CN107737201B

CN107737201B - Antiviral application of cloverleaf extract

Info

Publication number: CN107737201B
Application number: CN201711251250.2A
Authority: CN
Inventors: 闫滨; 邓华亮; 王丹丹; 高敏; 岳路路; 高荣
Original assignee: Shandong University of Traditional Chinese Medicine
Current assignee: Shandong University of Traditional Chinese Medicine
Priority date: 2017-12-01
Filing date: 2017-12-01
Publication date: 2021-05-18
Anticipated expiration: 2037-12-01
Also published as: CN107737201A

Abstract

本发明提供本发明提供了三叶青提取物在制药中的新用途：三叶青在制备抗RSV的药物中的应用。三叶青在制备抗HSV‑1的药物中的应用。所述三叶青的应用具体为：先将三叶青进行前处理，然后将前处理得到的三叶青提取物用于制备抗病毒的药物，如可将提取物与医学上可接受的辅料一起制成胶囊剂、片剂、颗粒剂、口服液制剂等，将提取物进一步纯化也可以制成注射制剂。The present invention provides the present invention provides a new use of cloverleaf extract in pharmacy: the application of cloverleaf in the preparation of anti-RSV medicines. Application of cloverleaf in the preparation of anti-HSV-1 drugs. The specific application of the cloverleaf is as follows: first, the cloverleaf is pretreated, and then the cloverleaf extract obtained by the pretreatment is used to prepare an antiviral drug. For example, the extract can be mixed with medically acceptable auxiliary materials. Together, they can be prepared into capsules, tablets, granules, oral liquid preparations, etc., and the extract can be further purified to prepare injection preparations.

Description

Application of radix tetrastigme extract in resisting virus

Technical Field

The invention relates to application of radix tetrastigme in preparing medicines for inhibiting Respiratory Syncytial Virus (RSV) and Herpes Simplex Virus type1 (HSV-1).

Background

Radix tetrastigme (Tetrastigmatis hemsleyani) is Vitaceae Tetrastigma hemsleyanum Diels et Gilg, the root tuber of the Tetrastigma hemsleyanum is named as snake monkshood, and the other names are stone monkey, stone broomcorn seed, tiger, ledebouriella root, stone breaking pearl, soil meridian pill, herba seu radix piperis, radix tetrastigme, herba Hedyotidis Diffusae, YINLINGZI, radix tetrastigme, herba Anoectochili Roxburghii, and rhizoma Solani Lyrati by Anoectochilus roxburghii. Mainly distributed in the southwest of China, Zhejiang, Guangdong, Hubei, Fujian and other areas, and originated from the forest beside the mountain slopes, ditches or brooks in the shade and wetness. It is preferably picked from winter to frightening period, and its medicinal parts are root tuber, fruit or whole herb. The whole plant of Adina triloba recorded in Chinese botanical records for medicine use, has the functions of promoting blood circulation, removing blood stasis, detoxifying and reducing phlegm, is clinically used for treating viral meningitis, Japanese encephalitis, viral pneumonia, icterohepatitis and the like, and particularly has special effect on high fever of children by root tubers. The medicine has slightly bitter taste, smooth meridian tropism and no toxicity, and has the functions of clearing away heat and toxic material, promoting blood circulation to disperse stagnation, diminishing inflammation, relieving pain, dispelling wind and eliminating phlegm, regulating vital energy, invigorating spleen, etc. And (3) character identification: climb the vine. The root is thick and strong, and is in a spindle shape or a block shape, and constant pieces are connected. Thin and weak stem, root on the lower node. Leaf intergrowth; palm-shaped compound leaves, 3 small leaves, from a narrow oval shape to an oval shape, wherein 1 large leaf in the center is 3-7 cm long and 2-3 cm wide, the tip is sharp or short, the base is gradually narrow, the edge is provided with thorn-shaped sparse teeth, and the small leaves on the two sides are small and asymmetric, 2-4 cm long and 1.3-2 cm wide; the length of the petiole is 3-5 mm, and the length of the total petiole is 2-4 cm; the curled beards and the leaves are opposite to each other and provided with a sucking disc. Flower mixed abnormal plants; axillary growth of an umbrella-shaped inflorescence; 4, opening the petals, and combining the flower disc with the ovary base; pistil 1, stigmas 4 are deeply fissured. Small berry, spherical. The seed is in the shape of an egg. The root is spindle-shaped, oval, gourd-shaped or oval, generally 1.5-6 cm long and 0.7-2.5 cm in diameter. The surface is tan, mostly smoother, or has wrinkles and a few nodules with skin pores, sometimes with depressions, in which there remain tan fine roots. Hard and brittle, flat and rough cross section, white-like, powdery, and brown color forming lamellar ring. No smell, sweet taste.

