CN112402429B - New use of corydalis edulis in preventing and treating human cytomegalovirus infection - Google Patents
New use of corydalis edulis in preventing and treating human cytomegalovirus infection Download PDFInfo
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- CN112402429B CN112402429B CN202011436864.XA CN202011436864A CN112402429B CN 112402429 B CN112402429 B CN 112402429B CN 202011436864 A CN202011436864 A CN 202011436864A CN 112402429 B CN112402429 B CN 112402429B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/66—Papaveraceae (Poppy family), e.g. bloodroot
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
Abstract
The invention belongs to the technical field of biology, and provides a new application of corydalis edulis in preparing a medicament for preventing and treating human cytomegalovirus infection. Experiments show that the corynolen has obvious HCMV resistance effect. The host cell of the human cytomegalovirus HCMV, namely the human embryonic lung diploid fibroblast WI-38, treated by the corynollin (1-20 mu M) alone has no obvious cytotoxicity, the corynollin can obviously improve the cytopathic effect formed by HCMV infection, inhibit the expression of HCMV characteristic immediate early protein IE1/2 and early protein UL44, effectively inhibit the DNA replication of the HCMV, and has application prospect in preparing anti-HCMV medicaments.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a new application of corydalis edulis in preventing and treating human cytomegalovirus infection.
Background
Human Cytomegalovirus (HCMV) belongs to the sub-family of herpes virus beta, is generally susceptible to people, and carries virus for life after infection, after infection is relieved, the virus remains in the body for a long time to form latent infection, and in patients with low body immunity or organ transplantation, secondary infection is easy to occur, so that injuries of a nervous system, a liver, a respiratory system, a blood system and the like are easily caused, and even the life is threatened in serious cases; meanwhile, in pregnant women, secondary infection of HCMV is a main pathogen causing congenital diseases of newborn, and causes multiple organ and multiple system diseases such as icterohepatitis, mental retardation, blindness, deafness and the like. Therefore, the positive and effective treatment of the cytomegalovirus infection of the human is an important means for improving the birth population quality and improving the life quality of the old with low immunity. At present, no approved vaccine is available on the market, clinically used symptomatic medicines are mainly antiviral medicines such as foscarnet, cidofovir, fomivirsen and the like, but the medicines have obvious side effects of bone marrow inhibition, nephrotoxicity, electrolyte disorder and the like, and can also generate drug resistance, so that the clinical application of the medicines is limited.
The corynolamine is an alkaloid derived from corynolamine buchneri of papaveraceae, and the application of the corynolamine in preparing a medicament for preventing and/or treating HCMV is not provided so far.
Disclosure of Invention
The invention adopts commercial corydaline to carry out a series of biological experiments, and provides a theoretical basis for the new application of the corydaline in preventing and treating human cytomegalovirus infection.
The invention provides a new application of corydalis edulis in preparing a medicament for preventing and treating human cytomegalovirus infection.
The corynoline can inhibit the expression of HCMV immediate early protein IE1/2 and early protein UL44, thereby playing a role in resisting HCMV infection.
The corynoline form comprises a salt form and a pharmaceutically acceptable carrier combination form.
The preparation form of the medicament for preventing and treating human cytomegalovirus infection is liquid preparation, granule, tablet, electuary, capsule, sustained-release agent, dripping pill or injection.
The results of the biological experimental study show that:
1. the corydalis yanhusuo alone treated HCMV host cells-human embryonic lung diploid fibroblasts WI-38 show no obvious cytotoxicity at 0-20 mu M;
2. the corynoline has good anti-HCMV effect, and particularly shows that the expression of immediate early protein IE1/2 and early protein UL44 is obviously inhibited; while inhibiting nucleic acid replication of HCMV.
The invention has the advantages and beneficial effects that a series of biological experiments find and prove that the corynolamine has obvious anti-HCMV effect under the condition of no obvious cytotoxicity dose, is safe and effective, and is expected to be developed into a drug for preventing and treating HCMV.
Drawings
FIG. 1: the MTT method measures the influence of the corynoline on the WI-38 cell viability.
