A phenolic acid coumarin hybrid compound and its separation and purification method and application

Abstract

The present invention provides a phenolic acid coumarin hybrid compound and its separation and purification method and application, belonging to the field of drug extraction. The effective substances are extracted from perilla stems by alcohol extraction, and the extract is separated and purified by silica gel column chromatography, dextran gel column chromatography, and preparative liquid chromatography to obtain a phenolic acid coumarin hybrid compound with anti-inflammatory activity. Its structure is analyzed by nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. Its chemical formula is C 18 H 12 O8, with an unsaturation degree of 13, was subjected to in vitro anti-inflammatory experiments, which confirmed that it has high anti-inflammatory activity and can be used to prepare anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of drug extraction, and in particular to a phenolic acid coumarin hybrid compound and a separation and purification method and application thereof. Background Art

[0002] Perilla (Perilla frutescens L.Britt.) is an annual herbaceous plant of the Lamiaceae family. It has a long history of use in my country, with its stems, leaves, fruits, and seeds all being used as medicine. my country is rich in Perilla resources, with both wild and cultivated species found in North, Central, and South China, as well as in Taiwan. Perilla stems (Perilla e Caulis) are a commonly used Traditional Chinese Medicine (TCM) for their qi-regulating, analgesic, and fetal health benefits. They are primarily used to treat chest and diaphragm discomfort, abdominal pain, vomiting due to heatstroke, and fetal restlessness.

[0003] Literature reports that the main chemical components of perilla include volatile oils, phenolic acids, flavonoids, anthocyanins, triterpenes, and polysaccharides. Modern research indicates that the biological activities of perilla stems include antioxidant, anti-inflammatory, antibacterial, anti-allergic, and anti-tumor properties. Perilla stems also contain a variety of vitamins and rich protein, possessing health benefits and possessing significant potential for development. We have systematically studied the chemical composition of perilla stems, laying the foundation for the discovery of active natural products and further drug development. Summary of the Invention

[0004] The purpose of the present invention is to provide a phenolic acid coumarin hybrid compound and its separation and purification method and application, and to extract the anti-inflammatory active ingredients from the perilla stems to provide a new development direction for the research and development of anti-inflammatory drugs.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a phenolic acid coumarin hybrid compound, the chemical structure of which is as follows:

[0007]

[0008] The present invention provides a method for separating and purifying the above-mentioned phenolic acid coumarin hybrid compound, comprising the following steps:

[0009] 1) extracting the stems of Perilla frutescens with alcohol and extracting with ethyl acetate in sequence to obtain an ethyl acetate extract of the stems of Perilla frutescens;

[0010] 2) separating the ethyl acetate extract of the stem of Perilla frutescens by silica gel column chromatography to obtain a fraction containing a phenolic acid coumarin hybrid compound;

[0011] 3) separating the fraction containing the phenolic acid coumarin hybrid compound by using dextran gel column chromatography to obtain subfractions containing the phenolic acid coumarin hybrid compound;

[0012] 4) Purifying the subfraction containing the phenolic acid coumarin hybrid compound by preparative liquid chromatography to obtain the phenolic acid coumarin hybrid compound.

[0013] Preferably, the alcohol extraction time in step 1) is 8 to 12 days, and the number of alcohol extractions is 2 to 4 times.

[0014] Preferably, in the alcohol extraction process of step 1), the mass volume ratio of perilla stem to alcohol is 1:3 to 1:8.

[0015] Preferably, the eluent for the silica gel column chromatography in step 2) is a mixed solution of petroleum ether and ethyl acetate, and the elution method is gradient elution.

[0016] The gradient elution procedure is a volume ratio of petroleum ether to ethyl acetate of 100-0:0-100.

[0017] Preferably, the eluent of the dextran gel column chromatography in step 3) is methanol.

