CN116637118A - Astragalin L, extracts including the compound, and use of the two as antiviral drugs - Google Patents

Abstract

The invention provides application of a known compound, namely, a white grass glycoside-free L (lobatosideL), in preparing an antiviral medicament, and discloses an zygotic ethanol extract containing the white grass glycoside-free and antiviral application of the zygotic ethanol extract. The zygotic grass ethanol extract and the zygotic grass extract provided by the invention have antiviral activity to H1N1 (A/PuertoRico/8/34; PR8) viruses through the measurement of a CCK-8 method.

Description

No astragalin L, extracts including this compound, and both as antiviral agents the use of

technical field

The invention belongs to the field of antiviral drugs, and in particular relates to a kind of astragaloside L, an extract including the compound, and the use of the two as antiviral drugs.

Background technique

Zygomyces, also known as zygote grass, celestial grass, no white grass, etc., is the whole grass, seeds and leaves of Actinostemmalobatum (Maxim.) Maxim., a plant of Cucurbitaceae. It is distributed in the north and south of my country, Korea, Japan, the Soviet Union , India, and Indochina Peninsula are also distributed. Zygonia is cold in nature, bitter in taste, slightly poisonous, diuretic, detumescent, heat-clearing and detoxifying, and dehumidifying.

Patent (CN108888649A) discloses the saponin extract extracted from Zygmus chinensis (the chemical structure of the specific components and molecules is not disclosed), and it is believed that this type of extract has anti-tumor activity, and the patent (CN102432666A) discloses the extraction of A method for isolating a compound without astragaloside L and disclosing the use of the compound in the preparation of antitumor drugs.

However, in the prior art, there is no report about the application of the extract and active ingredients of Zygonia chinensis in antiviral aspects.

Contents of the invention

For above technical problem, the present invention proposes a kind of compound as the purposes of preparing antiviral drug, and described compound has the chemical structure as formula (1):

The compound of formula (1) is no arugalin L.

The present invention also proposes an antiviral application of the extract of Zygmus chinensis, which comprises the compound of formula (1).

The present invention also proposes an antiviral use of the Zygomyces extract, which is the Zygosia ethanol extract prepared by the step of refluxing the Zygosia raw material medicine through 72-78% ethanol solvent extraction.

The present invention also proposes an antiviral use of the Zygomyces extract, which is prepared by the step of carrying out the steps of polar gradient extraction of the Zygothemus ethanol extract through organic solvents of different polarities. things.

The present invention also proposes an antiviral use of the Zygonia extract. The Zygogo extract is the Zygogo extract prepared through the steps of separating and purifying the Zygogo extract by high-performance liquid chromatography.

The present invention also proposes a preparation for preparing antiviral drugs, which includes the above-mentioned one or more extracts of Zygonia chinensis and pharmaceutical auxiliary materials.

The zygosinia ethanol extract, zygosinia extract and zygosinia essence extract all have antiviral activity to H1N1 (A/PuertoRico/8/34; PR8) virus after being measured by CCK-8 method.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

The raw materials of zygosinia used in the embodiments of the present invention include fresh or dried whole herb, seeds and leaves of zygosinia. A preferred embodiment uses dry feedstock.

Example 1 Preparation of Ethanol Extract of Zygonia chinensis

Example 1-1

Take 1.0 kg of the whole herb of Zygia chinensis, reflux extraction with 75% ethanol for 3 times, each time for at least 2 hours, combine the extracts, recover the ethanol under reduced pressure, and obtain 650 g of Zygosinia ethanol extract.

Example 1-2

Take 1.0 kg of the whole herb of Zygia chinensis, reflux extract with 72% ethanol for 3 times, each time for at least 2 hours, combine the extracts, recover the ethanol under reduced pressure, and obtain 665 g of Zygosinia ethanol extract.

Example 1-3

Take 1.0 kg of the whole herb of Zygia chinensis, reflux extraction with 78% ethanol for 3 times, each time for at least 2 hours, combine the extracts, recover ethanol under reduced pressure, and obtain 675 g of Zygosinia ethanol extract.

The moisture content of the ethanol extract of Zygonia chinensis in Example 1 is 20-25%.

