CN112759599B - Effective part of elephantopus scaber and application thereof in preparation of medicine with respiratory syncytial virus resisting effect - Google Patents
Effective part of elephantopus scaber and application thereof in preparation of medicine with respiratory syncytial virus resisting effect Download PDFInfo
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Abstract
The invention relates to the technical field of natural medicinal chemistry, and particularly discloses an effective part of elephantopus scaber and application thereof in preparing a medicament with an effect of resisting respiratory syncytial virus. The preparation method of the effective part of the elephantopus scaber comprises the following steps: (1) extracting herba elephantopi with ethanol to obtain herba elephantopi ethanol extract; (2) suspending the elephantopus scaber ethanol extract with water, and extracting with petroleum ether, ethyl acetate and n-butanol respectively to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract; (3) loading the petroleum ether extract on a silica gel column, eluting by using a mixed organic solvent A, collecting an elution part, concentrating and drying to obtain a silica gel elution part A; (4) and (3) putting the silica gel elution part A on a silica gel column again, eluting by adopting a mixed organic solvent B, and collecting the elution part to obtain the effective part of the elephantopus scaber. The effective part of the elephantopus scaber prepared by the method has excellent effect of resisting respiratory syncytial virus.
Description
Technical Field
The invention relates to the technical field of natural medicinal chemistry, in particular to an effective part of elephantopus scaber and application thereof in preparing a medicament with an effect of resisting respiratory syncytial virus.
Background
Respiratory Syncytial Virus (RSV) infection is widespread worldwide, is universally susceptible to populations, and can occur as a result of repeated infections and co-infections with multiple Respiratory viruses. Thus, RSV is a common human pathogen and poses a threat to the health of newborns worldwide. The infection routes of RSV are mainly divided into air transmission and contact transmission, and the RSV can enter a human body through eyes, nose and throat, so that people or objects carrying RSV by contacting at a short distance have a certain infection risk, especially infants or people with low immunity. The clinical manifestations are mainly high fever, chest distress, shortness of breath, wheezing, cough and other upper respiratory tract infection symptoms, and long-term infection can cause viruses to enter lower respiratory tract to cause bronchiolitis, pneumonia and the like. Infants are one of the main population with lower respiratory tract infection, wherein pneumonia and bronchiolitis are the causes of hospitalization of infants
And one of the leading causes of death. In addition, the elderly and people with low immunity are also the main pathogens of lower respiratory tract infections. RSV is reported by the WHO to infect about 6.4 million people per year with a mortality rate of about 0.25%.
The safe and effective RSV vaccine can prevent the lower respiratory tract diseases of high-risk people caused by RSV infection, and reduce the hospitalization rate and the death rate. The formalin fire vaccine studied in the 20 th century was the first RSV vaccine, but it eventually ended up failing. Although a variety of vaccines, such as live attenuated vaccines, vector vaccines, subunit vaccines, etc., have been developed in the late stage, no safe and effective RSV vaccine has been developed yet for clinical use.
At present, the clinical medicines for resisting RSV infection are few in types, mainly ribavirin, Palivizumab (Palivizumab) and some Chinese patent medicines with broad-spectrum antiviral effect. A large amount of clinical data show that the ribavirin has extremely strong side effects, is not suitable for pregnant women and children, and is mostly used for severe patients; the action mechanism of broad-spectrum antiviral Chinese patent medicines, such as lotus antipyretic granules/capsules, antipyretic and yanning capsules and the like, on RSV infection is not clear. Therefore, the further development of the medicine with the effect of resisting the respiratory syncytial virus has important significance.
The invention discloses a elephantopus scaber extract containing a series of caffeoylquinic acid components in patent CN102219687A, and experiments show that the elephantopus scaber extract has an IC50 value of less than 5.0 mu g/mL and has a good effect of resisting the respiratory syncytial virus. However, the inventor believes that the active substance basis of the elephantopus scaber for resisting the respiratory syncytial virus is still not completely understood at present, and a large number of effective parts with antiviral effect and effective components still exist in the elephantopus scaber to be further developed.
