Application of alkaloid compound in preparation of product with effect of resisting type II herpes simplex virus

Abstract

The invention relates to the technical field of medicinal chemistry, and particularly discloses application of an alkaloid compound in preparation of a product with a herpes simplex virus type II resisting effect. The alkaloid compound has a structure as shown in a formula I which is described in the specification. Researches show that the alkaloid compound with the structure as shown in the formula I has very remarkable anti-II-type herpes simplex virus activity, and the anti-II-type herpes simplex virus activity of the alkaloid compound is superior to that of a positive drug acyclovir; therefore, the compound has important application value when being used as an active ingredient for preparing medicines or health-care products with the effect of resisting the II-type herpes simplex virus.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemistry, and in particular to the application of an alkaloid compound in preparing a product having an anti-type II herpes simplex virus effect. Background Art

[0002] Herpes simplex virus type II (HSV-2) is a linear double-stranded DNA virus of the alphavirus subfamily of the herpesviridae family. It can easily cause chronic, inflammatory, recurrent, and difficult-to-cure sexually transmitted diseases, namely genital herpes, by infecting the skin and mucous membranes of the urinary tract, genitals, and perianal areas. According to WHO statistics, 536 million people aged 15 to 49 are infected with HSV-2 worldwide, and there are as many as 23 million new cases of infection each year. Both men and women are likely to be infected, with the HSV-2 infection rate in women as high as 30% to 80%, and in men 10% to 50%. In addition, the damage to the genital mucosa caused by HSV-2 virus infection can increase the risk of infection with other pathogens such as HIV and Treponema pallidum. In clinical practice, acyclovir is the main antiviral drug used for genital herpes. However, long-term use of this drug in clinical practice causes mutations in the UL23 gene encoding thymidine kinase, resulting in the emergence of drug-resistant virus variants. More and more patients have developed resistance to acyclovir, resulting in unsatisfactory treatment effects. Therefore, it is necessary to develop new alternative drugs against herpes simplex virus type 2.

[0003] Natural medicines have the advantages of wide range of effects, safety and reliability, and are gradually becoming a hot spot for research on antiviral drugs. Peganum harmala L. is the dried mature seeds of the plant Peganum harmala L. of the Tribulus family. It is also known as bitter vegetable, sand bean bean, Adi Resmang (Uyghur medicine name), Umushi-Ubus (Mongolian medicine name), etc. Peganum harmala has a long history of being used as medicine. It has the effects of promoting lung qi, relieving cough and asthma, removing rheumatism, and reducing swelling and toxins. It is clinically used to treat cough and asthma, rheumatic pain, unidentified swelling and toxins, and skin itching. Modern pharmaceutical research has shown that Peganum harmala extract has multiple pharmacological activities such as antiviral, anti-tumor, antibacterial, anti-inflammatory, and antioxidant. The rich alkaloid components are the main pharmacological material basis for its pharmacological effects. Summary of the invention

[0004] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides a new alkaloid compound; the alkaloid compound has a very excellent anti-herpes simplex virus type II effect, and its anti-herpes simplex virus type II effect is stronger than acyclovir.

[0005] The technical solution of the present invention is as follows:

[0006] An alkaloid compound having a structure shown in Formula I:

[0007]

[0008] The alkaloid compound of the structure shown in formula I (abbreviated as PF) is a new compound; studies have shown that the IC of the alkaloid compound of the structure shown in formula I against respiratory syncytial virus 50 0.90±0.10μM; significantly lower than the IC of positive drug acyclovir against respiratory syncytial virus 50 (1.12±0.15μM); This indicates that the alkaloid compound of the structure shown in Formula I has a very significant anti-type II herpes simplex virus activity, and its anti-type II herpes simplex virus activity is significantly better than the positive drug acyclovir; this technical effect is unpredictable by those skilled in the art.

[0009] The present invention also provides a method for preparing the alkaloid compounds, which uses Salvia salviae chinensis as a raw material and extracts and separates the alkaloid compounds therefrom.

[0010] The present invention separates the alkaloid compound with the structure shown in formula I from the fruit of camel thorn for the first time, and provides a brand-new method for preparing the alkaloid compound with the structure shown in formula I.

