CN112263603B - Efficient preparation method and antiviral application of active ingredients of honeysuckle leaves - Google Patents
Efficient preparation method and antiviral application of active ingredients of honeysuckle leaves Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/35—Caprifoliaceae (Honeysuckle family)
- A61K36/355—Lonicera (honeysuckle)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
The invention belongs to the technical field of honeysuckle extract, and particularly relates to a high-efficiency preparation method of active components of honeysuckle leaves and antiviral application of the active components. Honeysuckle is used as a large amount of medicinal materials commonly used in China, and has wide application in the aspects of medicines, foods and health care products, and because the honeysuckle has large demand and a tense supply-demand relationship, the related development of the honeysuckle leaves is developed in the field to be used as a substitute for the honeysuckle to relieve the current situation of insufficient market supply. The method is used for separating the honeysuckle leaf extract based on linear elution countercurrent chromatography, can separate components with different polarities in the extract to the maximum extent, and verifies that the components with better antiviral activity in the extract obtained by the separation method are obtained by a neuraminidase inhibition experiment.
Description
Technical Field
The disclosure belongs to the technical field of countercurrent chromatography separation of natural extracts, and particularly relates to a method for separating a honeysuckle leaf extract by gradient elution countercurrent chromatography, the obtained extract and antiviral application.
Background
The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Lonicera japonica Thunb (Lonicera japonica Thunb.) is a perennial evergreen wound vine plant of Lonicera of Caprifoliaceae, is an important traditional Chinese medicine resource in China and has various medicinal effects such as heat clearing, inflammation diminishing, bacteria inhibiting, virus resisting and the like. Honeysuckle (also known as honeysuckle flower, honeysuckle flower and the like) is a dried flower bud of plant honeysuckle or a flower which is just opened, is a large amount of medicinal materials and widely known antiviral medicinal materials commonly used in China, can be widely used as a medicine, can also be prepared into food or health food such as beverage, jelly, candy and the like, and has certain effects of improving human immunity, clearing heat and removing toxicity and preventing viral diseases, so the honeysuckle flower has huge demand, very tense supply-demand relationship and high market price. The inventor believes that providing raw materials with similar functions and components can help solve the current market situation of insufficient yield and short supply.
The contents of main active ingredients in honeysuckle flowers, stems and leaves are compared by a stoichiometric method, and the results prove that the honeysuckle and the honeysuckle leaves have similar active ingredients and contain rich organic acid substances and flavonoid substances. Therefore, the honeysuckle leaves are proved to be feasible in terms of pharmacological and biological activity to replace the honeysuckle, and are a potential substitute of the honeysuckle. At present, most of honeysuckle leaves as byproducts of honeysuckle harvesting are regarded as having no utilization value and are directly discarded, so that great waste on resources is caused. Therefore, the research and development of the honeysuckle leaves are accelerated, and the honeysuckle flower health care product has higher significance and value for relieving the shortage of supply and demand relationship in honeysuckle flower market, controlling the honeysuckle flower price to be stable and even optimizing biological resources for reasonable utilization.
The Countercurrent Chromatography (CCC) is a continuous liquid-liquid partition Chromatography without a solid carrier, has large single separation amount, can realize high-efficiency separation and preparation in a short time, and has important significance for separating natural active ingredients. The gradient elution is widely applied to countercurrent chromatography, comprises modes such as linear gradient, step gradient and pH gradient, can separate components with large polarity difference in a sample, and is widely applied to separation and preparation of active components of natural products.
Neuraminidase (NA), a glycoprotein ubiquitously distributed on the surface of influenza viruses, plays an important role in the cell cycle of influenza viruses and can help mature influenza viruses to replicate and infect uninfected cells. Neuraminidase, by its antigenicity, is specifically recognized and is of great significance for replication of influenza viruses, and therefore there is increasing interest in the study of inhibitors of its activity. Therefore, in recent years, more and more researches and researches are favored to extract and separate natural active ingredients which have neuraminidase inhibition capability, have small adverse reactions and are not easy to cause virus drug resistance.
