CN117466903A - Indole diterpenoid alkaloid compound and preparation method and application thereof - Google Patents
Indole diterpenoid alkaloid compound and preparation method and application thereof Download PDFInfo
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- CN117466903A CN117466903A CN202311457891.9A CN202311457891A CN117466903A CN 117466903 A CN117466903 A CN 117466903A CN 202311457891 A CN202311457891 A CN 202311457891A CN 117466903 A CN117466903 A CN 117466903A
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- Prior art keywords
- brefeldin
- indole
- bone
- fermentation culture
- preparation
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- -1 Indole diterpenoid alkaloid compound Chemical class 0.000 title claims abstract description 57
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Classifications
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/04—Ortho-condensed systems
- C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
- C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A—HUMAN NECESSITIES
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- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
- C07D491/18—Bridged systems
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Abstract
The invention provides an indole diterpenoid alkaloid compound, and a preparation method and application thereof, and relates to the field of marine medicaments. Two novel indole diterpenoid alkaloids are disclosed, which are named as brefeldin E and brefeldin F, the structural formulas of which are shown as formula (I) and formula (II) respectively, and the two compounds have remarkable inhibition effect on LPS-induced NF- κB luciferase at the concentration of 10 mu M, so that the BMMs of the bone-breaking precursor cells induced by RANKL are inhibited from differentiating into bone-breaking cells, and the bone-breaking precursor cells have no obvious cytotoxicity, can effectively replace the existing bisphosphate drugs and the denoximab, inhibit the bone absorption activity of the bone-breaking cells, and are ideal candidate compounds developed into novel bone-breaking cell differentiation inhibitor drugs for preventing and treating bone-dissolving diseases related to the bone-breaking cells.
Description
Technical Field
The invention relates to the field of ocean medicines, in particular to indole diterpenoid alkaloids compounds, and a preparation method and application thereof.
Background
Osteoporosis is a common degenerative disease of the bone, which is mainly manifested by increased bone fragility, reduced bone mass and destruction of bone microstructure, severely threatening the health of the aging population and postmenopausal women. Osteoclasts (OCs) are special cells formed by the fusion of monocyte/macrophage hematopoietic lineage precursor cells, the only cells in humans that have bone resorption function. The defect of osteoclast activity leads to bone sclerosis and bone marrow failure, and excessive activation can lead to osteolytic diseases such as osteoporosis, rheumatoid arthritis, tumor bone metastasis and the like, and inhibition of the formation and resorption functions of OCs is one of the main strategies for treating osteoporosis. The formation of osteoclasts is a stepwise process initiated by the binding of a receptor activator of the nuclear factor- κb receptor activator ligand (Receptor activator of nuclear kappaB ligand, RANKL) to its receptor RANK on the monocyte/macrophage precursor. The related inhibitors of osteoclast differentiation applied clinically at present are mainly denoxib and bisphosphonate drugs, but have certain complications and side effects. Therefore, a need exists for a safe, effective, quality-controllable, economical, targeted drug that inhibits osteoclast formation and bone resorption that effectively addresses the great clinical need for inhibitors of osteoclast differentiation.
The marine natural product has the unique advantages of novel structure and excellent activity, and is an important source of novel drug lead compounds. In the marine-source osteoclast differentiation related inhibitor drugs, only one cycloindole diterpenoid compound penidite rpenoid A has a related effect (see Chinese patent application 202310241947.0, marine fungus-source cycloindole diterpenoid compound penidite rpenoid A and a preparation method and application thereof) and other indole diterpenoid compounds have no related effect. The marine microorganism resources are rich, and the novel anti-osteoporosis lead compound is found as a novel ideal resource, so that the research and development of novel high-efficiency low-toxicity related medicines of osteoclast differentiation inhibitors from the marine microorganism resources are of great significance in preventing and treating osteolytic diseases.
Disclosure of Invention
The present invention addresses the above problems by providing a novel use of indole diterpene alkaloids (designated brefeldin E and brefeldin F).