[0018] Preferably, the eluent for the preparative liquid chromatography in step 4) is a methanol-water solution, and the elution method is a gradient elution;

[0019] The gradient elution procedure is that the volume concentration of the methanol aqueous solution is 45% to 100%.

[0020] The present invention also provides the use of the phenolic acid coumarin hybrid compound in the preparation of anti-inflammatory drugs.

[0021] The present invention has at least the following beneficial effects:

[0022] This study, based on a systematic study of the chemical composition of Perilla frutescens stems and combined with mass spectrometry and spectral data, isolated and identified a novel phenolic acid coumarin hybrid compound from Perilla frutescens stems. In vitro anti-inflammatory activity tests demonstrated that the phenolic acid coumarin hybrid provided by the present invention significantly inhibited the expression of the inflammatory factor IL-1β (interleukin) in primary bone marrow-derived cells of BMDM mice stimulated with LPS (lipopolysaccharide). This phenolic acid coumarin hybrid compound exhibits significant in vitro anti-inflammatory activity and may be used to develop new anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a structural diagram of the phenolic acid coumarin hybrid compound extracted from Example 1 of the present invention;

[0024] Figure 2 This is the HRESIMS graph of the phenolic acid coumarin hybrid compound extracted from Example 1 of the present invention;

[0025] Figure 3 The phenolic acid coumarin hybrid compound extracted from Example 1 of the present invention1 H NMR spectra;

[0026] Figure 4 The phenolic acid coumarin hybrid compound extracted from Example 1 of the present invention 13 C NMR spectrum;

[0027] Figure 5 The phenolic acid coumarin hybrid compound extracted from Example 1 of the present invention 1 H- 1 H COSY diagram;

[0028] Figure 6 This is the HSQC graph of the phenolic acid coumarin hybrid compound extracted from Example 1 of the present invention;

[0029] Figure 7 This is the HMBC chart of the phenolic acid coumarin hybrid compound extracted from Example 1 of the present invention;

[0030] Figure 8 1 is a high performance liquid chromatogram of fraction XII in Example 1 of the present invention;

[0031] Figure 9 This is a graph showing the in vitro anti-inflammatory test results of the phenolic acid coumarin hybrid compound extracted from Example 1 of the present invention. DETAILED DESCRIPTION

[0032] The present invention provides a phenolic acid coumarin hybrid compound, the chemical structure of which is as follows:

[0033]

[0034] The present invention provides a method for separating and purifying the above-mentioned phenolic acid coumarin hybrid compound, comprising the following steps:

[0035] 1) extracting the stems of Perilla frutescens with alcohol and extracting with ethyl acetate in sequence to obtain an ethyl acetate extract of the stems of Perilla frutescens;

[0036] 2) separating the ethyl acetate extract of the stem of Perilla frutescens by silica gel column chromatography to obtain a fraction containing a phenolic acid coumarin hybrid compound;

[0037] 3) separating the fraction containing the phenolic acid coumarin hybrid compound by using dextran gel column chromatography to obtain subfractions containing the phenolic acid coumarin hybrid compound;

[0038] 4) Purifying the subfraction containing the phenolic acid coumarin hybrid compound by preparative liquid chromatography to obtain the phenolic acid coumarin hybrid compound.

[0039] In the present invention, the alcohol extraction time in step 1) is 8 to 12 days, preferably 9 to 11 days, and more preferably 10 days; the number of alcohol extractions is 2 to 4 times, preferably 3 times.

[0040] In the present invention, during the alcohol extraction process in step 1), the mass volume ratio of perilla stems to alcohol is 1:3 to 1:8, preferably 1:4 to 1:7, and more preferably 1:5 to 1:6.

[0041] In the present invention, the step of evaporating and concentrating the extract is further included after the alcohol extraction.