The radix tetrastigme extract refers to an n-butanol extraction part of an ethanol extract of the radix tetrastigme and an ethyl acetate extraction part of the ethanol extract of the radix tetrastigme in the patent. The n-butanol extraction site can identify 9 compounds with structures, which are epigallocatechin, procyanidin B2, catechin, epicatechin, kaempferol-7-O-rhamnose-3-O-glucoside, quercetin-3-O-beta-D-glucoside, quercetin-7-O-beta-D-glucoside, kaempferol-3-0-rutinoside, and kaempferol-7-O-beta-D-glucoside. The structure of the compound which can be identified from the ethyl acetate extraction part comprises 12 compounds, namely beta-sitosterol, kaempferol, 5,7,4 '-trihydroxy-3' -methoxyflavone, quercetin, 5,7,3 ', 5' -tetrahydroxydihydroflavone, ethyl 3, 4-O-dicaffeoylquininate, kaempferol-7-O-beta-D-glucoside, quercetin-3-O-beta-D-glucoside, kaempferol-3-O-neohesperidoside, rutin, kaempferol-7-O-rhamnose-3-O-glucoside, wherein the 5,7,4 '-trihydroxy-3' -methoxyflavone, kaempferol and the 5,7,4 '-trihydroxy-3' -methoxyflavone, 5,7,3 ', 5' -tetrahydroxyflavanone and 3, 4-O-dicaffeoylquinic acid ethyl ester are obtained by separating ethyl acetate part of radix tetrastigme tuber for the first time; the other compounds (mainly alkaloids) have small content and are difficult to identify the structure.

RSV is the most important viral pathogen for viral lower respiratory tract infections in infants and young children worldwide. In recent years, RSV infection has been found to be an important pathogen in immunosuppressed adults and the elderly. Most RSV infections are upper respiratory tract infections, but many infants can develop pneumonia and lower respiratory tract infections.

HSV-1 is a virus that causes Herpes Simplex Encephalitis (HSE). Herpes simplex encephalitis is the most common viral infectious disease of the central nervous system, is seen in all parts of the world, has no seasonal tendency and sex difference, can be attacked at any age, is at risk and is high in fatality rate.

Disclosure of Invention

In order to overcome the defects, the invention provides a new application of the radix tetrastigme extract in pharmacy. Application of radix tetrastigme in preparing anti-RSV medicine is provided. Application of radix tetrastigme in preparing anti-HSV-1 medicines is provided. The application of the radix tetrastigme is as follows: the radix Apioris Fortunei is pretreated, and the pretreated radix Apioris Fortunei extract is used for preparing antiviral drugs, such as capsule, tablet, granule, oral liquid, etc. with pharmaceutically acceptable adjuvants, and the extract can be further purified or made into injection.

In order to achieve the purpose, the invention adopts the following technical scheme:

application of radix Apioris Fortunei ethanol extract n-butanol extract part or ethyl acetate extract part in preparing anti-RSV medicine is provided.