Example 2 Preparation of Zygmus chinensis extract

Zygomyces ethanol extract is prepared by the steps of polar gradient extraction with organic solvents of different polarities. Specifically, the Zygothemus alcohol extract obtained in Example 1-1 is suspended in water, and sequentially Extracted three times with equal volume of petroleum ether, ethyl acetate and n-butanol, combined the extracts with different polarities and recovered the corresponding solvent to obtain 20 g of Zygmus chinensis extract.

Embodiment 3 Zygmus chinensis extract

Zygomyces chinensis extract is subjected to high performance liquid chromatography to carry out the step of separation and purification of Zygomyces chinensis essence extract, specifically, carry out silica gel column chromatography after mixing the sample of 20 g of Zygomyces chinensis extract prepared in Example 2, dichloromethane - methanol-water (7:1:1-7:5:1) was eluted to obtain fraction 1-6; fraction 6 was subjected to silica gel column chromatography, dichloromethane-methanol (2:1) was eluted and separated to obtain 6.3; Fraction 6.3 was separated by gel chromatography to obtain fraction 6.3.1 and fraction 6.3.2, and fraction 6.3.2 was prepared again by high performance liquid phase to obtain the separated and purified Zygmus chinensis extract.

Next, the chemical structure of the obtained Zygmus chinensis extract was determined.

First, it was found to be recrystallized in methanol solvent as a white powder, mp. 320-322°C, [α] ²⁰ _D — 76° (c0.25, MeOH). 10% sulfuric acid ethanol solution was blue, Liebermann-Burchard reaction was positive, and Molish reaction was positive. The above phenomenon suggested that it might be a triterpenoid saponin compound. Glucose, arabinose and rhamnose were detected by 10% sulfuric acid hydrolysis. HR-ESI-MS gives: m/z1417.6249[M+Na]+ quasi-molecular ion peak (C65H102O32Na+, calculated value is 1417.6252), combined with NMR data, it can be speculated that its molecular formula is C65H102O32. In the infrared spectrum, there are strong absorption peaks at 3442cm-1 and 1734cm-1, indicating that there are hydroxyl groups and carbonyl groups in the structure.

In the 1H-NMR (600MHz, C5D5N) spectrum, the signals of the 8 methyl protons on the structure are 0.98 (3H, s), 1.02 (3H, s), 1.32 (3H, s), 1.44 (3H, d, J =6.0Hz), 1.52(3H, s), 1.69(3H, s), 1.88(3H, s), 1.92(3H, s); 1 hydrogen proton signal δ5.64(1H, br.s); Signals of 5 sugar anomeric protons δ5.12(1H, d, J=7.8Hz), 5.29(1H, d, J=7.2Hz), 5.98(1H, d, J=7.2Hz), 6.22(1H, br .s) and 5.20 (1H, d, J = 7.8 Hz). According to the cleavage of the methyl group and the analysis of 13C-NMR data, it is inferred that the extract of Zygonia chinensis is an oleanane-type pentacyclic triterpene saponin.

