Caffeic acid hemiterpenoid ester and its salt, preparation method and use

Abstract

The present invention discloses a caffeic acid hemiterpenoid ester having the structural formula (I): #imgabs0# The present invention also provides a pharmaceutically acceptable salt of the caffeic acid hemiterpenoid ester, an anti-inflammatory drug, and a preparation method of the caffeic acid hemiterpenoid ester and use of the caffeic acid hemiterpenoid ester in the preparation of an anti-inflammatory drug.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and more specifically to a caffeic acid hemiterpenoid ester and its salt, preparation method and use. Background Art

[0002] Jiubiying (Shoubiying) is the dried bark of Ilex rotunda Thunb., a plant of the Aquifoliaceae family. First recorded in the Lingnan Collection of Medicinal Herbs, it is primarily found in Guangdong, Guangxi, and other regions south of the Yangtze River. Its bitter flavor and cold nature are associated with clearing heat, detoxifying, promoting diuresis, and alleviating pain. Currently, there are compound preparations available, including Yanhouling (Yanhouling) tablets, Changyanling (Changyanling) tablets, and Weireqing (Weireqing) capsules. Studies have shown that Jiubiying (Shoubiying) primarily contains ursane-type pentacyclic triterpenes and phenylpropanoid compounds, with pedunculin, ferric acid, and syringin as the primary chemical components. It exhibits pharmacological activities including anti-inflammatory, analgesic, lipid-lowering, hypoglycemic, antibacterial, anti-tumor, and cardiovascular protective properties. It is commonly used clinically for sore throats, acute and chronic gastroenteritis, and traumatic injuries. To fully develop and utilize Jiubiying (Shoubiying) and explore the underlying chemical basis for its anti-inflammatory activity, our research group discovered and identified a new compound from Jiubiying (Shoubiying). This compound was identified as novel and has pharmaceutical applications. Summary of the Invention

[0003] The present invention provides a caffeic acid hemiterpenoid ester and a salt thereof, as well as a preparation method and use of the caffeic acid hemiterpenoid ester in preparing anti-inflammatory drugs.

[0004] In order to achieve these objects and other advantages according to the present invention, there is provided a caffeic acid hemiterpenoid ester having the structural formula (I):

[0005]

[0006] Pharmaceutically acceptable salts of caffeic acid hemiterpenyl esters, pharmaceutically acceptable salts are sulfates, phosphates, hydrochlorides, perchlorates, methanesulfonates, formates, acetates, propionates, butyrates, succinates or salicylates.

[0007] The anti-inflammatory drug comprises a therapeutically effective amount of the caffeic acid hemiterpenoid ester or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0008] Preferably, the anti-inflammatory drug is prepared into a pharmaceutically acceptable dosage form.

[0009] The preparation method of the caffeic acid hemiterpenoid ester comprises:

[0010] Step 1: Take the alcohol extract of the herb, grind it, suspend it in water, extract it with ethyl acetate and n-butanol in sequence, and recover the solvent under reduced pressure to obtain the ethyl acetate fraction and the n-butanol fraction;

[0011] Step 2: dissolve the n-butanol fraction in methanol and water, recover the solvent under reduced pressure until there is no alcohol smell, pour it into a D101 macroporous adsorption resin column for adsorption, and gradient elute with water, 10% methanol-water, 30% methanol-water, 50% methanol-water, 70% methanol-water and 90% methanol-water in sequence. Concentrate the above gradient eluates to obtain a total of 6 fractions Fr.N1 to N6. The 70% methanol-water elution fraction Fr.N5 is subjected to medium-pressure ODS column chromatography at detection wavelengths of 210nm and 254nm, and gradient eluted with 10% methanol-water, 30% methanol-water, 40% methanol-water, 60% methanol-water, and 70% methanol-water in sequence. The above gradient elutions were concentrated to obtain a total of 5 fractions Fr.N5-1 to N5-5. The 60% methanol-water elution fraction Fr.N5-4 was subjected to medium-pressure ODS column chromatography and isocratically eluted with 45% methanol-water. After TLC and HPLC analysis, the same fractions were combined to obtain a total of 21 fractions Fr.N5-4-1 to N5-4-21. The isocratic elution fraction Fr.N5-4-15 with a retention time of 36.2 to 43.8 min was subjected to semi-preparative high-performance liquid chromatography at detection wavelengths of 210 nm and 254 nm and isocratically eluted with 50% methanol-water to obtain the caffeic acid hemiterpenoid ester with a retention time of 12 to 14 min.

