A compound extracted and separated from Rhizoma Dioscoreae and its preparation method and use

Abstract

The present invention provides a compound extracted and isolated from the herb Rhizoma Coptidis, with a structure shown in Formula I. It is a new coumarin compound that has the activity of inhibiting the growth of lung cancer cells, and provides a new strategy for the further development of the herb Rhizoma Coptidis and the further research and development of cancer therapeutics. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of separation and purification of active ingredients of traditional Chinese medicines, and particularly relates to a compound extracted and separated from Rhizoma Dioscoreae, and a preparation method and use thereof. Background Art

[0002] Wikstroemia indica (Linn. CA Meyer) is a plant of the genus Wikstroemia Endl., Thymelaeaceae, also known as Nanling Wikstroemia, ground cotton bark, mountain cotton bark, etc. It is a commonly used folk Chinese medicine. It tastes bitter, slightly spicy, and is cold in nature. It enters the lung and liver meridians.

[0003] At present, it has been identified that Kingia gewang contains more than 120 compounds including lignin, flavonoids, coumarins, steroids, volatile oils, acids, esters, alcohols and terpenes. It has a wide range of pharmacological effects, including antibacterial, antiviral, cytotoxic, anti-inflammatory, and tumor cell growth inhibition. It is clinically used to treat respiratory diseases, and is also used to treat a series of diseases such as neuralgia, chronic hepatitis, cirrhosis, and gynecological inflammation.

[0004] Among the many active ingredients in Wikstroemia indica, the anti-tumor effects of flavonoids, coumarins, volatile oils, unsaturated fatty acids, terpenes, steroids and other ingredients have been reported. For example, Shao et al. have shown that a new type of chalcone-flavonoid combined biflavonoid component 3'-hydroxydaphnodorin A isolated from Wikstroemia indica root has obvious cytotoxic activity against human liver cancer HepG2 and human nasopharyngeal cancer CNE2 cells (Shao Meng et al. A new cytotoxic biflavonoid from the rhizome of Wikstroemia indica. [J]. Natural product research, 2016, 30 (12): 1417-22.); For example, as a major chemical component of Wikstroemia indica, a biflavonoid: daphnodorin, is also a hot topic in the study of anti-tumor pharmacological effects. Yang Zhenyu et al. observed that different concentrations of daphnodorin had a significant effect on in vitro cultured human lung adenocarcinoma cells AGZY-83-a, human laryngeal cancer cells Hep2 and human liver cancer cells HepG 2 have obvious inhibitory effects, and are concentration-dependent (Yang Zhenyu, Guo Wei, Wu Dongyuan, Du Zhimin. Extraction and separation of daphneline from Rhizoma leucoderma and its anti-tumor effect [J]. Natural Product Research and Development, 2008(03):522-526.DOI:10.16333 / j.1001-6880.2008.03.039.); the research results of Gu et al. also showed that daphneline can block the proliferation of human osteosarcoma HOS cells (Gu S, He J. Daphnoretin includes cell cycle arrest and apoptosis in humanosteosarcoma HOS cells [J]. Molecules, 2012, 17(01):598-612).

[0005] However, due to the complex chemical composition of Rhizoma Lucidum, the types and efficacy of other active ingredients in Rhizoma Lucidum still need further exploration. Summary of the Invention

[0006] The object of the present invention is to provide a new compound discovered from Rhizoma Dioscoreae.

[0007] The present invention provides a compound represented by formula I or a derivative thereof, or a salt thereof, or an optical isomer thereof, or a tautomer thereof, or a solvate thereof:

[0008]

[0009] Furthermore, the above-mentioned compound or its derivative, or its salt, or its optical isomer, or its tautomer, or its solvate is extracted, separated and purified from King Dalbergia glutinosa.

[0010] The present invention also provides a method for preparing the above-mentioned compound or its derivative, or its salt, or its optical isomer, or its tautomer, or its solvate, comprising the following steps:

[0011] (1) Crush the medicinal material of Rhizoma Cibotii and extract it with ethanol solution, concentrate it, and extract it with ethyl acetate;

[0012] (2) The aqueous phase after ethyl acetate extraction is enriched by macroporous adsorption resin, washed with water, and analyzed; concentrated and filtered to obtain a filtrate;

[0013] (3) The filtrate is separated by high pressure liquid phase preparation to obtain the product.

[0014] Furthermore, the extraction times in step (1) is 2 to 4 times, preferably 3 times.

[0015] Furthermore, the ethanol aqueous solution in step (1) is 75% to 85% v / v ethanol, preferably 80% v / v ethanol.