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the novel indole diterpenoid alkaloids are named as brefeldin E and brefeldin F respectively; the structural formula of the brefeldin E is shown as a formula (I), and the structural formula of the brefeldin F is shown as a formula (II):
another object of the present invention is also to protect a strain of Penicillium brefeldianum (Penicillium brefeldianum) GXIMD 02511, deposited at the microorganism strain collection (GDMCC) of Guangdong province, month 09 of 2022, address: the Guangzhou City of Guangdong province, first, the middle road No. 100 Guangdong province microorganism research No. 59 building five, the Guangdong province institute of microorganisms, accession number: GDMCC No.62843.
The invention also aims at protecting a method for preparing indole diterpenoid alkaloids compounds, wherein the indole diterpenoid alkaloids compounds are prepared and separated from a fermentation culture of a strain of penicillium brefeldianum (Penicillium brefeldianum) GXIMD 02511.
Further, the method comprises the following steps:
s1 preparation of fermentation culture: preparing a fermentation culture of the strain penicillium brefeldii (Penicillium brefeldianum) GXIMD 02511;
s2, extracting: soaking the fermentation culture in ethyl acetate, cutting or pulverizing the fermentation culture, performing ultrasonic extraction, and filtering to obtain filter residue and filtrate respectively; extracting the filtrate and the filter residue with ethyl acetate respectively, concentrating to remove ethyl acetate, and mixing the extracts;
s3, separation and purification: subjecting the extract to medium pressure normal phase liquid chromatography with petroleum ether/dichloromethane as eluent, wherein the volume ratio is 100:0 to 0:10, gradient elution is carried out, and petroleum ether is collected: the volume ratio of dichloromethane is 80:20 gradient eluted fraction, continuing to pass medium-pressure reversed phase C 18 Column chromatography, using methanol/water as eluent, from a volume ratio of 10: 90-100: 0, gradient elution, collecting methanol: water volume ratio 58:42 gradient eluting, purifying the fraction to obtain indole diterpenoid alkaloids.
Further, in the preparation of the S1 fermentation culture, the fermentation culture is prepared by fermentation by the following method: inoculating activated strain Penicillium brefeldianum (Penicillium brefeldianum) GXIMD 02511 into seed culture medium, and dynamically culturing at 25deg.C and 180rpm for 72 hr to obtain seed solution; inoculating the seed solution into a fermentation culture medium in an inoculum size of 5%, and statically culturing for 30 days at 25 ℃ to obtain a fermentation culture; the seed culture medium formula comprises the following components in each 1L of culture medium: 15g of malt extract powder, the balance of water and pH 7.5; the formula of the fermentation medium comprises the following components in each 1L of triangular flask medium: 200g of rice, 2g of sea salt, 200mL of water and pH 7.5.
The invention also aims at protecting the application of the indole diterpenoid alkaloids compound in preparing an osteoclast differentiation inhibitor.
Further, the osteoclast differentiation inhibitor is a medicament for treating osteolytic diseases caused by overactivation of osteoclasts.
Further, the osteoclast differentiation inhibitor is a medicament for treating osteoporosis, rheumatoid arthritis and tumor metastasis bone destruction.
The invention also aims at protecting the application of the indole diterpenoid alkaloids compound in preparing NF- κB nuclear factor expression inhibitor.
It is a further object of the present invention to provide a pharmaceutical composition which may be an NF- κb nuclear factor expression inhibitor or an osteoclast differentiation inhibitor drug, comprising an effective amount of the active ingredient and a pharmaceutically acceptable carrier or adjuvant. Wherein the pharmaceutical activity becomes indole diterpenoid alkaloids compound and/or medicinal salt thereof, and the specific pharmaceutical activity can be brefeldin E and/or medicinal salt thereof and/or brefeldin F and/or medicinal salt thereof.
The invention is obtained by test: indole diterpenoid alkaloids compounds brefeldole E and brefeldole F have inhibition effect (p < 0.01) on LPS-induced NF- κB luciferase at the concentration of 10 mu M, can be used as a lead compound for developing NF- κB nuclear factor expression inhibitor, can remarkably inhibit the differentiation of RANKL-induced osteoclast precursor cells BMMs (Bone marrow macrophage cells, bone marrow macrophages) into osteoclasts, and has no obvious cytotoxicity, so the novel safe and effective osteoclast differentiation inhibitor medicine is expected to be developed.