Disclosure of Invention
Based on the research background, the present disclosure is directed to the extraction of active ingredients from honeysuckle leaves,
the linear gradient countercurrent method constructed in the experiment is a new method imitating binary high performance liquid chromatography, so that two solvent systems used for one sample can completely elute substances with different polarities in the sample as far as possible, namely the polarity range of bioactive substances in the sample is contained as far as possible. In the experiment, suitable LGCCC solvent systems are respectively constructed according to the polar characteristics of the ethyl acetate extract and the n-butanol extract.
In a first aspect of the present disclosure, there is provided an ethyl acetate extract of honeysuckle leaves, the preparation method of the extract is as follows: adding an ethanol solution into the honeysuckle leaves, refluxing to obtain a crude ethanol extract of the honeysuckle leaves, filtering and concentrating to obtain an ethanol extract, and sequentially distributing and extracting by adopting petroleum ether, ethyl acetate and n-butanol, wherein the part obtained by ethyl acetate extraction is an ethyl acetate extract of the honeysuckle leaves.
In a second aspect of the present disclosure, there is provided a n-butanol extract of honeysuckle leaves, the extract being prepared by the following steps: adding an ethanol solution into the honeysuckle leaves, refluxing to obtain a crude ethanol extract of the honeysuckle leaves, filtering and concentrating to obtain an ethanol extract, and sequentially adopting petroleum ether, ethyl acetate and n-butanol to perform distributed extraction, wherein the n-butanol extract is obtained by n-butanol extraction.
In a third aspect of the present disclosure, a high-efficiency preparation method of an active ingredient of honeysuckle leaves is provided, the method uses the ethyl acetate extract of the first aspect as a separation object, and the honeysuckle leaves extract is separated by gradient elution and counter-current chromatography; separating to obtain ethyl acetate extract A, ethyl acetate extract B, ethyl acetate extract C, ethyl acetate extract D, ethyl acetate extract E, ethyl acetate extract F, ethyl acetate extract G and ethyl acetate extract tail-blown part.
Preferably, the solvent system of the counter current chromatography is: petroleum ether: ethyl acetate: and (3) water.
Further, the petroleum ether: ethyl acetate: the volume ratio of the water is 4-6: 8-12.
Experiments of the disclosure prove that the solvent system can give consideration to good stationary phase retention effect and distribution effect of different polar components in the extract.
Preferably, the rotating speed of the countercurrent chromatography is 700-900 rpm; under the condition of the rotating speed, the retention rate of the stationary phase reaches more than 70 percent.
Preferably, the flow rate of the counter-current chromatography mobile phase is 1.8-2.2 mL/min.
Preferably, the gradient elution procedure is as follows: 0min, 100% A1；50min，0％A1；220min， 0％A1。
A is described1As mobile phase, petroleum ether: ethyl acetate: water 5:5:10 upper phase.
Preferably, the sample injection amount is 230-270 mg.
In a fourth aspect of the present disclosure, a further method for efficiently preparing an active ingredient of honeysuckle leaves is provided, wherein the n-butanol extract of the second aspect is used as a separation target, the honeysuckle leaves are separated by gradient elution and counter-current chromatography, and n-butanol extract a, n-butanol extract B, n-butanol extract C, n-butanol extract D, n-butanol extract E, n-butanol extract F, n-butanol extract G, n-butanol extract H, n-butanol extract I, n-butanol extract J and n-butanol extract tail-blown fraction are obtained by separation.
Preferably, the solvent system of the counter current chromatography is: ethyl acetate: n-butanol: and (3) water.
Further, the ratio of ethyl acetate: n-butanol: the volume ratio of the water is 4-6: 8-12.
Experiments of the disclosure prove that the solvent system can give consideration to good stationary phase retention effect and distribution effect of different polar components in the extract.