[0012] The caffeic acid hemiterpenoid ester is used in the preparation of anti-inflammatory drugs.

[0013] The present invention has at least the following beneficial effects:

[0014] The present invention isolated a new caffeic acid hemiterpenoid from the n-butanol extraction fraction of a 70% ethanol extract of the herb Caulis Sinensis, identified as (E)-4-O-caffeoyl-3-methyl-but-2-en-1-ol, and named caffeic acid hemiterpenoid A. In vitro anti-inflammatory activity tests showed that caffeic acid hemiterpenoid A significantly inhibited LPS-induced nitric oxide (NO) release in RAW264.7 cells at a concentration of 40 μmol / L without significant cytotoxicity. Therefore, caffeic acid hemiterpenoid A has potential anti-inflammatory activity.

[0015] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the UV spectrum of caffeic acid hemiterpenoid ester A;

[0017] Figure 2 is the IR spectrum of caffeic acid hemiterpenoid ester A;

[0018] Figure 3 is the HR-ESI-MS spectrum of caffeic acid hemiterpenoid ester A;

[0019] Figure 4 Caffeic acid hemiterpenoid ester A 1 H-NMR spectrum;

[0020] Figure 5 Caffeic acid hemiterpenoid ester A 13 C-NMR spectrum;

[0021] Figure 6 DEPT spectrum of caffeic acid hemiterpenoid ester A;

[0022] Figure 7 Caffeic acid hemiterpenoid ester A 1 H– 1 H COSY spectrum;

[0023] Figure 8 is the HSQC spectrum of caffeic acid hemiterpenoid ester A;

[0024] Figure 9 Caffeic acid hemiterpenyl ester A 1 H– 1 H COSY is related to the key HMBC;

[0025] Figure 10 is the HMBC spectrum of caffeic acid hemiterpenoid ester A;

[0026] Figure 11 is the NOESY spectrum of caffeic acid hemiterpenoid ester A;

[0027] Figure 12 The effect of caffeic acid hemiterpenoid ester A on LPS-induced NO release in RAW264.7 cells;

[0028] Figure 13 The toxicity of caffeic acid hemiterpenoid ester A to RAW264.7 cells. DETAILED DESCRIPTION

[0029] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0030] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0031] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0032] 1 Instruments and Materials