Wherein: ordinate-cell viability (setting control group to 1)
The abscissa: 1-Control group (Control)
2-Violin (1. mu.M) alone for 2 days
3-Violin (2. mu.M) alone for 2 days
4-Violin (5. mu.M) alone for 2 days
5-Violin (10. mu.M) alone for 2 days
6-Violin (20. mu.M) alone for 2 days
FIG. 2: western-blot method for detecting the influence of corydalis on HCMV immediate early protein IE1/2 and early protein UL44 (GAPDH is an internal reference)
Wherein in FIG. 2A: 1-HCMV (MOI 0.01) was inoculated for 1 day
2-Violin (10. mu.M) + HCMV (MOI 0.01) for 1 day
3-DMSO + HCMV (MOI 0.01) was inoculated for 1 day
4-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 1 day
5-HCMV (MOI 0.01) was inoculated for 4 days
6-Violin (10. mu.M) + HCMV (MOI 0.01) for 4 days
7-DMSO + HCMV (MOI 0.01) were inoculated for 4 days
8-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 4 days
9-HCMV (MOI 0.01) were inoculated for 7 days
10-Violin (10. mu.M) + HCMV (MOI 0.01) for 7 days
11-DMSO + HCMV (MOI 0.01) were inoculated for 7 days
12-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 7 days
Wherein FIG. 2B: 1-HCMV (MOI 0.01)
2-DMSO+HCMV(MOI 0.01)
3-Violalin (10. mu.M) + HCMV (MOI 0.01)
4-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01)
P <0.5, P <0.05, P <0.001, compared to control group
FIG. 3: IFA method for detecting influence of corydaline on expression of HCMV immediate early protein IE1/2
Wherein: 1-DMSO + HCMV (MOI 0.01) was inoculated for 7 days
2-Violin (1. mu.M) + HCMV (MOI 0.01) for 7 days
3-Violin (5. mu.M) + HCMV (MOI 0.01) for 7 days
4-Violin (10. mu.M) + HCMV (MOI 0.01) for 7 days
5-Violin (20. mu.M) + HCMV (MOI 0.01) for 7 days
6-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 7 days
FIG. 4: qPCR method for detecting influence of corynolen on HCMV DNA amplification
Wherein: 1-HCMV (MOI 0.01) was inoculated for 1 day
2-DMSO + HCMV (MOI 0.01) was inoculated for 1 day
3-Violin (10. mu.M) + HCMV (MOI 0.01) for 1 day
4-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 1 day
5-HCMV (MOI 0.01) was inoculated for 4 days
6-DMSO + HCMV (MOI 0.01) were inoculated for 4 days
7-Violin (10. mu.M) + HCMV (MOI 0.01) for 4 days
8-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 4 days
9-HCMV (MOI 0.01) were inoculated for 7 days
10-DMSO + HCMV (MOI 0.01) were inoculated for 7 days
11-Violin (10. mu.M) + HCMV (MOI 0.01) for 7 days
12-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 7 days
P <0.5, P <0.001, compared to HCMV group
Detailed Description
The human embryonic lung diploid fibroblast strain WI-38 and the Human Cytomegalovirus (HCMV) Towne strain related to the present invention are both from American Type Culture Collection (ATCC).
Example 1 Effect of treatment with Violin alone on WI-38 cell proliferation Activity
Cell viability was measured by the MTT method as follows: the human embryonic lung diploid fibroblast WI-38 of PD30 was seeded in 96-well cell culture plates at a cell density of 3000 cells per well, and after 24h of culture, different concentrations of violaxyl were added, 3 parallel wells were provided for each concentration, and a drug-free blank control and a cell-free solvent control were provided. After 48 hours in the incubator, 20. mu.l of MTT (5mg/ml, prepared in serum-free DMEM medium) was added to each well at 37 ℃ with 5% CO2Culturing for 4h, completely sucking the liquid in the cell culture well, adding 150 μ l DMSO into each well, shaking gently on a shaking table for 10min to dissolve the crystal completely, measuring the light absorbance at 570nm, and calculating relative cell activity, wherein the cell activity of the blank control group without drug is 100%. The results of the experiment are shown in FIG. 1.
Example 2 Violin treatment and inoculation of HCMV
Human embryonic lung fibroblast WI-38 cells using PD30 in 2X 10 medium containing 10% FBS4/cm2The cell amount of (2) is plated on a six-well cell culture plate, after 24 hours, the culture medium of 0.2% FBS is replaced to continue culturing for 48 hours, and the cell cycle is synchronized with G0/G1 by a serum starvation method, so that HCMV infection is facilitated. Thereafter, inoculation of HCMV virus (Towne strain) was performed at an inoculation dose of 0.01MOI (multiplicity of infection), and the culture was continued for a prescribed period of time for relevant detection. When the anti-HCMV activity of the corydaline is detected, the corydaline with a certain concentration is added into a culture medium 2 hours ahead of time, then the HCMV (MOI 0.01) is inoculated, and further the changes of viral protein expression, DNA amplification and the like are observed.
