A lignan compound with anti-inflammatory activity, preparation method and application thereof

Abstract

The present invention relates to a lignan compound with anti-inflammatory activity, a preparation method and an application thereof, and relates to the field of medicine. The compound has a structural formula as shown in formula (I) or a pharmaceutically acceptable salt of the compound has a structural formula as shown in formula (I): the lignan compound provided by the present invention has been confirmed by in vitro pharmacological experiments that when its concentration is 10 μM, the cell proliferation rate is 120%, and the NO generation amount is 6.7299, indicating that it has good anti-inflammatory activity. Therefore, the lignan compound, its derivative, or its pharmaceutically acceptable salt can be used in anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a lignan compound with anti-inflammatory activity, a preparation method and application thereof. Background Art

[0002] Inflammation is a defensive response of the body to external irritants and injuries, manifested as redness, swelling, heat, pain and dysfunction. Short-term inflammation helps to remove inflammatory substances and promote recovery, while excessive or long-term inflammation can lead to diseases such as cancer, arthritis, and sepsis, which seriously threaten human health. The treatment drugs for inflammatory diseases mainly include steroidal and non-steroidal anti-inflammatory drugs. These drugs currently used have more or less side effects. For example, non-steroidal anti-inflammatory drugs cause changes in the hematopoietic system, cardiovascular damage, asthma, etc., which have caused serious harm to human health. Therefore, finding safe and efficient anti-inflammatory drugs from natural products is of great significance to protecting human life and health.

[0003] Lignans are a class of natural compounds formed by the polymerization of two molecules of phenylpropanoid derivatives (i.e. C6-C3 monomers). Most of them are in a free state, and a few are combined with sugar to form glycosides and exist in the wood and resin of plants, hence the name. Modern pharmacological research shows that lignans have great potential in the treatment of inflammatory diseases.

[0004] Castanopsis chinensis, also known as Guilin Castanopsis, Castanopsis chinensis, and small chestnut, is a plant of the genus Castanopsis in the Fagaceae family. It is widely distributed in Guangdong, Guangxi, southwest Guizhou, and southeast Yunnan. It is mainly used for the treatment of diarrhea, hemostasis, and gastroenteritis and other diseases among the people. At present, there is no report on extracting a new lignan component, Chinensin C, with anti-inflammatory activity from Castanopsis chinensis. In view of this, the present invention provides a lignan compound with anti-inflammatory activity, a preparation method, and an application thereof. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a lignan compound with anti-inflammatory activity, a preparation method and application thereof.

[0006] In order to solve the above technical problems, the first object of the present invention is to provide a lignan compound with anti-inflammatory activity, which is a compound with a structural formula as shown in formula (I) or a pharmaceutically acceptable salt of the compound with a structural formula as shown in formula (I):

[0007]

[0008] Among them, the new lignan component with anti-inflammatory activity provided by the present invention is isolated from the leaves of Castanopsis chinensis for the first time, and its molecular formula is C 38 H 37O 10 The novel lignan compound can be named as Chinensin C. The pharmaceutically acceptable salt is selected from the salts of the novel lignan and organic or inorganic acids. It can also be a pharmaceutically acceptable esterified derivative of the lignan compound of the present invention.

[0009] The beneficial effect of the present invention is that the lignan compound Chinensin C provided by the present invention has been confirmed by in vitro pharmacological experiments that when its concentration is 10 μM, the cell proliferation rate is 120%, and the NO generation amount is 6.7299, indicating that it has good anti-inflammatory activity. Therefore, the lignan compound Chinensin C, or its derivatives, or its pharmaceutically acceptable salts can be used in anti-inflammatory drugs.

[0010] Based on the above technical solution, the present invention can also be improved as follows.

[0011] The second object is to provide a method for preparing a lignan compound having anti-inflammatory activity, comprising the following steps:

[0012] (1) taking cone leaves, crushing the cone leaves, adding an aqueous solution containing an alcohol compound to extract the leaves, and concentrating the extract obtained by the extraction under reduced pressure to obtain a water extract;

[0013] (2) degreasing the water extract with petroleum ether, and concentrating the degreasing solution under reduced pressure to obtain a degreased water extract;

[0014] (3) subjecting the defatted water extract to Sephadex LH-20 gel column chromatography, using a methanol-water mixed solution for gradient elution, and collecting eluate I containing lignan compounds; subjecting the collected eluate I containing lignan compounds to Diaion HP20SS column chromatography, using a methanol-water mixed solution for gradient elution, and again collecting eluate II containing lignan compounds;

