CN114907346A - Alkaloid compound, extract and application thereof in preparation of products with respiratory syncytial virus resisting effect - Google Patents
Alkaloid compound, extract and application thereof in preparation of products with respiratory syncytial virus resisting effect Download PDFInfo
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- CN114907346A CN114907346A CN202210545561.4A CN202210545561A CN114907346A CN 114907346 A CN114907346 A CN 114907346A CN 202210545561 A CN202210545561 A CN 202210545561A CN 114907346 A CN114907346 A CN 114907346A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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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
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of pharmaceutical chemistry, and particularly discloses an alkaloid compound, an extract and application thereof in preparation of a product with an effect of resisting respiratory syncytial virus. The alkaloid compound has a structure shown in a formula I. The research shows that: the alkaloid compound with the structure shown in the formula I has very obvious activity of resisting respiratory syncytial virus, and the activity of resisting the respiratory syncytial virus is superior to that of a positive medicament ribavirin; therefore, the compound has important application value when being used as an active ingredient for preparing the medicine or the health care product with the effect of resisting the respiratory syncytial virus.
Description
Technical Field
The invention relates to the technical field of pharmaceutical chemistry, in particular to an alkaloid compound, an extract and application thereof in preparing a product with an effect of resisting respiratory syncytial virus.
Background
Human Respiratory Syncytial Virus (RSV) is an enveloped negative strand RNA virus of the paramyxoviridae family. Respiratory syncytial virus infection is the leading cause of respiratory disease, resulting in over 300 million respiratory syncytial virus-associated respiratory disease cases and 20 million respiratory syncytial virus-associated deaths worldwide each year. Currently, antiviral therapy is an important option for the treatment of respiratory syncytial virus infection. Ribavirin is an FDA approved antiviral drug for treatment of RSV infection and is a nucleoside analog directed to RNA transcription and replication. However, excessive use of such drugs has led to the emergence of drug-resistant strains. Therefore, there is a need to develop new alternative antiviral molecules against respiratory syncytial virus.
The semen Pegani Harmalae is dried mature seed of Peganum harmala L of Tribulus (Zygophylolaceae) of Peganum. The peganum harmala is mainly distributed in arid grassland and desert areas such as Xinjiang, inner Mongolia, Ningxia, Qinghai and Gansu. Peganum harmala seed is a famous drug with a long history of use and is listed in the drug Standard of Ministry of health of the people's republic of China. It is widely used as traditional Chinese medicine, Uighur medicine and Mongolian medicine for treating arthralgia, rheumatoid arthritis, sciatica, etc. Previous chemical component researches on peganum harmala seeds show that the plant contains a large amount of alkaloid components and has wide biological activities including antiviral activity, antitumor activity, antioxidant activity, antibacterial activity and the like. However, no alkaloid fraction isolated from Peganum harmala has been reported to have activity against Respiratory Syncytial Virus (RSV).
In addition, the structure and the variety of the alkaloid are various, and the antiviral effects of the alkaloids with different structures are completely different; for example, an alkaloid may have an anti-influenza effect, but it need not have an anti-respiratory syncytial virus effect; even though it has the effect of resisting the respiratory syncytial virus, the effect of the alkaloid with different structures on resisting the respiratory syncytial virus is different; especially, alkaloids with stronger respiratory syncytial virus resistance than ribavirin are rarely reported. Therefore, the alkaloid with the effect of resisting the respiratory syncytial virus, particularly the alkaloid with the effect of resisting the respiratory syncytial virus stronger than that of ribavirin, has important application value.
Disclosure of Invention
In order to overcome at least one technical problem in the prior art, the invention provides a brand new alkaloid compound; the alkaloid compound has excellent effect of resisting respiratory syncytial virus, and the effect of resisting the respiratory syncytial virus is stronger than that of ribavirin.
The technical scheme of the invention is as follows:
an alkaloid compound having the structure shown in formula I:
the alkaloid compound (abbreviated as PG) with the structure shown in the formula I is a brand new compound; the research shows that: IC of alkaloid compound with structure shown as formula I on respiratory syncytial virus 50 5.01 +/-0.14 mu M; is significantly less than the IC of the positive drug ribavirin for the respiratory syncytial virus 50 (8.71. + -. 0.95. mu.M); the alkaloid compound with the structure shown in the formula I has very obvious activity of resisting respiratory syncytial virus, and the activity of resisting the respiratory syncytial virus is obviously superior to that of a positive medicament ribavirin; this technical effect is unexpected by the person skilled in the art.