[0011] Preferably, the preparation method comprises the following steps:

[0012] (1) taking Peganum harmala seeds and crushing them, and cold-immersing them with ethanol to obtain a total extract;

[0013] (2) subjecting the extract to an acid extraction and alkali precipitation treatment step to obtain the total alkaloid component;

[0014] (3) separating the total alkaloids on a D101 macroporous resin column to obtain a macroporous resin elution fraction;

[0015] (4) subjecting the macroporous resin elution fraction to an ODS column for separation to obtain an ODS elution fraction;

[0016] (5) The ODS eluted fraction was subjected to HPLC again to prepare the alkaloid compound with the structure shown in Formula I.

[0017] The present invention also provides an extract containing an alkaloid compound with a structure shown in formula I.

[0018] Since the alkaloid compound with the structure shown in Formula I has excellent anti-herpes simplex virus type II effect, those skilled in the art can expect that the extract containing the structure shown in Formula I also has anti-herpes simplex virus type II effect.

[0019] Preferably, the mass fraction of the alkaloid compound with the structure shown in formula I in the extract is 1% to 80%.

[0020] Preferably, the mass fraction of the alkaloid compound of the structure shown in formula I in the extract is 5% to 50%;

[0021] More preferably, the mass fraction of the alkaloid compound with the structure shown in formula I in the extract is 10% to 30%.

[0022] Preferably, the extract is an extract prepared using Salvia salsa as raw material.

[0023] The present invention also provides an application of the above alkaloid compound or extract in preparing a product having an anti-type II herpes simplex virus effect.

[0024] Preferably, the product is a medicine or a health product.

[0025] Preferably, the medicine or health product contains a therapeutically effective amount of a compound having a structure shown in Formula I and a pharmaceutically acceptable carrier.

[0026] Preferably, the dosage form of the medicine or health product is powder, pill, tablet, capsule, oral liquid, aerosol or injection.

[0027] Beneficial effects: The present invention provides an alkaloid compound with a completely new structure; studies have shown that the alkaloid compound with the structure shown in formula I has a very significant anti-type II herpes simplex virus activity, and its anti-type II herpes simplex virus activity is significantly better than the positive drug acyclovir; in addition, the SI value of the alkaloid compound with the structure shown in formula I reaches 86.2, which has a larger safety range. Therefore, it has important application value to use it as an active ingredient to prepare drugs or health products with anti-type II herpes simplex virus effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the chemical structural formula of compound PF.

[0029] Figure 2 is the molecular number of compound PF.

[0030] Figure 3 The figure is the experimental result of the inhibitory effect of compound PF on herpes simplex virus type Ⅱ (HSV-2); Figure 3 A is the cytotoxic effect of compound PF on Vero cells; Figure 3 B and Figure 3 C is the inhibitory effect of compound PF and positive control drug acyclovir on herpes simplex virus type Ⅱ (HSV-2) at different concentrations; Figure 3 D is the effect of compound PF on the proliferation of herpes simplex virus type Ⅱ (HSV-2) at different times;

[0031] Figure 4This is the high-resolution mass spectrum of compound PF.

[0032] Figure 5 This is the UV spectrum of compound PF (dissolved in methanol).

[0033] Figure 6 This is the infrared spectrum of compound PF (KBr pellet).

[0034] Figure 7 The NMR of compound PF 1 H spectrum (600 MHz, dissolved in DMSO).

[0035] Figure 8 The NMR of compound PF 13 C spectrum (150 MHz, dissolved in DMSO).

[0036] Fig. 9 The DEPT-135 nuclear magnetic resonance spectrum of compound PF (150 MHz, dissolved in DMSO) is shown in FIG.

[0037] Fig.10 The NMR of compound PF 1 H- 1 H COSY spectrum (150 MHz, dissolved in DMSO).

[0038] Fig.11 The NMR HSQC spectrum of compound PF (150 MHz, dissolved in DMSO) is shown in FIG.

[0039] Fig.12 The HMBC NMR spectrum of compound PF (150 MHz, dissolved in DMSO).

