CN113816866B - Organic acid compound and preparation method and application thereof - Google Patents

Abstract

The invention provides an organic acid compound, a preparation method and application thereof, wherein the organic acid compound is (2R) -2-amino-2-hydroxymethyl-3- [ (4-hydroxy-3-methoxybenzoyl) -O- ] -propionic acid, and has an inhibition effect on macrophage foaming, so that the occurrence and development of atherosclerosis are inhibited.

Description

Organic acid compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to an organic acid compound and a preparation method and application thereof.

Background

The pericarpium Trichosanthis is dried mature pericarp of fructus Trichosanthis (Trichosanthes kirilowii Maxim) or fructus Trichosanthis (Trichosanthes rosthornii Harms) of Cucurbitaceae (Cucurbitaceae). The earliest recorded in Shennong Ben Cao Jing (Shennong's herbal meridian), mainly distributed in Shandong and Henan, the Shandong Changqing and Feicheng are the areas of the road and the land. It is sweet, slightly bitter and cold in nature, and has the effects of clearing heat and resolving phlegm, relieving chest stuffiness and resolving masses, and relaxing bowel. The ancient books are often used for treating cough, chest distress, hypochondriac pain and other diseases caused by lung heat; modern clinic is often used for treating various cardiovascular diseases, such as coronary heart disease, hyperlipidemia, angina pectoris, etc. Modern pharmacological researches have proved that the pericarpium Trichosanthis has various pharmacological activities such as lipid metabolism regulation, atherosclerosis resistance, vascular endothelial protection, platelet aggregation resistance, anoxia resistance, inflammation resistance, phlegm elimination and bacteria inhibition. The chemical components of the snakegourd peel comprise flavone, saponin, alkaloid, amino acid, polysaccharide and the like. The snakegourd peel flavonoid has the function of protecting vascular endothelial injury, the snakegourd peel total saponins have a certain antibacterial function, the snakegourd peel total amino acids have a good phlegm eliminating function, and the research of the snakegourd peel oligosaccharides has the angiotensin converting enzyme inhibiting function. However, the researches on the chemical components and the biological activity of the organic acids in the snakegourd peel are less, and the researches show that the organic acid compound has the physiological activities of anti-inflammatory, antibacterial, anticancer, antioxidation and the like.

Disclosure of Invention

The invention aims to provide an organic acid compound.

Another technical problem to be solved by the present invention is to provide a method for preparing the above organic acid compound.

Another technical problem to be solved by the present invention is to provide an application of the above organic acid compound.

The technical scheme adopted by the invention is as follows:

an organic acid compound is (2R) -2-amino-2-hydroxymethyl-3- [ (4-hydroxy-3-methoxybenzoyl) -O- ] -propionic acid, and has a structure shown in a structural formula (I):

preferably, the organic acid compound has hydrogen spectrum data [ ] ¹ H-NMR(DMSO-d ₆ ,600MHz):δ _H 7.60(1H,dd,J＝7.8,1.8Hz,H-6')，7.48(1H,d,J＝1.8Hz,H-2')，6.94(1H,d,J＝7.8Hz,H-5')，δ _H 4.42(1H,d,J＝11.4Hz,H-3a)，4.37(1H,d,J＝11.4Hz,H-3b)，3.74(1H,d,J＝11.4Hz,H-4a)，3.70(1H,d,J＝11.4Hz,H-4b)，3.80(1H,S,H-8’)。

Preferably, the organic acid compound and the carbon spectrum data are [ (] ¹³ C-NMR(DMSO-d ₆ ,150MHz):δ _C 119.8(C-1')，113.3(C-2')，147.4(C-3')，152.3(C-4')，115.2(C-5')，124.3(C-6')，165.5(C-7')，55.8(C-8')，63.5(C-1)；δ _C 65.4 (C-3), 62.3 (C-4) are two methylene carbon signals; delta _C 169.0 (C-1) is a carboxyl carbon signal.

Preferably, the organic acid compound is white powder and is easily dissolved in methanol.