The invention also provides application of the alkaloid compound in preparation of a product with an effect of resisting respiratory syncytial virus.
The alkaloid compound with the structure shown in the formula I has excellent anti-respiratory syncytial virus activity; therefore, the compound has important application value when being used as an active ingredient for preparing products with the effect of resisting the respiratory syncytial virus.
Preferably, the product is a medicament or health product.
Preferably, the medicine or health care product contains a therapeutically effective amount of the compound with the structure shown in the formula I and a pharmaceutically acceptable carrier.
Preferably, the medicament or the health-care product is in the form of powder, pills, tablets, capsules, oral liquid, aerosol or injection.
The invention also provides an extract which contains the alkaloid compound with the structure shown in the formula I.
Preferably, the mass fraction of the alkaloid compound with the structure shown in the formula I in the extract is 1-80%.
Further preferably, the mass fraction of the alkaloid compounds with the structure shown in the formula I in the extract is 5-50%.
More preferably, the mass fraction of the alkaloid compounds with the structure shown in the formula I in the extract is 10-30%.
Preferably, the extract is prepared from peganum harmala serving as a raw material.
Because the alkaloid compound with the structure shown in the formula I has excellent effect of resisting respiratory syncytial virus; therefore, one skilled in the art would expect that the alkaloid compounds containing the structure shown in formula I would also have anti-respiratory syncytial virus activity.
The invention also provides application of the extract in preparing a product with the effect of resisting the respiratory syncytial virus.
Has the advantages that: the invention provides an alkaloid compound with a brand-new structure; the research shows that: the alkaloid compound with the structure shown in the formula I has very obvious activity of resisting respiratory syncytial virus, and the activity of resisting the respiratory syncytial virus is obviously superior to that of a positive medicament ribavirin; in addition, the SI value of the alkaloid compound with the structure shown in the formula I reaches 15.1, and the safety range is wider. Therefore, the compound has important application value when being used as an active ingredient for preparing the medicine or the health care product with the effect of resisting the respiratory syncytial virus.
Drawings
FIG. 1 shows the chemical structure of compound PG.
FIG. 2 is the molecular number of Compound PG.
FIG. 3 is a graph showing the results of an experiment on the inhibitory effect of compound PG on Respiratory Syncytial Virus (RSV); wherein, FIG. 3A shows the cytotoxic effect of compound PG on HEp-2; FIGS. 3B and 3C are graphs showing the inhibitory effect of compound PG and the positive control drug ribavirin on Respiratory Syncytial Virus (RSV) at various concentrations; figure 3D is the effect of compound PG on Respiratory Syncytial Virus (RSV) proliferation at different times; FIG. 3E shows the effect of immunofluorescence on the expression of F protein representative of viruses at various concentrations of compound PG.
Fig. 4 is a high resolution mass spectrum of compound PG.
FIG. 5 shows the UV spectrum (in methanol) of compound PG.
FIG. 6 shows an infrared spectrum (KBr pellet) of Compound PG.
FIG. 7 shows NMR of Compound PG 1 H spectrum (600MHz in DMSO).
FIG. 8 shows NMR of Compound PG 13 C spectrum (150MHz in DMSO).
FIG. 9 shows the nuclear magnetic resonance DEPT-135 spectrum (150MHz in DMSO) of compound PG.
FIG. 10 shows NMR of Compound PG 1 H- 1 H COSY spectra (150MHz in DMSO).
FIG. 11 shows the NMR HSQC spectra (150MHz in DMSO) of compound PG.
FIG. 12 shows the NMR HMBC spectra (150MHz in DMSO) of compound PG.
FIG. 13 shows the NOESY spectrum (150MHz in DMSO) of compound PG.
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method without specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition suggested by an instrument manufacturer.
Example 1
Pulverizing dried 15.0kg semen Pegani Harmalae, and cold extracting with 95% ethanol at room temperature to obtain total extract 1.6 kg. In order to enrich the alkaloid components in the extract, the total extract is treated by an acid-alkali precipitation method to obtain 378.0g of total alkaloid components. Subjecting the total alkaloid components to 200-mesh and 300-mesh silica gel column chromatography, and purifying with CH 2 Cl 2 Gradient MeOH system elution (1000:0 → 0:1), combined according to TLC inspection to give 7 fractions (Fr.A-G). Performing ODS column chromatography on Fr.D (27.5g) fraction with methanol/water (0:100 → 100:0) as mobile phase, performing gradient elution, combining according to TLC inspection to obtain 5 sub-fractions (Fr.D1-5), performing isocratic elution on Fr.D3(5.1g) with preparative HPLC (methanol-water-ammonia water as mobile phase at volume ratio of 60:40: 0.01), collecting retention time t R The fraction corresponding to the chromatographic peak at 16.2 min) to yield compound PG, an alkaloid compound of the structure shown in formula I.
