CN108358878B - Isoflavone compound in ficus auriculata as well as preparation method and application thereof - Google Patents

Isoflavone compound in ficus auriculata as well as preparation method and application thereof Download PDF

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CN108358878B
CN108358878B CN201810022626.0A CN201810022626A CN108358878B CN 108358878 B CN108358878 B CN 108358878B CN 201810022626 A CN201810022626 A CN 201810022626A CN 108358878 B CN108358878 B CN 108358878B
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isoflavone
ethyl acetate
ficus
isoflavone compound
compound
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CN108358878A (en
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韩长日
邵泰明
宋小平
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Hainan Normal University
Hainan Institute of Science and Technology
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Hainan Institute of Science and Technology
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    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
    • C07D311/36—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only not hydrogenated in the hetero ring, e.g. isoflavones
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    • C07—ORGANIC CHEMISTRY
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    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
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Abstract

The invention discloses a preparation method and application of a novel isoflavone compound in ficus auriculata, wherein the novel isoflavone compound is prepared by the following steps: crushing ficus microcarpa root, sieving, cold soaking and extracting with alcohol solution, vacuum concentrating to obtain extract, adding distilled water into the extract for suspension, and sequentially extracting with petroleum ether and ethyl acetate to obtain extracts of various parts; and carrying out column chromatography and high performance liquid chromatography on the ethyl acetate extract to obtain the new isoflavone compound. The isoflavone new compound extracted from ficus auriculata has good effect of inhibiting the proliferation of three tumor cells.