[0033] Bruker AVANCE III 500 MHz nuclear magnetic resonance instrument (Bruker, Germany); Agilent G6230 time-of-flight mass spectrometer (Agilent, USA); Waters G2-XS time-of-flight mass spectrometer (Waters, USA); Waterse 2695 analytical high-performance liquid chromatograph (Waters, USA); Shimadzu LC-20AD XR high-performance liquid chromatograph infusion pump and SPD-20A ultraviolet detector (Shimadzu, Japan); Kromasil 100-5C18 (10 mm × 250 mm, 5 μm) chromatographic column (Akzo Nobel, Sweden); HT7200S medium-pressure preparative chromatograph (Suzhou Bencao Tiancheng Biotechnology Co., Ltd.); Nicolet iS10 infrared spectrometer (Thermo Scientific, USA); UV-5200PC ultraviolet spectrometer (Shanghai Yuanxi Instrument Co., Ltd.); SB-800DTD ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); RV3 An ecoS096 rotary evaporator (IKA, Germany); an HH-8 constant temperature water bath (Changzhou Yichen Instrument Manufacturing Co., Ltd.); a DLSB-5 / 20 low-temperature cooling circulation pump (Zhengzhou Great Wall Science and Technology Co., Ltd.); a Milli-Q IQ7000 ultrapure water analyzer (Millipore, USA); a ZF-7 darkroom triple-purpose UV analyzer (Shanghai Jiapeng Technology Co., Ltd.); silica gel G thin-layer chromatography plates and column chromatography silica gel (100-200 and 200-300 mesh, Qingdao Ocean Chemical Co., Ltd.); D101 macroporous adsorption resin (Xi'an Lanxiao Technology New Materials Co., Ltd.); chromatographic grade methanol and acetonitrile (Thermo Fisher Scientific, USA); all other reagents were of analytical grade. Phosphate-buffered saline (PBS), fetal bovine serum (FBS), and Dulbecco's modified Eagle's medium (DMEM) were all from Gibico, USA; LPS, MTT, and Griess reagent were all from Sigma-Aldrich, USA.

[0034] RAW264.7 cells were obtained from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0035] Jiubiying was purchased from Zhencaotang Chinese Medicine Pieces Co., Ltd. in Yulin, Guangxi Province, in August 2019. It was identified by Professor Wang Liuping of Guangxi University of Chinese Medicine as the dried bark of Ilex rotunda Thunb. (Aquifoliaceae). Sample No. 20190813 is stored at the Scientific Experimental Center of Guangxi University of Chinese Medicine.

[0036] 2 Extraction and separation

[0037] 100 kg of dried Jiubiying medicinal materials were crushed and extracted with 8 times the amount of 70% (v / v) ethanol under heating and reflux. The filtrate was concentrated under reduced pressure to obtain 50 kg of dry extract, which was crushed into powder with a grinder, suspended in water, and extracted with ethyl acetate and n-butanol in sequence. The solvent was recovered under reduced pressure to obtain 400 g of the ethyl acetate fraction and 1000 g of the n-butanol fraction.

[0038] The extract of the n-butanol fraction was dissolved in methanol and water, and the solvent was recovered under reduced pressure until the alcohol odor disappeared. The sample solution was poured onto a D101 macroporous adsorption resin column and adsorbed overnight. It was then eluted with pure water, 10%, 30%, 50%, 70%, and 90% methanol-water, respectively. Each gradient eluate was concentrated to obtain six fractions (Fr. N1 to N6). The fraction Fr. N5 (45 g) eluted with 70% methanol was subjected to medium-pressure ODS column chromatography using a methanol-water gradient (10%, 30%, 40%, 60%, 70%, 50 mL / min) at detection wavelengths of 210 nm and 254 nm to obtain five fractions (Fr. N5-1 to N5-5). The fraction eluted with 60% methanol, Fr.N5-4 (8 g), was subjected to medium-pressure ODS column chromatography, followed by isocratic elution with 45% methanol-water (10 mL / min). The same fractions were combined for TLC and HPLC analysis to obtain 21 fractions (Fr.N5-4-1 to N5-4-21). Fr.N5-4-15 (50 mg) was subjected to semi-preparative HPLC, detected at 210 nm and 254 nm wavelengths, and isocratic elution with methanol-water (50:50, 2 mL / min) to obtain the compound caffeic acid hemiterpenoid ester (5 mg, t R =12.5min), having the structural formula (I):

[0039]

