CN111228354A

CN111228354A - Anti-virus active site in cortex dictamni, preparation method, content determination method and application thereof

Info

Publication number: CN111228354A
Application number: CN202010134725.5A
Authority: CN
Inventors: 王磊; 刘贵栋
Original assignee: Harbin Vocational and Technical College
Current assignee: Harbin Vocational and Technical College
Priority date: 2020-03-02
Filing date: 2020-03-02
Publication date: 2020-06-05

Abstract

The invention relates to an antiviral active site in cortex dictamni, a preparation method, content measurement and application thereof. Experiments show that the cortex dictamni effective part obtained by the method has a very good antiviral effect, and experiments further show that the higher the total content of the phellodendron ketone and the fraxinellone is, the stronger the antiviral effect is.

Description

Anti-virus active site in cortex dictamni, preparation method, content determination method and application thereof

Technical Field

The invention relates to a traditional Chinese medicine extract with antiviral activity, in particular to an antiviral active part in cortex dictamni, a preparation method, content measurement and application thereof.

Background

Influenza not only brings great threat to the health of human beings and animals, but also causes great economic loss and disruption of social order. Seasonal influenza can cause 300 to 500 million cases of severe illness each year, resulting in the death of 25 to 50 million people. Influenza virus can be divided into three types, i.e., influenza a virus, influenza b virus and influenza c virus, and influenza a virus is a major factor causing seasonal influenza in humans and can infect various animals including poultry, pigs and horses. Influenza a viruses can be divided into many subtypes according to their antigenic structures of surface Hemagglutinin (HA) and Neuraminidase (NA). To date, 18 different HA subtypes have been found, of which H1 and H3 are responsible for most human influenza, while 11 NA subtypes, the main subtypes in human influenza today being N1, N2 and N9.

The main current control measure for influenza viruses is vaccination, but the vaccines themselves have certain limitations: the immunization rate is low, and effective group immunity is difficult to generate; the effective immunity is difficult to generate for high risk group; and because influenza viruses are susceptible to mutation, it is difficult to produce a corresponding vaccine in a short time at an early stage when a newly mutated virus is rapidly circulating. Thus, antiviral drugs play an important role in the prevention and treatment of influenza.

The traditional Chinese medicine has the characteristics of multiple components and multiple links, not only has direct antiviral effect, but also can regulate the complex process of immunopathological injury caused by viruses, and has unique advantages in the treatment of influenza. The medicinal biological resources of China are very rich, and the physiologically active substances of the medicinal biological resources are natural treasury for researching and discovering new medicines and developing new medicines. At present, our country extracts active substances from natural products, and few new drugs with good safety and low toxicity are developed for treating antiviral drugs, and extracts active substances from natural products and develops new drugs with antiviral efficacy, so that the new drugs have important application values and wide development prospects.

Dittany bark, alias: cortex Dictamni Radicis, herba Ardisiae Japonicae, caulis et folium Paeoniae suffruticosae, and herba Dictamni Radicis are root bark of perennial herb Dictamni and Dictamnus angustifolia of Rutaceae. Mainly produced in Liaoning, Hebei, Sichuan and Jiangsu provinces. The main components are as follows: dictamnolide, phellodendron ketoacid, trigonelline, choline, dictamnine, phellodendrone, thujavanine, procambranone, isopulegone, limonin, phellodendrone, fraxinellone, campesterol, fraxinellone, sitosterol, acidic substances, saponin, etc.

The functional indications are as follows: 1, clearing away heat and toxic materials, removing dampness and relieving itching: can be used for treating damp-heat sores and rashes, pus or yellow water dripping, skin damp and rot, skin pruritus and rubella scabies. 2, clearing away heat and toxic materials and removing dampness: it can be used for treating jaundice due to damp-heat and arthralgia due to damp-heat.

Disclosure of Invention

The invention aims to research the antiviral effect of the cortex dictamni, further discovers an active part of the antiviral effect of the cortex dictamni, optimizes the preparation method of the antiviral active part of the cortex dictamni to obtain a part with better antiviral activity, and performs content measurement on the main component of the antiviral active part.

In view of the above, the present invention provides an antiviral active site of cortex dictamni, wherein the active site contains more than 50% of total limonoids, and contains limonin, phellodendron ketone, fraxinellone and procambricin.

Further, the active fraction contains more than 80% of total limonoids, and contains limonin, obacunone, fraxinellone and pre-obacunone.

The invention also provides the application of the active site in the preparation of antiviral drugs.

Furthermore, the active site is applied to the preparation of antiviral drugs.

The invention also provides a preparation method of the cortex dictamni antiviral active site, which comprises the following steps:

(1) extraction: crushing cortex dictamni, adding 6-10 times of petroleum ether, carrying out reflux extraction for 2 times in a water bath at 70 ℃ for 1 hour each time, and combining filtrates. Filtering, adding 6-10 times of dichloromethane into the dregs of a decoction, performing reflux extraction for 2 times in a water bath at 85 ℃ for 1 hour each time, filtering, and combining the filtrates. Concentrating to obtain concentrated extract.

(2) Removing impurities: performing macroporous resin column chromatography, dissolving the concentrated extract obtained by extraction with 1: 1-1: 2 pure water, performing macroporous resin adsorption, and eluting with 1-5 times of column volume of pure water to remove water-soluble impurities; then eluting with 60-80% ethanol water solution with volume fraction of 1-6 times of column volume; collecting the elution part of 60-80% by volume of ethanol water solution, concentrating and drying to obtain the macroporous resin column chromatography active part.

