CN114213428A - Indole alkaloid compound and preparation method and application thereof - Google Patents
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Abstract
The invention discloses an indole alkaloid compound and a preparation method and application thereof, belonging to the technical field of marine fungus active ingredient analysis. The invention extracts and separates 4 indole alkaloid compounds with novel structures from fermentation culture of Aspergillus cf. terreus CXX-158-20, and the structural formulas are shown in formulas (I) to (IV). The indole alkaloid compound has good antitumor activity and good development prospect in the field of pharmacy.
Description
Technical Field
The invention relates to the technical field of marine fungus active ingredient analysis, and particularly relates to an indole alkaloid compound and a preparation method and application thereof.
Background
The marine microorganism is an important biological resource in the sea, participates in the whole process of the decomposition and transformation of marine substances, and has complex and important ecological functions. The marine microorganisms can tolerate the extreme conditions peculiar to the sea, such as high salt, high pressure, low oxygen, low light and the like, and the specificity of the living environment causes the diversity of the marine microorganisms in species, genetic composition and ecological functions.
The unique living environment of the marine microorganisms makes the marine microorganisms have great potential for producing novel bioactive substances, and the screening of medicinal active substances from secondary metabolites of the marine microorganisms becomes a current research hotspot. The marine fungi in the marine microorganisms are rich sources of active secondary metabolites, and have the characteristics of strong vitality, vigorous metabolism and the like, 70-80% of the secondary metabolites of the marine fungi have biological activity, and discovery of natural products with specific structure types by taking the marine fungi as raw materials has important significance for research and development of marine medicaments.
The Aspergillus fungus Aspergillus terreus (Aspergillus cf. terreus) belongs to the deuteromycetes, order chitinales, family californiaceae. At present, the research aiming at the secondary metabolites of the aspergillus terreus mainly focuses on polyketides, terpenoids, grease compounds and the like, and the aspergillus terreus has abundant and full-potential biological activities such as anti-inflammatory, antibacterial and anti-tumor and the like. For example, the compounds 1, 2-dihydrohemic acid and fumagillolic acid which are separated from the endophyte Aspergillus PC-038 secondary metabolite by the rambutan and the like have obvious inhibiting effect on Staphylococcus aureus (Staphylococcus aureus) ATCC 25923, and lay a foundation for the research and development of novel antibiotics [ the research of antibacterial chemical components in the endophyte Aspergillus PC-038 secondary metabolite, chemical reagents, 2021,43(08), 1143-containing 1150 ].
Compounds such as neoaspergillic acid, ferrieospermilin, (2 ' S) -4-methoxy-3- (2 ' -methyl-3 ' hydroxyl) propylhydroxy-methyl benzoate, aflatoxin and the like are separated from metabolites of aspergillus 16-02-1 from deep sea, and the compounds have certain inhibiting effects on human chronic myelocytic leukemia K562 cells, human promyelocytic leukemia HL-60 cells, human cervical carcinoma HeLa cells and human gastric adenocarcinoma BGC-823 cells [ preliminary evaluation of metabolites of aspergillus 16-02-1 from deep sea and antitumor and antifungal activities thereof, Chinese marine drugs, 6 months in 2013, No. 32, No. 3 ].
In recent years, the compounds separated and reported from the aspergillus terreus metabolites are various in types and have better pharmacological activity, but the indole alkaloid compounds with antitumor activity separated from the aspergillus terreus metabolites have not been reported in documents to date.