Preferably, the rotating speed of the countercurrent chromatography is 600-800 rpm; under the condition of the rotating speed, the retention rate of the stationary phase can reach 80 percent.
Preferably, the flow rate of the counter-current chromatography mobile phase is 1.8-2.2 mL/min.
Preferably, the gradient elution procedure is as follows: 0min, 100% A2；15min，90％A2；110min， 90％A2；135min，80％A2；225min，80％A2；300min，0％A2；500min，0％A2。
A is described2As mobile phase, ethyl acetate: n-butanol: water 10:0:10 upper phase.
Preferably, the sample injection amount is 90-110 mg.
In a fifth aspect of the present disclosure, an extract of honeysuckle leaves obtained by the separation method of the third aspect is provided, and the extract includes an ethyl acetate extract a, an ethyl acetate extract B, an ethyl acetate extract C, an ethyl acetate extract D, an ethyl acetate extract E, an ethyl acetate extract F, an ethyl acetate extract G, and an ethyl acetate extract tail-blown fraction.
In a sixth aspect of the present disclosure, there is provided a honeysuckle leaf extract obtained by the separation method of the fourth aspect, comprising n-butanol extract a, n-butanol extract B, n-butanol extract C, n-butanol extract D, n-butanol extract E, n-butanol extract F, n-butanol extract G, n-butanol extract H, n-butanol extract I, and n-butanol extract J and n-butanol extract tail-blown fraction.
In a seventh aspect of the present disclosure, there is provided an ethyl acetate extract of honeysuckle leaves of the first aspect, an n-butanol extract of honeysuckle leaves of the second aspect, an extract of the fifth aspect, and an extract of the sixth aspect as antiviral active ingredients.
Preferably, the antiviral active ingredient includes use as a neuraminidase inhibitor.
The extract according to the fifth aspect is more preferably ethyl acetate extract B, ethyl acetate extract C, ethyl acetate extract E and ethyl acetate extract tail-blown fraction.
The extract according to the sixth aspect is more preferably an n-butanol extract a, an n-butanol extract B, an n-butanol extract C, an n-butanol extract D, an n-butanol extract I, and an n-butanol extract J.
Compared with the prior art, the beneficial effect of this disclosure is:
1. the disclosure provides a method for separating honeysuckle leaf extract, which can realize the effect of fully separating ethyl acetate extract and n-butanol extract by linear elution and countercurrent chromatography separation.
2. The extract obtained by separating the honeysuckle leaf extract by the method disclosed by the invention contains higher neuraminidase activity inhibitory components, including ethyl acetate extract B, ethyl acetate extract C, ethyl acetate extract E and ethyl acetate extract tail-blowing part, n-butanol extract A, n-butanol extract B, n-butanol extract C, n-butanol extract D, n-butanol extract I, n-butanol extract J and the like. The inhibition rate of the substance on neuraminidase presents an S-shaped curve, and the inhibition effect is more obvious along with the increase of concentration.
3. The separation method provides various honeysuckle leaves and extracts with neuraminidase inhibitory activity, and is expected to provide a research basis for screening antiviral components.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.
FIG. 1 is the HPLC analysis result of the ethyl acetate extract of Lonicera japonica Thunb of example 1;
FIG. 2 is the HPLC analysis result of n-butanol extract of Lonicera japonica Thunb of example 1;
FIG. 3 is a histogram of the solid phase retention in different operating modes of example 1;
wherein, fig. 3A is the stationary phase retention rate of the petroleum ether-ethyl acetate-water solvent system;
FIG. 3B shows the stationary phase retention of an ethyl acetate-n-butanol-water solvent system;
FIG. 4 is a histogram showing the influence of the separation rotation speed on the stationary phase retention rate in example 1;
wherein, fig. 4A is the stationary phase retention rate of the petroleum ether-ethyl acetate-water solvent system at different rotation speeds;
FIG. 4B shows the stationary phase retention of the ethyl acetate-n-butanol-water solvent system at different rotational speeds.