Modern pharmacological action research indicates that: radix tetrastigme has pharmacological activity of resisting virus infection and regulating autoimmunity. However, the antiviral action mechanism of radix tetrastigme is not yet clarified, and the active parts of the radix tetrastigme alcohol extract extracted by different solvents are various, so that the existing radix tetrastigme medicine has weak inhibition effect on specific viruses and poor pertinence. Therefore, on the basis of deeply researching the antiviral action mechanism of the radix tetrastigme, the invention discovers through large-scale experimental screening that: the n-butanol extraction part or the ethyl acetate extraction part of the radix tetrastigme alcohol extract has obvious inhibition effect on RSV and HSV-1.

Preferably, the preparation method of the n-butanol extraction part of the radix tetrastigme alcohol extract comprises the following steps:

extracting the radix tetrastigme ethanol total extract suspension with n-butanol, concentrating the extractive solution under reduced pressure, oven drying to constant weight, and weighing.

Preferably, in the extraction process, demulsification is carried out by adopting a heating ultrasonic method.

Preferably, the mass ratio of the n-butanol to the total ethanol extract suspension of radix tetrastigme is 1: 1.

preferably, the preparation method of the total ethanol extract of radix tetrastigme comprises the following steps: in the material-liquid ratio of 15:1, the ethanol concentration is 60 wt%, ultrasonic extraction is carried out for 20min, and then reflux extraction is carried out for 20 min.

The invention also provides application of the ethyl acetate extraction part or the n-butanol extraction part of the radix tetrastigme ethanol extract in preparing the anti-HSV-1 medicament.

Preferably, the preparation method of the ethyl acetate extraction part of the radix tetrastigme alcohol extract comprises the following steps:

extracting the radix tetrastigme ethanol total extract suspension with ethyl acetate, concentrating the extract under reduced pressure, drying to constant weight, and weighing.

Preferably, the mass ratio of the ethyl acetate to the total ethanol extract suspension of radix tetrastigme is 1: 1.

The invention has the advantages of

(1) The radix tetrastigme extract has obvious inhibition effect on RSV and HSV-1 viruses, which indicates that the radix tetrastigme not only has the functions of promoting blood circulation, removing blood stasis, detoxifying and reducing phlegm, but also has potential application value in the field of treating RSV and HSV-1 virus infectious diseases. The invention provides experimental basis for the clinical application of the radix tetrastigme in treating virus infectious diseases, provides certain guiding significance for developing anti-RSV and HSV-1 virus medicaments, and has important reference value.

(2) The extraction method is simple, high in inhibition efficiency, strong in practicability and easy to popularize.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Example 1 study of the effect of tetrastigma hemsleyanum extract on viruses:

(1) preparing a radix tetrastigme extract:

smashing dried radix tetrastigme into coarse powder, crushing the coarse powder into 100-mesh fine powder by using a high-speed medicine crusher, performing heating ultrasonic extraction on all the powder in batches by using 15 times of 60 wt% ethanol for 20min, performing heating reflux extraction for 20min, filtering an extracting solution, performing reduced pressure concentration, extracting filter residues for 2 times by using the same method, suspending the concentrated extracting solution by using water to obtain a suspension, sequentially extracting the suspension by using petroleum ether, ethyl acetate and n-butyl alcohol (the volume ratio of an extracting solvent to the radix tetrastigme extracting solution suspension is 1.5: 1), demulsifying an emulsion layer generated in the extraction process by using a heating ultrasonic method until the extracting solution is almost colorless, stopping extraction, and respectively performing reduced pressure concentration on each part of the extracting solution and drying the extracting solution to constant weight.

(2) Cell and virus strains:

are provided by the institute for basic medicine of Shandong provincial academy of medical sciences: MA104 (rhesus monkey embryonic kidney cells, RSV and HSV-1 sensitive cells); RSV (respiratory syncytial virus, long strain, introduced in viral institute of preventive medicine institute of Chinese institute of preventive medicine in 10 months in 2000), HSV-1 (herpes simplex virus type1, strain F, introduced in 1996 in the institute of microbiology, university of Shandong medical sciences laboratory).