Disclosure of Invention
The invention aims to extract natural active substances with medicinal value from metabolites of marine aspergillus terreus.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention separates and obtains Aspergillus fungus Aspergillus terreus (Aspergillus cf. terreus) CXX-158-20 from Taiwan ocean sediment, carries on the intensive research to the chemical component and the biological activity of the secondary metabolite produced by the culture in rice solid culture medium, separates and purifies to obtain 4 new monomer indole alkaloid compounds, the structural formula of the compound is selected from the formulas (I) - (IV),
the 4 compounds are indole alkaloid compounds with indole and polysubstituted benzene ring, and the formula of the compound A (compound I) is C24H20N2O5The formula of the compound II is C22H19NO6The formula of the compound III is C25H20N2O6The molecular formula of the compound VI of the tularenine D (compound VI) with the structural formula shown as the formula (IV) is C26H28N2O6。
The invention also provides a preparation method of the indole alkaloid compound, which comprises the following steps:
(1) the preservation number is CCTCC NO: activating M20211214 Aspergillus fungus (Aspergillus cf. terreus) CXX-158-20, inoculating into rice solid culture medium, and statically culturing at 20-30 deg.C for 10-40 days;
(2) after the fermentation culture is finished, mycelium and fermentation liquor are obtained through separation;
(3) adding the mycelium into methanol for leaching, separating to obtain a leaching liquor, concentrating the leaching liquor, suspending the leaching liquor with distilled water to obtain an aqueous suspension, extracting the aqueous suspension with ethyl acetate, and separating and purifying the extract to obtain an indole alkaloid compound;
extracting the fermentation liquor by using ethyl acetate, and separating and purifying the extract liquor to obtain an indole alkaloid compound;
the separation and purification comprises the following steps: performing normal-phase silica gel column chromatography on the extract, performing gradient elution by using petroleum ether/ethyl acetate mixed liquor with the volume ratio of 100:0, 98:2, 95:5, 90:10, 80:20, 70:30, 40:60 and 0:100 in sequence, collecting 80:20 fractions, and recrystallizing by using methanol to obtain a compound I; collecting 70:30 fractions, and recrystallizing with a methanol/ethyl acetate mixed solution with a volume ratio of 3:1 to obtain a compound II;
performing reverse phase silica gel column chromatography on the extract, performing gradient elution by using 30-100% methanol/water solution by volume ratio, and collecting 85-90% fraction to obtain a compound III; collecting 90-95% of fractions to obtain a compound VI;
or carrying out normal phase silica gel column chromatography on the extract, carrying out gradient elution by using a petroleum ether/ethyl acetate mixed solution and ethyl acetate in volume ratios of 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:3 and 1:9 in sequence, and collecting a sub-fraction a eluted from the petroleum ether/ethyl acetate mixed solution in volume ratio of 4:1 and a sub-fraction b eluted from 3: 1; performing preparative high performance liquid chromatography on the sub-fraction a to obtain a compound I and a compound II; and the sub-fraction b is subjected to reverse phase silica gel column chromatography and preparative high performance liquid chromatography to obtain III and a compound VI.
In the step (1), Aspergillus terreus CXX-158-20 is subjected to fermentation culture.
Preferably, the strain activation adopts PDA culture medium, and comprises the following raw materials in volume of 1L: 200g of potato, 20g of glucose, 20g of agar and the balance of H2O, pH is natural.
The fermentation culture adopts a rice solid culture medium, and specifically comprises the following raw materials: 75mL of water was added to 50g of rice, and the pH was adjusted to a natural level.
Preferably, the temperature of the fermentation culture is 22-26 ℃. More preferably, the culture is carried out at 25 ℃ for 20 days.
And (2) separating to obtain mycelium and fermentation liquor, wherein the mycelium and the fermentation liquor can be extracted and separated to obtain the indole alkaloid compound.
Wherein, when the indole alkaloid compound is obtained by using mycelium, the mycelium is soaked in methanol for 7-14 days, and the mycelium is broken fully, so that intracellular substances are dissolved out effectively. And extracting the target product by using ethyl acetate.
When the indole alkaloid compound is obtained by using fermentation liquor, the fermentation liquor and diatomite are stirred and then extracted by adopting ethyl acetate reflux.
Separating and purifying the target product by using the technical means of normal phase silica gel column chromatography, reverse phase silica gel column chromatography, recrystallization, high performance liquid chromatography separation and the like. The alkaloid compound with high purity can be obtained by multi-step separation and purification.
