CN112675211B - Cyclocarya paliurus extract and application thereof in preparation of anti-coronavirus medicines - Google Patents
Cyclocarya paliurus extract and application thereof in preparation of anti-coronavirus medicines Download PDFInfo
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Abstract
The invention discloses a cyclocarya paliurus extract and application thereof in preparing an anti-coronavirus medicament. In the cyclocarya paliurus extract, the content of flavone is 20-40 wt.%, the content of polysaccharide is 5-20 wt.%, and the content ratio of flavone to polysaccharide is more than or equal to 1.5: 1. experimental results show that the cyclocarya paliurus extract with specific flavone and polysaccharide contents has the SARS-CoV-2 virus neutralization activity, can effectively inhibit cytopathic effect caused by SARS-CoV-2 virus, and can protect cells from being infected by SARS-CoV-2 virus. The cyclocarya paliurus extract provided by the invention has wide application prospect in preparing anti-coronavirus (particularly SARS-CoV-2 virus) medicines with good safety and low preparation cost.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a cyclocarya paliurus extract and application thereof in preparing an anti-coronavirus medicament.
Background
The coronaviruses which can infect human and are discovered at present are 7 types: HcoV-229E, HcoV-NL63, HcoV-HKU1, HcoV-OC43, Severe acute respiratory syndrome coronavirus (SARS-CoV), Zhongdong respiratory syndrome coronavirus (MERS-CoV), and 2019 novel coronavirus (2019-nCoV, SARS-CoV-2). Among them, SARS-CoV-2 is a novel beta coronavirus belonging to single-stranded RNA virus, which has extremely strong infectivity and is generally susceptible to the population. At present, although there is a certain understanding on the epidemiology, clinical characteristics, diagnosis, treatment and prevention outcome of the novel coronavirus pneumonia caused by SARS-CoV-2, the treatment still can not meet the clinical requirements, and the treatment is lack of specific drugs, and the form is still very severe.
Existing antiviral drugs are mostly ineffective against SARS-CoV-2, for example, the anti-HIV drug kresoxim (lopinavir and ritonavir) has poor effect and great side effect in clinical trials for treating the novel coronavirus pneumonia. Antibodies, small proteins, and lipopeptides targeting spike S protein, while effective in blocking coronavirus entry into cells, are expensive. Therefore, there is a need for the development of a novel drug having good safety, low production cost and excellent anti-SARS-CoV-2 virus activity.
The traditional Chinese medicine extract has received wide attention due to the advantages of good safety and low preparation cost. Cyclocarya paliurus, cyclocarya paliurus (Batal.) Iljinskaja, also called Qingqian plum, money tree, sweet tea tree, etc., is a unique single species of plants of cyclocarya paliurus of Juglandaceae, is unique to China, and is widely distributed in Jiangxi, Guangxi, etc. Cyclocarya paliurus is a high-speed growing broad-leaf tree, and is named as cyclocarya paliurus because the tree shape is similar to that of the cyclocarya paliurus, the fruit is round and similar to copper cash, and the color is blue and drooping. In recent years, the study of physical and chemical components and health care functions of cyclocarya paliurus at animal level and clinical tests by scholars at home and abroad shows that the cyclocarya paliurus and related health care products have various beneficial pharmacological effects and health care effects, including the effects of reducing blood pressure and blood sugar, losing weight, resisting tumors and allergy, enhancing body immunity and the like. However, there has been no report on the use of cyclocarya paliurus extract for preparing a drug against SARS-CoV-2 virus.
Disclosure of Invention
In order to solve the problems of good safety, low preparation cost and excellent drug shortage of resisting SARS-CoV-2 virus, the invention aims to provide a cyclocarya paliurus extract and application thereof in preparing drugs for resisting coronavirus (particularly SARS-CoV-2 virus).
