H&E) staining, the pathological changes of lung tissue and the morphology and structure of lung tissue cells can be directly observed. Fig.13 It can be seen that the epithelial cells of the mice in the control group were normal in morphology, with normal alveolar structure, no inflammation, edema and infiltration in the tissues, and no obvious pathological changes; compared with the control group, a large number of inflammatory cells were observed to gather in the bronchi, around blood vessels and in the alveolar area of ​​the lung tissue in the model group, leading to pathological changes such as alveolar structure collapse and pulmonary fibrosis. Compared with the model group, the mice in the Infundilide A group had partial alveolar structure destruction, smaller inflammatory mass area, and significantly reduced edema infiltration area, indicating that after the intervention of Infundilide A, the pathological changes in the lungs of ALI mice were alleviated and improved.

[0133] 7.4 Effect of Infundilide A on IL-6, IL-1β and TNF-α levels in BALF of ALI mice

[0134] ELISA kit was used to detect the changes of IL-6, IL-1β and TNF-α inflammatory factors in mouse BALF. Fig.14As shown in AC, compared with those in the control group, the contents of inflammatory cytokines IL-6, IL-1β and TNF-α in BALF of mice in the model group were significantly increased (P<0.01); compared with the model group, the contents of IL-6, IL-1β and TNF-α in BALF of the Infundilide A group were significantly decreased (P<0.01).

[0135] Effects of Infundilide A on COX-2, IL-6 and TNF-α levels in lung tissues of ALI mice

[0136] The results of immunofluorescence analysis showed that ( Fig.14 DG), compared with the control group, the expression levels of COX-2, IL-6 and TNF-α in the lung tissue of the mice in the model group were significantly increased (P<0.01); compared with the model group, the expression levels of COX-2, IL-6 and TNF-α in the lung tissue of the mice in the Infundilide A group were significantly decreased (P<0.01).

[0137] 7.6 Effect of Infundilide A on the Expression of TLR4, MyD88, and NF-κB Proteins in Lung Tissues of ALI Mice

[0138] The results are as follows Fig.15 As shown, compared with those in control group, the expressions of TLR4, MyD88 and NF-κB proteins in lung tissue of mice in model group were significantly increased (P<0.01); compared with those in model group, the expressions of TLR4, MyD88 and NF-κB proteins in lung tissue of mice in Infundilide A group were significantly decreased (P<0.01).

[0139] In this study, we established an LPS-induced ALI mouse model to explore the role and mechanism of Infundilide A, a new steroidal saponin component in Panax notoginseng, in alleviating inflammatory response and lung injury. The results showed that Infundilide A could significantly reduce the number of inflammatory cells and total protein content in mouse BALF, effectively inhibit the infiltration and accumulation of inflammatory cells, reduce the increase in alveolar capillary permeability and lung tissue edema, and significantly reduce the wet / dry weight (W / D) ratio of lung tissue, further verifying its protective effect on lung injury. At the molecular mechanism level, Infundilide A can significantly adjust the levels of proinflammatory cytokines IL-6, IL-1β and TNF-α in BALF and lung tissue, adjust the expression of COX-2, and inhibit the expression of TLR4, MyD88 and NF-κB proteins in lung tissue by regulating the TLR4 / MyD88 / NF-κB signaling pathway activated by LPS, thereby exerting an anti-inflammatory effect.

[0140] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. The compound represented by formula I or its pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer:

2. The method for preparing the compound according to claim 1, comprising the following steps: extracting Panax notoginseng with an ethanol solution having a volume fraction of 50% to 95%, collecting the extract; and separating the compound of formula I from the extract.

3. The preparation method according to claim 2, characterized in that: The extraction is performed at least once; during each extraction, the solid-liquid ratio of the Chinese ginseng ethanol solution is 1:10-20, and the time of each extraction is 5-10 days.

4. The preparation method according to claim 2 or 3, characterized in that: The method further comprises the steps of treating the extract as follows: concentrating the extract under reduced pressure to obtain a crude extract; adding distilled water to the crude extract to disperse it uniformly, then extracting it with petroleum ether, ethyl acetate and water-saturated n-butanol in sequence, collecting the water-saturated n-butanol extract and concentrating it under reduced pressure to obtain an n-butanol extraction fraction; and separating the compound shown in formula I from the n-butanol extraction fraction.

5. The preparation method according to claim 4, characterized in that: The ratio of the crude extract to distilled water is 1:10; the petroleum ether is extracted twice, the ethyl acetate is extracted three times, and the water-saturated n-butanol is extracted five times; the amount of each extraction solvent used in each extraction is the same as the amount of distilled water used.

6. Use of the compound of claim 1 or its pharmaceutically acceptable salt, ester, solvate, stereoisomer, or tautomer in the preparation of at least one of the following products: 1) Products for preventing and / or alleviating and / or treating inflammation; 2) Products for the prevention and / or treatment of acute lung injury.

7. The use according to claim 6, characterized in that: The acute lung injury is caused by inflammation; And / or, the product is a medicine, a composition, a health product or other biological product.

8. The use according to claim 6, characterized in that: The prevention and / or treatment of acute lung injury is embodied in at least one of the following aspects: a) Inhibit the increase in the number of inflammatory cells and the increase in total protein content in bronchoalveolar lavage fluid caused by acute lung injury; b) Reduce lung tissue edema caused by acute lung injury; c) Alleviate and / or improve lung pathological changes caused by acute lung injury; d) inhibiting the increased expression levels of IL-6, IL-1β and TNF-α in bronchoalveolar lavage fluid caused by acute lung injury; e) inhibiting the increased expression levels of COX-2, IL-6 and TNF-α in lung tissue caused by acute lung injury; f) Inhibit the increased expression levels of TLR4, MyD88 and NF-κB proteins in lung tissue caused by acute lung injury.

9. A product comprising the compound of formula I according to claim 1 or a pharmaceutically acceptable salt, ester or solvate, stereoisomer or tautomer thereof; The product has at least one of the following effects: 1) preventing and / or alleviating and / or treating inflammation; 2) preventing and / or treating acute lung injury.

10. The product according to claim 9, characterized in that: The product is a medicine, a composition, a health product or other biological product.

Images