Specifically, performing high performance liquid chromatography separation on a sub-fraction a collected after the extract is subjected to normal phase silica gel column chromatography, wherein a mobile phase adopts 63% acetonitrile-water, the flow rate is 3mL/min, and a fraction with the retention time of 17.8min is collected to obtain a compound I; collecting the fraction with retention time of 19.1min to obtain compound II;
performing ODS open column chromatography on the sub-fraction b, performing gradient elution by using methanol-water (30-100%), collecting a 60-100% eluted sub-fraction Fr.3, performing high performance liquid chromatography separation, collecting a fraction with retention time of 13.0min at a flow rate of 3mL/min by using 68% methanol-water as a mobile phase, and thus obtaining a compound III; the fraction with retention time of 16.6min was collected to obtain compound VI.
The research of the invention shows that the indole alkaloid compound separated from the fermentation culture by the method has better anti-tumor activity and aims at tumor cells Hela and IC of the compound I50Value of 10.3. mu.M, IC of Compound II50Value 13.5. mu.M, IC of Compound III50Value 34.8. mu.M, IC of Compound IV50The value was 43.7. mu.M; IC for Compound I against tumor cell A54950Value 32.8. mu.M, IC of Compound II50Value 24.9. mu.M, IC of Compound III50Value 41.0. mu.M, IC of Compound IV50The value was 25.7. mu.M.
Therefore, the invention also provides the application of the indole alkaloid compound in preparing antitumor drugs.
Further, the tumor is cervical cancer or lung cancer.
The invention also provides a pharmaceutical composition, which comprises an effective dose of indole alkaloid compound and a pharmaceutically acceptable carrier.
The pharmaceutical composition is prepared by taking the indole alkaloid compound as a main active ingredient and adding pharmaceutically acceptable auxiliary materials, and can be prepared into preparations according to preparation methods recorded in pharmaceutics. The preparation can be injection, infusion solution, powder injection, granule, tablet, granule, powder, oral liquid, sugar coated tablet, film coated tablet, enteric coated tablet, buccal preparation, granule, pill, unguent, pellet, spray, dripping pill, disintegrant, orally disintegrating tablet, pellet, etc.
The invention has the following beneficial effects:
(1) the indole alkaloid compound with a novel structure is extracted and separated from the fermentation culture of marine fungi, and the method has the advantages of simple and convenient operation, high extraction yield and high product purity, and is suitable for large-scale production.
(2) In vitro anti-tumor tests show that the indole alkaloid compound provided by the invention has better anti-tumor activity, can be used for preparing anti-tumor drugs, and has good development prospect.
Drawings
FIG. 1 is a drawing of Compound I of the present invention1H NMR spectra (in CD)3OD)。
FIG. 2 is a drawing of Compound I of the present invention13C NMR spectra (in CD)3OD)。
FIG. 3 is a drawing of Compound II of the present invention1H NMR spectra (in CD)3OD)。
FIG. 4 is a drawing of Compound II of the present invention13C NMR spectra (in CD)3OD)。
FIG. 5 is a drawing of Compound III of the present invention1H NMR spectra (in CD)3OD)。
FIG. 6 shows the preparation of compound III according to the invention13C NMR spectra (in CD)3OD)。
FIG. 7 is a drawing showing the preparation of Compound IV of the present invention1H NMR spectra (in CD)3OD)。
FIG. 8 is a drawing of Compound IV of the present invention13C NMR spectra (in CD)3OD)。
Detailed Description
The present invention is further illustrated by the following specific examples. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit or essential characteristics thereof.
The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
The fungus Aspergillus terreus (Aspergillus cf. terreus) CXX-158-20 used in the examples was isolated from marine sediments in taiwan and identified as the fungus Aspergillus terreus by molecular biology and colony morphology. And (3) the culture is preserved in China center for type culture Collection at 24/9/2021, with the preservation address: china, Wuhan and Wuhan university, the preservation number is: CCTCC NO: m20211214, tested as viable at 10 months and 9 days 2021.