The invention provides a cyclocarya paliurus extract, wherein the content of flavone is 20-40 wt%, the content of polysaccharide is 5-20 wt%, and the content ratio of flavone to polysaccharide is more than or equal to 1.5: 1.
further, the content ratio of the flavone to the polysaccharide is (2.0-3.0): 1. furthermore, in the cyclocarya paliurus extract, the content of flavone is 25 wt.% to 26 wt.%, and the content of polysaccharide is 10 wt.% to 11 wt.%.
Furthermore, in the cyclocarya paliurus extract, the content of flavone is 25.357 wt.%, and the content of polysaccharide is 10.206 wt.%.
The invention also provides a preparation method of the cyclocarya paliurus extract, which comprises the following steps:
(1) weighing cyclocarya paliurus leaves, adding ethanol for soaking, removing supernatant, and leaving precipitate;
(2) adding water into the precipitate, soaking, and collecting supernatant;
(3) concentrating or drying the supernatant obtained in the step (2) to obtain the cyclocarya paliurus extract.
Further, in the step (1), the cyclocarya paliurus leaves are crushed cyclocarya paliurus leaves; the weight ratio of the cyclocarya paliurus leaves to the ethanol is 1: (4-6), wherein the ethanol is 70% -90% ethanol; the soaking temperature is normal temperature, and the soaking time is 2-6 h; centrifuging to remove the supernatant, wherein the centrifuging speed is 3000-5000 r/min, and the centrifuging time is 5-15 min;
and/or, in the step (2), the weight ratio of the precipitate to the water is 1: (3-5); the soaking temperature is normal temperature to 90 ℃, and the soaking time is 2 to 6 hours; the supernatant is taken by centrifugation at a speed of 3000-5000 r/min for 5-15 min.
Further, in the step (1), the cyclocarya paliurus leaves are dried leaves or fresh leaves of cyclocarya paliurus (Batal.) Iljinskaja; the weight ratio of the cyclocarya paliurus leaves to the ethanol is 1:5, the ethanol is 80% ethanol; the soaking temperature is normal temperature, and the soaking time is 4 hours; the centrifugation speed is 4800r/min, and the time is 10 min;
and/or, in the step (2), the weight ratio of the precipitate to the water is 1: 4; the soaking temperature is 85 ℃, and the soaking time is 4 hours; the speed of centrifugation is 4800r/min, and the time is 10 min.
The invention also provides application of the cyclocarya paliurus extract in preparation of anti-coronavirus medicines.
Further, the drug has coronavirus neutralizing activity; and/or, the medicament is capable of inhibiting coronavirus-induced cytopathic effects.
Further, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HcoV-229E, HcoV-NL63, HcoV-HKU1 or HcoV-OC43, preferably SARS-CoV-2.
The invention also provides an anti-coronavirus medicine, which is a preparation prepared by taking the cyclocarya paliurus extract as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
Preferably, in the unit preparation of the medicine, the content of the cyclocarya paliurus extract is 1.8-2.0 mg, and 1.9064mg is preferred.
"Normal temperature" means 25. + -. 2 ℃.
An agent "having coronavirus neutralizing activity" refers to an agent capable of protecting more than 50% of cells from infection by a virus.
Experimental results show that the cyclocarya paliurus extract with flavone and polysaccharide contents has SARS-CoV-2 virus neutralization activity, can effectively inhibit cytopathic effect caused by SARS-CoV-2 virus, and can protect cells from being infected by SARS-CoV-2 virus. The cyclocarya paliurus extract provided by the invention has a wide application prospect in preparing anti-coronavirus medicines with good safety and low preparation cost.
The cyclocarya paliurus extract disclosed by the invention is simple in preparation method, low in cost and suitable for industrial production.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
Example 1: preparation of cyclocarya paliurus leaf extract
(1) Weighing 100kg of cyclocarya paliurus leaves dried in the shade at room temperature, pulverizing, adding 80% ethanol with a material-liquid ratio of 1:5(w/w), soaking at room temperature for 4h, centrifuging at 4800r/min for 10min, and removing supernatant. This step was repeated 2 times.