[0016] Furthermore, the analysis in step (2) is performed using 75% to 85% v / v methanol, preferably 80% v / v methanol.

[0017] Furthermore, the filtration in step (2) is performed by filtering through a 0.45 μm organic membrane.

[0018] Furthermore, the high pressure liquid phase preparative separation chromatography conditions in step (3) are as follows:

[0019] Mobile phase: a mixture of methanol and water, wherein the volume ratio of methanol to water is (45-50):(50-55), preferably 50:50;

[0020] Detection wavelength: 346 nm; chromatographic column: C18 reverse phase column.

[0021] The present invention also provides use of the above-mentioned compound or its derivative, or its salt, or its optical isomer, or its tautomer, or its solvate in the preparation of a drug for treating cancer.

[0022] Furthermore, the above-mentioned medicine is a medicine for treating lung cancer.

[0023] Beneficial effects of the present invention: The present invention provides a new compound discovered from Rhizoma Lucidum, which has the activity of inhibiting the growth of lung cancer cells, and provides a new strategy for the further development of Rhizoma Lucidum medicinal materials and the further research and development of cancer treatment drugs.

[0024] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0025] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The figure is a schematic diagram of the process for isolating and purifying the compound of the present invention from Rhizoma Dioscoreae.

[0027] Figure 2 The compound of the present invention 1 H-NMR results.

[0028] Figure 3 To fill the compound of the present invention 13 C-NMR results.

[0029] Figure 4 This is the HSQC result of the compound of the present invention.

[0030] Figure 5 This is the HMBC result diagram of the compound of the present invention. DETAILED DESCRIPTION

[0031] Unless otherwise specified, the raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0032] Example 1. Preparation of the compound of the present invention

[0033] The compounds of the present invention can be chemically synthesized by those skilled in the art according to conventional techniques in the art, or can be obtained by separation and purification from the medicinal material of Rhizoma Dioscoreae by the following process:

[0034] 5 kg of dried stems and leaves of the plant were crushed, then extracted three times with 10 times the weight of 80% ethanol, heated under reflux, and combined. The extracts were concentrated under reduced pressure until the alcohol was no longer present, yielding 10 L of concentrated extract. The extract was then allowed to stand at room temperature. The extract was then extracted twice with an equal volume of ethyl acetate. The aqueous phase was then enriched with AB-8 macroporous resin and washed until the effluent was clear. Desorption was then performed with 80% methanol to yield a red elution solution. The extract was then concentrated under reduced pressure at 60°C to approximately 50% alcohol content. The filtrate was filtered through a 0.45 μm organic membrane and subjected to high-pressure preparative separation on C18 reverse-phase chromatography using a mobile phase consisting of A:methanol and B:water in a 50:50 (v / v) ratio. The detection wavelength was 346 nm, and the corresponding peaks were collected. The product was concentrated under reduced pressure at 45°C to dryness, yielding 0.15 g of an off-white solid.

[0035] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0036] Experimental Example 1: Characterization of the structure of the compound of the present invention

[0037] The electrospray ionization mass spectrum of the compound showed that the positive ion was 667.70 [M+Na] + , negative ion 643.81[MH] - ; The molecular weight of the compound is 644. The quasi-molecular ion peak given by high-resolution mass spectrometry is: 667.7934[M+Na] + , 643.8015[MH] - ; The calculated value is (C 33 H 24 O 14 +Na)667.7945, (C 33 H 24 O 14 -H) 643.8026, it can be determined that its molecular formula is C 33 H 24 O 14 .

[0038] Combining plant origin, NMR data, and mass spectrometry data, it is preliminarily determined that this compound is a polymeric coumarin. Its molecular weight is identical to that of [8,8′-Bi-2H-1-benzopyran]-2,2′-dione,7′-(α-D-glucopyranosyloxy)-7-hydroxy-3-[(2-oxo-2H-1-benzopyran-7-yl)oxy] (CAS: 924282-19-9), indicating that it is an isomer. Comparison of the carbon spectra revealed that the sugar chemical shifts are essentially identical, indicating that they share the same sugar structural unit. HSQC was used to assign all carbon and hydrogen atoms. HMBC revealed that δH 4.95 (H-1″′) correlated with δC 158.2 (C-7″), indicating that the sugar unit was attached to C-7″. At the same time, δH 6.95 (H-6) correlated with δC 106.8 (C-8) in addition to δC 159.4 (C-7); δH 7.42 (H-6″) correlated with δC 158.2 (C-9″) and 113.8 (C-8″). The structure is as follows:

[0039]

[0040] The attribution and analysis of NMR data are shown in Tables 1 and 2:

[0041] Table 1

[0042]

[0043]

[0044] Table 2

[0045]

[0046]

[0047] Document 1: CHEMICAL CONSTITUENTS OF Edgeworthia chrysantha. Chemistry of Natural Compounds, Vol.45, No.1, 2009.