The 13C-NMR (150MHz, C5D5N) spectrum gives a total of 65 carbon signals, of which 30 carbons are carbon signals on the oleanane core structure, 29 carbons are carbon signals on sugar, and 6 carbons are 3- Hydroxy-3-methylglutarate group signal. Among the 30 carbon signals on the core structure, there are 6 methyl carbon signals δ15.4(C-24), 17.6(C-25), 17.8(C-26), 27.4(C-27), 33.2( C-29), 24.4 (C-30); 4 oxycarbon signals 70.2 (C-2), 83.1 (C-3), 73.5 (C-16), 64.5 (C-23); 2 olefinic carbons Signal δ 123.4 (C-12), 145.3 (C-13); 1 ester carbonyl signal δ 176.4 (C-28). Combining with 1H-NMR, comparing the above data with the data of polygalacic acid reported in the literature, except for the differences in the chemical shifts of the 2-position, 3-position and 28-position, the other data are basically consistent. Therefore, it is determined that in the molecular structure of the extract of Zygonia chinensis, the parent nucleus of aglycone is polygalacic acid, and according to the law of glycosylation displacement, it can be determined that the sugar group is substituted at the 3-position and 28-position. Among the 29 sugar signals, there are 23 oxycarbon signals δ84.2, 78.9, 78.0, 78.0, 77.9, 77.8, 77.3, 77.3, 77.3, 75.9, 75.0, 74.9, 73.5, 72.2, 71.6, 71.1, 71.0, 69.5, 68.1, 67.5, 65.1, 62.7, 62.5; 5 dioxo-carbon signals δ105.8, 105.6, 103.8, 102.6, 94.7; 1 methyl carbon signal δ18.5, which is the 6-carbon signal of rhamnose. Combining the corresponding sugar anomeric proton signals in 1H-NMR with the results of acid hydrolysis, it can be determined that there are three glucose, one rhamnose and one arabinose in the extract of Zygomycinia chinensis. The carbon signals of the three groups of glucose are Glc: δ103.8, 84.2, 77.3, 71.6, 78.0, 62.7; Glc': δ105.6, 77.3, 78.0, 71.1, 75.9, 65.1; Glc": δ105.8, 74.9, 77.9 , 71.0, 77.8, 62.5; the carbon signals of a group of rhamnose are δ102.6, 72.2, 78.9, 73.5, 68.1, 18.5; the carbon signals of a group of arabinose are δ94.7, 77.3, 75.0, 69.5, 67.5. The 6 carbon signals of 3-hydroxy-3-methylglutarate group are δ171.4(C-1'), 47.1(C-2'), 70.3(C-3'), 47.1(C-4 '), 171.9 (C-5'), 25.9 (C-6'). The carbon spectrum data of this zygosinum extract is compared with the data of the reported compound Tubeimoside III (tubeimoside III) in the literature, and find Except for the data of the outer xylose, other data are basically consistent. Therefore, it is determined that the connection of the inner sugar of this compound is consistent with tubeimosideⅢ, the difference is that the outer xylose is replaced by glucose.

In the HMBC spectrum, the anomeric proton signal δ5.12 (1H, d, J=7.8Hz, Glc H-1) of glucose is remotely correlated with the 3-position δ83.1 (C-3) signal of the mother nucleus, and the Glc' The terminal group proton signal δ5.29 (1H, d, J=7.2Hz, Glc'H-1) has a long-range correlation with the 2-position carbon signal δ84.2 (GlcC-2) of Glc, indicating that Glc is connected to the C of the mother nucleus At the -3 position, Glc' is connected to the 2 position of Glc; the anomeric proton signal of arabinose is δ5.98 (1H, d, J=7.2Hz, AraH-1) and the C-28 position of the mother nucleus is δ176.4 The signals are remotely correlated, indicating that Ara is connected to the C-28 position of the mother nucleus; the terminal group proton signal of Rha is δ6.22 (1H, br.s, RhaH-1) and the 2-position of Ara is δ77.3 (AraC-2 ) signals have a long-range correlation, indicating that Rha is linked to AraC-2; the anomeric proton signal of Glc” δ5.20 (1H, d, J=7.8Hz, Glc”H-1) and the 3-position carbon signal of Rha δ78.9 (RhaC-3) has a long-range correlation, indicating that Glc" is connected to the C-3 position of Rha; the 6-bit proton signal of Glc' is δ5.02 (1H, d, J=11.4Hz, Glc'H-6 ) is remotely correlated with the 1-position carbon signal δ171.4 (C-1') of the hydroxymethylglutarate group, and the 4-position proton signal of Rha is δ6.05 (1H, t, J=9.6Hz, RhaH-4) There is a long-range correlation with the 5' carbon signal δ171.9 (C-5') of the hydroxymethylglutarate group, indicating that the two ends of the 3-hydroxy 3-methylglutarate group (1' and 5' ) are connected respectively on the 6-position of Glc' and the 4-position of Rha. The absolute configuration of sugar can be determined by gas chromatography. This compound is carried out acid hydrolysis, derivatization, and its gas chromatography is compared with standard sugar derivatives, Proved to be D-glucose, L-rhamnose and L-arabinose, respectively, combined with their chemical shift values and coupling constants for terminal hydrogens identified as β-D-glucose, α-L-rhamnose and α-L, respectively -Arabic candy.

In summary, the structure of the compound is identified as lobatoside L, and the structure is formula (1):

Test Example 1 Antiviral Test

The CCK-8 method was used to measure the activity of the following test items on H1N1 (A/Puerto Rico/8/34; PR8) virus respectively.