(2) Adding water with a feed-liquid ratio of 1:4(w/w) into the precipitate left after centrifuging to remove the supernatant, soaking at 85 deg.C for 4h, centrifuging at 4800r/min for 10min, and collecting the supernatant. This step was repeated 2 times.
(3) The supernatant obtained in step (2) was concentrated and then dried under vacuum at 50 ℃ for 24 hours to obtain 12.2kg of a powdery solid (named extract 1).
Measuring the content of flavone in the extract 1 to be 25.357 wt% by using ultraviolet-visible spectrophotometry (the general rule 0401 of 2015 edition in Chinese pharmacopoeia) and rutin as a control; determining the content of polysaccharide in the extract 1 as 10.206 wt.% by using D-glucose as control
Comparative example 1: preparation of control cyclocarya paliurus leaf extract
(1) Weighing 100kg of cyclocarya paliurus leaves dried in the shade at room temperature, pulverizing, adding 80% ethanol with a material-liquid ratio of 1:5(w/w), soaking at room temperature for 4h, centrifuging at 4800r/min for 10min, and collecting the supernatant. This step was repeated 2 times.
(2) Concentrating the supernatant obtained in the step (1), adding 5 times of hot water at 80 ℃, uniformly stirring, and standing at normal temperature for 24 hours. Centrifuging at 4800r/min for 10min, concentrating the supernatant, vacuum drying at 50 deg.C for 24 hr to obtain powdery solid 9kg (named as extract 2), and storing at 4 deg.C.
Measuring the content of flavone in the extract 2 by ultraviolet-visible spectrophotometry (according to the general rule 0401 in 2015 th edition of Chinese pharmacopoeia) with rutin as control to obtain 26.615 wt%; the content of polysaccharide in the extract 2 was determined to be 22.782 wt.% based on D-glucose as a control.
The beneficial effects of the present invention are demonstrated by the following experimental examples.
Experimental example 1: neutralization test of cyclocarya paliurus leaf extract on 2019 novel coronavirus (SARS-CoV-2)
1. Viral strains and drugs
Test virus strains: SARS-CoV-2 virus (BetacoV/JS02/human/2020, New isolated virus strain of clinical cases of coronary pneumonia in Jiangsu province).
And (3) culturing the virus: SARS-CoV-2 virus was isolated by culturing in VeroE6 cell line. VeroE6 cells were seeded in 24-well cell culture plates in Dulbecco's Minimal Essential Medium (DMEM) medium containing 10% fetal bovine serum and antibiotics at 37 deg.C with 5% CO2Culturing for 3-4 days until 80% of the bottom of the hole is full of monolayer cells. 50ul of the virus stock was inoculated into 24-well plates and cultured for 3 days until the cells became diseased. Digesting the adherent single-layer VeroE6 cells by using 0.5% papain, performing ultrasonication, centrifuging to remove precipitates, separating supernatant, and storing at-80 ℃. The virus amount was determined by standard plaque counting method.
The tested drugs are: separately, 19.064mg and 19.064mg of cyclocarya paliurus leaf extract powders prepared in example 1 (extract 1) and comparative example 1 (extract 2) were precisely weighed, dissolved in 10ml of DMEM medium (without serum), and filtered and sterilized by a 0.22 μm filter to obtain 1.9064mg/ml extract 1 stock solution and 1.9064mg/ml extract 2 stock solution, which were stored at 4 ℃ for further use.
2. Test procedure
1) Vero E6 cells (Vero cells) were plated at 5X 104Each cell/well was inoculated in 96-well plates and cultured overnight;
2) the prepared extract stock solutions are all diluted according to the ratio of 1:1, 1:2, 1:4 and 1: 8, diluting, and incubating 100 μ L of each of the obtained solutions with 100TCID50 SARS-CoV-2 virus at 37 deg.C for 1 hr;
3) adding the incubated extract and SARS-CoV-2 mixture into Vero E6 cells, culturing for 3 days, setting multiple wells for each dilution;
4) after 3 days, the viral cytopathic effect (CPE) was observed by light microscopy, with 0-25% cellular CPE changes designated "+", 26-50% cellular CPE changes designated "+", 51-75% cellular CPE changes designated "+ +", 76-100% cellular CPE changes designated "+ + + + +", and normal cell morphology designated "-", and the results were recorded.