Description

Isoflavone compound in ficus auriculata as well as preparation method and application thereof
Technical Field
The invention relates to a natural plant extract, in particular to a novel isoflavone compound in ficus auriculata, a preparation method and application thereof.
Background
Ficus auriculata (A. Mey.) (Ficus auriculatalour is Ficus of Moraceae (genus Ficus)Ficus) The plant, named as steamed bun fruit, is sweet and edible in fruit flavor, is mainly distributed in tropical and subtropical regions, is mainly distributed in Hainan, Guangxi, Yunnan, Guizhou, Sichuan and other places in China, and the Chinese patent records that the fruits of Ficus benjamina have the effects of dispelling wind, ventilating the lung, tonifying the kidney and benefiting the essence, and are mainly used for treating cough due to lung heat, spermatorrhea and hematemesis. In recent years, research on chemical components and biological activity of ficus auriculata shows that the ficus auriculata contains components such as isoflavone and the like and has better biological activity. Qicui et al (A new isovalvone from the roots ofFicus auriculata[J]Natural Product Research, 2018, 32(1): 43) isolated 4 isoflavonoids with antibacterial activity from Ficus auriculata root. Antioxidant activity and antioxidant activity of alcohol extract of Ficus microcarpa fruitαGlucosidase and acetylcholinesterase inhibitory Activity [ J]Food science 2016.37 (13): 77) for the content of polyphenol in ficus microcarpa fruit and its antioxidation,αThe research on the inhibitory activity of glucosidase and acetylcholinesterase shows that ficus microcarpa fruit polyphenol has good oxidation activity,αGlucosidase and a certain acetylcholinesterase inhibitory activity. At present, the systematic research of the ficus auriculata isoflavone components is lacked, and the research of the antitumor activity of the ficus auriculata isoflavone components is yet to be further researched.
Disclosure of Invention
The invention aims to solve the problems and provides a novel isoflavone compound with anti-tumor activity in ficus auriculata, and a preparation method and application thereof.
The technical scheme for realizing the purpose of the invention is as follows: a new isoflavone compound has a structure shown in formula (I):
Figure 88604DEST_PATH_IMAGE002
（Ⅰ）。
the preparation method of the isoflavone new compound comprises the following steps:
① naturally drying Ficus benjamina tissue in the shade, pulverizing, extracting with alcoholic solution, concentrating to obtain extract, suspending with distilled water, and extracting with petroleum ether and ethyl acetate to obtain petroleum ether and ethyl acetate fractions;
② subjecting the ethyl acetate part obtained in step ① to silica gel column chromatography to obtain 7 components A-G, subjecting component C to gradient elution with petroleum ether-ethyl acetate system, and mixing similar fractions to obtain 5 components Fr 1-5;
③ the Fr3 from step ② is separated by high performance liquid chromatography to obtain isoflavone noval compound.
The ficus auriculata tissues in the step ① are roots, stems, leaves or fruits of ficus auriculata, the alcohol solution is 75-95% of alcohol solution in volume fraction, and the volume ratio of the distilled water to the extractum is 1: 1.
In the step ②, the column chromatography eluent is petroleum ether-ethyl acetate at a volume ratio of 100:0-0: 100.
The high performance liquid solvent system described in step ③ above is 45% methanol to 55% water.
The isoflavone new compound is applied to inhibiting tumor cells, wherein the tumor cells are cervical cancer Hela cells, breast cancer MCF-7 cells or lung adenocarcinoma A549 cells.
The invention has the following positive effects: provides a novel isoflavone compound with anti-tumor activity in ficus auriculata and a preparation method thereof.
Drawings
FIG. 1 shows isoflavone compounds of the present inventionIs/are as follows1H-NMR spectrum (hydrogen nuclear magnetic resonance spectrum).
FIG. 2 shows the preparation of isoflavone compound of the present invention13C-NMR spectrum (nuclear magnetic resonance carbon spectrum) chart.
FIG. 3 is an HSQC spectrum of the isoflavone compound of the present invention.
FIG. 4 is an HMBC spectrum of the isoflavone compound of the present invention.
FIG. 5 shows isoflavone compounds of the present invention1H-1H COSY spectrum (hydrogen-hydrogen correlation spectrum) diagram.
FIG. 6 is a high resolution electrospray ionization mass spectrometry (HRESIMS) spectrum of the isoflavone compound of the present invention.
Detailed Description
The preparation process of the present invention is further illustrated with reference to the following specific examples, but the scope of the present invention is not limited thereto.
(examples)
The structural formula of the isoflavone compound of the embodiment is shown as the formula (I):
Figure DEST_PATH_IMAGE003
（Ⅰ）。
the preparation method of the isoflavone compound comprises the following steps:
① pulverizing and sieving 20kg of naturally dried Ficus microcarpa root, soaking and extracting with 95% ethanol at room temperature for 3 times (6 days each time), concentrating under reduced pressure to recover ethanol to obtain total extract 460g, adding equal volume of distilled water suspension extract, extracting with 3L petroleum ether for 3 times to obtain petroleum ether extract 175g, and extracting with 3L ethyl acetate for several times until the color of the upper layer solution is obviously lightened to obtain ethyl acetate 70 g.
② gradient-eluting 70G of ethyl acetate part in step ① with petroleum ether-ethyl acetate (100: 0-0:100, V/V), collecting fractions per 500mL, detecting with TCL dot plate, mixing similar components to obtain 7 components A-G, subjecting component C to petroleum ether-ethyl acetate (3:1, V/V) column chromatography, detecting with TCL dot plate, mixing similar components to obtain 5 components Fr 1-5;
③ Fr3 of step ② was purified by high performance liquid chromatography (methanol/water, 45:55, 3 mL/min, t)R=17.6 min) to obtain 5.6 mg of isoflavone new compound.