FIG. 5 is a line graph of the stationary phase retention for the solvent system at different flow rates in example 1;
wherein, fig. 5A shows the stationary phase retention rate of the petroleum ether-ethyl acetate-water solvent system at different flow rates;
FIG. 5B shows the stationary phase retention for different flow rates of ethyl acetate-n-butanol-water solvent system;
FIG. 6 is a graph showing the separation result of ethyl acetate extract LGCCC of Lonicera japonica Thunb in example 1;
FIG. 7 is a graph showing the separation result of an n-butanol extract LGCCC of Lonicera japonica Thunb in example 1;
FIG. 8 is a graph showing the inhibition rate of neuraminidase by the positive drug in example 2;
FIG. 9 is a graph showing the inhibition rate of neuraminidase by each extract of Japanese honeysuckle leaves in example 2;
FIG. 10 is a graph showing the inhibition rate of neuraminidase by each fraction of the ethyl acetate extract of lonicera japonica thunb of example 2 after separation by countercurrent chromatography;
FIG. 11 is a graph showing the inhibition rate of neuraminidase by n-butanol extract of Lonicera japonica Thunb of example 2 by countercurrent chromatography.
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 disclosure belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
Term interpretation section
As described in the background, in order to solve the above technical problems, the present disclosure proposes a method for separating a honeysuckle leaf extract based on linear elution counter-current chromatography.
In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.
Fresh honeysuckle leaves were used in the following examples and were supplied by Shandong Asia Biotechnology Ltd (Lonicera japonica Thunb., variety: Asia Te)
Example 1
1. Preparation of crude samples
The purchased dried honeysuckle leaves are selected, and the mixed honeysuckle and honeysuckle stem are discarded. Weighing 1kg of selected dried honeysuckle leaves, crushing, putting into a round-bottom flask, and mixing according to the weight ratio of 1: 10L of 95 percent ethanol is added into the mixture according to the feed-liquid ratio of 10, and the mixture is heated, refluxed and extracted for three times, and each time lasts for 2 hours. The crude ethanol extract of the honeysuckle leaves obtained by extraction is filtered, decompressed and concentrated to obtain 386g of brown viscous ethanol extract.
The ethanol extract is prepared into suspension by using distilled water, then petroleum ether, ethyl acetate and n-butanol with the same volume are sequentially used for partition extraction for three times, and after decompression concentration and freeze drying, 22g, 19g and 135g of honeysuckle leaf petroleum ether extract, honeysuckle leaf ethyl acetate extract and honeysuckle leaf n-butanol extract are respectively obtained, and the extraction rates are respectively 5.7%, 4.9% and 35.0%.
According to the related research results, the active ingredients of the honeysuckle leaves are mainly concentrated in the ethyl acetate extract and the n-butanol extract thereof, so that the ethyl acetate extract and the n-butanol extract of the honeysuckle leaves are selected as the samples of this example for the subsequent LGCCC methodology experiment.
2. High Performance Liquid Chromatography (HPLC) analysis
2.1 HPLC analysis of ethyl acetate extract of Lonicera japonica leaves
Analytical liquid chromatography of ethyl acetate extract of lonicera japonica leaves adopts the following analytical method: HPLC analysis was performed using Waters e2695 high performance liquid chromatography on SYMMETRY-C18(250 mm. times.4.6 mm, 5.0. mu.m); mobile phases of a (methanol) and B (0.3% aqueous acetic acid); the gradient elution conditions were as follows: 0min, 30% A; 5min, 40% A; 20min, 45% A; 35min, 100% A; 45min, 100% of A. The flow rate is 1.0 mL/min; the sample volume is 10 mu L; the detector wavelength is 254 nm.