5) The experiment is simultaneously provided with a normal cell control group (no virus and no extract), a virus control group (100 TCID50 SARS-CoV-2 virus and no extract) and a neutralizing antibody control group (100 TCID50 SARS-CoV-2 virus and neutralizing antibody); in addition, to ensure the accuracy of the experiment, a virus titer back-drop group was set, and the virus titer back-drop 1TCID50/0.1mL was 10 e-5.0.
The neutralizing antibody is the corresponding antibody produced when SARS-CoV-2 virus invades the body, and the neutralizing antibody can protect cells from infection by SARS-CoV-2 virus.
3. Result determination and analysis
The concentration at which 50% or more of the cells are protected from virus infection is determined as a concentration at which virus-neutralizing activity is exhibited, i.e., an effective concentration.
Statistical analysis was performed using the IBM SPSS statistics 25 software using the Chi-Square test, with P <0.05 indicating that the differences were statistically significant.
4. Results of the experiment
TABLE 1 test results of viral cytopathic Effect
Note: the P value represents the P value compared to the extract 1 group.
TABLE 2 test results of virus neutralizing Activity
The results of the experiment are shown in tables 1 and 2. The cells of the normal cell control group grew well; the neutralizing antibody control group had no cytopathic effect; the virus control group showed cytopathic effects of ++++ - +++++ respectively.
Extract 2 at each concentration had no virus-neutralizing activity and failed to protect cells from infection by SARS-CoV-2 virus; however, extract 1 (stock solution, concentration 1.9064mg/ml) has virus-neutralizing activity and protects more than 50% of cells from infection by SARS-CoV-2 virus. The difference between the virus neutralization of extract 1 (stock solution) and extract 2 (stock solution) was statistically significant (P < 0.05). The above experimental results show that only the cyclocarya paliurus leaf extract (extract 1) of example 1 of the present invention has virus-neutralizing activity, can significantly inhibit cytopathic effect caused by SARS-CoV-2 virus, and protect cells from SARS-CoV-2 virus infection.
In conclusion, the invention provides a cyclocarya paliurus extract and application thereof in preparing an anti-coronavirus medicine. Experimental results show that the cyclocarya paliurus extract with specific flavone and polysaccharide contents has the SARS-CoV-2 virus neutralization activity, can effectively inhibit cytopathic effect caused by SARS-CoV-2 virus, and can protect cells from being infected by SARS-CoV-2 virus. The cyclocarya paliurus extract provided by the invention has a wide application prospect in preparing anti-coronavirus medicines with good safety and low preparation cost.
Claims (11)
1. A cyclocarya paliurus extract is characterized in that: in the cyclocarya paliurus extract, the content of flavone is 20-40 wt.%, the content of polysaccharide is 5-20 wt.%, and the content ratio of flavone to polysaccharide is more than or equal to 1.5: 1;
the preparation method of the cyclocarya paliurus extract comprises the following steps:
(1) weighing cyclocarya paliurus leaves, adding ethanol for soaking, removing supernatant, and leaving precipitate; the ethanol is 70-90% ethanol;
(2) adding water into the precipitate, soaking, and collecting supernatant;
(3) concentrating or drying the supernatant obtained in the step (2) to obtain the cyclocarya paliurus extract.
2. The cyclocarya paliurus extract of claim 1, wherein: the content ratio of the flavone to the polysaccharide is (2.0-3.0): 1.
3. the cyclocarya paliurus extract of claim 2, wherein: in the cyclocarya paliurus extract, the content of flavone is 25-26 wt%, and the content of polysaccharide is 10-11 wt%.