(3) Cytotoxicity assay:

referring to the literature of Wang Xiaoyan, Zhang Meiying, Wang Yafeng, etc., the betel nut extract is used for in vitro anti-coxsackie virus B3 and research of herpes simplex virus type1 and hepatitis B virusShizhen Chinese medicine, 2008; 19(12): 2954-2955, spotting cells on a 96-well plate to form a monolayer, diluting a drug-containing cell maintenance solution 2-fold from the 1 st well to 12 concentrations, plating the diluted solution on the 96-well plate, repeating the steps for 3 wells at each dilution, and incubating the diluted solution at 37 ℃ with 5% CO₂Culturing under the condition, observing CPE under a microscope, and continuously observing drug toxicity. CPE greater than 50% was considered toxic. Then MTT staining is carried out, the determination wavelength is set to be 570nm, the OD value is determined, the cell survival rate is the result of comparing the OD value of the experimental group with the OD value of the cell control group, and the half poisoning concentration (50 percent of toxic concentration, TC) is calculated by a Reed-Muench method₅₀)。TC₅₀Either [ Antilog (log higher than 50% of the value of the lesion rate drug dilution-pd)]X C first-well drug concentration.

(4) And (3) toxicity inhibition test:

referring to the literature of Wang Xiaoyan, Zhang Meiying, Wang Yafeng and the like, the research of in vitro anti-coxsackie virus B3, herpes simplex virus type1 and hepatitis B virus of betel nut extract, Shizhen Chinese medicine, 2008; 19(12): 2954 to 2955, cells spotted on a 96-well plate are grown into a monolayer, the drug-containing cell maintenance solution is diluted 2-fold from the 1 st well to 12 concentrations, and the diluted solution is seeded on the 96-well plate (50 μ L/well), 3 wells are repeated for each dilution, and a cell control and a virus control are set, and ribavirin is used as a positive control (10 mg/mL). 100 TCIDs were added to each well in addition to the cell controls₅₀50 μ L of the virus of (4). 37 ℃ and 5% CO₂Incubations were performed under conditions, and microscopic observations of cytopathic effects continued until 90% virus control CPE was reached. The absorbance A was measured by a microplate reader, and the dilution of CPE 50% was regarded as the half Effective Concentration (EC) of the drug₅₀)。EC₅₀Either [ Antilog (log higher than 50% viability drug dilution-pd)]×C_{First hole drug concentration}. According to the half Toxic Concentration (TC) of the tested medicine₅₀) And half Effective Concentration (EC) of the drug₅₀) The two are compared to obtain a toxicity inhibiting index (TI), and if the TI is more than 4, the medicine is judged to be effective.

(5) As a result:

firstly, the concentration TC of the drug in the median poisoning₅₀The determination of (1): the cells of the control group are good in morphology when observed under a microscopeGood and compact adherent growth. Breaking the medicinal cells, displaying toxic form, measuring OD value, and calculating TC of the medicine according to formula₅₀。

② the drug antiviral test: observation under a microscope shows that the cells of the control group have good morphology and compact adherent; the RSV and HSV-1 virus control groups have more than 90 percent of CPE in 24h, and the RSV is mainly characterized in that the CPE is rounded in wall thickness and is adhered and broken in MA 104; HSV-1 has the main characteristic that CPE is mainly rounded off and occasionally fused in MA 104.

Staining a 96-well plate to be tested by 5mg/mL MTT, measuring absorbance A at 570nm by an enzyme labeling instrument, subtracting A value of a virus control group from A value of each group, and comparing A value of each experimental group with A value of a cell control group to obtain EC₅₀，EC₅₀And TC₅₀And obtaining TI:

a. inhibiting RSV from generating CPE Effect (CPE) on MA104 cells, wherein the toxicity inhibiting index TI of n-butanol part is 128, and the toxicity inhibiting index TI of ethyl acetate part is 64;

b. the inhibition effect of HSV-1 on the CPE of MA104 cells is realized, the toxicity inhibition index TI of the n-butanol part is 64, and the toxicity inhibition index TI of the ethyl acetate part is 64.