Disclosure of Invention
Aiming at the defects of the prior art in developing effective parts of the elephantopus scaber for resisting the respiratory syncytial virus, the invention firstly provides a preparation method of the effective parts of the elephantopus scaber. The herba elephantopi effective part prepared by the method has excellent effect of resisting respiratory syncytial virus.
The technical scheme of the invention is as follows:
a preparation method of an effective part of elephantopus scaber comprises the following steps:
(1) extracting herba elephantopi with ethanol to obtain herba elephantopi ethanol extract;
(2) suspending the elephantopus scaber ethanol extract with water, and extracting with petroleum ether, ethyl acetate and n-butanol respectively to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract;
(3) loading the petroleum ether extract on a silica gel column, eluting by using a mixed organic solvent A, collecting an elution part, concentrating and drying to obtain a silica gel elution part A;
(4) and (3) putting the silica gel elution part A on a silica gel column again, eluting by adopting a mixed organic solvent B, and collecting the elution part to obtain the effective part of the elephantopus scaber.
Preferably, the ethanol in the step (1) is ethanol with a volume fraction of 80-100%.
Preferably, the extraction in step (1) is heating reflux extraction.
Preferably, the mixed organic solvent A in the step (3) consists of petroleum ether and ethyl acetate;
the specific elution conditions of step (3) are: putting the petroleum ether extract into a silica gel column, and eluting by using a mixed organic solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 95-90: 5-10; then eluting with a mixed organic solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 85-80: 15-20; and collecting the elution part eluted by the mixed organic solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 85-80: 15-20, and concentrating and drying to obtain the silica gel elution part A.
Further preferably, the specific elution conditions of step (3) are: loading the petroleum ether extract on a silica gel column, and eluting by using a mixed organic solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 92: 8; then eluting with a mixed organic solvent consisting of petroleum ether and ethyl acetate according to the volume ratio of 85: 15; collecting the eluted part eluted by the mixed organic solvent consisting of petroleum ether and ethyl acetate with the volume ratio of 85:15, concentrating and drying to obtain the silica gel eluted part A.
Preferably, the mixed organic solvent B in the step (4) consists of chloroform and methanol;
the specific elution conditions of step (4) are: putting a silica gel eluting part A on a silica gel column, and eluting by using a mixed organic solvent consisting of chloroform and methanol in a volume ratio of 99-97: 1-3; then eluting with a mixed organic solvent consisting of chloroform and methanol in a volume ratio of 95-92: 5-8; collecting the eluted part eluted by the mixed organic solvent consisting of chloroform and methanol in a volume ratio of 95-92: 5-8, concentrating and drying to obtain the effective part of the elephantopus scaber.
Further preferably, the mixed organic solvent B in the step (4) consists of chloroform and methanol;
the specific elution conditions of step (4) are: applying a silica gel column on the silica gel elution part A, and eluting by using a mixed organic solvent consisting of chloroform and methanol in a volume ratio of 98: 2; then eluting with a mixed organic solvent consisting of chloroform and methanol in a volume ratio of 94: 6; collecting the eluted part eluted by a mixed organic solvent consisting of chloroform and methanol with the volume ratio of 94:6, concentrating and drying to obtain the effective part of the elephantopus scaber.
The inventor shows through a large number of experimental studies that whether the effective part with the effect of resisting respiratory syncytial can be prepared or not in the process of preparing the effective part of the elephantopus scaber, which is closely related to the silica gel elution conditions of the steps (3) and (4); studies have shown that steps (3) and (4) cannot be reversed, i.e.: the effective part of the elephantopus scaber with excellent effect of resisting the respiratory syncytial virus can be obtained only by adopting the mixed organic solvent A consisting of petroleum ether and ethyl acetate under the condition of the step (3) for elution and then adopting the mixed organic solvent B consisting of chloroform and methanol under the condition of the step (4) for elution. If the steps (3) and (4) are reversed or the ratio of the organic solvent in the steps (3) and (4) is different, the effective part of the elephantopus scaber with excellent anti-respiratory syncytial virus effect cannot be obtained, or the effective part of the elephantopus scaber with the anti-respiratory syncytial virus effect cannot be obtained at all.