[0015] (4) injecting the collected eluate II containing the lignan compound into a reverse HPLC for separation, using a 42% volume fraction methanol aqueous solution for isocratic elution, collecting the eluate with a retention time of 8.93 min of the chromatographic peak, concentrating and drying the eluate with a retention time of 8.93 min to obtain a compound represented by structural formula (I), which is a lignan compound;

[0016]

[0017] The beneficial effects of adopting the above further scheme are: the invention uses cone leaves as raw materials to extract and separate the potent anti-inflammatory component Chinensin C, the preparation method is simple, easy to operate, and the leaves are used as raw materials for extraction, which is rich in resources and has good potential economic benefits, and the new anti-inflammatory component Chinensin C is stable in nature and easy to store. It has good anti-inflammatory activity and is expected to be used as a lead compound to develop new inflammatory therapeutic drugs.

[0018] Furthermore, the alcohol compound in the aqueous solution containing alcohol compounds in step (1) includes any one of ethanol, methanol, and propanol or a mixture of at least two thereof, and the volume ratio of the alcohol compound to water in the aqueous solution containing alcohol compounds is (70-90): (30-10);

[0019] Step (1) specifically comprises: taking cone leaves, crushing the cone leaves, adding an aqueous solution containing alcohol compounds for leaching, wherein the ratio of the volume of the aqueous solution containing alcohol compounds to the weight of the cone leaves is 3 to 8 L / Kg, filtering, and again leaching the filtered residue, wherein the number of leaching times is 2 to 5 times, and the time of each leaching is 2 to 4 days, and the extract obtained by leaching is concentrated under reduced pressure at 45 to 70° C. to completely separate the alcohol compounds therein, thereby obtaining a water extract.

[0020] Furthermore, step (2) is specifically as follows: the water extract is degreased using petroleum ether, the volume ratio of the water extract to petroleum ether is 0.5 to 1, the number of degreasing is 2 to 4 times, and after degreasing, the water extract is concentrated under reduced pressure at 30 to 80° C. to obtain the degreased water extract.

[0021] Furthermore, step (3) is specifically as follows: subjecting the defatted water extract to Sephadex LH-20 gel column chromatography, using a methanol-water mixed solution with a volume ratio of 0 to 100% for gradient elution in sequence, and collecting the eluate I containing the lignan compounds; subjecting the collected eluate I containing the lignan compounds to Diaion HP20SS column chromatography, using a methanol-water mixed solution with a volume ratio of 0 to 100% for gradient elution in sequence, and again collecting the eluate II containing the lignan compounds.

[0022] Furthermore, in step (4), the parameters for separation by high performance liquid chromatography are: Matena Dimensions C18 chromatographic column, the particle size of the filler of the Matena Dimensions C18 chromatographic column is 5 μm, and the column temperature of the Matena Dimensions C18 chromatographic column is 40°C.

[0023] The third object is to provide a pharmaceutical composition comprising the lignan compound or pharmaceutically acceptable salt as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0024] The actual dosage of the active ingredient (lignan compound or pharmaceutically acceptable salt of the present invention) in the pharmaceutical composition should be determined according to a variety of relevant factors, including the severity of the disease to be treated, the route of administration, the age, sex, and weight of the patient, and therefore, the above dosage should not limit the scope of protection of the present invention in any way. The pharmaceutically acceptable carrier and / or excipient include pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, which depend on the mode of administration and the designed dosage form.

[0025] Furthermore, the dosage form of the pharmaceutical composition includes tablets, capsules, granules, oral liquids, buccal tablets, suspensions, suppositories, injections, powder injections, pills, sustained-release preparations or controlled-release preparations.

[0026] The pharmaceutical composition may be in any pharmaceutically acceptable dosage form. The suitable dosage of the pharmaceutical composition may be prescribed in a variety of ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, morbidity, diet, administration time, administration route, and reaction sensitivity. A skilled physician can usually easily determine the prescription and the dosage that is effective for the desired treatment.

[0027] The fourth purpose is to provide a use of a pharmaceutical composition, wherein the pharmaceutical composition as described above is used to prepare a drug with anti-inflammatory activity.