2.2 HPLC analysis of n-butanol extract of Lonicera japonica Thunb
The analytical liquid chromatography of the n-butanol extract of the honeysuckle leaves adopts the following analytical method: analyzing by Waters e2695 high performance liquid chromatography, wherein the chromatographic column is SYMMETRY-C18(250 mm. times.4.6 mm, 5.0. mu.m); mobile phases of a (methanol) and B (0.3% aqueous acetic acid); the gradient elution conditions were as follows: 0min, 20% A; 40min, 60% A; 48min 100% A; 58min, 100% A. The flow rate is 1.0 mL/min; the sample volume is 10 mu L; the detector wavelength is 254 nm.
The distribution coefficient determination by HPLC method is as follows: preparing 10mL of solvent according to the proportion of the solvent system, fully shaking, standing and layering. Adding 2mL of lower phase into a test tube, adding about 5mg of crude sample to be detected, and measuring the peak area of each target peak in the lower phase by HPLC, and recording as P1Adding 2mL of upper phase, sufficiently shaking, standing for layering, and measuring the peak area of each target peak in the lower phase, and recording as P2. Calculating the distribution coefficient K of the sample in different solvent systems by using a formulaD，KDThe range of 0.5-2.0 shows that the separation effect of the target peak of the sample under the solvent system is better.
Distribution coefficient K of sampleDCalculated according to the following formula:
KD＝P2/(P1-P2)
2.3 determination of the solvent System
The solvent system of the honeysuckle leaf ethyl acetate extract is a petroleum ether-ethyl acetate-water solvent system prepared from petroleum ether, ethyl acetate and water.
The results of HPLC analysis of the ethyl acetate extract of lonicera japonica thunb are shown in FIG. 1, where the target peak is assigned to the counter-current solvent system. Through detection, the distribution coefficient value detection of each test counter-current solvent system of the ethyl acetate extract of the honeysuckle leaves is shown in table 1:
TABLE 1 partition coefficient values of ethyl acetate extract solvent system from Lonicera japonica Thunb
As shown in the results in Table 1, the partition coefficients of the 4 target peaks in the solvent system (petroleum ether: ethyl acetate: water, v/v) are all in the range of 0.5-2.0, and good separation effect can be achieved.
The solvent system of the n-butyl alcohol extract of the honeysuckle leaves is an ethyl acetate-n-butyl alcohol-water solvent system prepared from ethyl acetate, n-butyl alcohol and water. The results of the HOLC analysis of the n-butanol extract are shown in FIG. 2, where the distribution target peak is identified and the distribution coefficient value of the solvent system of the n-butanol extract of honeysuckle leaves is shown in Table 2:
TABLE 2 partition coefficient values of n-butanol extract solvent system of Lonicera japonica Thunb
As shown in the results in Table 2, the partition coefficients of the 8 target peaks in the solvent system (ethyl acetate: n-butanol: water, v/v) are all in the range of 0.5-2.0, and good separation effect can be achieved.
2.4 preparation of solvent System and sample solution
Preparation of a solvent system: and fully oscillating and uniformly mixing the prepared two-phase solvent system in a separating funnel, discharging gas in the separating funnel, standing and layering, and respectively taking an upper phase and a lower phase, wherein the lower phase of the initial gradient solvent system is a stationary phase, and the upper phases of the two solvent systems are mobile phases.
Preparation of sample solution: the sample extract is dissolved by 3-6mL of each of the upper phase and the lower phase of the initial gradient solvent system, the volumes of the upper phase and the lower phase are the same, and the specific volume is determined according to the size of the sample amount and the sample dissolution condition.
2.5 Linear gradient countercurrent chromatography methodological experiment
Since the major problem faced by the LGCCC is that the stationary phase will be lost due to the change of the polarity of the mobile phase during the operation, the methodological separation result in this embodiment focuses more on whether the loss of the stationary phase can be effectively controlled, i.e., whether the retention rate is high enough, besides focusing on the separation effect. The retention rate is calculated as follows:
Sf(％)＝(Vc-Vl)/Vc×100％
Sf(%) represents the stationary phase retention, VcDenotes the high-speed countercurrent chromatography column volume, VlIndicating the volume of fixed phase loss.