(3) And (3) purification: continuing to separate the obtained eluent by using preparative liquid chromatography under the following separation conditions: the chromatographic column is an Agilent preparative column Zorba x SB-C18; 21.2mm X250 mm, the mobile phase being methanol-water solution, and carrying out gradient elution, wherein the gradient elution procedure is as follows: the methanol phase rises from 10% to 40% for 0-30min, and 40-60min, and the methanol phase rises from 40% to 95%. The flow rate is 10ml/min, and the column temperature is room temperature; dissolving the sample with chromatographic grade methanol, separating by preparative liquid chromatography, collecting the solution within 5-15 minutes, concentrating and drying the collected solution to obtain the cortex Dictamni antiviral active fraction.

Specifically, the macroporous resin column type number in the impurity removing step can be DH101, DM-130, AB-8.

The invention also provides a method for measuring the content of the antiviral active site in the cortex dictamni, which comprises the following steps:

(1) preparing a test sample: precisely weighing 10.0mg of antiviral active site in cortex Dictamni Radicis, dissolving with chromatographic pure methanol, diluting to a constant volume of 10ml volumetric flask, and filtering with 0.45 μm microporous membrane to obtain cortex Dictamni Radicis antiviral active site sample;

(2): preparation of a reference solution: precisely weighing dried limonin, phellodendron ketone, fraxinellone and pre-phellodendron ketone reference substances respectively at 5.0mg, placing in a 10mL measuring flask, dissolving with methanol and diluting to scale to obtain a cortex Dictamni Radicis limonin mixed reference substance stock solution, and placing in a refrigerator at-4 deg.C for use.

(3): performing high performance liquid chromatography determination on the sample prepared in the step (1), and performing content determination on the reference substances of limonin, phellodendron ketone, fraxinellone and pre-phellodendron ketone prepared in the step (2); the HPLC conditions were as follows: the chromatographic column packing is Aglient C18, the specification is 250mm multiplied by 4.6mm 5 μm, the wavelength is 210nm, the mobile phase is methanol-water solution, and the gradient elution is as follows: 0-30min, the methanol phase rises from 10% to 65%, 30-35min, the methanol phase keeps 65%, 35-40min, the methanol phase rises from 65% to 95%, 40-60min, the methanol phase elutes isocratically at 95%; the flow rate was 1ml/min and the column temperature was 30 ℃.

The invention has the beneficial effects that: 1. the extraction and separation process can effectively remove impurities with low polarity, improve the content of effective components and quickly and accurately obtain the effective components. 2. The cortex dictamni active ingredient provided by the invention has simple and clear chemical components, is easier to clarify the action mechanism in pharmacological research, and is easier to control the quality of the medicine in production. 3. The method provided by the invention obtains the limonin compound parts containing limonin, phellodendron ketone, fraxinellone, pre-phellodendron ketone and the like from the cortex dictamni medicinal material, and screens the antiviral drug effect of the limonin compound parts for the first time, so that the method is definite in components, clear in content, convenient and fast in preparation process, good in activity and suitable for being developed into new antiviral traditional Chinese medicine.

Drawings

FIG. 1 is a liquid phase chromatogram prepared from an ethanol water solution elution part with 75% in a cortex dictamni purification step of the invention;

FIG. 2 is an HPLC analysis chart of the antiviral active site of cortex Dictamni Radicis of the present invention; wherein, 1 is limonin; 2, pre-phellodendron ketone 3: phellodendron ketone 4: Fraxinellone 5: fraxinellone.

Detailed Description

As mentioned above, the present invention aims at providing a preparation method, a content determination method and an application of an antiviral active site in cortex dictamni. The following will specifically describe the embodiments with reference to the contents of the examples.

It is specifically noted that similar alternatives and modifications will be apparent to those skilled in the art, which are also intended to be included within the present invention. It will be apparent to those skilled in the art that the techniques of the present invention may be implemented and applied by modifying or appropriately combining the methods and applications described herein without departing from the spirit, scope, and content of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention.

If the specific conditions are not indicated, the method is carried out according to the conventional conditions or the conditions suggested by manufacturers, and the used raw material medicines or auxiliary materials and the used reagents or instruments are the conventional products which can be obtained commercially.

Example 1:

an antiviral active fraction of cortex Dictamni Radicis contains limonin, obacunone, fraxinellone and pre-obacunone, and has a total limonin content of more than 80%.

Application of cortex Dictamni Radicis antiviral active fraction in preparing antiviral medicine is provided.

As shown in figure 1, the preparation method of the antiviral active site of the cortex dictamni comprises the following steps:

(1) extraction: pulverizing cortex Dictamni Radicis, adding 10 times of petroleum ether, reflux-extracting in 70 deg.C water bath for 2 times, each for 1 hr, and mixing filtrates. Filtering, adding 10 times of dichloromethane into the residue, extracting under reflux in 85 deg.C water bath for 2 times, each time for 1 hr, filtering, and mixing filtrates. Concentrating to obtain concentrated extract.