Further research shows that the effective part of the elephantopus scaber prepared by the method contains isodeoxyelephantopus scaber lactone and deoxyelephantopus scaber lactone.
The effective part of the elephantopus scaber prepared by the method is detected by adopting a silica gel thin-layer plate, and is developed by using sulfuric acid-vanillin, so that the effective part of the elephantopus scaber mainly contains two purple components and three other components with weak purple; further loading the effective part of herba Ajugae onto silica gel column, continuously eluting with mixed organic solvent composed of chloroform and methanol at volume ratio of 96:4, separating to obtain 2 main components, and identifying the two main components by structure to obtain isodeoxyelephantolide and deoxyelephantolide. Therefore, the effective part of the elephantopus scaber prepared by the method mainly contains isodeoxyelephantopus scaber lactone, deoxyelephantopus scaber lactone and other small-amount elephantopus scaber lactone compounds.
The invention also provides the effective part of the elephantopus scaber prepared by the preparation method.
The invention also provides application of the effective part of the elephantopus scaber in preparing a medicament with the effect of resisting respiratory syncytial virus.
Has the advantages that: the invention provides a brand-new method for preparing the effective part of elephantopus scaber, and the effective part of elephantopus scaber prepared by the method has excellent effect of resisting respiratory syncytial virus; experiments show that the IC50 value of the extract on the respiratory syncytial virus is superior to the IC50 value of the extract on the respiratory syncytial virus, which contains a series of caffeoylquinic acid components and is disclosed in the patent CN 102219687A.
Detailed Description
The present invention is further explained below with reference to specific examples, which are not intended to limit the present invention in any way.
Example 1 preparation of effective fractions of elephantopus scaber
(1) Taking 400g of dried elephantopus scaber herb, carrying out heating reflux extraction on the dried elephantopus scaber herb with 4L of ethanol with the volume fraction of 95%, wherein the extraction time is 1.5h and the extraction times are 2 times, combining the two extracting solutions, concentrating and drying to obtain the elephantopus scaber herb ethanol extract;
(2) suspending the elephantopus scaber ethanol extract with 10 times of water, extracting with petroleum ether, ethyl acetate and n-butanol respectively, and concentrating the extractive solution to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract;
(3) applying the petroleum ether extract to a silica gel column (the weight of the silica gel filler in the silica gel column is 30 times of that of the ethyl acetate extract), and eluting with a mixed organic solvent of petroleum ether and ethyl acetate at a volume ratio of 92:8 of 5 times of the column volume (the part is discarded); then eluting with a mixed organic solvent which is 8 times of the column volume and consists of petroleum ether and ethyl acetate with the volume ratio of 85: 15; collecting the elution part eluted by the mixed organic solvent consisting of petroleum ether and ethyl acetate with the volume ratio of 85:15, concentrating and drying to obtain a silica gel elution part A;
(4) applying silica gel column to the silica gel elution part A, eluting with 5 times of mixed organic solvent composed of chloroform and methanol at a volume ratio of 98:2 (the part is discarded); then eluting with a mixed organic solvent consisting of chloroform and methanol with the volume ratio of 94:6, wherein the volume ratio of the mixed organic solvent is 8 times of the column volume; collecting the eluted part eluted by a mixed organic solvent consisting of chloroform and methanol with the volume ratio of 94:6, concentrating and drying to obtain the effective part of the elephantopus scaber.
The effective part of the elephantopus scaber prepared in the embodiment is detected by adopting a silica gel thin-layer plate, and is developed by using sulfuric acid-vanillin, so that the effective part of the elephantopus scaber mainly contains two purple components and three other components with weak purple; further loading the effective part of herba Ajugae onto silica gel column, continuously eluting with mixed organic solvent composed of chloroform and methanol at volume ratio of 96:4, separating to obtain 2 main components, and identifying the two main components by structure to obtain isodeoxyelephantolide and deoxyelephantolide. Therefore, the effective part of the elephantopus scaber prepared by the embodiment mainly contains the isodeoxyelephantopus scaber lactone and the deoxyelephantopus scaber lactone, and also contains other small-amount elephantopus scaber lactone compounds.