And determining the operation mode most suitable for the stationary phase retention of a petroleum ether-ethyl acetate-water solvent system by measuring the retention rate of the stationary phase when the stationary phase reaches the equilibrium state. The results are shown in FIG. 3.
As can be seen from the experimental results shown in fig. 3A and fig. 3B, the "FWD-OUT" operation mode is selected to have a better stationary phase retention rate and less damage to the pipeline.
For the study of the flow rate, the present embodiment selects 6 rotation speeds of 400rpm, 500rpm, 600rpm, 700rpm, 800rpm and 900rpm for experimental tests. The operation mode is 'FWD-OUT', the flow rate is set to be 2mL/min, and the test time is 220 min. During the test, the loss of the stationary phase during the balance and the loss of the stationary phase every 5min after the balance are recorded, and a line graph is drawn after data are summarized for comparing the stationary phase retention effect. The results of the experiment are shown in FIG. 4.
As shown in FIG. 4A, 800rpm is the separation speed most suitable for LGCCC separation in a petroleum ether-ethyl acetate-water solvent system.
As shown in FIG. 4B, 700rpm is the optimum separation speed for the LGCCC separation in the ethyl acetate-n-butanol-water solvent system.
The present example also examined the separation flow rate, and selected 5 different flow rates of 1.0mL/min, 2.0mL/min, 3.0mL/min, 5.0mL/min, and 7.0mL/min for experimental tests. During the test, the amount of loss of the stationary phase during the equilibration and the amount of loss of the stationary phase in every 10mL of the effluent liquid after the equilibration were recorded, and after the data were summarized, a line graph was drawn for comparing the stationary phase retention effects, and the results are shown in fig. 5.
As shown in FIG. 5A, 5.0mL/min was used as the separation flow rate most suitable for LGCCC separation in the petroleum ether-ethyl acetate-water solvent system.
As shown in FIG. 5B, 3.0mL/min was used as the separation flow rate most suitable for the separation of the ethyl acetate-n-butanol-water solvent system LGCCC.
2.6 determination of gradient conditions and sample amount in Lonicera japonica leaf linear gradient countercurrent chromatography
(1) The honeysuckle leaf ethyl acetate extract is subjected to experimental tests on different sample volumes and different gradient conditions in the optimum operation mode (FWD-OUT), the optimum rotation speed (800rpm) and the optimum flow rate (5.0mL/min) determined in the foregoing, and the sample volumes and the gradient conditions are continuously adjusted and optimized to determine the most appropriate sample volumes and gradient conditions. After optimizing the sample volume and gradient conditions of the ethyl acetate extract of the honeysuckle leaves, the separation sample volume and gradient conditions of the ethyl acetate extract of the honeysuckle leaves are confirmed as follows:
sample introduction amount: 250mg of
Gradient conditions: 0min, 70% A1；200min，0％A1；400min，0％A1
(2) The n-butanol extract of honeysuckle leaves is subjected to experimental tests on different sample volumes and different gradient conditions in the optimum operation mode (FWD-OUT), the optimum rotation speed (700rpm) and the optimum flow rate (3.0mL/min) determined in the foregoing, so that the sample volumes and the gradient conditions are continuously adjusted and optimized to determine the most suitable sample volumes and gradient conditions. The experimental test and adjustment optimization of different sample volumes and different gradient conditions of the n-butanol extract of the honeysuckle leaves are carried out, and the LGCCC separation sample volume and the gradient conditions of the n-butanol extract of the honeysuckle leaves are determined as follows:
sample introduction amount: 100mg of
Gradient conditions: 0min, 100% A2；15min，90％A2；110min，90％A2；135min， 80％A2；225min，80％A2；300min，0％A2；500min，0％A2。
3. Detection of separated materials
The ethyl acetate extract of the honeysuckle leaves is separated by LGCCC, and the collected extract comprises 7 segments of separated substances and tail-blown substances, and the total amount of the separated substances is 8 segments. The mass of each part of the substances obtained by separating 250mg of ethyl acetate extract of honeysuckle leaves once is respectively as follows: a (6.3mg), B (11.3mg), C (1.7mg), D (2.1mg), E (2.6mg), F (14.9mg), G (12.4mg), and tail-blown (43.3 mg). The results of the LGCCC separation of the ethyl acetate extract of honeysuckle leaves are shown in FIG. 6.