The preparation method of the organic acid compound comprises the following specific steps:

(1) Soaking dried pericarpium Trichosanthis in ethanol at room temperature, filtering, and concentrating under reduced pressure to obtain extract;

(2) The extract is dispersed by D101 macroporous resin to obtain a water layer, a 30% ethanol layer, a 70% ethanol layer and a 95% ethanol layer;

(3) The 30% ethanol layer is eluted by an ODS chromatographic column with methanol-water to obtain three fractions F1, F2 and F3, F1 is eluted by a liquid phase methanol-water 9:91 to obtain three fractions F1-1, F1-2 and F1-3, and F1-3 is purified by a liquid phase methanol-water 7:93 to obtain the novel compound.

The organic acid compound has an inhibitory effect on macrophage foaming.

The application of the organic acid compound in preparing atherosclerosis inhibiting medicine.

The beneficial effects of the invention are as follows:

the organic acid compound is (2R) -2-amino-2-hydroxymethyl-3- [ (4-hydroxy-3-methoxybenzoyl) -O- ] -propionic acid, has an inhibiting effect on macrophage foaming, and has a preventing and treating effect on cardiovascular and cerebrovascular diseases such as coronary heart disease with atherosclerosis as pathological characteristics.

Drawings

FIG. 1 is a HMBC correlation diagram of compound TP 1;

FIG. 2 is a comparison of TP1 experimental ECD spectra with TP1 enantiomer calculated ECD spectra;

FIG. 3 is the effect of compound TP1 on cell viability;

FIG. 4 is the effect of compound TP1 on macrophage foaming;

FIG. 5 shows lipid content of compound TP 1.

Fig. 6 is a graph showing the inhibitory effect of aortic atherosclerosis in rats, wherein the three graphs from left to right correspond to a blank control group, a model group and an administration group respectively.

Detailed Description

To further illustrate the invention, the following examples are provided in connection with:

example 1 preparation of Compounds

Experimental instrument and reagent

Fourier transform Nuclear magnetic resonance spectrometer (type AVIII; bruker, switzerland); mass spectrometer (Waters map G2 mass; waters, USA); automatic polarimeter (Autopol type III; rudolph Research, USA); analytical high performance liquid chromatography (Agilent 1260; agilent technologies Co., ltd., U.S.); preparation high performance liquid chromatograph (Agilent 1260; agilent technologies Co., ltd., U.S.); one ten thousandth balance (AL 204; METTER TOLEDOO Instrument Shanghai Co., ltd.); one ten million balance (XP 6; METTER TOLEDOPO Instrument Shanghai Co., ltd.); rotary evaporator (RE-52A; shanghai Asia Biochemical instruments Co.); temperature-regulating electrothermal sleeve (ZDHW; beijing Zhongxing Wei industry instruments Co., ltd.); dark box ultraviolet analyzer (ZF-20D; shanghai Gu Cun electro-optical instruments factory); thin layer heater (TH-II type; shanghai family hucho technology Co., ltd.); the display agent comprises: 2%

FeCl ₃ -K ₃ [Fe(CN) ₆ ]An ethanol solution; bismuth potassium iodide.

Specific preparation method

The dried snakegourd peel (30 kg) is added with 130L of 70% ethanol to be soaked for three times at room temperature, each time for one week, filtered, decompressed and concentrated to obtain extract, and D101 macroporous resin is dispersed with proper amount of water to obtain a water layer, a 30% ethanol layer, a 70% ethanol layer and a 95% ethanol layer. Subjecting 30% ethanol layer to ODS chromatography column, eluting with methanol-water (0:100-100:0) to obtain three fractions (F1, F)2, f 3). F1 is eluted by preparing liquid phase methanol-water (9:91) to obtain three fractions (F1-1, F1-2, F1-3); f1-3 is purified by preparing liquid phase methanol-water (7:93) to obtain new compound TP1 (t) _R ＝19min，16mg)。

