阅读说明：本技术 一种间苯三酚杂萜类化合物的抗新冠用途及制备方法 (Anti-neocoronarism application and preparation method of phloroglucinol heteroterpenoid ) 是由 侯博 胡江苗 于 2021-07-29 设计创作，主要内容包括：本发明公开了一种间苯三酚杂萜类化合物的抗新冠用途及制备方法,该间苯三酚杂萜类化合物具有抗新冠病毒活性,所述新冠病毒包括：SARS-CoV-2。该间苯三酚杂萜类化合物为大羽鳞毛蕨的提取物。本发明首次发现大羽鳞毛蕨提取物具有抗新冠作用,提取物间苯三酚杂萜类化合物对新冠病毒感染的VeroE6及Calu-3均具明显的抑制作用,同步的毒性测试表明显示出低毒性,可以用于作为制备治疗抗新冠病毒的药物。(The invention discloses an anti-new-corona application and a preparation method of a phloroglucinol heteroterpenoid compound, wherein the phloroglucinol heteroterpenoid compound has the activity of resisting new corona viruses, and the new corona viruses comprise: SARS-CoV-2. The phloroglucinol heteroterpenoid is extract of Dryopteris macrocarpa. The invention discovers for the first time that the large-feather dryopteris longissima extract has the effect of resisting new corona, the phloroglucinol heteroterpenoid of the extract has the obvious inhibition effect on VeroE6 and Calu-3 infected by new corona viruses, and a synchronous toxicity test table obviously shows low toxicity, so that the extract can be used for preparing medicines for treating the new corona viruses.)

1. The anti-neocoronarium application of the phloroglucinol triterpenoid is characterized in that the structural formula of the phloroglucinol triterpenoid is as follows:

the phloroglucinol heteroterpenoids have activity against novel coronaviruses including: SARS-Cov-2.

2. The anti-neocorona use according to claim 1, wherein the phloroglucinol triterpenoid is an extract of Dryopteris macrocarpa.

3. The anti-neocoronarium use according to claim 1, wherein the phloroglucinol heteroterpenoid is used for the preparation of a medicament for the treatment of anti-neocoronaviruses.

4. The anti-neocorolla use according to claim 3, wherein the phloroglucinol triterpenoid is formulated with pharmaceutically acceptable adjuvants for use in the preparation of a medicament for treating anti-neocoronaviruses.

5. The anti-neocrown use according to claim 4, wherein said formulation comprises: capsule, tablet, granule, gel, sustained release preparation, oral liquid, dripping pill and nanometer preparation.

6. A method of preparing phloroglucinol triterpenoids according to claims 1 to 5, comprising:

extracting Dryopteris macrocarpa with organic solvent as extraction solvent by one of soaking extraction, reflux extraction, percolation extraction, microwave extraction and ultrasonic extraction, and concentrating the extractive solution to obtain concentrated solution;

performing silica gel column chromatography on the concentrated solution, performing gradient elution by using petroleum ether-ethyl acetate, detecting and combining components by TLC, and taking eluent obtained by eluting the petroleum ether-ethyl acetate with the volume ratio of 100: 0-1: 1 as a component I;

removing impurities chlorophyll and xanthophyll from the first component, and purifying by using an HPLC normal phase column to obtain a compound 3 and a compound 4, wherein an eluent adopted by the HPLC normal phase column is n-hexane-ethyl acetate containing 0.5% of acetic acid or petroleum ether-ethyl acetate containing 0.5% of acetic acid.

The method according to claim 6, wherein the Dryopteris macrocephala is extracted by dipping.

7. The method according to claim 6, wherein the organic solvent is one or more selected from the group consisting of diethyl ether, acetone, ethyl acetate, ethanol, and methanol.

8. The method according to claim 6, wherein the gradient elution is carried out using petroleum ether-ethyl acetate at a volume ratio of from 100:0 to 0: 100.

9. The method according to claim 6, wherein the volume ratio of n-hexane or petroleum ether to ethyl acetate in the n-hexane-ethyl acetate containing 0.5% of acetic acid or the petroleum ether-ethyl acetate containing 0.5% of acetic acid is 95: 5.