The n-butanol extract of the honeysuckle leaves is separated by LGCCC, and the extract is collected to obtain the separated substance which comprises 10 segments and tail blowing, and the total amount of the separated substance is 11 segments. The mass of each part of the substances obtained by separating 100mg of the n-butanol extract of the honeysuckle leaves once is respectively as follows: a (2.6mg), B (1.7mg), C (0.9mg), D (10.2mg), E (8.5mg), F (5.2mg), G (2.3mg), H (12.9mg), I (6.0mg), J (5.6 mg), and Dow (7.4 mg). The results of the LGCCC separation of n-butanol extract of honeysuckle leaves are shown in FIG. 7.
Example 2
1.1 concentration gradient formulation of test sample solutions
Separating each part substance obtained by separating ethyl acetate extract and n-butanol extract of honeysuckle leaves, ethanol extract of honeysuckle leaves, petroleum ether extract of honeysuckle leaves, ethyl acetate extract of honeysuckle leaves, n-butanol extract of honeysuckle leaves and residual water-soluble substance of honeysuckle leaves from LGCCC, dissolving the substances respectively with 50% ethanol aqueous solution to prepare 1mg/mL test sample solution, and continuously diluting 1mg/mL test sample solution with 50% ethanol aqueous solution for 9 times to obtain 10 test sample solutions with different concentrations and 2 times of adjacent concentrations.
The method comprises the steps of selecting zanavir as a positive control group of an experiment, selecting chlorogenic acid as a natural component positive control group of the experiment, preparing 0.005mg/mL zanavir solution by using 50% ethanol aqueous solution, and continuously diluting the zanavir solution by using 50% ethanol aqueous solution for 9 times to obtain 10 solutions with different concentrations and 10 times of adjacent concentrations.
1.2 test samples on neuraminidase IC50Measurement of (2)
The inhibition rate of the test sample solution and the two groups of positive control solutions with different concentrations on neuraminidase is measured by using an enzyme labeling instrument. Using a microplate reader, the excitation wavelength was set to 355nm, and the luminescence was measuredThe emission wavelength was 460nm, and the fluorescence absorptions of the sample group at the set wavelength were measured (A)s) Fluorescence absorption of control group (A)c) And fluorescence absorption of blank group (A)b). The inhibition rate of the sample on neuraminidase is shown as follows:
inhibition ratio (%) ═ ac–As)/(Ac–Ab)×100％
Plotting the obtained inhibition rate and the corresponding sample concentration by using Origin 8.0 software to obtain an inhibition rate curve of each sample; calculating the obtained inhibition rate and the corresponding sample concentration by GraphPad 5 software to obtain the IC of each sample50The value is obtained.
The inhibition rate curve of neuraminidase by the positive control and each extract of Lonicera japonica is shown in FIG. 8.
The inhibition rate curve of each part of the ethyl acetate extract of honeysuckle leaves after LGCCC separation on neuraminidase is shown in FIG. 9.
The inhibition rate curve of each part of the n-butanol extract of honeysuckle leaves on neuraminidase after LGCCC separation is shown in FIG. 10.
As can be seen from the results shown in FIGS. 8-10, the inhibition of neuraminidase by the positive drug showed an "S" curve. As can be seen from FIG. 9, the ethyl acetate and n-butanol extract fractions of Lonicera japonica Thunb have better neuraminidase inhibitory activity.