[0048] Experimental Example 2: The structure of the compound of the present invention inhibits the growth of lung cancer cells

[0049] 1. Experimental methods

[0050] 1.1 Cell Culture: Culture cells using 10% complete medium at 37°C, 5% CO2. Digest with 0.25% trypsin solution for 2 min each. Subculture at a 1:3 ratio.

[0051] 1.2 Drug preparation: Dissolve the compound prepared in Example 1 in DMSO solution to prepare a stock solution with a concentration of 200 μmol / L. Take 1 μL each time and dilute it to 1 ml with serum-free culture medium at a dilution ratio of 1:4.

[0052] 1.3 MTT assay to detect the inhibitory effect of the compounds of the present invention on the growth of A549 cells

[0053] Take A549 cells in the logarithmic growth phase, digest them with 0.25% trypsin solution for 2 minutes, and add complete medium with 10% serum to stop digestion. Place in a centrifuge at 1200r / min and centrifuge for 3 minutes. Resuspend the cell clumps with 5ml of 10% complete medium. Count with a hemocytometer, add 10% complete medium at a concentration of 1×10^4 cells / ml, mix them in layers and pour them into the liquid adding tank, mix them 15 times with a 12-well spray gun and add them to a 96-well plate. Mix them about 10 times before inoculating each row of cells. The entire inoculation process is completed within 2 minutes. After inoculation, place the 96-well plate in a clean bench and let it stand for 5 minutes before transferring it to a CO2 constant temperature incubator and incubate for 24 hours. Discard the original medium in the wells, wash once with PBS, add drugs and blank medium (set up 3 replicates for each group) and place them in a CO2 constant temperature incubator and incubate for 24 hours. Add 20 μL of MTT solution and continue to incubate for more than 4 hours. Use a 1 ml syringe to aspirate the liquid in the wells, add 150 μL of DMSO solution to each well, place in a microplate reader and shake for 5 minutes, and then detect at a wavelength of 490 nm.

[0054] 2. Experimental results

[0055] As shown in Table 3:

[0056] Table 3

[0057]

[0058] Note: ** indicates extremely significant differences.

[0059] It can be seen that the compound of the present invention has a significant inhibitory effect on the growth of lung cancer cells.

[0060] In summary, the present invention provides a new coumarin compound that has the activity of inhibiting the growth of lung cancer cells, and provides a new strategy for the further development of Gewang medicinal materials and the further research and development of cancer therapeutic drugs.

Claims

1. A compound represented by formula I, or a salt thereof, or a tautomer thereof:

2. A method for preparing the compound according to claim 1, or a salt thereof, or a tautomer thereof, characterized in that: The steps include: (1) Crush the medicinal material of Rhizoma Cibotii and extract it with ethanol solution, concentrate it, and extract it with ethyl acetate; (2) The aqueous phase after ethyl acetate extraction is enriched by AB-8 macroporous adsorption resin, washed with water, and analyzed; concentrated and filtered to obtain a filtrate; (3) separating the filtrate by high pressure liquid phase preparation to obtain the product; The chromatographic conditions for the high pressure liquid preparative separation are as follows: Mobile phase: a mixture of methanol and water, with a volume ratio of methanol to water of (45-50):(50-55); Detection wavelength: 346 nm; chromatographic column: C18 reverse phase column.

3. The preparation method according to claim 2, wherein The extraction times in step (1) is 2 to 4 times.

4. The preparation method according to claim 3, wherein The extraction times in step (1) is 3 times.

5. The preparation method according to claim 2, wherein The ethanol aqueous solution in step (1) is 75% to 85% v / v ethanol.

6. The preparation method according to claim 5, wherein The ethanol aqueous solution in step (1) is 80% v / v ethanol.

7. The preparation method according to claim 2, wherein The analysis in step (2) is performed using 75% to 85% v / v methanol.

8. The preparation method according to claim 7, wherein The analysis in step (2) is performed using 80% v / v methanol.

9. The preparation method according to claim 2, wherein The filtration in step (2) is performed by filtering through a 0.45 μm organic membrane.

10. The preparation method according to claim 2, wherein In the chromatographic conditions for the high pressure liquid preparative separation in step (3), the volume ratio of methanol to water is 50:

50.

11. Use of the compound according to claim 1, or a salt thereof, or a tautomer thereof in the preparation of a medicament for treating lung cancer.

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