Technical Field

The invention relates to a Dryopteris macrocarpa extract, in particular to an anti-neocoronal application of phloroglucinol heteroterpenoid and a preparation method thereof.

Background

Dryopteris macrocephala is the dry root, aced and petiole residue of Dryopteridaceae (Dryopteridaceae) plant Dryopteridaceae (Dryopteris wallichiana). The Dryopteris plant usually has bitter taste, slight cold, and small toxicity, and has effects of clearing heat, detoxicating, and expelling parasites.

The Severe Acute Respiratory Syndrome caused by SARS-CoV-2 virus (Severe Acute Respiratory Syndrome Coronavir 2, Severe Acute Respiratory Syndrome Coronavirus 2) that has appeared in recent years, i.e., COVID-19(CoronaVirus Disease 2019, a novel Coronavirus pneumonia), causes economic and social damages, and antiviral drugs that are not currently effective have been approved for the prevention or treatment of highly contagious SARS-CoV-2 and middle east Respiratory Coronavirus Syndrome (MERS-CoV).

Some precursors and potential drug candidates, such as Reidesvir and chloroquine, have been considered as treatment regimens for COVID-19. Reidesciclovir is a nucleoside analog, an RNA-dependent RNA polymerase (RdRP) inhibitor that reduces viral replication (EC) by targeting viral RNA polymerase₅₀0.77 μ M) was effective in inhibiting SARS-CoV-2 infection of Vero E6 cells. One experiment found that no significant therapeutic effect was observed in the short-term treatment with zirvir given the same conditions. Chloroquine is widely used in the treatment of autoimmune diseases and malaria, and can be used to combat certain viral infections. The drug can prevent viral infection by increasing the pH mediated by fusion of virus and cell or by late interaction with virus replication, and recently it has been shown to inhibit SARS-CoV-2 (EC) in Vero E6 virus cells₅₀1.13 μ M)). Some clinical trials have shown that treatment of patients with COVID-19 with chloroquine shows partial clinical efficacy, but most of the clinical data remains preliminary. To date, no clinical antiviral drug against SARS-CoV-2 has been developed.

Disclosure of Invention

The invention aims to provide an anti-new crown application and a preparation method of a phloroglucinol heteroterpenoid compound, which solve the problem that no SARS-CoV-2 antiviral drug exists at present, the phloroglucinol heteroterpenoid compound has obvious inhibition effect on Vero E6 and Calu-3 infected by new crown viruses, and a synchronous toxicity test table obviously shows low toxicity.

In order to achieve the aim, the invention provides an anti-neocrown application of a phloroglucinol triterpenoid, and the structural formula of the phloroglucinol triterpenoid is shown as follows:

the phloroglucinol heteroterpenoids have activity against novel coronaviruses including: SARS-Cov-2.

Preferably, the phloroglucinol heteroterpenoid is an extract of Dryopteris macrocarpa.

Preferably, the phloroglucinol heteroterpenoids are used for preparing medicaments for treating anti-new coronavirus.

Preferably, the phloroglucinol heteroterpenoid compound and pharmaceutically acceptable auxiliary materials are prepared into a preparation for preparing a medicine for treating the anti-new coronavirus.

Preferably, the formulation comprises: capsule, tablet, granule, gel, sustained release preparation, oral liquid, dripping pill and nanometer preparation.

Another object of the present invention is to provide a method for preparing the phloroglucinol triterpenoid, which comprises the following steps: extracting Dryopteris macrocarpa with organic solvent as extraction solvent by one of soaking extraction, reflux extraction, percolation extraction, microwave extraction and ultrasonic extraction, and concentrating the extractive solution to obtain concentrated solution; performing silica gel column chromatography on the concentrated solution, performing gradient elution by using petroleum ether-ethyl acetate, detecting and combining components by TLC, and taking eluent obtained by eluting the petroleum ether-ethyl acetate with the volume ratio of 100: 0-1: 1 as a component I; removing impurities chlorophyll and xanthophyll from the first component, and purifying by using an HPLC normal phase column to obtain a compound 3 and a compound 4, wherein an eluent adopted by the HPLC normal phase column is n-hexane-ethyl acetate containing 0.5% of acetic acid or petroleum ether-ethyl acetate containing 0.5% of acetic acid.