Structural identification of TP1

TP1 white powder (methanol), readily soluble in methanol. Dark spots at UV 254nm by silica gel thin layer chromatography, 2% FeCl ₃ -K ₃ [Fe(CN) ₆ ]Positive color development of ethanol shows that the compound is an organic acid compound; the bismuth potassium iodide shows positive color development, which indicates that the compound is a nitrogen-containing compound. HR-ESI-MS gives an excimer ion peak [ M+H ] at M/z 286.0932] ⁺ (molecular formula C ₁₂ H ₁₆ NO ₇ Calculated as 286.0927), combined with ¹ H NMR ¹³ C NMR to confirm that the compound is of formula C ₁₂ H ₁₅ NO ₇ . Comparing the experimental ECD spectrum of compound TP1 with the calculated ECD spectrum of the TP1 enantiomer, as shown in FIG. 2, (2R) -TP1 calculated spectrum is characterized at 285 and 214nm, qualitatively consistent with the experimental spectrum, and incorporates the optical rotation value [ alpha ]]2 D0= -2.00 (c 0.40, meOH) and literature (Yajima T, aizawa Y, nishida, et al preparation of optically active-aminobutanoic acid via ptical resolution by replacing crystallization. Bioscience, biotechnology, and biochemistry.2007,71 (5): 1338-1341)

(c 1.00, methanol) symbols are identical, so the 2-position is defined as the R configuration.

¹ H-NMR(DMSO-d ₆ 600 MHz) hydrogen spectral data delta _H 7.60(1H,dd,J＝7.8,1.8Hz,H-6')，7.48(1H,d,J＝1.8Hz,H-2')，6.94(1H,d,J＝7.8Hz,H-5')，δ _H 4.42(1H,d,J＝11.4Hz,H-3a)，4.37(1H,d,J＝11.4Hz,H-3b)，3.74(1H,d,J＝11.4Hz,H-4a)，3.70(1H,d,J＝11.4Hz,H-4b)，3.80(1H,S,H-8’)。

¹³ C-NMR(DMSO-d ₆ 150 MHz) carbon spectrum data:δ _C 119.8(C-1')，113.3(C-2')，147.4(C-3')，152.3(C-4')，115.2(C-5')，124.3(C-6')，165.5(C-7')，55.8(C-8')，63.5(C-1)；δ _C 65.4 (C-3), 62.3 (C-4) are two methylene carbon signals; delta _C 169.0 (C-1) is a carboxyl carbon signal. Specific nuclear magnetic spectrum data are shown in table 1.

HMBC Spectrum methine Signal delta _H 7.48(H-2')，δ _H 7.60 (H-6') and delta _C 165.5 (C-7') correlation; methyl hydrogen signal delta _H 3.80 (H-8') and delta _C 147.4 (C-3') correlation; methylene hydrogen signal delta _H 4.42 (H-3 a) and 4.37 (H-3 b) and delta _C 165.5 (C-7') and 169.0 (C-1); methylene hydrogen signal delta _H 3.74 (H-4 a), 3.70 (H-4 b) and delta _C 65.4 (C-8) and 169.0 (C-1). As shown in FIG. 1, the structure of the compound TP1 was determined as (2R) -2-amino-2-hydroxymethyl-3- [ (4-hydroxy-3-methoxybenzoyl) -O-]Propionic acid.

TABLE 1 Compound TP1 ¹ H NMR and ¹³ C NMR data

^a measured in DMSO-d ₆

Example 2 macrophage foaming assay

And (3) cells: RAW264.7 (China academy of sciences of Shanghai life sciences cell resource center)

Experimental apparatus and reagent

Carbon dioxide incubator (IL-161HI; STIK); desk-top refrigerated centrifuges (D-375200 Osterode; thermo); inverted fluorescence microscope (Nikon Ti-u; nikon); microscope (CKX 31SF; OLYMPUS); biosafety cabinet (HR 40-IIA 2; haier); a microplate reader (SPARK; tecan); cell scraping (Corning); 96 well plates (Corning); DMEM high sugar medium (Gibco); foetal Bovine Ser μm (Gibco); diabody (Hyclone); PBS (bi yun tian); DMSO (Solarbio); CCK8 ( Bao Bio); oil red O dye powder (Sigma); isopropyl alcohol (conrad); simvastatin (aletin); OX-LDL (Yiyuan).

Experimental method

1 drug toxicity screening

(1) Cell seed plates: scraping cells in logarithmic phase to obtain cell suspension, adding into 96-well plate, adding 100 μl of the suspension into each well to obtain density of 1×10 ⁴ The edge wells were filled with sterile PBS, 5% CO ₂ Culturing in an incubator at 37 ℃ for 24 hours.