[0028] The fifth purpose is to provide an application of a lignan compound having anti-inflammatory activity, wherein the lignan compound or a pharmaceutically acceptable salt as described above is used to prepare a drug having anti-inflammatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The compound Chinensin C of the present invention 1 H-NMR (500MHz, CD3OD) spectrum;

[0030] Figure 2 The compound Chinensin C of the present invention 13 C-NMR (125MHz, CD3OD) spectrum;

[0031] Figure 3 is the HSQC spectrum of the compound Chinensin C of the present invention;

[0032] Figure 4 is the HMBC spectrum of the compound Chinensin C of the present invention;

[0033] Figure 5 It is the compound Chinensin C 1 H- 1 H COSY spectrum;

[0034] Figure 6 is the HREIMS spectrum of the compound Chinensin C of the present invention;

[0035] Figure 7 This is the ECD diagram of the compound Chinensin C of the present invention. DETAILED DESCRIPTION

[0036] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0037] Example 1: Preparation and identification of its structure

[0038] 1. A method for preparing a lignan compound with anti-inflammatory activity

[0039] The Chinensin C of the present invention is a new lignan compound extracted, separated and purified from the leaves of Castanopsis chinensis, and the separation and purification process is as follows:

[0040] (1) 7.5 kg of dried cone leaves were crushed and then soaked in 40 L of 80% methanol for 3 times, each time for 3 days, and filtered. The filtrates were combined and concentrated under reduced pressure to obtain 2.1 L of water extract.

[0041] (2) The water extract obtained in step (1) was degreased three times with petroleum ether (3.0 L), and the degreased water extract was concentrated under reduced pressure to remove the petroleum ether to obtain a degreased water extract;

[0042] (3) The defatted water extract was separated by Sephadex LH-20 gel column (9.5cm×32.0cm) chromatography, and gradient eluted with methanol: water (0%, 20%, 40%, 60%, 80%, 100%, 3L each), and the same components were combined by TLC detection to obtain 9 fractions (Fr.1-9). Among them, Fr.7 (28.0g) was further separated by Diaion HP20SS (5.5cm×30.0cm) column chromatography, and gradient eluted with methanol: water (0%, 20%, 40%, 60%, 80%, 100%, 1.5L each), and the same components were combined by TLC detection to obtain 12 fractions (Fr.7.1-7.12); the eluate II containing lignan compounds was collected (Fr.7.11 (2.4g));

[0043] (4) The collected eluate II (Fr.7.11 (2.4 g)) containing lignan compounds was subjected to reverse HPLC with 42% methanol aqueous solution as the mobile phase, and the eluate with a chromatographic peak retention time of 8.93 min was collected. The eluate with a retention time of 8.93 min was concentrated and dried to obtain a compound represented by structural formula (1), namely, the neolignan compound Chinensin C (30 mg).

[0044] 2. Identification of Chinensin C:

[0045] in, Figure 1 The compound Chinensin C of the present invention 1 H-NMR (500MHz, CD3OD) spectrum; Figure 2 The compound Chinensin C of the present invention 13 C-NMR (125MHz, CD3OD) spectrum; Figure 3 is the HSQC spectrum of the compound Chinensin C of the present invention; Figure 4 is the HMBC spectrum of the compound Chinensin C of the present invention; Figure 5 It is the compound Chinensin C 1 H- 1 HCOSY spectrum; Figure 6 is the HREIMS spectrum of the compound Chinensin C of the present invention; Figure 7 This is the ECD diagram of the compound ChinensinC of the present invention.

[0046] The lignan compound Chinensin C of the present invention is a white amorphous powder, with bright spots under 254nm ultraviolet light, and reacts with 1% FeCl3-EtOH color developer to form brown spots. HREIMS spectrum ( Figure 6 ) shows that its quasi-molecular ion peak is m / z: 653.2329 [MH] - (Calculated value 653.2392, C 38 H 37 O 10 ), indicating that the molecular formula is C 38 H 38 O 10 , the unsaturation is 20. 1 H-NMR (500MHz, CD3OD), 13 The data of C-NMR (125 MHz, CD3OD) are shown in Table 1 below.