Preferably, the Dryopteris macrocarpa is extracted by a dipping extraction method.

Preferably, the organic solvent is selected from one or more of diethyl ether, acetone, ethyl acetate, ethanol and methanol.

Preferably, the gradient elution is performed with petroleum ether-ethyl acetate in a volume ratio of from 100:0 to 0: 100.

Preferably, the volume ratio of the n-hexane or the petroleum ether to the ethyl acetate in the n-hexane-ethyl acetate containing 0.5% of acetic acid or the petroleum ether-ethyl acetate containing 0.5% of acetic acid is 95: 5.

The invention relates to an anti-neocoronarism application and a preparation method of phloroglucinol heteroterpenoid compounds, which solves the problem that no SARS-CoV-2 antiviral drug exists at present, and has the following advantages:

the phloroglucinol heteroterpenoid compound has obvious inhibition effect on Vero E6 and Calu-3 infected by new coronavirus, a synchronous toxicity test table obviously shows low toxicity, and the EC of the compound 3 in an experiment of resisting the Vero E6 cell infected by the new coronavirus is adopted as the active compound 3-4₅₀Value of 4.5. mu.M, EC of Compound 4₅₀EC for compounds 3 and 4 with a value of 12.1. mu.M₅₀The value was 6.8. mu.M; EC of Compound 3 in an experiment against Calu-3 cells infected with New coronavirus₅₀Value of 20.2. mu.M, EC of Compound 4₅₀EC for compounds 3 and 4 with a value of 30.0. mu.M₅₀At a value of 24.8. mu.M, lung cells having no activity on chloroquine also showed a clear inhibitory activity.

The phloroglucinol heteroterpenoid compound is obtained by extracting the large-feather dryopteris crassipes, and the anti-new-crown active compound is obtained by separating and purifying by using various chromatographic methods (normal phase silica gel, reverse phase silica gel, gel and High Performance Liquid Chromatography (HPLC)).

Drawings

FIG. 1 shows the inhibition rate of the activity of compounds 1 to 6 of the present invention against the infection of Vero E6 cells by new coronavirus at a final concentration of 10. mu.M.

FIG. 2 shows the activity EC of the active compound 3-4 in resisting the new coronavirus infection Vero E6 cell₅₀The value is obtained.

FIG. 3 shows the activity EC of the active compounds 3-4 of the present invention in resisting Calu-3 cell infected by new coronavirus₅₀The value is obtained.

FIG. 4 shows the cytotoxicity of active compounds 3 to 4 of the present invention on CC₅₀The value is obtained.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Reagents and instruments used in the following experiments, etc., were as follows:

1. test materials: yunan produced in 2019 in 12 months, identified as dry bulb of Dryopteris macrocarpa by Kunming plant research institute of Chinese academy of sciences. Pulverizing dried bulb of Dryopteris davidii by pulverizer, sieving with 40 mesh sieve, sealing and storing.

2. Reagent: the methanol, the ether and the acetonitrile are all chromatographically pure products, and are products of chemical reagent companies of national drug group.

3. The instrument comprises the following steps: agilent 1260HPLC high performance liquid; a KERN electronic analytical balance (Beijing Saedodus Instrument systems, Inc.); heidolp rotary evaporator (Hei-VAP, Germany); column ZORBAX SB-C18 (4.6X 250mm, Agilent, USA) and HPLC positive phase column.

Experimental example 1 Dryopteris macrocarpa extract

Pulverizing dried tuber of Dryopteris davidii 4.7kg, soaking in diethyl ether (or acetone) at room temperature for 3 times, and concentrating under reduced pressure to obtain extract. Performing silica gel column chromatography on the part, and performing gradient elution on petroleum ether-ethyl acetate (the volume ratio is 100: 0-0: 100), wherein the component collected at the volume ratio of 100: 0-1: 1 of the petroleum ether-ethyl acetate is Fr.A, and the component collected at the volume ratio of 1: 1-0: 100 of the petroleum ether-ethyl acetate is Fr.B).