[0042] In the present invention, the eluent for the silica gel column chromatography in step 2) is a mixed solution of petroleum ether and ethyl acetate, and the elution method is gradient elution;

[0043] The gradient elution procedure is to sequentially use mixed solutions of petroleum ether and ethyl acetate with volume ratios of 10:0, 10:1, 8:2, 7:3, 6:4, 5:5, 3:7, 1:9, and 0:10 for elution, and the volume of each mixed solution is 2 L.

[0044] In the present invention, during the separation of the ethyl acetate extract of the stem of Perilla frutescens by silica gel column washing method, high performance liquid chromatography is used to detect the fractions, and the fractions containing the phenolic acid coumarin hybrid compound of the present invention are collected.

[0045] In the present invention, the eluent of the dextran gel column chromatography in step 3) is methanol.

[0046] In the present invention, the eluent for the preparative liquid chromatography in step 4) is a methanol-water solution, and the elution method is a gradient elution;

[0047] The gradient elution procedure is 0-30 min: 45%-100% methanol aqueous solution.

[0048] The present invention also provides the use of the phenolic acid coumarin hybrid compound in the preparation of anti-inflammatory drugs.

[0049] The application of the phenolic acid coumarin hybrid compound in the preparation of anti-inflammatory drugs is specifically described as follows:

[0050] The phenolic acid coumarin hybrid compound is prepared into capsules: the phenolic acid coumarin hybrid compound and corn starch and / or lactose as a carrier are uniformly mixed, granulated, and filled into capsules to prepare capsules.

[0051] The phenolic acid coumarin hybrid compound is made into tablets: the phenolic acid coumarin hybrid compound is evenly mixed with one or more of corn starch, lactose and magnesium stearate, granulated and compressed into tablets.

[0052] When the phenolic acid coumarin hybrid compound is made into powder injection or injection solution, a carrier is added and prepared according to conventional pharmaceutical methods.

[0053] The phenolic acid coumarin hybrid compound is prepared into granules: the phenolic acid coumarin hybrid compound and a diluent such as corn starch are uniformly mixed, granulated, and dried to prepare granules.

[0054] When the phenolic acid coumarin hybrid compound is prepared into dosage forms such as fat emulsion, ointment or transdermal controlled release patch, a carrier is added and prepared according to conventional pharmaceutical methods.

[0055] In the present invention, the inflammation includes enteritis, nephritis and gastritis.

[0056] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0057] Example 1

[0058] 2 kg of dried perilla stems were cut into small pieces and extracted with 10 L of ethanol for 10 days each time, for a total of two extractions. The extracts were combined, filtered, and concentrated under reduced pressure to obtain an ethanol extract of the perilla stems. The ethanol extract was then extracted with 500 mL of ethyl acetate, and the ethyl acetate fraction was separated and concentrated under reduced pressure to obtain the ethyl acetate extract of the perilla stems. The ethyl acetate extract of Perilla stem was separated by silica gel column chromatography, and the gradient elution was carried out with a petroleum ether-ethyl acetate system. The specific elution procedure was to sequentially use 2L of a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 10:0, 2L of a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 10:1, 2L of a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 8:2, 2L of a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 7:3, 2L of a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 6:4, 2L of a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:5, 2L of a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3:7, 2L of a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:9, and 2L of a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 0:10 for elution; the eluent was combined with high performance liquid chromatography detection to obtain 14 fractions I to XIV. Fraction XII (the high performance liquid chromatography of fraction XII is as follows Figure 8 The product (shown in FIG) was separated by dextran gel column chromatography and isocratically eluted with methanol to obtain 11 subfractions XII-A to XII-K. Component XII-H was purified by preparative liquid chromatography using gradient elution. The gradient elution program was set to 0-30 min, 45% to 100% (volume fraction) methanol aqueous solution (i.e., the volume concentration of methanol gradually increased from 45% to 100% in 0-30 min), to obtain 3 mg of the phenolic acid coumarin hybrid compound (named Perihybridine).

