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

Abstract

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

[0002] Jiubiying is the dried bark of IIex rotunda Thunb., a plant of the genus Ilex of the Aquifoliaceae family. Ilex rotunda was first recorded in the "Record of Medicinal Herbs in Lingnan" and is mainly distributed in Guangdong, Guangxi and other areas south of the Yangtze River. It tastes bitter and is cold in nature, and has the effects of clearing away heat and detoxifying, promoting dampness and relieving pain. Currently, there are compound preparations such as Yanhouling Decoction Tablets, Changyanling Tablets and Weireqing Capsules. Studies have shown that Jiubiying mainly contains ursane-type pentacyclic triterpenes and phenylpropanoid compounds, among which long-stalked holly glycosides, ferric holly acid and lilac glycosides are its main chemical components. It has pharmacological activities such as anti-inflammatory, analgesic, hypolipidemic, hypoglycemic, antibacterial, anti-tumor and cardiovascular protection. It is often used clinically for sore throat, acute and chronic gastroenteritis and traumatic injuries. In order to fully develop and utilize Jiubiying and explore the material basis of its anti-tumor activity, our research group discovered and identified a new compound from Jiubiying. After searching, it was found that the compound is a new compound and has pharmaceutical use. Summary of the invention

[0003] The invention provides a caffeic acid hemiterpenoid ester and a salt thereof, and provides a preparation method thereof and use thereof in preparing an anti-tumor drug.

[0004] In order to achieve these purposes 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] An anti-tumor drug comprising a therapeutically effective amount of the caffeic acid hemiterpenoid ester or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier.

[0008] Preferably, the anti-tumor 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, crush 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 with methanol and water, recover the solvent under reduced pressure until there is no alcohol taste, 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 respectively, and obtain a total of 6 fractions Fr.N1~N6, and subject the 70% methanol-water elution fraction Fr.N5 to medium-pressure ODS column chromatography at 210nm and 254nm detection wavelengths, and use 10% methanol-water, 30% methanol-water, 40% methanol-water, 60% methanol-water, 70% methanol-water gradient elution in sequence, and the fraction with the following content: The above gradient eluates 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. The same fractions were combined by TLC and HPLC analysis to obtain a total of 21 fractions Fr.N5-4-1 to N5-4-21. The isocratic elution fraction Fr.N5-4-19 with a retention time of 56.2 to 62.5 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 hemiterpene ester with a retention time of 12 to 16 min.

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

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

[0014] The present invention isolates a new caffeic acid hemiterpenoid ester from the n-butanol extraction part of the 70% ethanol extract of the herb, identifies it as (E)-4-O-caffeoyl-2-methyl-but-2-ene-1-ol, and names it as caffeic acid hemiterpenoid ester B. The results of in vitro anti-tumor activity test show that caffeic acid hemiterpenoid ester B has a significant inhibitory effect on the activity of A549 cells at a concentration of 20 μmol / L without obvious cytotoxicity. Therefore, caffeic acid hemiterpenoid ester B has potential anti-tumor activity.

[0015] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part 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 B;

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

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

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

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

[0021] Figure 6 This is the DEPT spectrum of caffeic acid hemiterpenoid ester B;

[0022] Figure 7 Caffeic acid hemiterpenoid B 1 H- 1 H COSY spectrum;

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

[0024] Fig. 9 Caffeic acid hemiterpenoid ester B 1 H- 1 H COSY is related to the key HMBC;

[0025] Fig.10 is the HMBC spectrum of caffeic acid hemiterpenoid ester B;

[0026] Fig.11 This is the NOESY spectrum of caffeic acid hemiterpenoid ester B;

[0027] Fig.12 The toxicity of caffeic acid hemiterpenoid ester B to A549 cells. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0029] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0030] 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.

[0031] 1. Instruments and Materials

[0032] Bruker AVANCE III 500MHz 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); Waterse2695 analytical high performance liquid chromatograph (Waters, USA); Shimadzu LC-20AD XR high performance liquid chromatograph infusion pump, SPD-20A ultraviolet detector (Shimadzu, Japan); Kromasil 100-5C18 (10mm×250mm, 5μm) 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 cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.); RV3 ecoS096 rotary evaporator (IKA, Germany); HH-8 constant temperature water bath (Changzhou Yichen Instrument Manufacturing Co., Ltd.); DLSB-5 / 20 low temperature cooling circulation pump (Zhengzhou Great Wall Science and Technology Co., Ltd.); Milli-Q IQ7000 ultrapure water instrument (Millipore, USA); ZF-7 dark box three-purpose UV analyzer (Shanghai Jiapeng Technology Co., Ltd.); silica gel G thin layer chromatography plate, column chromatography silica gel (100-200, 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 Technology Co., Ltd., USA); other reagents were of analytical grade. Phosphate buffered saline (PBS), fetal bovine serum (FBS), DMEM medium (Gibico, USA); MTT reagent (Sigma-Aldrich, USA).