Need testing product: the compound that the zygote grass ethanol extract that embodiment 1-1, 1-2, 1-3 makes, the zygote grass extract that embodiment 2 makes and embodiment 3 obtain, acyclovir as For the positive control, phosphate buffer solution was used to prepare four groups of test products containing no astragalin concentration gradients of 1.25, 2.5, 5, 10, and 20 μmol/L, respectively.

Test Methods:

step 1)

Store the virus host cells (A549) in RPMI1640 medium, add 100 μg/mL penicillin and streptomycin and 10% fetal bovine serum, and cultivate them in a cell incubator at 37°C and 5% CO2. After the cells adhere to the wall, remove For the old medium, wash the cells in the medium with phosphate buffer, add an appropriate amount of trypsin to digest the cells, stop the digestion when the intercellular space increases and the cytoplasm retracts, suck out the digestion solution, and then wash the cells with phosphate buffer , add medium, transfer the cells to a centrifuge tube for centrifugation, remove the supernatant, add fresh medium and pipette to form a cell suspension. Inoculate 5×10 ⁴ cells (100 μL culture medium) in each well of a 96-well plate and incubate for 24 hours, infect the virus on A549 cells in good growth state, add to the culture medium containing 10% fetal bovine serum, 37°C, Cultivate in a cell incubator with 5% CO ₂ for 24 hours, stop the cultivation of virus cells when the lesion effect reaches 90%, and complete the virus amplification.

step (2)

Add 100 μL of the above-mentioned test substances to each well of the 96-well plate, set 3 duplicate holes for each concentration of the test substances in each group, then add 100 times the virus suspension of the half-infection amount of the cells, at 37 ° C, 5% Cultivate under CO ₂ conditions for 4 hours, when about 80% of the cells are damaged, discard the supernatant, add 100 μL of 5 mg/mL CCK-8 solution, and incubate in a cell incubator for 2 hours, discard the supernatant, add 100 μL DMSO to shake and shake well , Measure the A value of each well with a microplate reader at a wavelength of 530nm, and calculate the virus inhibition rate and IC ₅₀ of the compound.

Table 1 Antiviral test results

The test result shows: the zygosinia extract of embodiment 1-1, 1-2, 1-3 shows inhibitory action to H1N1 virus when the concentration of astragalin L is 150 μmol/L; The alcohol extract showed inhibitory effect on the H1N1 virus when the concentration of no rhizoside L was 50 μmol/L; The inhibitory effect was equivalent to the antiviral effect of the positive control acyclovir.

Although, the present invention has been described in detail with general description, specific implementation and test above, but on the basis of the present invention, some modifications or improvements can be made to it, which will be obvious to those skilled in the art . Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. Use of a compound for the preparation of an antiviral drug, wherein the compound has a chemical structure according to formula (1):

2. use of a zygotic herb extract for the preparation of an antiviral medicament, wherein the zygotic herb extract comprises a compound according to claim 1.

3. The application of the zygotic herb extract in preparing antiviral drugs is characterized in that the zygotic herb extract is an ethanol extract of the zygotic herb prepared by the step of reflux-extracting the zygotic herb with 72-78% ethanol solvent; preferably, the zygotic ethanol extract comprises a compound according to claim 1.

4. The use of a zygotic herb extract for preparing an antiviral drug, which is characterized in that the zygotic herb extract is prepared by the step of extracting the ethanol extract of the zygotic herb according to claim 3 by organic solvents with different polarities in a polar gradient manner; preferably, the zygotic grass extract comprises a compound according to claim 1.

5. The application of the zygotic herb extract in preparing antiviral drugs is characterized in that the zygotic herb extract is prepared by the steps of separating and purifying the zygotic herb extract in a high performance liquid chromatography.

6. The use according to any one of claims 2 to 5, wherein the antiviral agent is an anti-H1N 1 virus agent.

7. A formulation for antiviral use, characterized in that it comprises a compound according to claim 1 or an extract according to any one of claims 2 to 5, and pharmaceutical excipients.

8. The formulation of claim 7, wherein the formulation is a liquid formulation.

9. The formulation of claim 8, wherein said liquid formulation comprises 50 to 150 μmol/L of said compound per liter of said liquid formulation.

10. The formulation according to any one of claims 7 to 9, for use in the preparation of a medicament against H1N1 virus.