(2) Removing impurities: performing macroporous resin column chromatography, dissolving the concentrated extract obtained by extraction with 1:1 pure water, performing macroporous resin adsorption, and eluting with 2 times of column volume of pure water to remove water-soluble impurities; then eluting with 80% ethanol water solution with 6 times of column volume; collecting the elution part of 80% ethanol water solution, concentrating and drying to obtain the macroporous resin column chromatography active part. The macroporous resin model used is DH 101.

(3) And (3) purification: continuing to separate the obtained eluent by using preparative liquid chromatography under the following separation conditions: the chromatographic column is an Agilent preparative column Zorba x SB-C18; 21.2mm X250 mm, the mobile phase being methanol-water solution, and carrying out gradient elution, wherein the gradient elution procedure is as follows: the methanol phase rises from 10% to 40% for 0-30min, and 40-60min, and the methanol phase rises from 40% to 95%. The flow rate is 10ml/min, and the column temperature is room temperature; dissolving the sample with chromatographic grade methanol, separating by preparative liquid chromatography, collecting the solution for 8-13 min, concentrating and drying the collected solution to obtain cortex Dictamni Radicis antiviral active fraction with a total content of cortex Dictamni Radicis limonoids of 82.1%, and showing the liquid phase chromatogram in figure 1.

Example 2

An antiviral active fraction of cortex Dictamni Radicis contains limonin, obacunone, fraxinellone and pre-obacunone, and has a total limonin content of more than 50%.

A preparation method of an antiviral active part of cortex dictamni, which comprises the following steps:

(1) extraction: pulverizing cortex Dictamni Radicis, adding 6 times of petroleum ether, reflux-extracting in 70 deg.C water bath for 2 times, each for 1 hr, and mixing filtrates. Filtering, adding 8 times of dichloromethane into the residue, extracting under reflux in 85 deg.C water bath for 2 times (1 hr each time), filtering, and mixing filtrates. Concentrating to obtain concentrated extract.

(2) Removing impurities: performing macroporous resin column chromatography, dissolving the concentrated extract obtained by extraction with 1:2 pure water, performing macroporous resin adsorption, and eluting with 1 column volume of pure water to remove water-soluble impurities; then eluting with 70% ethanol water solution with 6 times of column volume; collecting the elution part of 70% ethanol water solution, concentrating and drying to obtain the macroporous resin column chromatography active part. The macroporous resin is AB-8.

(3) And (3) purification: continuing to separate the obtained eluent by using preparative liquid chromatography under the following separation conditions: the chromatographic column is an Agilent preparative column Zorba x SB-C18; 21.2mm X250 mm, the mobile phase being methanol-water solution, and carrying out gradient elution, wherein the gradient elution procedure is as follows: the methanol phase rises from 10% to 40% for 0-30min, and 40-60min, and the methanol phase rises from 40% to 95%. The flow rate is 10ml/min, and the column temperature is room temperature; dissolving the sample with chromatographic grade methanol, separating by preparative liquid chromatography, collecting the solution within 8-13 min, concentrating and drying the collected solution to obtain the cortex Dictamni antiviral active fraction. The total content of the cortex dictamni limonin compounds is 76.8 percent.

Example 3

(1) extraction: pulverizing cortex Dictamni Radicis, adding 6 times of petroleum ether, reflux-extracting in 70 deg.C water bath for 2 times, each for 1 hr, and mixing filtrates. Filtering, adding 6 times of dichloromethane into the residue, extracting under reflux in 85 deg.C water bath for 2 times (1 hr each time), filtering, and mixing filtrates. Concentrating to obtain concentrated extract.

(2) Removing impurities: performing macroporous resin column chromatography, dissolving the concentrated extract obtained by extraction with 1:2 pure water, performing macroporous resin adsorption, and eluting with 5 times of column volume of pure water to remove water-soluble impurities; then eluting with 60% ethanol aqueous solution with volume fraction of 1 column volume; collecting the elution part of the ethanol water solution with the volume fraction of 60%, concentrating and drying to obtain the macroporous resin column chromatography active part. The macroporous resin is DM-130.

(3) And (3) purification: continuing to separate the obtained eluent by using preparative liquid chromatography under the following separation conditions: the chromatographic column is an Agilent preparative column Zorba x SB-C18; 21.2mm X250 mm, the mobile phase being methanol-water solution, and carrying out gradient elution, wherein the gradient elution procedure is as follows: the methanol phase rises from 10% to 40% for 0-30min, and 40-60min, and the methanol phase rises from 40% to 95%. The flow rate is 10ml/min, and the column temperature is room temperature; dissolving the sample with chromatographic grade methanol, separating by preparative liquid chromatography, collecting the solution within 5-15 minutes, concentrating and drying the collected solution to obtain cortex Dictamni Radicis antiviral active fraction with a total content of 56.3% of cortex Dictamni Radicis limonoids.

Example 4

(1) extraction: pulverizing cortex Dictamni Radicis, adding 8 times of petroleum ether, reflux-extracting in 70 deg.C water bath for 2 times, each for 1 hr, and mixing filtrates. Filtering, adding 8 times of dichloromethane into the residue, extracting under reflux in 85 deg.C water bath for 2 times (1 hr each time), filtering, and mixing filtrates. Concentrating to obtain concentrated extract.