(test example)
Product analysis of the isoflavones obtained in the examples:
(1) of the isoflavone compound1The H-NMR spectrum is shown in FIG. 1 (solvent acetone-d 6，400Hz）。
(2) Of the isoflavone compound13The C-NMR spectrum is shown in FIG. 2 (solvent acetone-d 6，100MHz）。
The specific data are shown in Table 1.
TABLE 1 NMR data of Ficauisoflapine A (400/100 MHz, acetone-d 6)
Figure 179313DEST_PATH_IMAGE004
(3) The HSQC spectrum of the isoflavone compound is shown in FIG. 3 (solvent acetone-d 6）。
(4) The HMBC spectrum of the isoflavone compound is shown in FIG. 4 (solvent acetone-d 6）。
(5) Of the isoflavone compound1H-1The H COSY spectrum (hydrogen-hydrogen correlation spectrum) is shown in figure 5 (solvent acetone-d 6）。
(6) The high resolution electrospray ionization mass spectrum of the isoflavone compound is shown in FIG. 6.
Isoflavone new compound: light yellow powder, easily soluble in acetone and methanol, m.p. 157-α]25 D+26.5 (c0.1, CH3OH); UV (CH3OH)λ max(log ε) 260 (2.52) nm, 201 (3.56) nm. HRESI-MS gives the compound with the molecular formula C20H18O6（m/z355.11771 [M + H]+Calculated 355.11761), the unsaturation was 12. In the UV spectrum, absorption at 260 nm indicates that the compound contains a benzene ring, suggesting that the compoundMay be flavonoid compounds.1The low field region in the H NMR spectrum gives 1 typical hydroxyl signal of the C-5 site of flavone and intramolecular carbonyl forming hydrogen bondδ H12.99 (1H, s), 1 typical isoflavone C-2 site alkene hydrogen signalδ H8.20(1H, s), 1C-6 alkenyleneδ H6.28 (1H, d,J= 2.2 Hz), 1C-8 olefinic hydrogenδ H6.42 (1H, d,J= 2.2 Hz), 3 groups of 1,3, 4 substituted phenylcyclohexane signalsδ H7.36 (1H, dd,J= 8.3, 2.2 Hz)、7.30(1H, d,J= 2.2 Hz) and 6.91 (1H, d,J= 8.3 Hz), further proving that the compound is an isoflavonoid compound, and further,1the H NMR spectrum also shows 2 cis-alkene hydrogen signalsδ H6.44 (1H,J= 9.8Hz) and 5.77 (1H,J= 9.8Hz), 2 identical methyl signalsδ H1.43 (6H, s)；13C NMR spectrum of the product gives 1 tetra-substituted benzene ring: (δ C165.5, 163.9, 159.1, 106.1, 100.0, 94.6), 1,3, 4-substituted benzene ring ((B)δ C154.0, 130.6,128.0, 123.8, 122.0, 116.8), 1α,βUnsaturated ketone (C)δ C181.5, 154.5, 124.5), which corresponds exactly to the 15 carbon skeleton of isoflavones, and furthermore,13the C NMR spectrum also shows 2 olefinic carbon signals (δ C132.0, 122.8), 2 identical methyl carbon signals: (δ C28.3). The above information suggests that the compound is an isoflavone compound substituted at the C-3' position. The mode of linkage of the cis double bond is determined by HMBC, in the HMBC spectrumδ H6.44 (1H,J= 9.8Hz) and 5.77 (1H,J= 9.8Hz) are all in contact withδ C122.0 (C-3') indicates a double bond at the C-3' position, 2 methyl groupsδ H1.43 (6H, s) are all in contact withδ C132.0 (C-2'') and 77.2 (C-3''), indicating that 2 methyl groups are attached to C-3 ''. In conclusion, the compound is a novel isoflavone compound and is named as: ficauseoflavone A.
Figure DEST_PATH_IMAGE005
Structural formula of Ficauisoflavanone A
Figure DEST_PATH_IMAGE007
HMBC correlation of Ficauisoflavone A
(test examples, evaluation of in vitro antitumor Activity)
The test method comprises the following steps: the isoflavone compound separated in the example was used for the antitumor activity study.
Tumor cell lines: hela cells of cervical cancer, MCF-7 cells of breast cancer and A549 cells of lung adenocarcinoma (provided by college of pharmacy of Hebei university).
Positive control: doxorubicin.
The instrument comprises the following steps: refrigerator, sterilizer, microplate reader (BioTek ELx 800), microscope, freeze dryer, CO2A constant temperature culture box, a 96-pore plate, a pipettor, an electric heating constant temperature blast drying box, a micro-filter, a 0.22 mu m filter membrane and a high-speed centrifuge.
Reagent: RPMI1640 culture medium, pancreatin, MTT, DMSO and high-grade newborn bovine serum.
The specific experimental method comprises the following steps:
(1) inoculating cells: single cell suspensions were prepared in medium (DMEM) containing 10% fetal bovine serum at 5X 10 per well4、5×105Each/mL was inoculated into a 96-well plate at 100. mu.L per well volume, and adherent cells were inoculated for culture 12 hours in advance.
(2) A solution of a test compound (a primary screen with a fixed concentration of 40 mug/mL, 5 concentrations of the compound which inhibits the growth of tumor cells to be around 50% are set to enter a gradient secondary screen, namely, the detection concentrations are 5, 1, 0.2, 0.04 and 0.008 mug/mL respectively, and a test sample is diluted by a culture solution) is added into the solution, 100 mug is obtained, the final volume of each well is 100 mug, and 3 secondary wells are arranged in each treatment.
(3) Color development: after 48 hours incubation at 37 degrees Celsius, 20. mu.L of MTT solution was added to each well. Incubation was continued for 4 hours, the culture was terminated, and the culture supernatant in each well was aspirated off, and 150. mu.L of DMSO solution was added to each well to allow the crystals to be fully thawed.
(4) Color comparison: the 490nm wavelength was selected and each read by a microplate reader (BioTek ELx 800)Pore light absorption values and the results are recorded. Calculating IC of Compounds50The value is obtained.
And (3) data analysis:
calculating IC by using the cell inhibition rate as ordinate and the logarithm of the concentration as abscissa50The value is obtained. Wherein, the inhibition rate = (add the OD value of PBS control-OD value of test group)/add the OD value of PBS control × 100%.
The results of the test of the antitumor activity of the compound are as follows:
TABLE 2 isoflavone compound inhibition of tumor cell proliferation results
Figure DEST_PATH_IMAGE009
AdriamycinaIs a positive control.
The results show that the isoflavone compound has better function of inhibiting the proliferation of three tumor cells.