FIG. 10 shows the A-G fractions obtained by countercurrent chromatography of ethyl acetate extract, wherein the B, C, D, E and tail gas components have better inhibitory activity.
FIG. 11 shows that the fraction A-J is obtained by separating n-butanol extract by countercurrent chromatography, wherein the fractions A, B, C, G, I, J and the tail gas have good inhibitory activity.
1.3 Neurosidase IC in test samples50Measurement of (2)
IC of zanavir positive control and chlorogenic acid positive control on neuraminidase50The results are shown in Table 3.
TABLE 3 inhibition of neuraminidase by Positive control group: (
n＝3)
IC of extracts of honeysuckle leaves on neuraminidase50The results are shown in Table 4. From the experimental results, the honeysuckle leaves have good antiviral biological activity, and the antiviral activity is best by using the n-butanol extract and the ethyl acetate extract.
TABLE 4 inhibition of neuraminidase by extracts of Lonicera japonica leaves: (
n＝3)
Note: "-" represents the IC of the corresponding sample for neuraminidase50Values greater than 1.0000 mg/mL.
IC of each part of ethyl acetate extract of honeysuckle leaves on neuraminidase after LGCCC separation50The value results are shown in table 5: the ethyl acetate extract B, C, E and the tail-blown part have better antiviral activity, wherein the inhibition effect of the B part with the best activity is obviously higher than that of other components.
TABLE 5 inhibition of neuraminidase by the isolated fractions of ethyl acetate extract: (
n＝3)
Note:"-" represents the IC of the corresponding sample for neuraminidase50Values greater than 1.0000 mg/mL.
IC of each part of honeysuckle leaf n-butanol extract on neuraminidase after LGCCC separation50The value results are shown in table 6: the n-butanol extract A, B, C, D, I, J has good antiviral activity.
TABLE 6 inhibitory Effect of n-butanol extract on neuraminidase
n＝3)
Note: "-" represents the IC of the corresponding sample for neuraminidase50Values greater than 1.0000 mg/mL.
The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.
Claims (2)
1. An application of an ethyl acetate extract B of honeysuckle leaves in preparing a medicine with an antiviral active ingredient is characterized in that the preparation method of the ethyl acetate extract B of the honeysuckle leaves is as follows:
adding an ethanol solution into the honeysuckle leaves, refluxing to obtain a crude ethanol extract of the honeysuckle leaves, filtering and concentrating to obtain an ethanol extract, and sequentially distributing and extracting petroleum ether, ethyl acetate and n-butanol, wherein the part obtained by ethyl acetate extraction is an ethyl acetate extract of the honeysuckle leaves;
separating the ethyl acetate extract of the honeysuckle leaves by gradient elution and counter-current chromatography to obtain an ethyl acetate extract A, an ethyl acetate extract B, an ethyl acetate extract C, an ethyl acetate extract D, an ethyl acetate extract E, an ethyl acetate extract F, an ethyl acetate extract G and an ethyl acetate extract tail-blown part;
wherein, the gradient elution procedure of the countercurrent chromatography is as follows: 0min, 100% A1; 50 min, 0% A1; 220min, 0% A1, wherein A1 is a mobile phase and is petroleum ether: ethyl acetate: water =5:5:10 upper phase, petroleum ether: ethyl acetate: the lower phase of water =5:5:10 is the stationary phase;
the sample injection amount is 230-270 mg;
the flow rate of the counter-current chromatography mobile phase is 1.8-2.2 mL/min;
the rotating speed of the countercurrent chromatography is 700-900 rpm;
the time for the ethyl acetate extract B to flow out was 68 min to 131 min.
2. The use of ethyl acetate extract B of lonicera japonica thunb as claimed in claim 1 for the preparation of an antiviral active ingredient drug, wherein the antiviral active ingredient drug is a neuraminidase inhibitor drug.
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