(2) Removing impurities: performing macroporous resin column chromatography, dissolving the concentrated extract obtained by extraction with 1:15 pure water, performing macroporous resin adsorption, and eluting with 2 times of column volume of pure water to remove water-soluble impurities; then eluting with 75% ethanol water solution with 3 times of column volume; collecting the elution part of 75% ethanol water solution by volume fraction, concentrating and drying to obtain the macroporous resin column chromatography active part. Specifically, the type number of the macroporous resin column in the impurity removal step may be DH 101.

(3) And (3) purification: continuing to separate the obtained eluent by using preparative liquid chromatography under the following separation conditions: the chromatographic column is an Agilent preparative column Zorba x SB-C18; 21.2mm X250 mm, the mobile phase being methanol-water solution, and carrying out gradient elution, wherein the gradient elution procedure is as follows: the methanol phase rises from 10% to 40% for 0-30min, and 40-60min, and the methanol phase rises from 40% to 95%. The flow rate is 10ml/min, and the column temperature is room temperature; dissolving the sample with chromatographic grade methanol, separating by preparative liquid chromatography, collecting the solution within 7-15 minutes, concentrating and drying the collected solution to obtain cortex Dictamni Radicis antiviral active fraction with a total content of 63.4% of cortex Dictamni Radicis limonoids.

Example 5

(1) extraction: pulverizing cortex Dictamni Radicis, adding 10 times of petroleum ether, reflux-extracting in 70 deg.C water bath for 2 times, each for 1 hr, and mixing filtrates. Filtering, adding 8 times of dichloromethane into the residue, extracting under reflux in 85 deg.C water bath for 2 times (1 hr each time), filtering, and mixing filtrates. Concentrating to obtain concentrated extract.

(2) Removing impurities: performing macroporous resin column chromatography, dissolving the concentrated extract obtained by extraction with 1:2 pure water, performing macroporous resin adsorption, and eluting with 2 times of column volume of pure water to remove water-soluble impurities; eluting with 80% ethanol water solution with 4 times of column volume; collecting the elution part of 80% ethanol water solution, concentrating and drying to obtain the macroporous resin column chromatography active part. Specifically, the type number of the macroporous resin column in the impurity removal step can be AB-8.

(3) And (3) purification: continuing to separate the obtained eluent by using preparative liquid chromatography under the following separation conditions: the chromatographic column is an Agilent preparative column Zorba x SB-C18; 21.2mm X250 mm, the mobile phase being methanol-water solution, and carrying out gradient elution, wherein the gradient elution procedure is as follows: the methanol phase rises from 10% to 40% for 0-30min, and 40-60min, and the methanol phase rises from 40% to 95%. The flow rate is 10ml/min, and the column temperature is room temperature; dissolving the sample with chromatographic grade methanol, separating by preparative liquid chromatography, collecting the solution within 5-15 minutes, concentrating and drying the collected solution to obtain cortex Dictamni Radicis antiviral active fraction with a total content of 71.3% of cortex Dictamni Radicis limonoids.

Example 6

(2) Removing impurities: performing macroporous resin column chromatography, dissolving the concentrated extract obtained by extraction with 1:1 pure water, performing macroporous resin adsorption, and eluting with 4 times of column volume of pure water to remove water-soluble impurities; then eluting with 70% ethanol water solution with 5 times of column volume; collecting the elution part of 70% ethanol water solution, concentrating and drying to obtain the macroporous resin column chromatography active part. Specifically, the type number of the macroporous resin column in the impurity removal step can be DM-130.

(3) And (3) purification: continuing to separate the obtained eluent by using preparative liquid chromatography under the following separation conditions: the chromatographic column is an Agilent preparative column Zorba x SB-C18; 21.2mm X250 mm, the mobile phase being methanol-water solution, and carrying out gradient elution, wherein the gradient elution procedure is as follows: the methanol phase rises from 10% to 40% for 0-30min, and 40-60min, and the methanol phase rises from 40% to 95%. The flow rate is 10ml/min, and the column temperature is room temperature; dissolving the sample with chromatographic grade methanol, separating by preparative liquid chromatography, collecting the solution within 5-15 minutes, concentrating and drying the collected solution to obtain cortex Dictamni Radicis antiviral active fraction with a total content of 65.9% of cortex Dictamni Radicis limonoids.

Therefore, the preparation method of the extract has simple process, adopts organic solvent for full extraction and then carries out impurity removal and purification, is easy to operate, controllable in quality and easy to reproduce, and the total limonin content in the obtained cortex dictamni antiviral active part is more than 50 percent and can reach 82.1 percent at most.

Method for measuring content of limonin components and main components in antiviral active part of cortex dictamni

1. Instruments and reagents

UV-300 type ultraviolet-visible spectrophotometer, Shimadzu corporation, Japan; a constant temperature water bath, an electronic analytical balance, a limonin reference, Sichuan Veckqi Biotech limited; p-dimethylaminobenzaldehyde, Nanjing chemical reagents Ltd; sulfuric acid, Nanjing chemical reagents, Inc.; 95% ethanol, absolute ethanol, Nanjing chemical reagents Ltd; ferric chloride, Nanjing chemical reagents, Inc.

2. Content determination of limonin substances

2.1 solution preparation:

2.1.1 preparation of control solutions

Accurately weighing 8.119mg of limonin reference substance in a 10mL measuring flask, adding methanol to dissolve and dilute to scale, and shaking up to obtain the limonin reference substance.