Comparative example 1 preparation of effective fractions of elephantopus scaber
(1) Taking 400g of dried elephantopus scaber herb, carrying out heating reflux extraction on the dried elephantopus scaber herb with 4L of ethanol with the volume fraction of 95%, wherein the extraction time is 1.5h and the extraction times are 2 times, combining the two extracting solutions, concentrating and drying to obtain the elephantopus scaber herb ethanol extract;
(2) suspending the elephantopus scaber ethanol extract with 10 times of water, extracting with petroleum ether, ethyl acetate and n-butanol respectively, and concentrating the extractive solution to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract;
(3) loading petroleum ether extract on silica gel column, eluting with 5 times column volume of mixed organic solvent composed of chloroform and methanol at volume ratio of 98:2 (the part is discarded); then eluting with a mixed organic solvent consisting of chloroform and methanol with the volume ratio of 94:6, wherein the volume ratio of the mixed organic solvent is 8 times of the column volume; collecting the eluted part eluted by the mixed organic solvent of chloroform and methanol with the volume ratio of 94:6, concentrating and drying to obtain the silica gel eluted part A.
(4) Applying silica gel column to silica gel elution part A (the weight of silica gel filler in silica gel column is 30 times of that of ethyl acetate extract), and eluting with 5 times of mixed organic solvent composed of petroleum ether and ethyl acetate at volume ratio of 92:8 (the part is discarded); then eluting with a mixed organic solvent which is 8 times of the column volume and consists of petroleum ether and ethyl acetate with the volume ratio of 85: 15; collecting the eluted part eluted by the mixed organic solvent consisting of petroleum ether and ethyl acetate with the volume ratio of 85:15, concentrating and drying to obtain the effective part of the elephantopus scaber.
Comparative example 1 differs from example 1 in that comparative example 1 reverses steps (3) and (4) of example 1; the method comprises the steps of eluting by adopting a mixed organic solvent composed of chloroform and methanol, and then eluting by adopting a mixed organic solvent composed of petroleum ether and ethyl acetate.
Comparative example 2 preparation of effective fractions of elephantopus scaber
(1) Taking 400g of dried elephantopus scaber herb, carrying out heating reflux extraction on the dried elephantopus scaber herb with 4L of ethanol with the volume fraction of 95%, wherein the extraction time is 1.5h and the extraction times are 2 times, combining the two extracting solutions, concentrating and drying to obtain the elephantopus scaber herb ethanol extract;
(2) suspending the elephantopus scaber ethanol extract with 10 times of water, extracting with petroleum ether, ethyl acetate and n-butanol respectively, and concentrating the extractive solution to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract;
(3) applying the petroleum ether extract to a silica gel column (the weight of the silica gel filler in the silica gel column is 30 times of that of the ethyl acetate extract), and eluting with a mixed organic solvent of petroleum ether and ethyl acetate at a volume ratio of 85:15 of 5 times of the column volume (the part is discarded); then eluting with a mixed organic solvent which is 8 times of the column volume and consists of petroleum ether and ethyl acetate with the volume ratio of 75: 25; collecting the elution part eluted by the mixed organic solvent consisting of petroleum ether and ethyl acetate with the volume ratio of 75:25, concentrating and drying to obtain a silica gel elution part A;
(4) applying silica gel column to the silica gel elution part A, eluting with 5 times of mixed organic solvent composed of chloroform and methanol at a volume ratio of 98:2 (the part is discarded); then eluting with a mixed organic solvent consisting of chloroform and methanol with the volume ratio of 94:6, wherein the volume ratio of the mixed organic solvent is 8 times of the column volume; collecting the eluted part eluted by a mixed organic solvent consisting of chloroform and methanol with the volume ratio of 94:6, concentrating and drying to obtain the effective part of the elephantopus scaber.
Comparative example 2 is different from example 1 in that the elution conditions of the mixed organic solvent consisting of petroleum ether and ethyl acetate in step (3) of comparative example 2 are different.