(2) Administration: after 24 hours of incubation, quan Pei was aspirated, and the drug to be tested was added and incubated for 24 hours. Divided into blank groups, control group, and dosing group (TT 1 (0.1 μm,1 μm,10 μm)).

(3) After the cell culture and treatment, CCK8 reagent was added rapidly, and 10. Mu.L of CCK8 reagent was added to 100. Mu.L of medium per well.

(4) The plates were returned to the incubator and incubated at 37℃for 2h.

(5) OD was measured at 490 nm.

(6) Analysis of results: cell viability% = (dosing cell OD-blank OD)/(control cell OD-blank OD) ×100%

2 foam cell formation

(1) Cell seed plates: scraping cells in logarithmic phase to obtain cell suspension, adding into 96-well plate, adding 100 μl of the suspension into each well to obtain density of 1×10 ⁴ The edge wells were filled with sterile PBS, 5% CO ₂ Culturing in an incubator at 37 ℃ for 24 hours;

(2) Administration: after 24h incubation, quan Pei was aspirated, the drug to be tested was added and divided into blank (basal) model (OX-LDL 50 μm) and the drug-dosed (drug+OX-LDL) was incubated for 48h;

(3) Preparing an oil red O dye liquor: 500mg of oil red O is dissolved in 100mL of 70% ethanol to prepare 0.5%,

filtering the oil red O dye liquor with a 0.22 mu m filter membrane, sealing and keeping the oil red O dye liquor away from light at 4 ℃ for 24 hours to form saturated liquor, and then using the saturated liquor;

(4) Gently sucking the liquid medicine, and gently rinsing with PBS for 3 times;

(5) Adding 4% paraformaldehyde, and fixing at room temperature for 30min;

(6) Stock solution of diluent oil red O: diluting according to the volume ratio of the oil red O stock solution to the ultrapure water of 3:2, filtering with a 0.2 mu m filter membrane after dilution, and avoiding light in the preparation process;

(7) After the fixation, the fixation solution was discarded, rinsed gently 3 times with PBS, 100. Mu.L of 60% isopropyl alcohol solution was added to each well, and immersed for 15s;

(8) The isopropanol solution was aspirated and 80 μl of the diluted oil red O dye was added directly. Performing whole-process shading operation, and dyeing for 1h at 37 ℃;

(9) Decolorizing, absorbing oil red O dye, rinsing with PBS for 3 times, absorbing, adding 100 μl PBS solution, and directly observing with fluorescent inverted microscope;

(10) After photographing, PBS was removed from the wells, 100% isopropanol was added, the wells were immersed for 5min, the liquid was transferred to another 96-well plate, and OD was measured at 492 nm.

Experimental results

1 cytotoxicity

As shown in fig. 3, according to the cytotoxicity results of the compound TP1, the cell viability of TP1 at three concentrations was not significantly different from that of the control group at a medium concentration of 1 μm, and both low and high concentrations reduced the cell viability, and finally 1 μm was selected for subsequent activity test experiments.

Inhibition of macrophage foaming by Compounds 2

As shown in FIG. 4, intracellular lipids can be stained with oil red when the intracellular lipid droplets exceed 10 or more, and foam cell molding can be considered successful (ref: goswami R, michael M, sheta S, et al TRPV4calcium-permeable channel is a novel regulator of oxidized LDL-induced macrophage foam cell formation. Free radio Biol Med,2017,110:142-150;Tumurkhuu G,Jargalsaikhan D,Rebecca A,et al.Chlamydia pneumoniae Hijacks a Host Autoregulatory IL-1beta Loop to Drive Foam Cell Formation and Accelerate Atherosclerosis.Cell Metab,2018,28 (3): 432-448e 4). Compared with a normal group, the cell lipid drops in the model group are dense, and the number of the cell lipid drops is far more than 10, which indicates that the modeling is successful; both positive drug group (simvastatin), TP1 resulted in decreased intracellular lipid droplets relative to the model group. As shown in fig. 5, it is seen from the OD value results that both positive drug groups (simvastatin, P < 0.001) and TP1 (P < 0.05) significantly reduced intracellular lipid content relative to the model group. The results show that TP1 can inhibit foam cell formation and reduce lipid content.