2.1.2 preparation of test solutions

Weighing a certain amount of cortex dictamni antiviral active site powder, adding 6 times of 50% ethanol, extracting in water bath at 60 ℃ for 2h, filtering, and concentrating the filtrate for later use.

Color developing agent A liquid: weighing 125mg of p-dimethylaminobenzaldehyde, dissolving in 100mL of 65% sulfuric acid ethanol solution, and cooling for later use.

Color developing agent B liquid: 0.9g of ferric trichloride is taken and dissolved in water, and the volume is determined in a 100mL measuring flask.

3. Maximum absorption wavelength and calibration curve determination

Precisely measuring 0.4mL of reference solution, diluting to 2mL with anhydrous ethanol, adding developer A5mL and developer B0.5mL, standing for 30min, taking 1mL of test solution, and performing the same method. When full-wavelength scanning is carried out, the absorption curves of the reference substance and the test substance have maximum absorption at 500nm, so that 500nm is used as the measurement wavelength in the experiment.

Adding 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2mL of limonin control solution into 6 test tubes respectively, adding absolute ethyl alcohol to dilute to 2mL, adding 0.5mL of color developing agent A5mL and color developing agent B, standing for 30min, measuring absorbance (A) at 500nm, taking A as an abscissa and taking mass concentration (C) as an ordinate to obtain a regression equation C of 0.803A-0.0003(r of 0.9996), wherein the control solution has a good linear relation with the absorbance within the concentration range of 0.08119-0.48714 mg/mL.

4. Preparation of test solution

Taking a proper amount of the components to be measured of the antiviral active site of the cortex dictamni obtained in the above embodiments, precisely weighing, extracting with 50% ethanol in water bath at 60 ℃ for 2h, filtering, concentrating the filtrate, transferring to a 10ml volumetric flask, and diluting to scale marks.

5. Purity determination method

Precisely sucking 1mL of the test solution, precisely measuring 0.4mL of the reference solution, diluting to 2mL with anhydrous ethanol, adding 0.5mL of color developing agent A5mL and color developing agent B, and standing for 30 min. And (5) measuring the absorbance at 500nm, and calculating the total content of the limonin compounds in the sample according to the standard curve.

6. Examination of measurement method

Precision investigation: precisely sucking 0.4mL of the control solution prepared under item 2, diluting to 2mL with anhydrous ethanol, adding 0.5mL of color developing agent A5mL and color developing agent B, and standing for 30 min. After shaking up, the measurement was repeated 5 times at 500nm, and the absorbance value was recorded to calculate the RSD value. The result RSD was 0.89%, indicating good instrument precision.

And (3) stability investigation: precisely sucking 0.4mL of the control solution prepared under item 2, diluting to 2mL with anhydrous ethanol, adding 0.5mL of color developing agent A5mL and color developing agent B, and standing for 30 min. The absorbance values were recorded and the RSD values calculated at 500nm for 0, 15, 30, 60, 120min, respectively. The result RSD is 1.54%, which indicates that the test solution has good stability within 2 h.

And (3) repeatability inspection: precisely weighing 5 parts of the cortex dictamni active site obtained in the preparation example 2, transferring the cortex dictamni active site to a 10mL volumetric flask after dissolving, diluting to a scale mark, precisely sucking 1mL of sample solution, adding absolute ethyl alcohol to dilute to 2mL, adding 0.5mL of color developing agent A5mL and color developing agent B, and standing for 30 min. The absorbance was measured at 500nm to calculate RSD. The result shows that the average content of the total limonin is 76.8 percent, and the RSD is 1.05 percent, which indicates that the method has good reproducibility.

And (3) sample recovery rate investigation: accurately weighing 5 parts of the cortex dictamni active site obtained in the preparation example 2 by adopting a sample adding recovery method, adding a limonin reference substance according to the ratio of 1: 0.8, 1: 1.0 and 1: 1.2 respectively, adding absolute ethyl alcohol to dilute to 2mL, adding 0.5mL of a color developing agent A5mL and a color developing agent B, standing for 30min, and measuring the absorbance at 500 nm.

The method has good specificity, and the blank solvent has no interference to the absorbance of limonin components in the cortex dictamni under 500 nm; the linear relation is good within the range of 0.08119-0.48714 mg/ml, and the correlation coefficient is more than 0.999; the recovery rate is good, between 98% and 102%, and the RSD value is lower than 2.0%; the precision conforms to the specification, and the RSD is less than 1%; the solution has good stability within 2h, and the absorbance of the test solution and the reference solution is not obviously changed after the test solution and the reference solution are placed at room temperature for 2 h.

Method for measuring content of main component of antiviral active site of cortex dictamni

1 preparation of the solution

1.1 mixing control solutions

Respectively and precisely weighing the reference substances, placing the reference substances in a 50mL measuring flask, dissolving the reference substances by using methanol, diluting the reference substances to scale, shaking the reference substances uniformly, and preparing mixed reference substance solutions with the concentrations of 0.2013mg/mL of limonin, 0.1502mg/mL of phellodendron ketone, 0.1002mg/mL of fraxinellone, 0.1012mg/mL of fraxinellone and 0.2009mg/mL of pre-phellodendron ketone for later use.

1.2 test article solution

Taking 10mg of cortex dictamni antiviral active part, precisely weighing, dissolving with methanol, fixing the volume into a 10mL measuring flask, shaking up, filtering with a microporous filter membrane, and taking a subsequent filtrate as a test solution.