Comparative example 3 preparation of effective fractions of elephantopus scaber
(1) Taking 400g of dried elephantopus scaber herb, carrying out heating reflux extraction on the dried elephantopus scaber herb with 4L of ethanol with the volume fraction of 95%, wherein the extraction time is 1.5h and the extraction times are 2 times, combining the two extracting solutions, concentrating and drying to obtain the elephantopus scaber herb ethanol extract;
(2) suspending the elephantopus scaber ethanol extract with 10 times of water, extracting with petroleum ether, ethyl acetate and n-butanol respectively, and concentrating the extractive solution to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract;
(3) applying the petroleum ether extract to a silica gel column (the weight of the silica gel filler in the silica gel column is 30 times of that of the ethyl acetate extract), and eluting with a mixed organic solvent of petroleum ether and ethyl acetate at a volume ratio of 92:8 of 5 times of the column volume (the part is discarded); then eluting with a mixed organic solvent which is 8 times of the column volume and consists of petroleum ether and ethyl acetate with the volume ratio of 85: 15; collecting the elution part eluted by the mixed organic solvent consisting of petroleum ether and ethyl acetate with the volume ratio of 85:15, concentrating and drying to obtain a silica gel elution part A;
(4) applying silica gel column to silica gel eluting part A, eluting with 5 times of mixed organic solvent composed of chloroform and methanol at volume ratio of 95:5 (the part is discarded); then eluting with a mixed organic solvent consisting of chloroform and methanol with the volume ratio of 90:10 and the volume of 8 times of the column volume; collecting the eluted part eluted by the mixed organic solvent consisting of chloroform and methanol with the volume ratio of 90:10, concentrating and drying to obtain the effective part of the elephantopus scaber.
Comparative example 3 is different from example 1 in that the elution conditions of the mixed organic solvent consisting of chloroform and methanol in step (4) of comparative example 2 are different.
Experimental example 1 Studies on anti-RSV Activity of effective fractions of elephantopus scaber
The anti-RSV activity of the effective part of the elephantopus scaber is tested by adopting a plaque reduction experiment method, and the specific method is as follows: (1) HEp-2 cells were plated in 24-well cell culture plates (1 mL per well, cell density 2X 10)5cell/mL), followed by incubation at 37 ℃ with 5% CO2Culturing in an incubator until a cell monolayer grows; (2) 100 mu L of respiratory syncytial virus suspension with the concentration of 100 mu L and different drug concentration are added into each hole, and a cell control group and a virus control group are arranged at the same time; standing at 37 deg.C for 5% CO2Culturing in an incubator for 2 h. The cross is gently shaken every 15min to ensure that the virus is uniformly and fully adsorbed and penetrates into cells; (3) after the virus is adsorbed for 2 hoursRemoving virus liquid, washing for 2 times by PBS, adding 0.5mL of agarose gel covering liquid into each hole, adding 0.5mL of medicine with corresponding concentration after the covering liquid is solidified, and adding equal-volume maintenance liquid into a cell control group and a virus control group; (4) and (3) placing the 24-hole plate in a cell culture box for culturing for 4-5 days, adding 10% formaldehyde for fixing overnight, removing agarose gel, dyeing with 1% crystal violet for 30min, slightly washing the 24-hole plate with tap water, air-drying and counting the number of plaques. Calculating the plaque inhibition rate of the sample, and further calculating the median inhibition concentration IC 50; plaque inhibition rate ═ number of plaques of virogroup-number of plaques of drug group/number of plaques of virogroup]×100％。
The method is adopted to test the respiratory syncytial virus resistance activity of the following experimental group medicines; the test results are shown in Table 1.
Experimental group 1: the effective part of elephantopus scaber prepared in example 1;
experimental group 2: the effective part of elephantopus scaber prepared in comparative example 1;
experimental group 3: the effective part of elephantopus scaber prepared in comparative example 2;
experimental group 4: the effective part of elephantopus scaber prepared in comparative example 3;
experimental group 5: isodeoxyelephantolide;
experimental group 6: deoxycholic acid lactone.