Preparation of PDA solid culture medium: potato 200g, glucose 20g, agar 20g, dd H2Supplementing O to 1L, sterilizing at 121 deg.C for 30min, and naturally adjusting pH.
Preparation of a rice solid culture medium: adding 75mLdd H per 50g rice2O, sterilizing at 121 ℃ for 30min, and keeping the pH natural.
Example 1 isolation of the fungus Aspergillus terreus cf. terreus CXX-158-20
Firstly, putting a submarine sediment sample in a sterile culture dish, air-drying in a natural environment, then weighing 1g of the air-dried sediment sample, and dissolving the sediment sample by 10mL of 50% seawater to obtain 10-1Solution, further diluting to 10-2、10-3Obtaining three gradient sample liquids. Applying 100. mu.L of the suspension to separate culture2 separation culture mediums with different dilution concentrations are cultured on a GPY culture medium, a Martin culture medium, a CA culture medium, a PDA culture medium, a SDA culture medium and a Chongshi culture medium in parallel at 28 ℃ for 1-8 weeks. Observing and selecting single colony, inoculating on a Czochralski culture medium to obtain pure culture strain, and identifying as Aspergillus terreus by molecular biology and colony morphology, and is named as Aspergillus cf.
Example 2 fermentation culture of the fungus Aspergillus terreus cf
Aspergillus terreus CXX-158-20 is prepared into spore suspension, inoculated into PDA solid culture medium for activation, inoculated into rice solid culture medium for mass fermentation, and left to stand at room temperature for 20 days. Mycelia were extracted by soaking the mycelia with methanol overnight.
EXAMPLE 3 preparation of alkaloid Compound
After fermenting and culturing Aspergillus terreus CXX-158-20, taking 5L of fermentation culture solution, centrifuging and taking precipitate to obtain mycelium; soaking the mycelium in methanol for 1 week, concentrating the soaking solution, suspending with 1L distilled water, mixing the water suspensions, extracting with 6L ethyl acetate, and concentrating the ethyl acetate extractive solution to obtain extract 10 g; mixing the sample with normal phase silica gel (100 mesh, 100g), performing silica gel open column chromatography (phi 50X 640mm, 200-300 mesh, 500-700 g), and performing gradient elution with petroleum ether-ethyl acetate (100:0, 98:2, 95:5, 90:10, 80:20, 70:30, 40:60, 0:100) and ethyl acetate-methanol (95:5, 90:10) for 300mL each time; and detecting fractions by TLC. The petroleum ether-ethyl acetate 80:20 fraction was collected and recrystallized from methanol (room temperature, 100mL) to give crude crystals a of mainly Compound I. The crude crystals a were recrystallized from methanol (room temperature, 30mL) to give the pure compound I. The petroleum ether-ethyl acetate 70:30 fraction was collected with methanol: recrystallization from ethyl acetate (3:1) gives crude crystals b of mainly compound II. The crude crystals b were recrystallized from methanol (room temperature, 30mL) to give the pure compound II.
The mother liquor is eluted by methanol/water (30-100%) gradient through reversed phase silica gel ODS open column chromatography. The fractions of 85% and 90% (methanol/water) were combined to give crude compound III, which was recrystallized from methanol to give pure compound III. The fractions 90% and 95% (methanol/water) were combined to give crude compound IV, which was recrystallized from methanol to give pure compound IV.
EXAMPLE 4 preparation of alkaloid Compound
After Aspergillus terreus CXX-158-20 fermentation culture, 5L of fermentation culture solution is taken, centrifuged and supernatant is taken to obtain fermentation broth; concentrating the fermentation liquid, mixing with 10g of diatomite, refluxing with 1L of ethyl acetate, performing normal phase silica gel column chromatography (200 meshes, 300 meshes, 1 kg; silica gel column size L50 mm,
) Gradient elution is sequentially carried out on a petroleum ether/ethyl acetate mixed solution and ethyl acetate in the volume ratio of 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:3 and 1:9, and the sub-fraction a eluted from the petroleum ether/ethyl acetate mixed solution in the volume ratio of 4:1 and the sub-fraction b eluted in the volume ratio of 3:1 are collected.