2 chromatographic Condition System suitability test

Column Diamonsil C18 column (200 mm. times.4.6 mm), mobile phase: methanol-water with a gradient of 1.0m Lmin-1, a detection wavelength of 210nm and a sample size of 10 μ L.

Under the condition, the theoretical plate number is not less than 3000 calculated by limonin, phellodendron ketone, fraxinellone and pre-phellodendron ketone, and the separation of the limonin, phellodendron ketone, fraxinellone and pre-phellodendron ketone from other peaks is achieved, and the chromatogram is shown in figure 2:

3 investigation of Linear relationship

Accurately weighing mixed reference substance solution 0.3 mL, 1.0 mL, 1.5 mL, 3.0mL, 6.0 mL, and 9.0mL, respectively placing into 10mL measuring flask, adding methanol to dilute to scale, and shaking to obtain series of reference solutions. And (4) drawing a standard curve by taking the chromatographic peak area (Y) as a vertical coordinate and the mass concentration (X) as a horizontal coordinate, and calculating a regression equation.

4. Methodology investigation

4.1 precision test

Precisely sucking 3.0mL of the mixed reference solution, placing the mixed reference solution in a 10mL measuring flask, adding methanol to dilute the mixed reference solution to a scale, and shaking up the mixed reference solution. The sample injection determination is carried out according to the chromatographic conditions described in 2, and the determination is repeated for 6 times. RSD of chromatographic peak areas of the limonin, the phellodendron ketone, the fraxinellone and the pre-phellodendron ketone are all less than 3.0% (n is 6), and the instrument precision is good.

4.2 repeatability test

Taking 6 parts of cortex dictamni sample, preparing sample solution according to the method of 1.2, and carrying out sample injection measurement according to the chromatographic condition of 2. RSD of chromatographic peak areas of the limonin, the phellodendron ketone, the fraxinellone and the pre-phellodendron ketone are all less than 2.0 percent (n is 6), and the method is good in repeatability.

4.3 stability test

Taking 1 part of cortex Dictamni Radicis sample, preparing sample solution according to the method described in 1.2, and repeatedly injecting sample for 6 times within 12h according to the chromatographic condition described in 2. RSDs of chromatographic peak areas of the limonin, the phellodendron ketone, the fraxinellone and the pre-phellodendron ketone are all less than 3.0%, and the rsDs indicate that the limonin, the phellodendron ketone, the fraxinellone and the pre-phellodendron ketone are stable within 12 h.

4.4 sample application recovery test

Taking 6 parts of cortex dictamni antiviral active site with known content, respectively weighing 10mg, respectively adding 1, 1.3 and 1.6mL of reference substance solution with mass concentration of 80.0 mg.L < -1 > and 240.0 mg.L < -1 >, diluting to scale with methanol, shaking up, preparing test solution with low, medium and high mass concentration, and preparing 3 parts of sample with each mass concentration. Preparing test solution according to the method of 1.2, and calculating the recovery rate according to the chromatographic conditions of 2, wherein the RDS value is less than 3%.

5. Content determination of test sample

The antiviral active site 10mg of the cortex dictamni in each preparation example is precisely weighed, dissolved by methanol and fixed to 10ml, the operation is carried out according to the method 3, the content of 5 limonoids is calculated by measurement under the chromatographic condition 2, and the measurement results are shown in the following table.

Table 1: content determination of 5 limonoids in preparation example

Activity detection of cortex dictamni antiviral active site

The pharmaceutical activity is further illustrated by pharmacodynamic experiments below.

Experiment one: active site cytotoxicity test of cortex Dictamni Radicis

1. Experimental Material

The anti-virus active effective part of the cortex dictamni: sample 1 (prepared according to the method of preparation 1, having a total limonin content of dittany bark of 82.1%); sample 2 (prepared by the method of preparation 2, having a total limonin content of dittany bark of 76.8%); sample 3 (prepared by the method of preparation 3, having a total limonin content of dittany bark of 56.3%); sample 4 (prepared by the method of preparation 4, having a total limonin content of 63.4% in cortex dictamni); sample 5 (prepared according to the method of preparation 5, having a total limonin content of dittany bark of 71.3%); sample 6 (prepared according to the method of preparation 6, having a total limonin content of dittany bark of 65.9%). Dog kidney cell line MDCK, derived from ATCC, passage preserved by Wuhan virus institute of Chinese academy of sciences.

The instrument comprises the following steps: CO2 incubator, Thermo Fisher Scientific, USA; microplate readers, Molecular Devices; biosafety cabinets, available from Heal Force, Inc.

2. Principle and method of experiment

2.1 toxicity assay of samples on MDCK cells

The experimental principle is as follows: alamar was used for the experiment

(Invitrogen) the kit detects the toxic effect of the drug on the cells. alamar

Is a redox indicator which can generate absorbance change and fluorescence signals according to metabolic activity. alamar

Is easy to dissolve in water and has good water solubility,the oxidized form of the compound enters cells and is reduced by mitochondrial enzyme to generate measurable fluorescence and color change, and the compound is suitable for quantitative analysis of cell activity and cell proliferation and in vitro cytotoxicity research. Normal cells with metabolic activity are able to convert the agent to a stronger fluorescence and color change, while damaged or inactive cells have a lower natural metabolic activity and the corresponding signal is lower. Therefore, the fluorescence signal is strong and weak, and can reflect the activity of the cells.