[0040] Fig.13 The nuclear magnetic resonance NOESY spectrum of compound PF (150 MHz, dissolved in DMSO). DETAILED DESCRIPTION

[0041] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0042] Example 1

[0043] (1) 15.0 kg of dried Peganum harmala seeds were taken and crushed, and cold-extracted with 95% ethanol to prepare a total extract of 1.6 kg;

[0044] (2) In order to further concentrate the alkaloid components, the total extract was subjected to the "acid extraction and alkali precipitation" treatment step, and finally 378 grams of total alkaloid components were obtained; the "acid extraction and alkali precipitation" step is specifically as follows: the total extract was dispersed in 2L of water, the pH of the solution was adjusted to 2 with dilute hydrochloric acid, and then two volumes of dichloromethane were added, and the extract was extracted three times, and the extract was discarded. Then, the pH of the acid solution was adjusted to 9 with 25% ammonia water, and then two volumes of dichloromethane were added, and the extract was extracted three times to obtain the extract, i.e., the total alkaloids;

[0045] (3) The total alkaloids were loaded onto a D101 macroporous resin column, and eluted with a mixed solvent of ethanol / water in a volume ratio of 10:90, and the eluate was discarded; then eluted with a mixed solvent of ethanol / water in a volume ratio of 30:70, and the eluate was collected, concentrated and dried to obtain a macroporous resin elution fraction;

[0046] (4) The macroporous resin elution fraction is loaded onto an ODS column, and first eluted with a methanol / water solvent having a volume ratio of 80:20, and the eluate is discarded; then eluted with a methanol / water solvent having a volume ratio of 90:10, and the eluate is collected, concentrated, and dried to obtain an ODS elution fraction;

[0047] (5) The ODS eluted fraction was subjected to HPLC again (using a C18 reverse phase preparative column, methanol-water-ammonia water as the mobile phase, and isocratic elution at a volume ratio of methanol: water: ammonia water = 38:62:0.01). The retention time t R =21.2min) to prepare compound PF, i.e., an alkaloid compound with a structure shown in formula I.

[0048] Structural identification based on:

[0049] The structure of compound PF was obtained by analyzing high resolution mass spectrometry (HRESIMS), infrared spectroscopy (IR), one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy (NMR).

[0050] Molecular weight determined by high resolution mass spectrometry HRESIMS: m / z 355.1276 [M+Na] + (Theoretical value: 355.1264), showing the molecular formula is C 17 H 20 N2O5.

[0051] The infrared spectroscopy showed that there was an amino group (3392 cm -1 ) and benzene ring (1612,1464cm -1 ).

[0052] Table 1. 1D and 2D NMR spectra of compound PF (DMSO, δ in ppm, J in Hz)

[0053]

[0054] H NMR ( 1 H NMR) data and attribution are shown in Table 1. The data show that compound PF has an AMX spin system [δ H 7.20 (1H, d, J = 8.2), 6.52 (1H, dd, J = 8.2, 2.0), 6.39 (1H, d, J = 2.0)], a methoxyl hydrogen proton signal [δ H 3.86 (3H, s)] and two methyl proton signals [δ H 1.14(1H,s),1.12(1H,s)].

[0055] According to the carbon nuclear magnetic resonance spectrum ( 13 C NMR) data and attributions are shown in Table 1. The data show that compound PF has 8 quaternary carbons (including five connected oxygens), 3 tertiary carbons, 3 secondary carbons and 3 primary carbons (including one connected oxygen), for a total of 17 carbon signals.

[0056] The data of 2D NMR spectra and the binding relationships are shown in Table 1 .

[0057] The molecular number of compound PF is as follows Figure 2 shown.

[0058] Experimental Example 2: Anti-Herpes Simplex Virus Type II Activity Test of Compound PF

[0059] 1. The cytotoxicity of the compound was determined by MTT assay. 1.5×10 4 The cells were placed in a 96-well plate and cultured at 37°C in a 5% CO2 incubator for 18 h;

[0060] 2. Add agar overlay containing different concentrations of compounds (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM) for cultivation;

[0061] 3. After 48 hours of cell culture, the cells were treated with MTT method for 4 hours;

[0062] 4. Then, remove the supernatant and add dimethyl sulfoxide (DMSO) to dissolve the blue-purple crystalline formazan;

[0063] 5. Measure the absorbance at 570 nm and obtain the 50% cytotoxic concentration (CC) based on the nonlinear regression analysis of cell survival rate. 50 );

[0064] 6. Using the maximum non-cytotoxic concentration (MNCC) as the first concentration, the antiviral activity of the compound was detected;

[0065] 7. Cell suspension (1.5×10 per well 4 cells) were seeded into each well of a 96-well plate;

[0066] 8. After 18 hours, the monolayer Vero cells were treated with HSV-2 virus and different concentrations of compound PF and cultured for 72 hours;

[0067] 9. Observe the degree of syncytial pathology in each well under a microscope and evaluate the concentration that inhibits half of the CPE, which is the half-maximal inhibitory concentration (IC 50 ).