Subfraction A was subjected to preparative HPLC (YMC-Pack ODS-A, 63% acetonitrile-water, 3mL/min) to give Compound I (retention time Rt:17.8min) and Compound II (retention time Rt:19.1 min).
And (3) performing ODS open column chromatography on the sub-fraction b, and performing gradient elution by using methanol/water (30-100%) to obtain 3 sub-fractions: fr.1 (methanol/water (30-40%)), Fr.2 (methanol/water (40-60%)), and Fr.3 (methanol/water (60-100%)). Subfraction Fr.3 Compound III (retention time Rt:13.0min) and Compound VI (retention time Rt:16.6min) were obtained by preparative HPLC (YMC-Pack ODS-A, 68% methanol/water, 3 mL/min).
Example 5 structural identification of alkaloid Compound I
Performing purity identification on the prepared compound by HPLC, performing structure identification on a sample with the purity of more than 98% by mass spectrometry and nuclear magnetic resonance technology, wherein the nuclear magnetic resonance is measured by a JEOL 600MHz NMR secctrometer of Japan electronic company, and TMS is used as an internal standard; high resolution mass spectra were measured using an Agilent 6230TOF LC/MS Spectrometer.
Compound I is a black solid powder, and the molecular rotation is
+88.00(c 0.1, MeOH). The measurement result of the high resolution mass spectrum (HRESIMS) is M/z 417.1459[ M + H ]]+Is combined with13C-NMR data presume that the molecular formula is C24H20N2O5The unsaturation degree was 16.
By further analyzing the hydrogen spectrum, carbon spectrum (FIG. 1, FIG. 2, Table 1) and HSQC of the compound, it was judged that the structure may contain 2 methoxy groups [ delta ] inH 3.5(s,OCH3-6,δC 60.6)]And [ delta ]H 4.0(s, OCH3-3,δC 62.1)](ii) a 8 aromatic radicals H, each being [ delta ]H 7.75(d,J＝8.5Hz,H-4”，δC 126.6), δH 6.77(t,J＝7.9Hz,H-5”，δC 120.1),δH7.11–7.07(m,H-6”，δC 130.6),δH 6.66(d,J＝7.9Hz,H-7”，δC 110.6),δH 7.34(d,J＝8.0Hz,H-4’，δC 121.3), δH 6.98(t,J＝8.0Hz,H-5’，δC 120.1),δH 7.11–7.07(m,H-6’，δC122.4),δH 7.39(d,J＝8.1Hz,H-7’，δC 112.3)](ii) a 2 sp3Hybrid methine [ delta ]H 7.27(s,H-2’, δC 126.4),δH 6.11(s,H-2”,δC 107.0)]。
According to1H-1H COSY correlation and HMBC signals, [ delta ]C 126.6(C-4”)，120.1 (C-5”)，130.6(C-6”)，110.6(C-7”)，150.8(C-8”)，131.3(C-9”)]To a benzene ring, [ delta ]C 121.3(C-4’)，120.1(C-5’)，122.4(C-6’)，112.3(C-7’)，137.9 (C-8’)，128.6(C-9’)]The assignment to another benzene ring also identifies C4 '-C5', C5 '-C6', C6 '-C7', C4 '-C5', C6 '-C7' related fragments, and both benzene rings are 1, 2-disubstituted benzene rings. Six resonant [ delta ]C 146.4(C-1),123.4(C-2),142.0(C-3),143.6(C-4),119.7 (C-5),139.8(C-6)]A third hexa-substituted benzene ring is formed.
HMBC shows deltaH 6.11(s,H-2”,δC107.0) and deltaC 150.8(C-8”)，131.3(C-9 "), 146.4(C-1) with a relevant signal, thereby determining the linkage of the dihydrofuran ring to the dihydropyrrolidine ring and the hexasubstituted benzene ring.