Grouping samples: sample 1 (prepared according to the method of preparation 1, having a total limonin content of dittany bark of 82.1%); sample 2 (prepared by the method of preparation 2, having a total limonin content of dittany bark of 76.8%); sample 3 (prepared by the method of preparation 3, having a total limonin content of dittany bark of 56.3%); sample 4 (prepared by the method of preparation 4, having a total limonin content of 63.4% in cortex dictamni); sample 5 (prepared according to the method of preparation 5, having a total limonin content of dittany bark of 71.3%); sample 6 (prepared according to the method of preparation 6, having a total limonin content of dittany bark of 65.9%).

The experimental steps are as follows: and inoculating the MDCK cells into a 96-well cell culture plate, and keeping the cells attached to the wall for later use. The drug was serially diluted in DMEM medium for 6 gradients in 3-fold gradients from the 2-fold maximum concentration tested. Adding the drug to the cells at 37 deg.C CO₂Culturing in an incubator. After adding drugs and culturing for 24h, observing cytopathic effect (CPE) caused by the drugs under a microscope, and adding alamarBlue to detect the cell survival rate. The magnitude of the toxicity of a drug to a cell is inversely proportional to and reflected by the activity of the cell.

Calculating the formula: cell activity (%) ═ (drug group-blank)/(cell control-blank) × 100

TABLE 2 Densefruit Pittany root-bark limonin cytotoxicity test data

As a result, each sample group was not cytotoxic to MDCK cells at a concentration of 250. mu.g/ml, as shown in Table 1.

Experiment two: in vitro anti-influenza A virus H3N2 activity experiment of cortex dictamni active site

1. Experimental Material

The anti-virus active effective part of the cortex dictamni: sample 1 (prepared according to the method of preparation 1, having a total limonin content of dittany bark of 82.1%); sample 2 (prepared by the method of preparation 2, having a total limonin content of dittany bark of 76.8%); sample 3 (prepared by the method of preparation 3, having a total limonin content of dittany bark of 56.3%); sample 4 (prepared by the method of preparation 4, having a total limonin content of 63.4% in cortex dictamni); sample 5 (prepared according to the method of preparation 5, having a total limonin content of dittany bark of 71.3%); sample 6 (prepared according to the method of preparation 6, having a total limonin content of dittany bark of 65.9%).

Virus strain: influenza A virus (H3N2, A/hongkong/498/97) was amplified and preserved by the Wuhan virus institute, Chinese academy of sciences.

The culture conditions are as follows: DMEM + 10% fetal bovine serum, 37 ℃ and 5% CO₂。

2. anti-H3N 2 Activity assay of samples

The experimental principle is as follows: since the expression level of structural proteins of influenza virus is directly proportional to the replication of the virus, the experiment reflects the level of viral replication by measuring the expression level of influenza virus proteins. The laboratory adopts a reagent with high sensitivity to detect the change of fluorescence intensity and reflect the expression of influenza virus protein.

Grouping samples: group 1 (prepared by the preparation method of preparation example 1, the total limonin content of the cortex dictamni is 82.1%); group 2 (prepared by the preparation method of preparation example 2, the total limonin content of the cortex dictamni is 76.8%); group 3 (prepared by the preparation method of preparation example 3, the total limonin content of the cortex dictamni is 56.3%); group 4 (prepared by the preparation method of preparation 4, the total limonin content of the cortex dictamni is 63.4%); group 5 (prepared by the preparation method of preparation example 5, the total limonin content of the cortex dictamni is 71.3%); group 6 (prepared by the method of preparation 6, having a total limonin content of dittany bark of 65.9%). Group 7 was the positive drug Ribavirin. The initial concentration of each of the above-mentioned groups was 160. mu.g/ml.

The method comprises the following steps: a blank control hole (normal cells), a virus control hole (no medicine is added after virus infection) and a positive medicine control hole (ribavirin is added after infection) are arranged in the experiment, and according to the result of the cytotoxicity experiment, when the concentration is within 250 mu g/ml, the medicine has no toxicity to the cells.

MDCK cells were seeded in 96-well cell culture plates and cultured overnight at 37 ℃ for use. After washing MDCK cells twice with PBS, influenza virus solution and medicines with gradually diluted concentration are added at the same time. Culturing in a 37 deg.C cell culture box for 24 hr, and observing cytopathic effect (CPE) with microscope; and taking the supernatant of the culture solution to detect the expression level of the virus protein.

Inhibition (%) of 100- (sample well-blank)/(virus control-blank) 1003. assay of anti-H3N 2 activity of sample

The positive drug ribavirin and the sample have specific inhibition effect on influenza virus H3N2 and toxicity on MDCK cells.

TABLE 3 inhibition and cytotoxicity of samples against influenza virus H3N2

In the experiment, the result of detecting the replication level of the influenza virus shows that the positive drug ribavirin has strong dose-dependent inhibition effect on the influenza virus H3N2, and the concentration EC50 causing 50% inhibition effect is about 21.992 mug/ml. The cortex dictamni active site of a detected sample has antiviral activity on influenza virus H3N2, the cortex dictamni active site with 82.1 percent of content has no toxicity on MDCK under the concentration of 250 mu g/ml, the EC50 on H3N2 is about 18.818 mu g/ml, and the antiviral activity of the cortex dictamni active site is superior to that of a positive drug ribavirin. The experimental result shows that the cortex dictamni active site has good inhibitory action on influenza virus H3N2, and the inhibitory action on the influenza virus is obviously improved along with the increase of the content of the total limonin in the cortex dictamni active site, which indicates that the total limonin is the main active substance against the virus in the cortex dictamni.