[0040] 3 Structure identification

[0041] Compound 1: white amorphous powder, Figure 1 is the UV spectrum, 211(4.16),250(3.73),300(3.77),329(3.87). Figure 2 IR spectrum, IR vKBr max (cm -1 ):3371,2922,2851,1661,1633,1497,1385,1263,1179. Figure 3 This is the HR-ESI-MS spectrum. HR-ESI-MS shows the negative ion quasi-molecular ion peak m / z: 263.092 1[MH] - (Calculated value C 14 H 15O5, 263.0925), the molecular formula of the compound was determined to be C 14 H 16 O5. Figure 4 for 1 H-NMR spectrum (500 / 125MHz, CD3OD), Table 1 is 1 H-NMR and 13 C-NMR data, 1 H-NMR showed a group of ABX coupled aromatic proton signals δ H 7.03 (1H, d, J = 1.4 Hz, H-2′), 6.94 (1H, dd, J = 8.2, 1.4 Hz, H-6′), 6.77 (1H, d, J = 8.2 Hz, H-5′); 1 group of trans double bond proton signals δ H 7.55 (1H, d, J = 15.9 Hz, H-7′), 6.27 (1H, d, J = 15.9 Hz, H-8′); 1 olefin proton signal connected to the methylene group δ H 5.66 (1H, t, J = 6.4 Hz, H-2); 2 oxymethylene proton signals δ H 4.59 (2H, s, H-4), 4.15 (2H, d, J = 6.4 Hz, H-1) and one vinyl methyl proton signal δ H 1.72(3H,brs,H-5). Figure 5 for 13 C-NMR spectrum (500 / 125MHz, CD3OD), Figure 6 DEPT spectrum (500 / 125MHz, CD3OD), 13 C-NMR and DEPT showed that the compound had 14 carbon signals, including 5 quaternary carbon signals, 6 methine carbon signals, 2 oxymethylene carbon signals and 1 methyl carbon signal, among which δ C 168.9 (C-9') is an ester carbonyl carbon signal, δ C 149.7 (C-4') and 146.8 (C-3') are two aromatic carbon signals connected to the hydroxyl group, δ C 69.9 (C-4) and 59.0 (C-1) are two oxymethylene carbon signals, δ C 14.0 (C-5) is a methyl carbon signal.

[0042] Table 1

[0043]

[0044] Figure 7 for 1 H– 1H COSY spectrum (500 / 125MHz, CD3OD), Figure 8 is the HSQC spectrum (500 / 125MHz, CD3OD), Figure 9 Caffeic acid hemiterpenoid ester A 1 H– 1 H COSY is related to the key HMBC, Figure 10 is the HMB spectrum (500 / 125MHz, CD3OD), Figure 11 NOESY spectrum (500 / 125MHz, CD3OD), HMBC shows δ H 7.03 (H-2'), 6.77 (H-5') respectively with δ C 149.6 (C-4'), 146.8 (C-3') correlation and δ H 7.55(H-7') and δ C The long-range correlations at 115.1 (C-2'), 123.0 (C-6'), and 168.9 (C-9') indicate the presence of a caffeoyl fragment in this compound. H 4.15(H-1) and δ C 134.0(C-3), δ H 1.72(H-5) and δ C 128.3 (C-2), 69.9 (C-4) and δ H 4.59(H-4) and δ C The correlation of 128.3 (C-2) indicates that the compound contains a 3-methyl-but-2-en-1-ol or 2-methyl-but-2-en-1-ol hemiterpenoid fragment. H 4.59(H-4) and δ C The key HMBC correlation at 168.9 (C-9') and the singlet at H-4 confirmed that the C-4 position of the hemiterpene fragment was ester-bonded to the C-9' position of the caffeoyl fragment, and that the hemiterpene fragment was 3-methyl-but-2-en-1-ol. NOE correlations between H-2 and H-4, and H-5 and H-1 in the NOSEY spectrum, indicated that the double bond in the hemiterpene fragment had an E configuration. In summary, the compound was identified as (E)-4-O-caffeoyl-3-methyl-but-2-en-1-ol and designated as caffeic acid hemiterpene ester A.