NOESY signal shows, deltaH 4.0(s,OCH3-3) and δH7.75(d, J ═ 8.5Hz, H-4') is relevant, δH 3.5(s,OCH3-6) and δH7.27(s, H-2') is related, and δH 4.0(s, OCH3-4) and δH 3.5(s,OCH3-3) no correlation signal, determining that two methoxy groups are located at C-3 and C-6, respectively, and OH is located at C-4.
TABLE 1 NMR data for alkaloid Compounds I
It was thus established that the structure of compound I is as follows:
example 6 structural identification of alkaloid Compound II
Performing purity identification on the prepared compound by HPLC, performing structure identification on a sample with the purity of more than 98% by mass spectrometry and nuclear magnetic resonance technology, wherein the nuclear magnetic resonance is measured by a JEOL 600MHz NMR secctrometer of Japan electronic company, and TMS is used as an internal standard; high resolution mass spectra were measured using an Agilent 6230TOF LC/MS Spectrometer.
The compound II is brown oily liquid, and the molecule rotates optically
-3.6(c 0.1, MeOH). The measurement result of the high resolution mass spectrum (HRESIMS) is M/z 394.1281[ M + H ]]+Is combined with13C-NMR data presume that the molecular formula is C22H19NO6The unsaturation degree was 14.
NMR data for compound II (FIG. 3, FIG. 4, Table 2) show similar structural features to compound I, except that the C-5 substituted indole structure is missing, instead C-5 is shifted to the lower field and is substituted with a symmetrical para-substituted phenyl ring, shown by ROESY and HMBC signals, δH7.18(d, J ═ 8.5Hz, H-3') and δH6.80(d, J ═ 8.5Hz, H-2') is an adjacent and symmetrical aromatic H. The conjectures of H-2' and delta as shown by HMBCC126.1 (C-5).
TABLE 2 NMR data for alkaloid Compound II
| Position | δC,type | δH(J in Hz) |
| 1 | 144.5,C | - |
| 2 | 117.2,C | - |
| 3 | 140.6,C | - |
| 4 | 150.2,C | - |
| 5 | 126.1,C | - |
| 6 | 135.1,C | - |
| 2” | 107.3,CH | 6.11,s |
| 3” | 90.7,C | - |
| 4” | 126.8,CH | 7.84,d(7.4) |
| 5” | 120.1,CH | 6.74,t(7.5) |
| 6” | 130.5,CH | 7.08,t(7.7) |
| 7” | 110.5,CH | 6.65,d(7.9) |
| 8” | 150.7,C | - |
| 9” | 131.4,C | - |
| 1’ | 131.2,C | - |
| 2’ | 115.7,CH | 6.80,d(8.5) |
| 3’ | 132.7,CH | 7.18,d(8.5) |
| 4’ | 157.8,C | - |
| 5’ | 132.7,CH | 7.18,d(8.5) |
| 6’ | 115.7,CH | 6.80,d(8.5) |
| C3-OCH3 | 61.0,CH3 | 3.23,s |
| C6-OCH3 | 60.9,CH3 | 3.50,s |
It was thus determined that the structure of compound II is as follows:
example 7 structural characterization of alkaloid Compound III
Performing purity identification on the prepared compound by HPLC, performing structure identification on a sample with the purity of more than 98% by mass spectrometry and nuclear magnetic resonance technology, wherein the nuclear magnetic resonance is measured by a JEOL 600MHz NMR secctrometer of Japan electronic company, and TMS is used as an internal standard; high resolution mass spectra were measured using an Agilent 6230TOF LC/MS Spectrometer.
Compound III is brown solid powder, and the molecular rotation is
+82.00(c 0.1, MeOH). The measurement result of the high resolution mass spectrum (HRESIMS) is M/z 445.1403[ M + H ]]+Is combined with13C-NMR data presume that the molecular formula is C25H20N2O6The unsaturation degree was 17.