By adopting the technical scheme, the invention has the following beneficial effects:
in the research process of secondary metabolites of the rhizosphere bottom mud source penicillium brefeldianum (Penicillium brefeldianum) GXIMD 02511 in the protection zone of the mangrove forest in the northern Europe of Guangxi, two indole diterpenoid alkaloid compounds brefeldin E and brefeldin F with novel structures are separated and obtained, and the two compounds have remarkable inhibition effects on NF- κB luciferase induced by LPS, and all remarkably inhibit the differentiation of the bone-breaking precursor cells BMMs induced by RANKL into bone-breaking cells in a dose-dependent manner without cytotoxicity. Therefore, the compound is an ideal candidate compound developed into a novel NF- κB nuclear factor expression inhibitor or an osteoclast differentiation inhibitor drug.
Drawings
FIG. 1 is a graph comparing the inhibitory activity of indole diterpene alkaloids, brefeldin E and brefeldin F, against Lipopolysaccharide (LPS) -induced NF- κB luciferase in RAW264.7 cells (mouse mononuclear macrophages) at a concentration of 10. Mu.M, BAY being a positive control, ### p<0.001vs.control group；***p<0.001vs.LPS group；
FIG. 2 is a graph showing the effect of indole diterpene alkaloids, brefeldin E and brefeldin F, on cell viability of mouse Bone Marrow Macrophages (BMMs);
FIG. 3 is an experimental result of the effect of indole diterpenoid alkaloids, brefeldin E and brefeldin F, on the differentiation of osteoclast precursor cells BMMs into osteoclasts, wherein: RANKL is a ligand for activating nuclear factor NF- κb receptor, and # # # P <0.001 compared to the blank; p <0.05, P <0.01 compared to RANKL group;
Detailed Description
In order to make the objects, technical schemes and technical effects of the present invention more clear, the present invention will be further described in detail with reference to the following examples and the accompanying drawings. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
The indole diterpenoid alkaloid compounds are named as brefeldin E and brefeldin F respectively; the structural formula of the brefeldin E is shown as a formula (I), and the structural formula of the brefeldin F is shown as a formula (II):
EXAMPLE 2 strain Penicillium brefeldianum (Penicillium brefeldianum) GXIMD 02511
The strain Penicillium brefeldianum (Penicillium brefeldianum) GXIMD 02511 was isolated from the rhizosphere bottom sludge of the mangrove protection area of the mouth of Heteropap, guangdong province, china, deposited at the microbiological culture Collection center (GDMCC) at 2022, 09, 27, address: the Guangzhou City of Guangdong province, first, the middle road No. 100 Guangdong province microorganism research No. 59 building five, the Guangdong province institute of microorganisms, accession number: GDMCC No.62843.
The strain Penicillium brefeldin (Penicillium brefeldianum) GXIMD 02511 in this example can be used to prepare indole diterpenoid alkaloids, brefeldin E and brefeldin F in example 1.
EXAMPLE 3 preparation and isolation of indole diterpene alkaloids, brefeldin E and brefeldin F
1. Culture medium
1.1, seed culture medium: each 1L of the culture medium contains 15g of malt extract powder, the balance of water and has the pH of 7.5. Mixing the above components and contents, and sterilizing at 121deg.C for 30 min.
1.2, fermentation medium: each 1L of the flask medium contains: 200g of rice, 2g of sea salt, 200mL of water and pH 7.5. Mixing the above components and contents, and sterilizing at 121deg.C for 30 min.
2. Fermentation
2.1, seed culture: the activated strain Penicillium brefeldianum (Penicillium brefeldianum) GXIMD 02511 was inoculated into 1L triangular flask containing 300mL of seed medium per flask, and cultured at 25℃and 180rpm for 72 hours to obtain a seed solution.
2.2, fermentation culture: the seed solution was inoculated into 120 flasks of fermentation medium at an inoculum size (volume percent) of 5%, and statically cultured at 25℃for 100 days to prepare a fermentation culture.
3. Extracting: soaking the fermentation culture in ethyl acetate with volume of 2 times for about 24 hours, cutting or mashing, ultrasonic extracting in an ultrasonic instrument for 20min, and filtering with 8 layers of gauze to obtain filtrate and residue respectively. The filtrate was concentrated by rotary evaporator to remove the organic solvent and then extracted with ethyl acetate multiple times until the color of the aqueous phase became light, and concentrated to remove ethyl acetate. The residue was extracted with ethyl acetate several times and concentrated to remove ethyl acetate, and the two brown yellow extractum were combined to about 132g.