Example 3Western-blot detection of the Effect of Violin on HCMV Virus protein expression
To confirm the anti-HCMV effect of corynolamine, we examined the effect of corynolamine on the expression of HCMV immediate early protein IE1/2 and early protein UL44, cell culture and corynolamine treatment and HCMV vaccination as described in example 2 above. Setting a control group which is not subjected to any drug treatment and only inoculated with HCMV; solvent DMSO treatment, HCMV inoculated control group, HCMV inoculated drug control group treated with HCMV and clinically sodium foscarnet (200. mu.g/ml), and corydalis edulis (10. mu.M) treated and HCMV inoculated experimental groups at different times. 1, 4 and 7 days after HCMV inoculation, supernatant is discarded, cells are washed with PBS three times, cell samples are collected after the cells are lysed by intermediate-strength RIPA lysate, protein concentration is measured by a BCA kit, and Western-blot detection is carried out by adopting corresponding IE1/2 and UL44 antibodies, and the result shows that the violaxalin can obviously inhibit the expression of HCMV virus proteins IE1/2 and UL44 (fig. 2A and 2B).
Example 4 Indirect immunofluorescence assay (IFA) to determine the Effect of Violin on HCMV viral protein expression
As described in example 2 above, 7 days after drug treatment and HCMV inoculation, the supernatant was discarded, the cells were washed three times with PBS, fixed with pre-cooled methanol acetone (1:1) at-20 ℃ for 20 minutes, then the fixing solution was discarded, and left to air-dry in a fume hood. The conventional IFA procedure with an antibody against IE1/2 protein, followed by photographing under an inverted fluorescence microscope, showed that corynoline was able to significantly reduce the expression of HCMV immediate early protein IE1/2 and was dose-dependent (fig. 3).
Example 5qPCR determination of the Effect of Violin on HCMV DNA copy number
Cell culture, drug treatment and HCMV inoculation treatment were as described in example 2 above. The copy number of HCMV DNA was determined by qPCR method, 10ng of total DNA was taken according to the iQ SYBR Green Supermix kit (Bio-Rad) kit instructions, and the primers used were: upstream 5-TCTGCCAGGACATCTTTCTC-3 ' (SEQ ID NO.1) and downstream 5'-GTGACCAAGGCCACGACGTT-3' (SEQ ID NO. 2). Amplification conditions: at 95 ℃ for 5min, (95 ℃ for 5sec, 60 ℃ for 30 sec). times.40 cycles, run through
The results of the calculation show that the corynoline can obviously inhibit the copy number of the virus DNA (figure 4).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are included in the scope of the present invention.
Claims (4)
1. Use of corynolamine as the sole active ingredient in the manufacture of a medicament for the prevention and treatment of human cytomegalovirus infection, said corynolamine including salt forms thereof.
2. Use according to claim 1, characterized in that corynoline is used for preventing and treating human cytomegalovirus infection by inhibiting the expression of HCMV immediate early protein IE1/2 and early protein UL 44.
3. The use of claim 1, wherein said corydalis comprises a pharmaceutically acceptable carrier.
4. The use of claim 1, wherein the preparation form of the medicament for preventing and treating human cytomegalovirus infection is tablet, granule, capsule, dripping pill or injection.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0568001A2 (en) * | 1992-04-27 | 1993-11-03 | Showa Shell Sekiyu Kabushiki Kaisha | Antiviral agent containing crude drug |
| JP2011231077A (en) * | 2010-04-30 | 2011-11-17 | Dainippon Jochugiku Co Ltd | Pest-controlling agent |
| CN108642121A (en) * | 2018-04-26 | 2018-10-12 | 浙江医院 | A kind of method and its application of the anti-human cytomegalovirus drug of screening |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104940301B (en) * | 2015-05-31 | 2019-08-06 | 黑龙江童医生儿童生物制药有限公司 | A kind of antiviral oral pellet gel of Pudilan children and preparation method thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0568001A2 (en) * | 1992-04-27 | 1993-11-03 | Showa Shell Sekiyu Kabushiki Kaisha | Antiviral agent containing crude drug |
| JP2011231077A (en) * | 2010-04-30 | 2011-11-17 | Dainippon Jochugiku Co Ltd | Pest-controlling agent |
| CN108642121A (en) * | 2018-04-26 | 2018-10-12 | 浙江医院 | A kind of method and its application of the anti-human cytomegalovirus drug of screening |
Non-Patent Citations (2)
| Title |
|---|
| "地格达类"蒙药研究进展;张春红等;《中国中药杂志》;20131215;第38卷(第24期);表1倒数第2行 * |
| Biologically active isoquinoline alkaloids with drug-like properties from the genus Corydalis;M.Iranshahy等;《RSC Adv.》;20130219;第2014卷(第4期);第15900-15913页 * |
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