[0059] The above phenolic acid coumarin hybrid compound Perihybridine was analyzed by high resolution electrospray ionization mass spectroscopy (HRESIMS). The analysis results are as follows: Figure 2 As shown, its molecular ion peak is: m / z 355.0461[MH] - , it can be inferred that its molecular formula is C 18 H 12 O8, the degree of unsaturation is 13, combined with nuclear magnetic resonance spectroscopy analysis (solvent CD3OD), the analysis results are as follows Figures 3 to 7 As shown in Table 1, Figure 3 for 1 H NMR (500MHz) chart, Figure 4 for 13 C NMR (125MHz) Figure 5 for 1 H- 1 H COSY diagram, Figure 6 HSQC diagram, Figure 7 The HMBC diagram is shown in Table 1, and the NMR data are shown in Table 1. The structure of the phenolic acid coumarin hybrid compound Perihybridine can be analyzed by analyzing the above analysis results. 1 H NMR with 13 The C NMR data combined with HSQC can classify the carbon spectrum of the compound as follows: 8 unsaturated CH, 8 olefinic quaternary carbons and 2 carboxylic acids / esters. 1 H NMR and 13 C NMR data showed that there were five unsubstituted sites on the benzene ring [δ H 7.11(1H,s),δ H 6.80(1H,s),δ H 7.03 (1H, dd), δ H 6.95(1H,s),δ H 6.80(1H,s)], three olefinic hydrogen sites [δ H 6.45 (1H, d, J = 15.9 Hz), δ H 7.74 (1H, d, J = 15.9 Hz), δ H 7.66 (1H, s)]. By searching the literature, it was found that part of the structural fragment of this compound was close to the NMR data of 3,6,7-trihydroxy-coumarin (3,6,7-trihydroxycoumarin), indicating that it has the same coumarin mother core part. H 7.74) and H-2(δ H 6.45) Display existence1 H- 1 H COSY related, combined with H-3 (δ H 7.74) and C-1′(δ C 127.45), H-3(δ H 7.74) and C-6′(δ C 123.60), H-3(δ H 7.74) and C-1(δ C 166.58), H-2(δ H 6.45) and C-1′(δ C 127.45), H-2(δ H 6.45) and C-1(δ C 166.58) was correlated with the HMBC of the compound, confirming that the other half of the compound is a caffeic acid structure, and the two parts are connected by an ester bond between C-1 and C-1'. Through further literature comparison, it was determined that the compound is a new phenolic acid coumarin hybrid compound and was named Perihybridine. Its chemical structure is as follows Figure 1 shown.

[0060] Table 1 NMR spectrum data of compound Perihybridine

[0061] No. <![CDATA[δ C ]]> <![CDATA[δ H ]]> No. <![CDATA[δ C ]]> <![CDATA[δ H ]]> 1 166.58,C 1″ 134.56,C 2 112.99,CH 6.45d(15.9) 2″ 133.70,CH 7.66s 3 149.66,CH 7.74d(15.9) 3″ 112.04,C 1′ 127.45,C 4″ 112.92,CH 6.95s 2′ 115.39,C 7.11d(2.2) 5″ 145.00,C 3′ 146.97,C 6″ 151.37,C 4′ 150.30,C 7″ 103.62,CH 6.80* 5′ 116.58,CH 6.80* 8″ 148.53,C 6′ 123.60,CH 7.03dd(8.3,2.1) 9″ 159.57,C

[0062] The above-mentioned phenolic acid coumarin hybrid compound Perihybridine was subjected to an in vitro anti-inflammatory experiment. The specific steps are as follows:

[0063] 1×10 5 Cells / well BMDM primary macrophages were evenly seeded in 96-well plates. Blank, model, and different concentration sample groups were set up, with 6 replicates per group. The cells were incubated in a 37°C, 5% CO2 cell culture incubator for 24 hours. After the cells attached, the supernatant was discarded. The blank group was added with an equal volume of Dulbecco's modified eagle medium (DMEM), the model group was added with DMEM containing 100 ng / mL LPS, and the sample group was replaced with a DMEM solution containing 100 ng / mL LPS and the new compound Perihybridine isolated and purified in Example 1 of the present invention (final concentrations of 0.1, 1.0, 10, and 100 μg / mL, respectively). After incubation for 24 hours at 37°C, 5% CO2, 50 μL of the supernatant was added to a 96-well plate and the IL-1β content was detected according to the instructions of the interleukin-1β kit. The absorbance of each well was measured at 540 nm, and the IL-1β release inhibition rate (%) was calculated as follows: (OD value of model group - OD value of sample group) / (OD value of model group - OD value of blank group) × 100%.

[0064] The above in vitro anti-inflammatory effect test results are as follows Figure 9 As shown in the results, after 24 hours of LPS stimulation, the IL-1β content in the cell supernatant of the model group increased, which was significantly different from that of the blank group; while in the sample group, different concentrations of Perihybridine could reduce the release of IL-1β in BMDM primary macrophages induced by LPS in a dose-dependent manner, which was significantly different from that of the model group.

[0065] The above results show that Perihybridine can significantly inhibit the expression of inflammatory factor IL-1β in LPS-stimulated BMDM primary macrophages, and Perihybridine has obvious anti-inflammatory activity.

[0066] The phenolic acid coumarin hybrid compound Perihybridine obtained by separation and purification of the present invention can also be made into a drug. The specific preparation method is as follows:

[0067] Preparation of capsules: Take an appropriate amount of Perihybridine separated and purified in Example 1 above, add a certain amount of pharmaceutical excipient corn starch, mix thoroughly, and put into capsules to prepare capsules for oral use.

[0068] Preparation of tablets: Take an appropriate amount of Perihybridine separated and purified in Example 1 above, add a certain amount of pharmaceutical excipients such as starch and magnesium stearate, mix thoroughly, and press into tablets to prepare tablets for oral use.

[0069] Preparation of granules: Take an appropriate amount of Perihybridine separated and purified in Example 1 above, add a certain amount of pharmaceutical excipient corn starch, mix thoroughly, granulate, and dry to prepare granules for oral use.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A phenolic acid coumarin hybrid compound, characterized in that Its chemical structure is as follows: 。 2. The method for separating and purifying a phenolic acid coumarin hybrid compound according to claim 1, characterized in that: The following steps are involved: 1) Extracting the stems of Perilla frutescens with alcohol and ethyl acetate in sequence to obtain the ethyl acetate extract of the stems of Perilla frutescens; 2) Separating the ethyl acetate extract of Perilla stems by silica gel column chromatography to obtain fractions containing phenolic acid coumarin hybrid compounds, and eluting with mixed solutions of petroleum ether and ethyl acetate in volume ratios of 10:0, 10:1, 8:2, 7:3, 6:4, 5:5, 3:7, 1:9, and 0:10, in that order; 3) separating the fraction containing the phenolic acid coumarin hybrid compound by using dextran gel column chromatography to obtain a subfraction containing the phenolic acid coumarin hybrid compound, using methanol as the eluent; 4) Purify the subfraction containing the phenolic acid coumarin hybrid compound by preparative liquid chromatography to obtain the phenolic acid coumarin hybrid compound using a gradient elution program of 45% to 100% methanol in water for 0-30 min.

3. The method for separating and purifying a phenolic acid coumarin hybrid compound according to claim 2, characterized in that: The alcohol extraction time in step 1) is 8 to 12 days, and the number of alcohol extractions is 2 to 4 times.

4. The method for separating and purifying a phenolic acid coumarin hybrid compound according to claim 3, characterized in that: In the alcohol extraction process of step 1), the mass volume ratio of perilla stem to alcohol is 1:3 to 1:

8.

5. Use of the phenolic acid coumarin hybrid compound according to claim 1 in the preparation of anti-inflammatory drugs.

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