[0068] 10. Finally, the selectivity index (SI = CC) of the compound for inhibiting HSV-2 was calculated. 50 / IC 50 ).

[0069] Table 2. Anti-herpes simplex virus activity of compound PF

[0070]

[0071] From the experimental data in Table 2, it can be seen that the IC of the compound PF shown in Formula I against respiratory syncytial virus 50 0.90±0.10μM; less than the IC of positive drug acyclovir against respiratory syncytial virus 50 (1.12±0.15μM); This indicates that the compound PF with the structure shown in Formula I has a very significant anti-type II herpes simplex virus activity, and its anti-type II herpes simplex virus activity is significantly better than the positive drug acyclovir; this technical effect is unpredictable by those skilled in the art.

[0072] In addition, it can be seen from the experimental data in Table 2 that the SI value of compound PF against respiratory syncytial virus reached 86.2, which is far greater than the minimum standard of 10.0. This shows that the use of compound PF in the preparation of drugs against type II herpes simplex virus has a wider safety margin.

[0073] Further, Figure 3 A shows the cytotoxicity of compound PF to Vero cells (CC 50 ) was 77.59±1.33μM. Vero cells were infected with HSV-2 virus of different titers (MOI=0.1, 1 or 5) to evaluate the tolerance of compound PF to high titer virus infection ( Figure 3 B). Then, the plaque inhibition assay was performed to test the inhibitory effect of compound PF on RSV at a non-toxic concentration ( Figure 3 C). The results showed that compound PF and HSV-2 virus acted on Vero cells simultaneously, which could significantly reduce the virus yield in a concentration-dependent manner. Virus yield determination ( Figure 3D) shows that the number of progeny viruses reaches a plateau on the third day after infection with herpes simplex virus type Ⅱ (HSV-2). Compound PF (40μM) has a significant inhibitory effect on herpes simplex virus type Ⅱ (HSV-2) within 1d-4d. These data show that compound PF at different titers can significantly inhibit herpes simplex virus type Ⅱ infection.

[0074] It will be appreciated by those skilled in the art that the use of the present invention is not limited to the above-mentioned specific applications. The present invention is not limited to its preferred embodiments with respect to the specific elements and / or features described or depicted herein. It should be understood that the present invention is not limited to the disclosed embodiment examples or each embodiment, and that many rearrangements, modifications and substitutions can be made without departing from the scope of the present invention as set forth and defined by the following claims.

Claims

1. An alkaloid compound, characterized in that It has the structure shown in Formula I:

2. The method for preparing the alkaloid compound according to claim 1, characterized in that: It is obtained by extracting and separating Camel Pteris cochinchinensis from Camel Pteris cochinchinensis.

3. An extract, characterized in that An alkaloid compound having a structure shown in formula I.

4. The extract according to claim 3, characterized in that The mass fraction of the alkaloid compound with the structure shown in formula I in the extract is 1% to 80%.

5. The extract according to claim 4, characterized in that The mass fraction of the alkaloid compound of the structure shown in formula I in the extract is 5% to 50%; Further preferably, the mass fraction of the alkaloid compound with the structure shown in formula I in the extract is 10% to 30%.

6. The extract according to claim 3, characterized in that The extract is an extract prepared by taking camel thorn fruit as raw material.

7. Use of the alkaloid compound according to claim 1 or the extract according to any one of claims 3 to 6 in the preparation of a product having an anti-herpes simplex virus type II effect.

8. The use according to claim 7, characterized in that: The product is a medicine or a health product.

9. The use according to claim 7, characterized in that: The medicine or health product contains a therapeutically effective amount of a compound having a structure shown in formula I and a pharmaceutically acceptable carrier.

10. The use according to claim 7, characterized in that: The dosage form of the medicine or health product is powder, pill, tablet, capsule, oral liquid, aerosol or injection.

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