Fr.A (60.0g) is subjected to gel to remove impurities such as chlorophyll and lutein. Further purification by HPLC normal phase column (volume ratio 95:5 n-hexane-EtOAc, 0.5% HOAc, 2mL/min) group gave compound 3-6, compound 3 (yellow oil) content 320mg, compound 4 (yellow oil) content 260mg, compound 5 (yellow oil) content 50mg, compound 6 (yellow oil) content 30 mg;

further Fr.B (5.3g) was eluted with Sephadex LH-20 (chloroform-methanol in volume ratio of 1:1) and then chromatographed on silica gel column with petroleum ether-ethyl acetate (volume ratio of 1: 5-1:1), and then subjected to HPLC ZORBAX SB-C18 column (volume ratio of 30:35: 35H)₂O-MeOH-isoPropanol, 0.5% HCOOH, 2.0mL/min) to give 45mg of Compound 1 (yellow oil) and 10mg of Compound 2 (yellow oil).

The structures of the compounds 1 to 6 were determined by various spectroscopic analysis methods of nuclear magnetism, mass spectrometry, ultraviolet light, circular dichroism and optical rotation, as shown above.

The nuclear magnetic characterization data for compound 1 is shown below:

¹H NMR(600MHz,acetone-d₆,25℃):δ19.02(s,1H)；5.48(t,6.4,1H)；4.82(m,1H)；4.52(m,1H)；2.51(s,3H)；2.39–2.36(m,1H)；2.37–2.29(m,1H)；2.34-2.45(m,2H)；2.16(ddd,16.2,10.8,6.4,1H)；2.06-2.00(m,1H)；2.02(d,3.3Hz,2H)；1.98-1.83(m,2H)；1.85-1.87(m,1H)；1.81(m,1H)；1.79-1.87(1H)；1.73(s,3H)；1.58-1.60(m,1H)；1.58(m,2H)；1.46(td,12.8,4.3,1H)；1.38(m,1H)；1.34(s,3H)；1.30(s,3H)；1.23(td,12.9,3.5,1H)；1.14(s,3H)；0.79(s,3H)。

¹³C NMR(150MHz,acetone-d₆,25℃)δ200.6；196.8；189.1；180.0；174.9；148.9；133.2；129.4；83.8；47.8；108.358.0；50.4；47.8；39.639.1；38.4；37.8；27.8；27.3；25.8；25.2；24.9；23.1；19.2；17.9；17.1；15.0；12.4。

mass spectral characterization data for compound 1 were: ESI⁺:m/z 511[M+H]⁺。

The nuclear magnetic characterization data for compound 2 is shown below:

¹H NMR(600MHz,acetone-d₆,25℃):δ19.01(s,1H)；5.47(t,6.3,1H)；4.82(m,1H)；4.49(m,1H)；2.51(s,3H)；2.39–2.36(m,1H)；2.37–2.29(m,1H)；2.35-2.47(m,2H)；2.18(m,1H)；2.06-2.00(m,1H)；2.02(d,3.3Hz,2H)；1.98-1.83(m,2H)；1.86(m,1H)；1.81(m,1H)；1.79-1.87(1H)；1.73(s,3H)；1.58-1.60(m,1H)；1.58(m,2H)；1.46(td,12.5,3.8,1H)；1.38(m,1H)；1.34(s,3H)；1.30(s,3H)；1.24(td,12.8,3.9,1H)；1.14(s,3H)；0.79(s,3H)。

¹³C NMR(150MHz,acetone-d₆,25℃)δ200.7；197.0；189.1；180.1；175.0；149.0；133.3；129.8；84.0；47.8；108.358.0；50.4；47.8；39.639.1；38.4；37.8；27.9；27.3；25.8；25.2；24.8；23.1；19.2；18.0；17.2；15.0；12.3。

mass spectral characterization data for compound 2 were: ESI⁺:m/z 511[M+H]⁺。

The nuclear magnetic characterization data for compound 3 is shown below:

¹H NMR(600MHz,acetone-d₆,25℃):δ18.58(s)；16.49(s)；11.28(s)；10.17(s)；5.57(t,6.6,1H)；4.53(d,2.1,1H)；4.83(s,1H)；4.43(dd,10.3,2.3,1H)；3.52(s,2H)；2.70(s,3H)；3.03(ddd,15.1,8.3,6.4,1H)；2.94(ddd,15.6,8.3,6.6,1H)；2.74(ddd,16.64,5.57,2.65,1H)；2.56(m,1H)；2.37(m,1H)；2.02(m,1H)；1.96-2.03(m,1H)；2.37(m,1H)；2.17(ddd,16.3,10.7,6.7,1H)；1.91-1.84(overlapping signals),1.89-1.96(overlapping signals)；1.88(m,1H)；1.81(s,3H)；1.77-1.83(d,3.24,1H)；1.64(m,2H)；1.63-1.70(m,13.01,3.38,1H)；1.58-1.69^a(m,2H)；1.48(s,3H)；s 1.48(s,3H)；1.45(m,12.8,4.3,1H)；1.37(m,1H)；1.23(td,12.8,4.0,2H)；1.14(s,3H)；0.96(t,7.23,3H)；0.79(s,3H)。

¹³C NMR(150MHz,acetone-d₆,25℃)δ207.3；204.3；199.9；188.3；179.7；172.6；162.6；161.7；158.5；148.9；133.9；130.2；112.1；109.2；108.3；106.1；104.8；103.9；83.4；58.1；50.4；47.8；46.5；45.0；39.6；39.1；38.4；37.8；29.2；27.3；25.9；25.0；24.9；23.2；20.2；19.2；17.2；17.1；15.0；14.4；12.6。

mass spectral characterization data for compound 3 was: ESI^-:m/z 717[M-H]^-.

The nuclear magnetic characterization data for compound 4 is shown below:

¹H NMR(600MHz,acetone-d₆,25℃):δ18.58(s)；16.49(s)；11.28(s)；10.17(s)；5.55(t,6.41,1H)；4.54(s,1H)；4.83(d,2.2,1H)；4.44(dd,10.5,2.2,1H)；3.53(s,2H)；2.70(s,3H)；3.03(ddd,16.11,7.26,1H)；2.96(dt,16.11,7.26,1H)；2.74(ddd,16.52,5.54,2.57,1H)；2.57(m,1H)；2.35(m,1H)；2.03(m,1H)；1.96-2.03(m,1H)；2.35(m,1H)；2.20(ddd,16.3,10.85,6.93,1H)；1.91-1.84(overlapping signals),1.89-1.96(overlapping signals)；1.89(m,1H)；1.81(s,3H)；1.76-1.83(m,3.24,1H)；1.66(m,2H)；1.57-1.60(dd,13.01,3.38,1H)；1.58-1.69(m,2H)；1.48(s,3H)；1.48(s,3H)；1.45(m,12.8,4.1,1H)；1.38(m,1H)；1.23(td,12.9,3.80,2H)；1.15(s,3H)；0.95(t,3H)；0.80(s,3H)。

¹³C NMR(150MHz,acetone-d₆,25℃)δ207.3；204.3；199.9；188.3；179.7；172.6；162.6；161.6；158.5；148.9；133.9；130.4；112.1；109.2；108.4；106.1；104.8；103.9；83.4；58.1；50.4；47.8；46.4；45.0；39.6；39.1；38.4；37.8；29.2；27.3；26.0；25.0；24.9；23.2；20.2；19.0；17.2；17.1；15.0；14.3；12.6。

mass spectral characterization data for compound 4 was: ESI^-:m/z 717[M-H]^-.

The nuclear magnetic characterization data for compound 5 is shown below:

¹H NMR(600MHz,acetone-d₆,25℃):δ18.58(s)；14.17(s)；11.41(s)；9.11(s)；5.67(m,6.3)；4.63(dd,10.1,2.0)；4.86(d,1.2)；4.62(d,1.2)；3.48(d,16.4)；3.46(d,16.4)；3.16(dt,16.6,7.3)；3.11(dt,16.6,7.3)；2.72(ddd,16.7,5.7,3.1)；2.71(s)；2.51(ddd,16.7,11.3,5.7)；2.41(dd,15.6,6.3)；2.36(m,12.8,4.3,2.5)；2.25(ddd,15.6,11.0,6.3)；2.07–2.00(overlapping signals)；2.07–1.96(overlapping signals)；2.03(dd,2.5)；1.91–1.83(overlapping signals)；1.90(d,10.1)；1.85(s)；1.80(td,12.4,4.3)；1.70(m,7.3)；1.70–1.58(overlapping signals)；1.64–1.58(overlapping signals)；1.52(s)；1.50(s)；1.47(td,12.8,4.3)；1.39(m,12.8,5.3,2.5)；1.15(s)；1.23(td,12.8,3.9)；0.98(t,7.3)；0.81(s)。

¹³C NMR(150MHz,acetone-d₆,25℃)δ207.7,204.4,199.7,188.4,179.7,172.2,163.2,158.7,158.2,148.7,132.8,132.8,112.1,109.2,108.6,106.7,105.0,102.7,85.0,57.9,50.4,47.7,46.9,44.9,39.5,39.1,38.4,37.8,29.3,25.1,27.3,26.0,24.9,23.3,9.1,18.8,17.6,15.0,14.5。

mass spectral characterization data for compound 5 was: ESI^-:m/z 717[M-H]^-。

The nuclear magnetic characterization data for compound 6 is shown below:

¹H NMR(600MHz,acetone-d₆,25℃):δ18.55(s)；14.14(s)；11.46(s)；9.07(s)；5.72(m,6.3)；4.86(d,1.3)；4.62(dd,10.1,3.0)；4.59(d,1.4)；3.48(d,16.0)；3.46(d,16.0)；3.17(dt,16.6,7.3)；3.14(dt,16.6,7.3)；2.75(ddd,16.7,5.1,3.1)；2.70(s)；2.56-2.47(overlapping signals),2.39(m,12.8,4.3,2.8)；2.17(ddd,15.2,11.0,6.0)；210–2.00(overlapping signals)；2.04–1.96(overlapping signals)；2.07(dd,12.9,2.7)；1.93(overlapping signals)；2.04-1.96(overlapping signals)；1.85(s)；1.81(m,12.6,4.1)；1.70(m,7.3)；1.70–1.59(overlapping signals)；1.71–1.59(overlapping signals)；1.55(s)；1.51(s)；1.47(td,12.8,4.3)；1.39(m,12.8,5.3,2.7)；1.15(s)；1.27(td,12.8,4.0)；0.98(t,7.3)；0.81(s)。

¹³C NMR(150MHz,acetone-d₆,25℃)δ207.8,204.4,199.7,188.4,179.6,172.1,163.2,158.7,158.3,148.8,132.8,132.7,112.1,109.2,108.3,106.7,105.1,102.7,85.3,58.2,50.5,47.7,46.9,44.9,39.5,39.3,38.4,37.8,29.3,25.1,27.3,25.7,24.9,23.2,19.1,18.8,17.6,15.0,14.5。

mass spectral characterization data for compound 6 was: ESI^-:m/z 717[M-H]^-。

Experimental example 1 drug Activity test

(1) Vero E6 cells (or Calu-3 cells) were seeded into 48-well plates at approximately 5X 10 per well⁴The cells are tested the next day, and then put at 37 ℃ and 5% CO₂The culture was carried out overnight.

(2) Incubation of drug with cells: compounds 1-6 were diluted in DMEM medium containing 2% fetal bovine serum. Diluting the drug by three times, wherein 3 compound holes are arranged at each concentration, and 6 drug gradients are formed; DMSO was used as a control group, which was diluted with DMEM medium containing 2% fetal bovine serum in total volume and administered with the same volume of dimethyl sulfoxide. After removing the cell supernatant, 100. mu.L of the diluted compound was added to a 1.2 48-well plate, and the same volume of diluted DMSO was added to the control group, followed by incubation at 37 ℃ for 1 hour.

(3) SARS-Cov-2 infected cells: mu.L of a dilution of SARS-CoV-2 virus (MOI 0.01Vero E6 and MOI 0.05Calu-3) was added to each well of a 48-well plate and incubation was continued at 37 ℃ for lh, and the infectious agent supernatant was removed and washed once with 200. mu.L of LPBS (phosphate buffered saline). 200 mu L of culture medium containing the drug at the corresponding concentration is added into the wells again, the culture is continued for 24h, and 150 mu L of supernatant is collected for testing.