Claims (6)

1. An isoflavone compound having the structure shown in formula (I):
Figure DEST_PATH_IMAGE002
（Ⅰ）。
2. the process for preparing isoflavone compound according to claim 1, which comprises the steps of:
① naturally drying Ficus benjamina tissue in the shade, pulverizing, extracting with alcoholic solution, concentrating to obtain extract, suspending with distilled water, and extracting with petroleum ether and ethyl acetate to obtain petroleum ether and ethyl acetate fractions;
② subjecting the ethyl acetate part obtained in step ① to silica gel column chromatography to obtain 7 components A-G, subjecting component C to gradient elution with petroleum ether-ethyl acetate system, and mixing similar fractions to obtain 5 components Fr 1-5;
③ the obtained Fr3 from step ② is separated by high performance liquid chromatography to obtain isoflavone compound.
3. The method for preparing isoflavone compounds as claimed in claim 2, wherein the ficus auriculata tissue in the step ① is roots, stems, leaves or fruits of ficus auriculata, the alcohol solution is 75-95% of alcohol solution by volume fraction, and the volume ratio of the distilled water to the extract is 1: 1.
4. The process for preparing isoflavone compound according to claim 2, wherein the eluent for column chromatography in ② is petroleum ether-ethyl acetate at a volume ratio of 100:0-0: 100.
5. The process of claim 2, wherein the high performance liquid solvent system in step ③ is 45% methanol-55% water.
6. The use of the isoflavone compound of claim 1 in the preparation of a medicament for inhibiting tumor cells, wherein the tumor cells are cervical cancer Hela cells, breast cancer MCF-7 cells or lung adenocarcinoma A549 cells.
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