4. Separation and purification of indole diterpenoid alkaloids compounds brefeldin E and brefeldin F
Subjecting the extract (132 g) to medium pressure normal phase liquid chromatography (MPLC) with petroleum ether/dichloromethane as eluent, gradient eluting from volume ratio (100:0) - (0:100), collecting petroleum ether/dichloromethane volume ratio 80:20 gradient eluted fraction, continuing to pass medium-pressure reversed phase C 18 Column chromatography, gradient elution from volume ratios (10:90) - (100:0) using methanol/water as eluent, collecting methanol/water volume ratio 58:42 gradient elution, which is finally finely separated by semi-preparative high performance liquid phase, is purified by methanol/water (volume ratio 58:42, YMC-pack ODS-A column, 10X 250mm,5 μm,2 m/min) to obtain indole diterpene alkaloids, brefeldin E (2 mg) and brefeldin F (2 mg).
The indole diterpenoid alkaloids compounds brefeldin E and brefeldin F obtained by the extraction and separation are subjected to the following structural identification and experiment:
and (3) structural identification:
indole diterpenoid alkaloids compound brefeldin E is light yellow oily matter. The nuclear magnetic data shows that the indole diterpenoid alkaloids containing 3 aromatic protons are characterized by H-21 (delta) H 6.79,d,J＝7.0Hz)，H-22(δ H 6.94,t,7.6Hz)，H-23(δ H 7.16, d, j=8.1 Hz); 5 methines, H-14 (delta) H 6.17)，H-11(δ H 5.98)，H-6(δ H 5.76)，H-16(δ H 2.98 And H-9 (delta) H 4.30 A) is provided; 6 unimodal methyl groups, each H 3 -33(δ H 1.34)，H 3 -34(δ H 1.34)，H 3 -28(δ H 1.31)，H 3 -29(δ H 1.33)，H 3 -25(δ H 1.04)，H 3 -26(δ H 1.22 And 6 methylene groups. Analyze 13 32 carbons in the C nuclear magnetic resonance data are 6 methyl groups (delta) C 29.4,29.2,26.7,25.3,23.1,15.2), 6 methylene groups (delta) C 47.6,34.6,30.2,29.9,29.9,28.9) 7 methines (delta) C 132.6,122.2,121.5,119.5,110.5,79.3,46.8) and 13 quaternary carbons (delta) C 201.1,163.8,149.1,142.7,142.4,134.8,125.3,117.3,95.3,73.6,71.6,49.9,43.9). The data attribution is as follows: 1 H NMR(500MHz,CD 3 OD):δ H 2.44(2H,m,H-5,15),2.34(1H,dt,J＝18.5,5.0Hz,H-5),2.26(1H,td,J＝14.0,3.7Hz,H-6),2.17(1H,dt,J＝14.0,3.7Hz,H-6),4.30(1H,br s,H-9),5.98(1H,br s,H-11),6.17(1H,dd,J＝5.4,2.3Hz,H-14),2.03(1H,m,H-15),2.98(3H,m,H-16,17,30),2.65(1H,dd,J＝12.4,9.5Hz,H-17),6.79(1H,d,J＝7.0Hz,H-21),6.94(1H,t,J＝7.6Hz,H-22),7.16(1H,d,J＝8.1Hz,H-23),1.04(3H,s,H 3 -25),1.22(3H,s,H 3 -26),1.31(3H,s,H 3 -28),1.33(3H,s,H 3 -29),1.34(6H,s,H 3 -33,34),1.84(1H,m,H-31). 