The basis of structure identification:
the structure of compound PG was obtained by analysis of high resolution mass spectrometry (hreims), infrared spectroscopy (IR), one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy (NMR).
Determination of molecular weight 255.1126[ M + H ] by high resolution Mass Spectrometry HRESIMS: M/z] + (theoretical value: 255.1128) showing that the molecular formula is C 15 H 14 N 2 O 2 。
The data obtained by infrared spectroscopy showed the presence of an amino group (3441 cm) -1 ) And benzene rings (1625,1453 cm) -1 )。
TABLE 1 one-and two-dimensional NMR spectroscopic data (DMSO, delta in ppm, J in Hz) for Compound PG
Nuclear magnetic resonance hydrogen spectrum ( 1 H NMR) and assignments, see table 1. Data show that Compound PG has an AMX spin system [ delta ] H 8.15(1H,d,J＝8.7Hz),7.30(1H,d,J＝2.2Hz),6.91(1H,dd,J＝8.7,2.2Hz)]Two olefin proton signals [ delta ] H 8.44(1H,d,J＝4.9Hz),8.29(1H,d,J＝4.9Hz)]Hydrogen proton signal of one methoxy group [ delta ] H 3.86(3H,s)]And an ethyl proton signal [ delta ] H 3.33(2H,q,J＝7.3Hz),1.19(3H,t,J＝7.3Hz)]。
According to nuclear magnetic resonance carbon spectrum ( 13 C NMR) and assignments, see table 1. The data show that compound PG has 7 quaternary carbons (containing two vicinal oxygens), 5 tertiary carbons, 1 secondary carbon and 2 primary carbons (containing one vicinal oxygen), for a total of 15 carbon signals.
The data and the binding relationship of the two-dimensional nuclear magnetic resonance spectrum are shown in table 1.
The molecular number of compound PG is shown in FIG. 2.
EXAMPLE 2 test of PG Activity against respiratory syncytial Virus for Compound
1. The cytotoxicity of the compound was determined by MTT method, which was 1.5X 10 4 The individual cells were plated in 96-well plates at 37 ℃ in 5% CO 2 Culturing for 18h in an incubator;
2. agar overlay cultures containing different concentrations of compound (100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M) were added;
3. after 48h of cell culture, treating for 4h by using an MTT method;
4. subsequently, the supernatant was removed, and then dimethyl sulfoxide (DMSO) was added to dissolve blue-violet crystalline formazan;
5. absorbance was measured at 570nm and Cytotoxic Concentration (CC) of 50% was obtained by nonlinear regression analysis of cell viability 50 )；
6. Testing the antiviral activity of the compound at a first concentration of maximum non-cytotoxic concentration (MNCC);
7. cell suspension (1.5X 10 per well) 4 Individual cells) were seeded in each well of a 96-well plate;
after 8.18h, monolayer HEp-2 cells were treated with RSV virus and varying concentrations of compound PG and cultured for 72 h;
9. observing the pathological change degree of syncytia of each hole by a microscope, and evaluating the concentration of inhibiting half number of CPE by observation, namely the half maximum Inhibition Concentration (IC) 50 )。
10. Monolayer HEp-2 cells were treated with a mixture of equal volumes of RSV virus (MOI ═ 0.1) and 40 μ M compound PG. Ribavirin (40 μm) was used as a positive control. After 24h of culture, HEp-2 cells were stained with an anti-RSV F antibody and the fluorescence intensity of the cells was observed by a fluorescence microscope.
TABLE 2 anti-RSV Activity of Compound PG
As can be seen from the experimental data in Table 2, the IC of the compound PG with the structure shown in the formula I on the respiratory syncytial virus 50 5.01 +/-0.14 mu M; far less than the IC of positive drug ribavirin for respiratory syncytial virus 50 (8.71. + -. 0.95. mu.M); the compound PG with the structure shown in the formula I has very obvious activity of resisting respiratory syncytial virus, and the activity of resisting the respiratory syncytial virus is obviously superior to that of a positive medicament ribavirin; this technical effect is unexpected by the person skilled in the art.
In addition, as can be seen from the experimental data in table 2, the SI value of the compound PG against the respiratory syncytial virus reaches 15.1, which is far greater than the minimum standard 1.00, which indicates that the compound PG has a greater safety range when being used for preparing the medicament for resisting the respiratory syncytial virus.