(4) Viral RNA Extraction Using the Kit Takara MiniBEST Viral RNA/DNA Extraction Kit: virus was lysed and 50. mu.L of PBS solution was added to 150. mu.L of cell culture supernatant to make the total volume 200. mu.L. 200 mu L of Buffer VGB Buffer solution, 20 mu L of protease K and 1 mu L of Carrier RNA are added in sequence, evenly mixed by oscillation and placed in a water bath at 56 ℃ for 10min for full lysis. Then 200. mu.L of absolute ethyl alcohol is added, and the mixture is shaken and mixed evenly. And then obtaining virus RNA through column chromatography, washing and elution.

(5) Reverse transcription of viral RNA: the procedure is described in TakaraPrimeScript^TMRT reagent Kit with gDNA Eraser Kit. And removing DNA in the eluent, and then carrying out reverse transcription reaction for later use.

(6) Virus copy number was obtained using standard curve method: reference kit Takara TBPremix Ex Taq^TMII, using RBD plasmid with known copy number as standard, targeting RBD with specific primer, and calculating copy number of each sample. The inhibition rate of the compound treatment group is obtained by taking the copy number of the blank DMSO group as a reference. Fitting an inhibition rate curve by using prism6.0.1 software according to the inhibition rates of the compound treatment groups with different concentrations, and calculating the half effective concentration EC₅₀。

As shown in FIG. 1, the inhibition rate of the activity of compounds 1-6 of the present invention against the infection of Vero E6 cells by new coronavirus at the final concentration of 10. mu.M, it can be seen that compounds 3 and 4 (natural content is about 1.2:1 ratio) can inhibit the infection of Vero E6 cells by new coronavirus.

As shown in figure 2, the active compound 3-4 of the invention has the activity EC in resisting the new coronavirus infection Vero E6 cell₅₀Value, it can be seen that EC for Compound 3₅₀Value of 4.5. mu.M, EC of Compound 4₅₀EC for compounds 3 and 4 with a value of 12.1. mu.M₅₀The value was 6.8. mu.M.

As shown in FIG. 3, the EC activity of the active compound 3-4 of the invention in resisting the new coronavirus infection Calu-3 cell₅₀Value, it can be seen that EC for Compound 3₅₀Value of 20.2. mu.M, EC of Compound 4₅₀EC for compounds 3 and 4 with a value of 30.0. mu.M₅₀At a value of 24.8. mu.M, lung cells having no activity on chloroquine (Calu-3) also showed a clear inhibitory activity.

Experimental example 2 drug toxicity test

(1) Vero E6 cells were obtained in logarithmic growth phase, cell density was adjusted to 5X 10⁴One well, 100. mu.L/well of each well was inoculated into a 96-well plate and cultured overnight.

(2) Before administration, 8 concentration gradients were diluted in DMEM medium containing 2% fetal bovine serum in total volume at a rate of 2000. mu.M, 1000. mu.M, 500. mu.M, 125. mu.M, 62.5. mu.M, 31.25. mu.M, 15.625. mu.M, 7.8125. mu.M, and 100. mu.L of the diluted drug per well was added to Vero E6 cells in a 96-well plate of 1.2.1, respectively, to a final volume of 200. mu.L. Duplicate wells were set and DMSO solvent treated groups were blank.

(3) After incubation in an incubator for 48h, 10. mu.L of CCK-8 working solution was added to each well, incubation was continued for 3h, and absorbance at 450nm was measured.

(4) According to OD₄₅₀The viability of Vero E6 cells at each concentration of drug was calculated separately from the control.

As shown in FIG. 4, it is shown that the active compounds 3-4 of the present invention are cytotoxic CC₅₀Value, it can be seen that CC for Compound 3₅₀Value 160.0. mu.M, CC of Compound 4₅₀The value was 140.2. mu.M,CC of Compounds 3 and 4₅₀The value was 163.2. mu.M.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.