13 C NMR(125MHz,CD 3 OD):δ C 149.1(C,C-2),49.0(C,C-3),43.9(C,C-4),28.9(CH,C-5),34.6(CH 2 ,C-6),95.3(C,C-7),79.3(CH,C-9),201.1(C,C-10),122.2(CH,C-11),163.8(C,C-12),142.7(C,C-13),132.6(C,C-14),29.9(CH 2 ,C-15),46.8(CH,C-16),30.2(CH 2 ,C-17),117.3(C,C-18),125.3(C,C-19),134.8(C,C-20),119.5(CH,C-21),121.5(CH,C-22),110.5(CH,C-23),142.4(C,C-24),15.2(CH 3 ,C-25),23.1(CH 3 ,C-26),73.6(C,C-27),25.3(CH 3 ,C-28),26.7(CH 3 ,C-29),29.9(CH 2 ,C-30),47.6(CH 2 ,C-31),71.6(C,C-32),29.4(CH 3 ,C-33),29.2(CH 3 c-34). Identifying the compound asbrefeldole E. The structural formula of brefeldole E is shown as the following formula (I):
indole diterpenoid alkaloid compound brefeldin F is white amorphous powder. The nuclear magnetic data shows that the indole diterpenoid alkaloids containing 3 aromatic protons are characterized by H-21 (delta) H 6.74,dd,J＝7.2,0.9Hz)，H-22(δ H 6.88,dd,8.1,7.2Hz)，H-23(δ H 7.13, dd, j=8.1, 0.9 hz); 3 methines, H-11 (delta) H 5.81)，H-16(δ H 2.85 And H-9 (delta) H 4.30 A) is provided; 6 unimodal methyl groups, each H 3 -33(δ H 1.30)，H 3 -34(δ H 1.30)，H 3 -28(δ H 1.14)，H 3 -29(δ H 1.42)，H 3 -25(δ H 1.39)，H 3 -26(δ H 1.23 And 6 methylene groups. Analyze 13 32 carbons in the C nuclear magnetic resonance data are 6 methyl groups (delta) C 29.3,29.2,29.2,23.8,23.4,16.6), 7 methylene groups (delta) C 47.6,33.9,30.7,29.9,29.4,27.7,22.4), 6 methines (. Delta.) C 121.1,119.3,118.2,110.5,89.2,50.3) and 13 quaternary carbons (delta) C 199.5,172.4,153.2,142.0,134.6,125.7,116.1,106.2,79.5,77.7,71.6,52.5,40.9). The data attribution is as follows: 1 H NMR(700MHz,CD 3 OD):δ H 2.86(2H,m,H-5,6),2.63(1H,t,J＝11.2Hz,H-5),1.95(1H,m,H-6),4.30(1H,d,J＝1.3Hz,H-9),5.81(1H,br s,H-11),2.07(1H,m,H-14),1.78(1H,m,H-14),2.02(1H,m,H-15),1.94(1H,m,H-15),2.85(1H,m,H-16),2.86(1H,m,H-17),2.55(1H,m,H-17),6.74(1H,dd,J＝7.2,0.9Hz,H-21),6.88(1H,dd,J＝8.1,7.2Hz,H-22),7.13(1H,dd,J＝8.1,0.9Hz,H-23),1.39(3H,s,H 3 -25),1.23(3H,s,H 3 -26),1.14(3H,s,H 3 -28),1.42(3H,s,H 3 -29),2.89(1H,m,H-30),1.78(1H,m,H-31),1.30(6H,s,H 3 -33,34). 13 C NMR(175MHz,CD 3 OD):δ C 153.2(C,C-2),52.5(C,C-3),40.9(C,C-4),27.7(CH 2 ,C-5),29.4(CH 2 ,C-6),106.2(C,C-7),89.2(CH,C-9),199.5(C,C-10),118.2(CH,C-11),172.4(C,C-12),77.7(C,C-13),22.4(CH 2 ,C-14),33.9(CH 2 ,C-15),50.3(CH,C-16),30.7(CH 2 ,C-17),116.1(C,C-18),125.7(C,C-19),134.6(C,C-20),119.3(CH,C-21),121.1(CH,C-22),110.5(CH,C-23),142.0(C,C-24),16.6(CH 3 ,C-25),23.4(CH 3 ,C-26),79.5(C,C-27),23.8(CH 3 ,C-28),29.3(CH 3 ,C-29),29.9(CH 2 ,C-30),47.6(CH 2 ,C-31),71.6(C,C-32),29.2(CH 3 ,C-33),29.2(CH 3 c-34). The compound was identified as brefeldin F. The structure of brefeldin F is shown as the following formula (II):
experiment one: determination of LPS-induced NF- κB luciferase inhibitory Activity of indole diterpenoid alkaloids Compounds brefeldin E and brefeldin F obtained in example 3
NF-. Kappa.B luciferase inhibitory Activity assay the main reference (Marine Drugs,2022,20 (3): 178.).