Further, FIG. 3A shows cytotoxicity (CC) of Compound PG against Hep-2 cells 50 ) 75.81. + -. 1.75 μm. The tolerance of compound PG to high titer viral infection was evaluated by infecting Hep-2 cells with different titers of RSV virus (MOI ═ 0.1,1, or 5) (fig. 3B). Plaque inhibition assay then tested compound PG for inhibition of respiratory syncytial virus at non-toxic concentrations (fig. 3C). The results show that the compound PG and RSV viruses act on Hep-2 cells simultaneously, can obviously reduce the virus yield and have concentration dependence. The virus production assay (figure 3D) showed that,on day 3 post Respiratory Syncytial Virus (RSV) infection, progeny virus numbers reached a plateau. The compound PG (40 μm) has obvious inhibition effect on Respiratory Syncytial Virus (RSV) within 1d-4 d. These data indicate that the anti-respiratory syncytial virus effect of compound PG is not due to cytotoxicity. Green fluorescence represents the F protein of the virus, with high levels of F protein observed in the virus group. The green fluorescence associated with respiratory syncytial virus infection was significantly reduced upon addition of compound PG (fig. 3E). According to the data, different titers of compound PG can be shown to remarkably inhibit respiratory syncytial virus infection.
It will be appreciated by those skilled in the art that the use of the present invention is not limited to the specific applications described above. The invention is also not limited to the preferred embodiments thereof with respect to the specific elements and/or features described or depicted herein. It should be understood that the invention is not limited to the disclosed embodiment or embodiments, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.
Claims (10)
1. An alkaloid compound having a structure according to formula I:
2. use of the alkaloid compounds of claim 1 for the preparation of a product having an anti-respiratory syncytial virus effect.
3. The use according to claim 2, wherein the product is a pharmaceutical or nutraceutical product.
4. The use of claim 3, wherein the medicament or nutraceutical comprises a therapeutically effective amount of a compound having the structure of formula I and a pharmaceutically acceptable carrier.
5. The use of claim 3, wherein the pharmaceutical or nutraceutical is in the form of powder, pill, tablet, capsule, oral liquid, aerosol, or injection.
6. An extract characterized by containing an alkaloid compound having a structure represented by formula I.
7. The extract as claimed in claim 6, wherein the mass fraction of the alkaloid compounds with the structure shown in formula I in the extract is 1-80%.
8. The extract as claimed in claim 7, wherein the mass fraction of the alkaloid compound with the structure shown in formula I in the extract is 5-50%;
more preferably, the mass fraction of the alkaloid compounds with the structure shown in the formula I in the extract is 10-30%.
9. The extract according to claim 6, wherein the extract is prepared from harmel seeds.
10. Use of the extract according to any one of claims 6 to 9 for the preparation of a product having an anti-respiratory syncytial virus effect.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104838254A (en) * | 2012-11-30 | 2015-08-12 | 锡克拜控股有限公司 | Marking of material, marked material and process of authentication or dilution determination |
| CN112135613A (en) * | 2018-03-20 | 2020-12-25 | 西奈山伊坎医学院 | Kinase inhibitor compounds and compositions and methods of use |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104838254A (en) * | 2012-11-30 | 2015-08-12 | 锡克拜控股有限公司 | Marking of material, marked material and process of authentication or dilution determination |
| CN112135613A (en) * | 2018-03-20 | 2020-12-25 | 西奈山伊坎医学院 | Kinase inhibitor compounds and compositions and methods of use |
Non-Patent Citations (5)
| Title |
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| CHUN WU 等: "Three new alkaloids from the roots of Sophora tonkinensis", 《JOURNAL OF NATURAL MEDICINES》 * |
| KAI-BO WANG 等: "Structurally Diverse Alkaloids from the Seeds ofPeganum harmala", 《JOURNAL OF NATURAL PRODUCTS》 * |
| SHI-FEI LI 等: "b -Carboline alkaloids from the leaves ofTrigonostemon lii Y.T. Chang", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| YADI YANG 等: "Potent AChE and BChE inhibitors isolated from seeds of Peganum harmala Linn by a bioassay-guided fractionation", 《JOURNAL OF ETHNOPHARMACOLOGY》 * |
| ZHONG-NAN WU 等: "β‑Carboline Alkaloids from the Seeds of Peganum harmala and Their Anti-HSV ‑ 2 Virus Activities", 《ORGANIC LETTERS》 * |
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