Atherosclerosis is the primary cause of cardiovascular disease, and the formation of intimal macrophage foam is a hallmark of atherosclerosis. Can reduce the deposition of lipid in macrophage by inhibiting the foaming of macrophage, thereby inhibiting the occurrence and development of atherosclerosis, and achieving the effects of preventing and treating cardiovascular and cerebrovascular diseases. The mouse mononuclear macrophage RAW264.7 is easy to obtain and convenient to culture, is a common cell for researching macrophage foaming, adopts a RAW264.7 cell system to screen compounds for inhibiting macrophage foaming, and is a common technical means for developing medicaments for treating cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease and the like.

Example 3 rat aortic atherosclerosis inhibition experiments

Rats with atherosclerosis caused by high fat feed and vitamin D3 were studied for the pharmacodynamic effects of the compounds described in example 1.

The experimental process comprises the following steps: the wistar rats were acclimatized for 3 days and were divided into three groups, (1) blank control group: normal diet was 8 weeks. (2) The model group is fed with high-fat feed for 8 weeks, and is injected intraperitoneally before molding to administer 60 ten thousand IU/kg of vitamin D3, and then 30 ten thousand IU/kg of vitamin D3 is supplemented every two weeks, and physiological saline is administered by lavage every day. (3) The administration group was fed with the high-fat diet for 8 weeks, and was given 60 ten thousand IU/kg of vitamin D3 by intraperitoneal injection before molding, followed by 30 ten thousand IU/kg of vitamin D3 every two weeks, and the compound described in example 1 (10 mg/kg/D) was administered by daily intragastric administration.

Animal material selection: after the rats fasted for 12 hours, the body weight, 10% chloral hydrate and 5mL/kg of abdominal cavity injection anesthesia were weighed, one section of the aorta was taken and fixed in 4% paraformaldehyde solution, and the rest was transferred to-80 ℃ for HE staining after being quenched by liquid nitrogen, and histopathology was detected.

Experimental results: as shown in fig. 6, the vascular endothelium of the blank group (C) is complete, the inner membrane, the middle membrane and the outer membrane are clearly demarcated, no lipid is deposited under the inner membrane, the middle membrane is a fusiform smooth muscle cell, the structure of the elastic fiber layer is clear and complete, the elastic fiber layer is circularly arranged, and the outer membrane is loose connective tissue. The model group (Mod) has the advantages that the aortic intima is thickened, obvious calcification is visible, the tube wall protrudes into the tube cavity, and the protruding part contains a large amount of foam cells. The tunica media is atrophic, smooth muscle cells proliferate, arrangement disorder, elastin is denatured, broken and disintegrated, and inflammatory cells infiltrate. The administration group improved the pathological changes to some extent compared to the model group, indicating that the compound of example 1 has an inhibitory effect on aortic lesions in atherosclerotic rats.

The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the design of the present invention.

Claims (4)

1. An organic acid compound, characterized in that: is (2R) -2-amino-2-hydroxymethyl-3- [ (4-hydroxy-3-methoxybenzoyl) -O ]]Propionic acid having the structure of formula (I):

。

2. the method for producing an organic acid compound according to claim 1, wherein: the method comprises the following specific steps:

(1) Soaking dried pericarpium Trichosanthis in ethanol at room temperature, filtering, and concentrating under reduced pressure to obtain extract;

(2) The extract is dispersed by D101 macroporous resin to obtain a water layer, a 30% ethanol layer, a 70% ethanol layer and a 95% ethanol layer;

(3) The 30% ethanol layer is eluted by an ODS chromatographic column with methanol-water to obtain three fractions F1, F2 and F3, F1 is eluted by a liquid phase methanol-water 9:91 to obtain three fractions F1-1, F1-2 and F1-3, and F1-3 is purified by a liquid phase methanol-water 7:93 to obtain the novel compound.

3. Use of the organic acid compound according to claim 1 for the preparation of a medicament having an inhibitory effect on macrophage foaming.

4. Use of the organic acid compound according to claim 1 for the preparation of an atherosclerosis-inhibiting drug.

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