[0033] A549 cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences).

[0034] Jiubiying was purchased from Guangxi Yulin Zhencaotang Chinese Medicine Pieces Co., Ltd. in August 2019 and identified by Professor Wang Liuping of Guangxi University of Chinese Medicine as the dried bark of Ilex rotunda Thunb. of the genus Ilex of the Aquifoliaceae family. The sample (No. 20190813) is stored in the Scientific Experiment Center of Guangxi University of Chinese Medicine.

[0035] 2 Extraction and separation

[0036] 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, extracted with ethyl acetate and n-butanol in turn, and the solvent was recovered under reduced pressure to obtain ethyl acetate part (400 g) and n-butanol part (1000 g).

[0037] The extract of the n-butanol part was dissolved in methanol and water, and the solvent was recovered under reduced pressure until there was no alcohol taste. The sample solution was poured into a D101 macroporous adsorption resin column for adsorption overnight, and then eluted with pure water, 10%, 30%, 50%, 70% and 90% methanol-water in sequence. The above gradient eluents were concentrated to obtain 6 fractions (Fr.N1-N6). The 70% methanol elution part Fr.N5 (45g) was subjected to medium pressure ODS column chromatography, and at the detection wavelength of 210nm and 254nm, methanol-water (10%, 30%, 40%, 60%, 70%, 50mL / min) gradient elution was used to obtain 5 fractions (Fr.N5-1-N5-5). The 60% methanol elution fraction Fr.N5-4 (8 g) was subjected to medium pressure ODS column chromatography, 45% methanol-water isocratic elution (10 mL / min), TLC, HPLC analysis and merging of the same fractions to obtain 21 fractions (Fr.N5-4-1 to N5-4-21). Isocratic elution (56.2 to 62.5 min) Fr.N5-4-19 (30 mg) was prepared by 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 (2 mg, t R =14.1min), having the structural formula (I):

[0038]

[0039] 3 Structure identification

[0040] Caffeic acid hemiterpenoid ester B: white amorphous powder, Figure 1 is the UV spectrum, UV (nm):211(4.30),250(3.93),300(3.98),329(4.07)nm; Figure 2 is the IR spectrum, IR (cm -1 ):3359,2921,2851,1685,1599,1523,1448,1385,1261,1180,1118,1063,983. Figure 3 This is the HR-ESI-MS spectrum. HR-ESI-MS shows the cationic quasi-molecular ion peak m / z: 287.089 6[M+Na] + (Calculated value C 14H 16 O 5 Na, 287.089 0), the molecular formula is determined to be C 14 H 16 O 5 . Figure 4 for 1 H-NMR spectrum (500 / 125MHz, CD3OD), Table 1 is 1 H-NMR and 13 C-NMR data, 1H-NMR shows a group of ABX coupled aromatic proton signals δ H 7.02 (1H, d, J = 1.9 Hz, H-2′), 6.93 (1H, dd, J = 8.2, 1.9 Hz, H-6′), 6.76 (1H, d, J = 8.2 Hz, H-5′); 1 group of trans double bond proton signals δ H 7.52 (1H, d, J = 15.9 Hz, H-7′), 6.24 (1H, d, J = 15.9 Hz, H-8′); 1 olefin proton signal connected to the methylene group δ H 5.65 (1H, t, J = 7.0 Hz, H-2); 2 oxymethylene proton signals δ H 4.75 (2H, d, J = 7.0 Hz, H-4), 3.97 (2H, s, H-1) and 1 vinyl methyl proton signal δ H 1.74(3H,s,H-5). Figure 5 for 13 C-NMR spectrum (500 / 125MHz, CD3OD), Figure 6 DEPT spectrum (500 / 125MHz, CD3OD), 13C-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 169.2 (C-9') is an ester carbonyl carbon signal, δ C 149.6 (C-4') and 146.9 (C-3') are two aromatic carbon signals connected to the hydroxyl group. C 67.7 (C-1) and 61.8 (C-4) are two oxymethylene carbon signals, δ C 13.8 (C-5) is a methyl carbon signal.