RAW264.7 cells stably transfected with NF- κB luciferase reporter gene were inoculated in 96-well plates (1×10) 4 200 mu L of DMEM medium containing 10% of fetal bovine serum, 100IU/mL of penicillin and streptomycin and 0.1 mu G/mL of G418 is added into each hole, after cell attachment is stable, indole diterpenoid alkaloid compounds brefeldin E and brefeldin F are added, and 6 compound holes are arranged. After further incubation for 4h, each of the compound group (3 wells) and the positive control group (NF- κB inhibitor, BAY11-7082,5 μM) was added with LPS and RANKL respectively to a final concentration of 100ng/mL per well, after 8h of stimulation by both, the supernatant was discarded, 25 μL of cell lysate was added per well, shaking at low speed for 10min to lyse the cells sufficiently, then 20 μL was transferred to the white plate, 50 μL of fluorescein solution was added per well, and the Lucifer value was detected with a multifunctional microplate reader.
Conclusion of the test: as shown in the figure 1, the indole diterpene alkaloid compounds brefeldin E and brefeldin F have remarkable inhibition effect (p < 0.001) on LPS-induced NF- κB luciferase at 10 mu M compared with LPS blank group.
Experiment II: the indole diterpenoid alkaloids of example 3, brefeldin E and brefeldin F, have cytotoxic effects on the osteoclast precursor RAW264.7 cells and BMMs
Cell viability assay Main reference (Marine Drugs,2022,20 (3): 178.)
CCK-8 method for detecting vitality influence of indole diterpenoid alkaloids compounds brefeldin E and brefeldin F on mouse Bone Marrow Macrophages (BMMs)
a. Preparation of mouse Bone Marrow Macrophages (BMMs): under the aseptic condition, taking femur of C57BL/6 female mice with age of 8-12 weeks, cutting joint parts at two ends of femur, repeatedly flushing femur with phenol red-free alpha-MEM culture medium (containing 10% fetal calf serum, 100IU/mL penicillin and 100IU/mL streptomycin) until femur cavity blurs. Placing the washed bone marrow cavity cells of femur at 37deg.C and 5% CO 2 Incubating the cells in a cell incubator for 2 hours, absorbing the supernatant, lysing the red blood cells by using lysate, centrifuging and re-suspending to obtain the BMMs.
CCK-8 method to detect cell survival:
taking the BMMs (1×10) prepared in step a 5 0/well) was added to 200. Mu.L of phenol red-free alpha-MEM medium (containing 10% fetal bovine serum, 100IU/mL penicillin and 100IU/mL streptomycin) per well while macrophage colony stimulating factor (M-CSF, final concentration 50 ng/mL) was added per well, and then the 96-well plate was placed at 37℃with 5% CO 2 Is incubated overnight. After the cell adhesion is stable, indole diterpene alkaloid compounds brefeldin E and brefeldin F with different concentrations are respectively added, so that the final concentrations of the indole diterpene alkaloid compounds brefeldin E and brefeldin F in the holes are 10 mu M and 15 mu M, 3 compound holes are arranged in each group, and the cells are incubated for 4 days. After the incubation was completed, the supernatant (100. Mu.L) was discarded, 5. Mu.L of CCK-8 reagent (Cell Counting Kit-8 cell counting reagent) was added to each well, and the mixture was shaken and placed at 37℃in 5% CO 2 Incubation was continued for 3h in the environment, and the Optical Density (OD) at 450nm was measured with a TECANGENiosPro multifunctional microplate reader, and the cell viability of each group was calculated.
As shown in FIG. 2, there was no significant difference in BMMs cell viability after addition of 10. Mu.M and 15. Mu.M indole diterpene alkaloids brefeldin E and brefeldin F, indicating that indole diterpene alkaloids brefeldin E and brefeldin F were non-cytotoxic to BMMs in vitro assays.
Experiment III: effect of indole diterpene alkaloids, brefeldin E and brefeldin F, obtained in example 3 on RANKL-induced differentiation of osteoclast precursor cells BMMs into osteoclasts
RANKL-induced osteoclast precursor BMMs cell differentiation inhibitory activity was determined as the primary reference (medicine Drugs,2022,20 (3): 178.).