[0047] Table 1 NMR data of compound Chinensin C (in CD3OD)

[0048]

[0049]

[0050] Based on the comprehensive analysis of the above mass spectrometry, one-dimensional, two-dimensional NMR and ECD calculation spectral data, the structural formula of the compound was deduced as shown in formula (I), and named Chinensin C:

[0051]

[0052] Example 2: Activity Detection

[0053] 1. Experimental methods

[0054] 1.1 Cell culture and passaging

[0055] Cell culture and passage: RAW264.7 (purchased from China Center for Type Culture Collection) was cultured in a culture medium prepared with 10% fetal bovine serum, 100U / mL penicillin and streptomycin and DMEM stock solution, and placed in an incubator at 37°C and 5% CO2 for incubation and culture. The culture medium was changed every 24 hours, and the cell state was observed under a microscope. When the cells in the culture dish grow to about 80% of the total volume, the cells can be passaged. Because RAW264.7 cells are semi-adherent cells, they do not need to be digested with trypsin. After discarding the cell culture supernatant, wash the old culture medium with PBS buffer, then add fresh culture medium and separate the cells from the bottom of the culture dish by blowing to obtain a cell suspension. Finally, at an appropriate density, transfer the cells to a new culture dish for culture.

[0056] 1.2 CCK-8 method for the determination of monomeric compounds in RAW264.7 cell proliferation assay

[0057] CCK-8 method for determination of RAW264.7 cell proliferation test of monomeric compounds: the experimental groups were set as drug group, control group, and blank group. The drug-treated groups were 2.5, 5, 10, 20, 40, and 80 μM of the compound Chinensin C prepared in the example, and 3 replicates were set for each group. The cell density was adjusted to 2×10 4 / well. In a 96-well cell culture plate, culture medium and LPS were plated, incubated for 24 hours, and different concentrations of the compound prepared in Example 1 were added. After incubation for 48 hours, 10 μL of cell culture medium containing 10 μL of CCK-8 reagent was added per 100 μL, and after further incubation for 1 hour, the wavelength of the microplate reader was set to 450 nm to measure the OD value.

[0058] Cell proliferation rate (%) = [(O administration - O blank) / (O control - O blank)] × 100%

[0059] Wherein, O administration: sample + cell culture medium + CCK-8; O control: cell culture medium + CCK-8; O blank: sample + CCK-8.

[0060] 1.3 Griess method to detect the effect of monomeric compounds on NO release from macrophages

[0061] Griess method was used to detect the effect of monomer compounds on NO release in macrophages: the experimental groups were set as the drug group (sample + LPS + RAW264.7 cells), the LPS model group (LPS + RAW264.7 cells), and the blank group (RAW264.7 cells). RAW264.7 cells in the logarithmic growth phase were taken and 2×10 4 The cells were inoculated in a 96-well plate at a density of 100 / well. After the cells were placed in a CO2 incubator and adhered to the wall, the cell culture supernatant was discarded and samples of various concentrations containing 1μg / mL LPS (lipopolysaccharide) (2.5, 5, 10, 20, 40, 80μM of the compound Chinensin C prepared in the example) were added. After 24 hours of culture, 50μL of the cell culture supernatant was taken, and then 50μL of Griess ReagentⅠ and Griess ReagentⅡ solutions restored to room temperature were added to the 96-well plate. At the same time, different concentrations of NaNO2 solutions (0μM, 1μM, 2μM, 5μM, 10μM, 20μM, 40μM, 60μM, 100μM) were prepared with complete cell culture medium as standard solutions. 50μL of standard solutions of different concentrations were taken in a 96-well plate and equal volumes of Griess reagent were added in sequence. The OD value was measured at a wavelength of 540nm using an enzyme marker. Draw a standard curve with NaNO2 solution concentration as the horizontal axis and absorbance as the vertical axis. Calculate the NO2 in the sample solution according to the standard curve. - The experimental results are expressed as the amount of NO generated.

[0062] 2. Activity Results

[0063] LPS-induced NO production in macrophages RAW264.7: At a concentration of 10μM, the cell proliferation rate of Chinensin C was 120%, and the NO production was 6.7299, at which time the cell proliferation rate was the highest. The results showed that Chinensin C had a good anti-inflammatory effect. As the only active ingredient, it significantly reduced LPS-induced NO release from RAW264.7 cells, and can be effectively used to prepare anti-inflammatory drugs, opening up a new path for anti-inflammatory drugs and a new use of cone plants, with significant economic and social benefits.