[0041] Table 1

[0042]

[0043] Figure 7 for 1 H- 1H COSY spectrum (500 / 125MHz, CD 3 OD), Figure 8 HSQC spectrum (500 / 125MHz, CD 3 OD), Fig. 9 Caffeic acid hemiterpenoid B 1 H- 1 H COSY is related to the key HMBC, Fig.10 HMBC spectrum (500 / 125MHz, CD 3 OD), Fig.11 NOESY spectrum (500 / 125MHz, CD 3 OD), δ H 4.75(H-4) and δC 142.3(C-2), δ H 1.74(H-5) and δ C 119.6(C-3), 67.7(C-1) and δ H 3.97(H-1) and δ C The correlation of 119.6 (C-3) and 13.8 (C-5) suggests that the hemiterpene fragment of this compound may be 2-methyl-but-2-en-1-ol. H 4.75(H-4) and δ C The key HMBC correlation of 169.2 (C-9') and H-4 as a doublet peak confirmed that the C-4 position of the hemiterpene fragment was connected to the C-9' position of the caffeoyl fragment through an ester bond and confirmed that the hemiterpene fragment was 2-methyl-but-2-ene-1-ol. In the NOSEY spectrum, H-3 and H-1, H-5 and H-4 had NOE correlations, indicating that the double bond in the hemiterpene fragment was in E configuration. In summary, the compound was identified as (E)-4-O-caffeoyl-2-methyl-but-2-ene-1-ol and named caffeic acid hemiterpene ester B.

[0044] 4 Anti-tumor activity screening

[0045] The MTT method was used to detect the effects of the compounds on the activity of A549 cells. Fig.12 The toxicity of caffeic acid hemiterpenoid ester B to A549 cells ( n=3), ***P<0.001vs blank control group, indicating that at a concentration of 20 μmol / L, caffeic acid hemiterpenoid ester B has a significant inhibitory effect on the activity of A549 cells. Therefore, caffeic acid hemiterpenoid ester B has potential anti-tumor activity.

[0046] 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 Ilex rotunda Thunb., and uses HR-ESI-MS and NMR to identify the structures of the compounds. The anti-tumor effects of the compounds were evaluated by measuring their inhibitory ability on the release of the inflammatory mediator nitric oxide (NO) from lipopolysaccharide (LPS)-induced mouse RAW264.7 macrophages; the cytotoxicity of the compounds was determined by the MTT method. A new compound isolated from Ilex rotunda Thunb. in the present invention was identified as (E)-4-O-caffeoyl-2-methyl-but-2-en-1-ol, named caffeic acid sesquiterpene ester B. Caffeic acid sesquiterpene ester B has potential anti-tumor activity.

[0047] The equipment quantities and processing scales described herein are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.

[0048] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated examples described herein.

Claims

1. Caffeic acid hemiterpenoid esters, It is characterized in that With structural formula (I): （I）。 2. The method for preparing the caffeic acid hemiterpenoid ester according to claim 1, It is characterized in that include: Step 1: Take the alcohol extract of the herb, crush 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: add methanol and water to dissolve the n-butanol fraction, 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, and concentrate the above gradient eluents to obtain a total of 6 fractions Fr. N1~N6. The 70% methanol-water elution fraction Fr.N5 is subjected to medium-pressure ODS column chromatography. At the detection wavelengths of 210 nm and 254 nm, it is gradient eluted with 10% methanol-water, 30% methanol-water, 40% methanol-water, 60% methanol-water and 70% methanol-water in sequence, and the above gradient eluents are concentrated to obtain a total of 5 fractions Fr. N5-1~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. The same fractions were combined by TLC and HPLC analysis to obtain a total of 21 fractions Fr. N5-4-1 to N5-4-21. The isocratic elution fraction Fr. N5-4-19 with a retention time of 56.2 to 62.5 min was subjected to semi-preparative high performance liquid chromatography and isocratically eluted with 50% methanol-water at detection wavelengths of 210 nm and 254 nm to obtain the caffeic acid hemiterpenoid ester with a retention time of 12 to 16 min.

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