[0045] 4 Anti-inflammatory activity screening

[0046] LPS was used to induce the mouse RAW264.7 macrophage inflammation model. The NO content was determined by Griess method, and the effects of LPS on the activity of RAW264.7 cells were detected by MTT method. Figure 12 Effect of caffeic acid hemiterpenoid ester A on LPS-induced NO release in RAW264.7 cells The results showed that at a concentration of 40 μmol / L, caffeic acid hemiterpenoid ester A had a significant inhibitory effect on NO production. Figure 13 The toxicity of caffeic acid hemiterpenoid ester A to RAW264.7 cells Cell viability test results showed that caffeic acid hemiterpenoid ester A had no effect on cell growth when the concentration was less than 80 μmol / L, and the cell viability was greater than 90%. Therefore, caffeic acid hemiterpenoid ester A has potential anti-inflammatory activity.

[0047] In summary, the present invention uses silica gel column chromatography, ODS column chromatography, D101 macroporous adsorption resin column chromatography and semi-preparative high performance liquid chromatography to separate and purify the chemical components of the herb, and uses HR-ESI-MS and NMR to identify the compound structure. The anti-inflammatory effect of the compound was evaluated by measuring the compound's ability to inhibit the release of inflammatory mediator nitric oxide (NO) by mouse RAW264.7 macrophages induced by lipopolysaccharide (LPS); the cytotoxicity of the compound was determined by the MTT method. A new compound isolated from the herb of the present invention was identified as (E)-4-O-caffeoyl-3-methyl-but-2-ene-1-ol and named caffeic acid hemiterpenoid ester A. Caffeic acid hemiterpenoid ester A has potential anti-inflammatory activity.

[0048] The number of devices and processing scales described herein are intended to simplify the description of the present invention. Applications, modifications, and variations of the present invention will be readily apparent to those skilled in the art.

[0049] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. Caffeic acid hemiterpenoid ester, characterized in that Has the structural formula (I): （I）。 2. An anti-inflammatory drug, characterized in that The invention comprises a therapeutically effective amount of the caffeic acid hemiterpenyl ester or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier.

3. The method for preparing the caffeic acid hemiterpenoid ester according to claim 1, wherein include: Step 1: Take the alcohol extract of the herb, grind it, suspend it in water, extract it with ethyl acetate and n-butanol in sequence, and recover the solvent under reduced pressure to obtain the ethyl acetate fraction and the n-butanol fraction; Step 2: Dissolve the n-butanol fraction in methanol and water, recover the solvent under reduced pressure until the alcohol odor is eliminated, and apply the fraction to a D101 macroporous adsorption resin column for adsorption. Gradient elution is performed with water, 10% methanol-water, 30% methanol-water, 50% methanol-water, 70% methanol-water, and 90% methanol-water. Each gradient elution is concentrated to obtain six fractions, Fr. N1 to N6. The fraction, Fr. N5, eluted with 70% methanol-water, is subjected to medium-pressure ODS column chromatography. Detection wavelengths are 210 nm and 254 nm, and gradient elution is performed with 10% methanol-water, 30% methanol-water, 40% methanol-water, 60% methanol-water, and 70% methanol-water. Each gradient elution is concentrated to obtain five fractions, Fr. N5-1 to N5-5. The fraction, Fr. N5, eluted with 60% methanol-water, is concentrated. N5-4 was subjected to medium-pressure ODS column chromatography and isocratically eluted with 45% methanol-water. The same fractions were combined for TLC and HPLC analysis to obtain a total of 21 fractions Fr. N5-4-1 to N5-4-21. The isocratic elution fraction Fr. N5-4-15 with a retention time of 36.2 to 43.8 min was subjected to semi-preparative high-performance liquid chromatography with isocratic elution using 50% methanol-water at detection wavelengths of 210 nm and 254 nm to obtain the caffeic acid hemiterpenyl ester with a retention time of 12 to 14 min.

4. Use of the caffeic acid hemiterpenoid ester according to claim 1 in the preparation of anti-inflammatory drugs.

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