[0064] In summary, the present invention uses cone leaves as raw materials to extract and separate the potent anti-inflammatory ingredient Chinensin C, the preparation method is simple, easy to operate, and the leaves are used as raw materials for extraction, which is rich in resources and has good potential economic benefits. The new anti-inflammatory ingredient Chinensin C is stable in nature and easy to store. It has good anti-inflammatory activity and is expected to be used as a lead compound to develop new inflammatory therapeutic drugs.

[0065] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0066] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A lignan compound having anti-inflammatory activity, characterized in that: It is a compound represented by the structural formula (I) or a pharmaceutically acceptable salt of the compound represented by the structural formula (I): 。 2. A method for preparing a lignan compound with anti-inflammatory activity according to claim 1, characterized in that: The steps include: (1) taking cone leaves, crushing the leaves, adding an aqueous solution containing an alcohol compound to extract the leaves, and concentrating the extract obtained by the extraction under reduced pressure to obtain a water extract; (2) degreasing the water extract with petroleum ether, and concentrating the degreasing solution under reduced pressure to obtain a degreased water extract; (3) subjecting the defatted water extract to Sephadex LH-20 gel column chromatography, using a methanol-water mixed solution for gradient elution, and collecting eluate I containing lignan compounds; subjecting the collected eluate I containing lignan compounds to Diaion HP20SS column chromatography, using a methanol-water mixed solution for gradient elution, and again collecting eluate II containing lignan compounds; (4) injecting the collected eluate II containing the lignan compound into a reverse HPLC for separation, using a 42% volume fraction methanol aqueous solution for isocratic elution, collecting the eluate with a retention time of 8.93 min of the chromatographic peak, concentrating and drying the eluate with a retention time of 8.93 min, and obtaining a compound represented by structural formula (I), which is the lignan compound; 。 3. The method for preparing a lignan compound with anti-inflammatory activity according to claim 2, characterized in that: The alcohol compound in the aqueous solution containing alcohol compounds in step (1) comprises any one of ethanol, methanol, and propanol or a mixture of at least two thereof, and the volume ratio of the alcohol compound to water in the aqueous solution containing alcohol compounds is (70-90): (30-10); Step (1) specifically comprises: taking cone leaves, crushing the cone leaves, adding an aqueous solution containing an alcohol compound to extract, wherein the ratio of the volume of the aqueous solution containing the alcohol compound to the weight of the cone leaves is 3-8 L / Kg, the number of extractions is 2-5 times, and the time of each extraction is 2-4 days, and the extract obtained by extraction is concentrated under reduced pressure at 45-70° C. to obtain a water extract.

4. The method for preparing a lignan compound with anti-inflammatory activity according to claim 2, characterized in that: Step (2) specifically comprises: degreasing the water extract with petroleum ether, wherein the volume ratio of the water extract to the petroleum ether is 0.5-1, the degreasing times are 2-4 times, and after degreasing, the water extract is concentrated under reduced pressure at 30-80° C. to obtain the degreased water extract.

5. The method for preparing a lignan compound with anti-inflammatory activity according to claim 2, characterized in that: Step (3) is specifically as follows: subjecting the defatted water extract to Sephadex LH-20 gel column chromatography, using a methanol-water mixed solution with a volume ratio of 0-100% to perform gradient elution in sequence, and collecting eluate I containing lignan compounds; subjecting the collected eluate I containing lignan compounds to Diaion HP20SS column chromatography, using a methanol-water mixed solution with a volume ratio of 0-100% to perform gradient elution in sequence, and again collecting eluate II containing lignan compounds.

6. The method for preparing a lignan compound with anti-inflammatory activity according to claim 2, characterized in that: In step (4), the parameters for separation by high performance liquid chromatography are: Matena Dimensions C18 chromatographic column, the particle size of the filler of the Matena Dimensions C18 chromatographic column is 5 μm, and the column temperature of the Matena Dimensions C18 chromatographic column is 40°C.

7. A pharmaceutical composition, characterized in that It comprises the lignan compound as claimed in claim 1, and a pharmaceutically acceptable carrier and / or excipient.

8. A pharmaceutical composition according to claim 7, characterized in that: The dosage forms of the pharmaceutical composition include tablets, capsules, granules, oral liquids, suspensions, suppositories, injections, powder injections, dropping pills, sustained-release preparations or controlled-release preparations.

9. Use of the pharmaceutical composition according to claim 7 or 8 in the preparation of a drug having anti-inflammatory activity.

10. Use of the lignan compound according to claim 1 in the preparation of a drug with anti-inflammatory activity.

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