4. The cyclocarya paliurus extract of claim 1, wherein: in the step (1), the cyclocarya paliurus leaves are crushed cyclocarya paliurus leaves; the weight ratio of the cyclocarya paliurus leaves to the ethanol is 1: (4-6); the soaking temperature is normal temperature, and the soaking time is 2-6 h; centrifuging to remove the supernatant, wherein the centrifuging speed is 3000-5000 r/min, and the centrifuging time is 5-15 min;
and/or, in the step (2), the weight ratio of the precipitate to the water is 1: (3-5); the soaking temperature is normal temperature to 90 ℃, and the soaking time is 2 to 6 hours; the supernatant is taken by centrifugation at a speed of 3000-5000 r/min for 5-15 min.
5. The cyclocarya paliurus extract of claim 4, wherein: in the step (1), the cyclocarya paliurus leaves are dried leaves or fresh leaves of cyclocarya paliurus (Batal.) Iljinskaja; the weight ratio of the cyclocarya paliurus leaves to the ethanol is 1:5, the ethanol is 80% ethanol; the soaking temperature is normal temperature, and the soaking time is 4 hours; the centrifugation speed is 4800r/min, and the time is 10 min;
and/or, in the step (2), the weight ratio of the precipitate to the water is 1: 4; the soaking temperature is 85 ℃, and the soaking time is 4 hours; the speed of centrifugation is 4800r/min, and the time is 10 min.
6. A preparation method of cyclocarya paliurus extract is characterized by comprising the following steps: the preparation method comprises the following steps:
(1) weighing cyclocarya paliurus leaves, adding ethanol for soaking, removing supernatant, and leaving precipitate; the ethanol is 70-90% ethanol;
(2) adding water into the precipitate, soaking, and collecting supernatant;
(3) concentrating or drying the supernatant obtained in the step (2) to obtain the cyclocarya paliurus extract.
7. The method of claim 6, wherein: in the step (1), the cyclocarya paliurus leaves are crushed cyclocarya paliurus leaves; the weight ratio of the cyclocarya paliurus leaves to the ethanol is 1: (4-6); the soaking temperature is normal temperature, and the soaking time is 2-6 h; centrifuging to remove the supernatant, wherein the centrifuging speed is 3000-5000 r/min, and the centrifuging time is 5-15 min;
and/or, in the step (2), the weight ratio of the precipitate to the water is 1: (3-5); the soaking temperature is normal temperature to 90 ℃, and the soaking time is 2 to 6 hours; the supernatant is taken by centrifugation at a speed of 3000-5000 r/min for 5-15 min.
8. The method of claim 7, wherein: in the step (1), the cyclocarya paliurus leaves are dried leaves or fresh leaves of cyclocarya paliurus (Batal.) Iljinskaja; the weight ratio of the cyclocarya paliurus leaves to the ethanol is 1:5, the ethanol is 80% ethanol; the soaking temperature is normal temperature, and the soaking time is 4 hours; the centrifugation speed is 4800r/min, and the time is 10 min;
and/or, in the step (2), the weight ratio of the precipitate to the water is 1: 4; the soaking temperature is 85 ℃, and the soaking time is 4 hours; the speed of centrifugation is 4800r/min, and the time is 10 min.
9. Use of the cyclocarya paliurus extract of any one of claims 1 to 5 in the preparation of a medicament against coronavirus, wherein the coronavirus is SARS-CoV-2.
10. Use according to claim 9, characterized in that: the drug has coronavirus neutralizing activity; and/or, the medicament is capable of inhibiting coronavirus-induced cytopathic effects.
11. An anti-coronavirus agent characterized by: the medicine is a preparation prepared by taking the cyclocarya paliurus extract as an active ingredient according to any one of claims 1 to 5 and adding pharmaceutically acceptable auxiliary materials.
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| CN103694364A (en) * | 2013-12-10 | 2014-04-02 | 南昌大学 | Method for synchronously extracting, separating and purifying polysaccharides and flavones of cyclocarya paliurus |
| CN105713802A (en) * | 2015-12-10 | 2016-06-29 | 黔东南森林王红豆杉发展有限责任公司 | Preparation method of cyclocarya paliurus liquor |
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