TABLE 1 results of anti-RSV activity test of elephantolide compounds
| Experimental group | Composition (I) | IC50(μg/mL) |
| Experimental group 1 | Example 1 the effective part of elephantopus scaber prepared | 1.18 |
| Experimental group 2 | Effective part of elephantopus scaber prepared in comparative example 1 | 135.56 |
| Experimental group 3 | Effective part of elephantopus scaber prepared in comparative example 2 | 158.44 |
| Experimental group 4 | Effective part of elephantopus scaber prepared in comparative example 3 | 92.32 |
| Experimental group 5 | Isodeoxyelephantolide | 3.17 |
| Experimental group 6 | Deoxycholic acid lactone | 10.25 |
From the above experimental results, it is found that the IC50 value of the anti-respiratory syncytial virus activity of the effective part of elephantopus scaber prepared in example 1 is 1.18 μ g/mL, which is smaller than the IC50 value of the elephantopus scaber extract containing a series of caffeoylquinic acid components disclosed in patent CN102219687A against respiratory syncytial virus; the new method is adopted to prepare the effective part of the elephantopus scaber with more excellent respiratory syncytial virus resistance. And the IC50 value of the effective part of the elephantopus scaber prepared in the example 1 is smaller than that of the monomer compounds isodesoxyelephantolide and desoxyelephantopus scaber lactone, which is probably because the isodesoxyelephantopus scaber lactone and desoxyelephantopus scaber lactone generate the synergistic anti-respiratory syncytial virus activity with other small amount of elephantopus scaber lactone compounds in the effective part of the elephantopus scaber prepared in the example 1.
The experimental results also show that the IC50 value of the anti-respiratory syncytial virus activity of the effective part of the elephantopus scaber prepared in the comparative examples 1 to 3 is far greater than that of the effective part of the elephantopus scaber prepared in the example 1; the result shows that the silica gel elution conditions in the steps (3) and (4) play a decisive role in the respiratory syncytial virus resisting effect of the effective part of the elephantopus scaber; steps (3) and (4) cannot be reversed, i.e.: the effective part of the elephantopus scaber with excellent effect of resisting the respiratory syncytial virus can be obtained only by adopting the mixed organic solvent A consisting of petroleum ether and ethyl acetate under the condition of the step (3) for elution and then adopting the mixed organic solvent B consisting of chloroform and methanol under the condition of the step (4) for elution. When the steps (3) and (4) are reversed or the ratio of the organic solvent in the steps (3) and (4) is different, an effective part of elephantopus scaber having an excellent effect of resisting respiratory syncytial virus cannot be obtained.
Claims (3)
1. An application of the effective part of elephantopus scaber in preparing the medicine for treating respiratory syncytial virus,
the effective part of the elephantopus scaber is prepared according to the following steps:
(1) extracting herba elephantopi with ethanol to obtain herba elephantopi ethanol extract;
(2) suspending the elephantopus scaber ethanol extract with water, and extracting with petroleum ether, ethyl acetate and n-butanol respectively to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract;
(3) loading the petroleum ether extract on a silica gel column, eluting by using a mixed organic solvent A, collecting an elution part, concentrating and drying to obtain a silica gel elution part A;
(4) applying silica gel eluting part A to silica gel column again, eluting with mixed organic solvent B, and collecting the eluting part to obtain herba Ajugae Bracteosae effective part;
the specific elution conditions of step (3) are: putting the petroleum ether extract into a silica gel column, and eluting by using a mixed organic solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 95-90: 5-10; then eluting with a mixed organic solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 85-80: 15-20; collecting an elution part eluted by a mixed organic solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 85-80: 15-20, concentrating and drying to obtain a silica gel elution part A;
the specific elution conditions of step (4) are: putting a silica gel eluting part A on a silica gel column, and eluting by using a mixed organic solvent consisting of chloroform and methanol in a volume ratio of 99-97: 1-3; then eluting with a mixed organic solvent consisting of chloroform and methanol in a volume ratio of 95-92: 5-8; collecting the eluted part eluted by the mixed organic solvent consisting of chloroform and methanol in a volume ratio of 95-92: 5-8, concentrating and drying to obtain the effective part of the elephantopus scaber.
2. The use according to claim 1, wherein the ethanol in step (1) is ethanol with a volume fraction of 80-100%.
3. The use of claim 1, wherein the extraction in step (1) is heating reflux extraction.
Priority Applications (1)
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