The NMR data (FIG. 5, FIG. 6, Table 3) for compound III show structural features similar to those of compound I, and the hydrogen, carbon and HSQC of this compound show an additional methine delta compared to compound IC 163.1(C-8”)，δH8.81(s, H-8'), HMBC Signal display, δH8.81(s, H-8') and deltaC141.6 (C-9') and deltaH6.46(s, H-2') and deltaC163.1 (C-8') there is a correlation signal. Meanwhile, the molecular mass and chemical shift change shown by the high-resolution mass spectrum result is considered that 1-NH is changed into 1-NOH.
TABLE 3 NMR data for alkaloid Compound III
We have thus determined that the structure of compound III is as follows:
example 8 structural characterization of alkaloid Compound IV
Performing purity identification on the prepared compound by HPLC, performing structure identification on a sample with the purity of more than 98% by mass spectrometry and nuclear magnetic resonance technology, wherein the nuclear magnetic resonance is measured by a JEOL 600MHz NMR secctrometer of Japan electronic company, and TMS is used as an internal standard; high resolution mass spectra were measured using an Agilent 6230TOF LC/MS Spectrometer.
Compound IV is a brown oily liquid. The measurement result of the high resolution mass spectrum (HRESIMS) is M/z 467.1968[ M + H ]]+Is combined with13C-NMR data presume that the molecular formula is C26H28N2O6The unsaturation degree was 14.
By further analyzing the NMR spectrum of this compound (FIG. 7, FIG. 8, Table 4), it was found to have a similar skeleton to that of compound I, and by comparison with compound I, we found that H-2 ', H-2 ", H-3" disappeared instead of the double bond of C2 "-C3" and the isoprenyl group of C-2' of compound IV. According to H-14' (delta)H1.36, s,3H) and C-2' (delta)C 141.7),C-11’(δC146.9) existence of BC correlation, H-11' (delta)H6.11, dd, J ═ 17.4,10.6Hz,1H) and C-2' (δC 141.7)，C-13’(δC26.8) BC correlation exists, while H-11 '/H-12' exists1H-1H COSY spin coupling, the presence of prenyl groups was verified and substitutions were at C-2'.
TABLE 4 NMR data for alkaloid Compound IV
We therefore determined that the structure of compound IV is as follows:
example 9 analysis of antitumor Activity of alkaloid Compound
Hela and A549 cells were cultured in RP-MI 1640 medium containing 10% calf serum, 100IU/mL penicillin and 100g/mL streptomycin, and the medium was changed 1 time every 3 days and passaged 1 time every 5 days. The cells were all placed at 37 ℃. Taking logarithmic growth phase cells, diluting to 5 × 10 with RPMI 1640 medium4Single cell suspension/mL, seeded in 96-well cell culture plates, 3 wells per concentration, 180. mu.L per well. After incubation in incubator for 12h, 20 μ L of test solution with different concentrations was added to each well of drug group, and blank control group (equal volume of RPMI 1640 medium was used to replace the drug to be tested) and positive control group (5-FU) were set in parallel for 48h of co-culture. mu.L of 1mg/mL MTT solution was added to each well, after further incubation for 4h, the supernatant was aspirated off, and 150. mu.L of dimethyl sulfoxide (DMSO) was added to each well to dissolve the MTT-reduced product sufficiently. Measuring optical density (D) of each drug group and blank group at 492nm on enzyme labeling instrument, and calculating half Inhibition Concentration (IC) of drug on tumor cells according to formula50) And the drug effect is preliminarily evaluated.
IR (%) - (1-mean D value of dosing group/mean D value of control group) × 100%.
The experimental result shows that the 4 alkaloid compounds have better anti-tumor activity and aim at tumor cells Hela, and the IC of the compound I50Value of 10.3. mu.M, IC of Compound II50Value 13.5. mu.M, IC of Compound III50Value 34.8. mu.M, IC of Compound IV50IC of positive control 5-FU with a value of 43.7. mu.M50The value was 18.3. mu.M; IC for Compound I against tumor cell A54950Value 32.8. mu.M, IC of Compound II50Value 24.9. mu.M, IC of Compound III50The value was 41.0. mu.M,IC of Compound IV50IC of positive control 5-FU with a value of 25.7. mu.M50The value was 24.2. mu.M. The compound is shown to have better anti-tumor effect.