Taking BMMs (2×10) obtained in step a above 4 0/well) was added to 200. Mu.L of phenol red-free alpha-MEM medium (containing 10% fetal bovine serum, 100IU/mL penicillin and 100IU/mL streptomycin) per well while macrophage colony stimulating factor (M-CSF, final concentration 50 ng/mL) was added per well, and then the 96-well plate was placed at 37℃with 5% CO 2 Is incubated overnight. After the cell wall attachment is stable, indole diterpene alkaloid compounds brefeldin E and brefeldin F with different concentrations are respectively added, so that the final concentrations of the indole diterpene alkaloid compounds brefeldin E and brefeldin F in the holes are 5 mu M,10 mu M and 15 mu M, 3 compound holes are arranged in each group, and the cells are incubated for 4 hours. After the incubation, RANKL (final concentration 100 ng/mL) was added and incubated for 3-4d. TRAP staining is carried out on the cells after incubation, and the cells are photographed and counted under an inverted microscope, wherein TRAP positive cells with more than 5 nuclei are osteoclasts.
As shown in FIG. 3, indole diterpenoid alkaloids, brefeldin E and brefeldin F, can significantly inhibit the induction of BMMs by RANKL to generate osteoclasts. At the effective concentration of 10 mu M, the RANKL can be obviously inhibited to induce the BMMs to generate osteoclasts.
From the above experimental results, it can be seen that: indole diterpenoid alkaloids compounds brefeldole E and brefeldole F have remarkable inhibition effect on NF- κB luciferase induced by LPS, can remarkably inhibit the differentiation of bone-breaking precursor cells BMMs induced by RANKL into bone-breaking cells at the concentration of 10 mu M, can remarkably inhibit the generation and activation of the bone-breaking cells, have no remarkable toxic effect, can effectively replace the existing bisphosphate drugs and denoxib and inhibit the bone absorption activity of the bone-breaking cells. Can be used as a novel osteoclast differentiation inhibitor or NF- κB nuclear factor expression inhibitor for development and prevention and treatment of osteolytic diseases such as osteoporosis.
Based on the above experiments, the two indole diterpene alkaloid compounds brefeldin E and brefeldin F can be proved to be applied to the preparation of anti-osteoclast differentiation inhibitor or NF- κB nuclear factor expression inhibitor drugs. The osteoclast differentiation inhibitor or NF- κB nuclear factor expression inhibitor is in the form of oral preparation, injection or external preparation; comprises an active ingredient indole diterpenoid alkaloid compound brefeldin E and brefeldin F and pharmaceutically acceptable pharmaceutic adjuvant. Anti-osteoclast differentiation class drugs may be treated including, but not limited to: clinical indications for which known osteoclast inhibitors such as biphosphate and denoximab are approved for treatment, such as postmenopausal osteoporosis and tumor metastasis bone destruction.
Generally, the medicines are clinically applied after being prepared into preparations. The indole diterpenoid alkaloids compound serving as the effective active ingredient can be prepared according to a method known in the art. The indole diterpenoid alkaloids compound which is the effective active ingredient of the invention can be prepared into any dosage form suitable for human or animal use by combining with one or more pharmaceutically acceptable solid or liquid excipients and/or auxiliary agents. The indole diterpenoid alkaloids compound of the invention or the indole diterpenoid alkaloids compound containing the same can be administered in unit dosage form, and the administration route can be intestinal tract or parenteral tract, such as oral administration, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.
The oral preparation is preferably capsule. In order to prepare the administration unit into a capsule, the phenylacetic acid compound serving as an effective ingredient of the present invention may be mixed with a diluent and a glidant, and the mixture may be directly placed into a hard capsule or a soft capsule. The indole diterpenoid alkaloids compound which is the effective component of the invention can be prepared into particles or pellets by the diluent, the adhesive and the disintegrating agent, and then placed into hard capsules or soft capsules. The diluents, binders, wetting agents, disintegrants and glidants used for preparing the tablets of the indole diterpenoid alkaloids compounds serving as the effective active ingredients can also be used for preparing capsules of the indole diterpenoid alkaloids compounds serving as the effective active ingredients.