Example 2

The experiment obtains the antiviral active part of the radix tetrastigme by screening the antiviral activity of the radix tetrastigme through the water elution part and the ethanol elution part (30%, 50%, 70% and 95%) with different concentrations of the different active parts obtained by extracting the radix tetrastigme with n-butyl alcohol, ethyl acetate and petroleum ether respectively, and the sample obtained in each stage of preparing liquid phase separation and purification and the sample obtained in each stage of preparing liquid phase chromatographic separation and purification through silica gel column chromatography and preparing liquid phase chromatographic separation and purification, thereby providing reliable theoretical data for the deep antiviral research and the elucidation of the antiviral action mechanism of the radix tetrastigme.

The tested drugs are:

1-7 parts of petroleum ether, 2mg

1: petroleum ether: ethyl acetate 100:1 elution

2: petroleum ether: ethyl acetate 50:1 elution

3: petroleum ether: ethyl acetate 25:1 elution

4: petroleum ether: ethyl acetate 15:1 elution

5: petroleum ether: ethyl acetate 5:1 elution

6: petroleum ether: ethyl acetate 2:1 elution

7: petroleum ether: ethyl acetate 1:1 elution

8-11: ethyl acetate fraction, 2mg

8: methanol: 100 parts of chloroform: 0 elution

9: methanol: 100 parts of chloroform: 15 elution

10: methanol: 100 parts of chloroform: 30 elution

11: methanol: 100 parts of chloroform: 45 elution

12-16: n-butanol fraction, 20mg

12: eluting with water

13: eluting with 30% ethanol

14: eluting with 50% ethanol

15: eluting with 70% ethanol

16: eluting with 95% ethanol

17-21: water part, 20mg

17: eluting with water

18: eluting with 30% ethanol

19: eluting with 50% ethanol

20: eluting with 70% ethanol

21: eluting with 95% ethanol

Viral strains

EV 71: the gene is isolated from a patient with hand-foot-and-mouth disease in infectious disease hospital of Jinan City, and is proved to be EV71 by complete sequence determination, and is stored in the department.

RSV: long strain, introduced from virus institute virus chamber of China academy of preventive medicine in 2000 and stored in the same department, 10 months.

HSV-1: strain F, introduced in 1996 from the university of medical science, shandong, the institute of microbiology, where it was stored.

Cell line

The MA 104: rhesus monkey embryonic kidney cells are RSV and HSV-1 sensitive cells and are preserved in the department.

RD: human rhabdomyoma cells, EV71 sensitive cells, were stored in the current department.

Culture solution

Cell culture solution: 1640 cell culture medium, GIBCO product, containing 10% newborn bovine serum and 100U/mL penicillin and streptomycin, filtering, sterilizing, and packaging at 4 deg.C.

Cell maintenance solution: 1640 cell culture medium, GIBCO product, containing 2% newborn bovine serum and 100U/mL penicillin and streptomycin; MDCK cell maintenance solution: 1640 cell culture medium, GIBCO product, containing 100U/mL penicillin and streptomycin; filtering, sterilizing, and packaging at 4 deg.C.

PBS: pH7.2, containing 8g of NaCl, 0.2g of KCl and KH₂PO₄ 0.2g、Na₂HPO₄2.9g, adding double distilled water to 1000mL, filtering and sterilizing, and subpackaging at 4 ℃ for later use.

Cell digestive juice: pancreatin (product of Diffico Co.) 2.5g was dissolved in 1000mL of PBS, filtered and sterilized, and then dispensed into penicillin bottles for use in a refrigerator at-20 ℃.

Instrument for measuring the position of a moving object

CO₂A constant-temperature incubator: products of the Japan Sanyo corporation;

the biological safety cabinet: product of the national institute of technology, West, Inc.;

a centrifuge: a hercules table low temperature centrifuge;

-80 ℃ refrigerator: products of Thermo corporation;

-20 ℃ refrigerator: product of Mike Mitsubishi corporation;

an enzyme-labeling instrument: products of Thermo corporation.

Consumable material

Cell culture flasks: german Greiner kalina product, specification: 75cm²。

A sterilizing needle head filter: products of Pall Corporation, USA. Pore diameter: 0.22 μm, diameter 13 mm.