Claims (8)
1. An indole alkaloid compound, characterized in that the structural formula of the compound is selected from the formulas (I) to (IV),
2. the process for the preparation of indole alkaloid compounds according to claim 1, comprising the steps of:
(1) the preservation number is CCTCC NO: activating M20211214 Aspergillus fungus (Aspergillus cf. terreus) CXX-158-20, inoculating into rice solid culture medium, and statically culturing at 20-30 deg.C for 10-40 days;
(2) after the fermentation culture is finished, mycelium and fermentation liquor are obtained through separation;
(3) adding the mycelium into methanol for leaching, separating to obtain a leaching liquor, concentrating the leaching liquor, suspending the leaching liquor with distilled water to obtain an aqueous suspension, extracting the aqueous suspension with ethyl acetate, and separating and purifying the extract to obtain an indole alkaloid compound;
extracting the fermentation liquor by using ethyl acetate, and separating and purifying the extract liquor to obtain an indole alkaloid compound;
the separation and purification comprises the following steps: performing normal-phase silica gel column chromatography on the extract, performing gradient elution by using petroleum ether/ethyl acetate mixed liquor with the volume ratio of 100:0, 98:2, 95:5, 90:10, 80:20, 70:30, 40:60 and 0:100 in sequence, collecting 80:20 fractions, and recrystallizing by using methanol to obtain a compound I with the structural formula shown in the formula (I); collecting 70:30 fractions, and recrystallizing with methanol/ethyl acetate mixed solution at a volume ratio of 3:1 to obtain a compound II with a structural formula shown in a formula (II);
performing reverse phase silica gel column chromatography on the extract, performing gradient elution by using 30-100% methanol/water solution by volume ratio, and collecting 85-90% fraction to obtain a compound III with a structural formula shown in a formula (III); collecting 90-95% of fractions to obtain a compound VI with a structural formula shown in a formula (IV);
or carrying out normal phase silica gel column chromatography on the extract, carrying out gradient elution by using a petroleum ether/ethyl acetate mixed solution and ethyl acetate in volume ratios of 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:3 and 1:9 in sequence, and collecting a sub-fraction a eluted from the petroleum ether/ethyl acetate mixed solution in volume ratio of 4:1 and a sub-fraction b eluted from 3: 1; performing preparative high performance liquid chromatography on the sub-fraction a to obtain a compound I and a compound II; and the sub-fraction b is subjected to reverse phase silica gel column chromatography and preparative high performance liquid chromatography to obtain III and a compound VI.
3. The method according to claim 2, wherein in the step (1), PDA medium is used for the activation, and the PDA medium comprises the following raw materials in a volume of 1L: 200g of potato, 20g of glucose, 20g of agar and the balance of H2O, pH is natural.
4. The preparation method according to claim 2, wherein in the step (1), the rice solid medium comprises the following raw materials: 75mL of water was added to 50g of rice, and the pH was adjusted to a natural level.
5. The method according to claim 2, wherein the fermentation culture conditions in the step (1) are static culture at 25 ℃ for 20 days.
6. The use of the indole alkaloid compound of claim 1 in the preparation of an anti-tumor medicament.
7. The use of claim 6, wherein the neoplasm is cervical cancer or lung cancer.
8. A pharmaceutical composition comprising an effective amount of the indole alkaloid compound of claim 1 and a pharmaceutically acceptable carrier.
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| CN116478121A (en) * | 2023-04-23 | 2023-07-25 | 广西大学 | New compound extracted from golden camellia endophyte metabolite and preparation method thereof |
| CN116478121B (en) * | 2023-04-23 | 2024-05-14 | 广西大学 | New compound extracted from golden camellia endophyte metabolite and preparation method thereof |
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