In order to prepare the indole diterpenoid alkaloids compound serving as an effective active ingredient into injection, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as a solvent, and a proper amount of solubilizer, cosolvent, pH regulator and osmotic pressure regulator which are commonly used in the field can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-beta-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, mannitol, glucose, etc. can be added as propping agent for preparing lyophilized powder for injection.
In summary, the invention provides a new candidate compound for developing a novel osteoclast differentiation inhibitor or NF- κB nuclear factor expression inhibitor drug, and has important significance for developing new drugs with independent intellectual property rights.
The above description is of a detailed description of a preferred embodiment of the invention, but the embodiment is not intended to limit the scope of the invention. All equivalent changes or modification changes which are accomplished under the technical conception suggested by the invention are included in the scope of the patent covered by the invention.
Claims (10)
1. The novel indole diterpenoid alkaloids are characterized in that the indole diterpenoid alkaloids are named brefeldin E and brefeldin F respectively; the structural formula of the brefeldin E is shown as a formula (I), and the structural formula of the brefeldin F is shown as a formula (II):
2. a strain of penicillium brefeldii (Penicillium brefeldianum) GXIMD 02511 for use in the preparation of an indole diterpenoid alkaloid compound of claim 1, deposited with the accession number: GDMCC No.62843.
3. A process for the preparation of an indole diterpenoid alkaloid compound as defined in claim 1, wherein said indole diterpenoid alkaloid compound is isolated from a fermentation culture of the strain penicillium brefeldianum (Penicillium brefeldianum) GXIMD 02511.
4. A method according to claim 3, characterized in that the method comprises the steps of:
s1 preparation of fermentation culture: preparing a fermentation culture of the strain penicillium brefeldii (Penicillium brefeldianum) GXIMD 02511;
s2, extracting: soaking the fermentation culture in ethyl acetate, cutting or pulverizing the fermentation culture, performing ultrasonic extraction, and filtering to obtain filter residue and filtrate respectively; extracting the filtrate and the filter residue with ethyl acetate respectively, concentrating to remove ethyl acetate, and mixing the extracts;
s3, separation and purification: subjecting the extract to medium pressure normal phase liquid chromatography with petroleum ether/dichloromethane as eluent, wherein the volume ratio is 100:0 to 0:10, gradient elution is carried out, and petroleum ether is collected: the volume ratio of dichloromethane is 80:20 gradient eluted fraction, continuing to pass medium-pressure reversed phase C 18 Column chromatography, using methanol/water as eluent, from a volume ratio of 10: 90-100: 0, gradient elution, collecting methanol: water volume ratio 58:42 gradient eluting the fraction, purifying the fraction to obtain the indole diterpenoid alkaloid compound.
5. The method of claim 4, wherein in the preparation of the S1 fermentation culture, the fermentation culture is prepared by fermentation by the following method: inoculating activated strain Penicillium brefeldianum (Penicillium brefeldianum) GXIMD 02511 into seed culture medium, and dynamically culturing at 25deg.C and 180rpm for 72 hr to obtain seed solution; inoculating the seed solution into a fermentation culture medium in an inoculum size of 5%, and statically culturing for 30 days at 25 ℃ to obtain a fermentation culture; the seed culture medium formula comprises the following components in each 1L of culture medium: 15g of malt extract powder, the balance of water and pH 7.5; the formula of the fermentation medium comprises the following components in each 1L of triangular flask medium: 200g of rice, 2g of sea salt, 200mL of water and pH 7.5.
6. Use of an indole diterpenoid alkaloid compound according to claim 1 for the preparation of an osteoclast differentiation inhibitor.
7. The use according to claim 6, characterized in that: the osteoclast differentiation inhibitor is a medicine for treating osteolytic diseases caused by overactivation of osteoclasts.
8. The use according to claim 6, characterized in that: the osteoclast differentiation inhibitor is a medicament for treating osteoporosis, rheumatoid arthritis and tumor metastasis bone destruction.
9. The use of an indole diterpenoid alkaloid compound according to claim 1 for preparing an NF- κb nuclear factor expression inhibitor.
10. A pharmaceutical composition comprising an effective amount of a pharmaceutically active ingredient and a pharmaceutically acceptable carrier or adjuvant; the active ingredients of the medicine are indole diterpenoid alkaloids compounds and/or medicinal salts thereof.
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