A liquid transfer device: product of germany Eppendorf, type: 20-200 mul; 100-.

Pipette tips: a german Eppendorf product, which is fitted to a pipette.

A laboratory:

BSL-2 laboratory.

In vitro antiviral drug assay

The specific cytotoxicity assay and toxicity inhibition assay were as in example 1.

Drug inhibitory Effect (TI)

80-drug repeat experiment HSV-1

Further experiments show that antiviral activity screening is carried out on the radix tetrastigme alcohol extract, water elution parts and ethanol elution parts (30%, 50%, 70% and 95%) with different concentrations, which are obtained by respectively extracting different active parts and n-butyl alcohol parts with n-butyl alcohol, ethyl acetate and petroleum ether from the radix tetrastigme alcohol extract, and samples obtained by preparing liquid phase separation and purification stages and samples obtained by preparing liquid phase chromatography separation and purification stages from ethyl acetate parts through silica gel column chromatography and preparing liquid phase chromatography separation and purification stages, and the results are shown in table 2, table 3 and table 4.

TABLE 2 Hemsley leaf extract n-butanol fraction anti-RSV virus results

TABLE 3 Hemsley leaf extract n-butanol fraction results against HSV-1 virus

Gradient eluting the ethyl acetate part with petroleum ether-ethyl acetate (10: 1-1: 1), ethyl acetate and methanol, collecting eluate, concentrating under reduced pressure, spreading the eluate with a paved thin-layer silica gel plate, observing the generated fluorescence with an ultraviolet analyzer, guiding to combine the same or similar components to obtain 4 components (SY6-SY9), and performing color reaction and antiviral activity screening.

TABLE 4 radix tetrastigme alcohol extract ethyl acetate part antiviral experimental result (TI)

Based on the experimental results, the invention specifically selects the n-butanol extraction part or the ethyl acetate extraction part for inhibiting Respiratory Syncytial Virus (RSV) or inhibiting herpes simplex virus type1 (HSV-1); comparing TI in the above table, it can be seen that the n-butanol extract fraction or the ethyl acetate extract fraction has stronger antiviral activity than the other extract fractions.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims

1. The application of the n-butanol extraction part of the radix tetrastigme alcohol extract in preparing anti-RSV or anti-HSV-1 medicines is characterized in that the preparation method of the n-butanol extraction part of the radix tetrastigme alcohol extract comprises the following steps:

extracting the radix tetrastigme ethanol total extract suspension with n-butanol, concentrating the extract under reduced pressure, drying to constant weight, and weighing; the preparation method of the total ethanol extract of radix tetrastigme comprises the following steps: in the material-liquid ratio of 15:1, under the condition that the concentration of ethanol is 60 wt%, carrying out ultrasonic extraction for 20min, and then carrying out reflux extraction for 20 min.

2. The use of claim 1, wherein the extraction is carried out by thermal sonication.

3. The use of claim 1, wherein the mass ratio of the n-butanol to the total ethanol extract suspension of radix tetrastigme is 1: 1.

4. the application of the ethyl acetate extraction part of the radix tetrastigme alcohol extract in preparing anti-RSV or anti-HSV-1 medicines is characterized in that the preparation method of the ethyl acetate extraction part of the radix tetrastigme alcohol extract comprises the following steps:

extracting the radix tetrastigme ethanol total extract suspension with ethyl acetate, concentrating the extract under reduced pressure, drying to constant weight, and weighing;

the preparation method of the total ethanol extract of radix tetrastigme comprises the following steps: in the material-liquid ratio of 15:1, under the condition that the concentration of ethanol is 60 wt%, carrying out ultrasonic extraction for 20min, and then carrying out reflux extraction for 20 min.

5. The use of claim 4, wherein the extraction is carried out by thermal sonication.

6. The use of claim 4, wherein the mass ratio of the ethyl acetate to the total extract suspension of radix tetrastigme ethanol is 1: 1.