Experiment three: in vitro anti-influenza A virus H1N1 activity experiment of cortex dictamni active site

1. Experimental Material

Virus strain: influenza A virus (H1N1, A/puerto Rico/8/1934), was amplified and preserved by the Wuhan virus institute, Chinese academy of sciences.

2. anti-H1N 1 Activity assay of samples

The method comprises the following steps: MDCK cells were seeded in 96-well cell culture plates and cultured overnight at 37 ℃ for use. The MDCK cell is added with medicine and influenza virus liquid. Culturing in a 37 deg.C cell culture box for 24 hr, and observing cytopathic effect (CPE) with microscope; and taking the supernatant of the culture solution to detect the expression level of the virus protein.

The experiment was performed with a blank control well (normal cells), a virus control well (no drug added after virus infection), and a positive drug control well (ribavirin added after infection).

Inhibition (%) of 100- (sample well-blank)/(virus control-blank) × 100

3. Detection result of anti-H1N 1 activity of sample

The specific inhibitory effect of the positive drug ribavirin and samples on influenza virus H1N1 and toxicity results on MDCK cells are shown below:

TABLE 4 inhibition and cytotoxicity of samples on influenza H1N1

In the experiment, the result of detecting the replication level of the influenza virus shows that the positive drug ribavirin has strong dose-dependent inhibition effect on the influenza virus H1N1, and the concentration EC50 causing 50% inhibition effect is about 25.887 mug/ml. The cortex dictamni active part with the content of 82.1 percent of the tested sample has antiviral activity on influenza virus H1N1, has no toxicity on MDCK under the concentration of 250 mu g/ml, has EC50 of about 27.367 mu g/ml on H1N1, and has the antiviral activity equivalent to that of ribavirin group. The experimental result shows that the cortex dictamni active site has good inhibitory action on influenza virus H3N2, and the inhibitory action on the influenza virus is obviously improved along with the increase of the content of the total limonin in the cortex dictamni active site, which indicates that the total limonin is the main active substance against the virus in the cortex dictamni.

Based on the pharmacodynamic tests, the cortex dictamni active site obtained by the method has a very good antiviral effect, the higher the content of the total limonin is, the stronger the antiviral effect is, and the cortex dictamni limonin substances have stronger antiviral activity, wherein when the content of the total limonin in the cortex dictamni active site is more than 80%, the antiviral activity is higher than or equal to that of a ribavirin group. The cortex dictamni active site prepared by the invention can be used for preparing antiviral drugs, in particular to drugs for resisting influenza viruses.

Preparation of composition containing cortex Dictamni antiviral active fraction

Tablet formulation

Taking 10.5g of cortex dictamni antiviral active part, grinding into fine powder, adding about 8g of 10% starch paste, 7g of medicinal starch and 0.8g of superfine silica gel powder, mixing, granulating by using a 14-mesh sieve, drying at 80 ℃, granulating, adding 0.05g of magnesium stearate, mixing uniformly, and pressing into 100 tablets to obtain the traditional Chinese medicine. The weight of each tablet is 0.2g, and the tablet is orally taken 1 tablet at a time and three times a day.

Hard capsules

Taking 15.5g of cortex dictamni antiviral active part, grinding into fine powder, 3.5g of medicinal starch and 0.01g of magnesium stearate, uniformly mixing, sieving, and encapsulating to prepare 100 granules. The content of each capsule weighs 0.2g, and is orally administered 1 capsule at a time twice a day.

Soft capsule

Taking 10.5g of cortex dictamni antiviral active fraction, adding 0.2g of propylene glycol, 0.9g of lecithin and 18g of soybean oil, vibrating and grinding for ultramicro 20 minutes, mixing uniformly, and preparing into 100 soft capsules. The content of each capsule weighs 0.3g, and is orally administered 1 capsule at a time three times a day.

Granules

Taking 15.5g of cortex dictamni antiviral active part, grinding into fine powder, adding 25g of dextrin and 458g of lactose, uniformly mixing, granulating, drying at 80 ℃, grading, subpackaging and making 100 bags to obtain the traditional Chinese medicine preparation. The weight of each bag is 5g, and the oral administration is carried out for 1 bag each time and twice a day.

Sustained-release tablet

Taking 25.5g of cortex dictamni antiviral active part, grinding into fine powder, sieving with a 100-mesh sieve, sieving with HPMC10.3g, lactose 16g and sodium dodecyl sulfate 1g, sieving with a 100-mesh sieve, mixing uniformly, adding 2% polyvidone (95% ethanol) adhesive to prepare a soft material, sieving with a 20-mesh sieve, granulating, drying at 50 ℃, sieving with a 18-mesh sieve, adding 2% magnesium stearate into the dry granules by an external method, mixing uniformly, tabletting, and preparing into 100 sustained-release tablets. Each tablet weighs 0.5 g. It is administered orally 1 tablet at a time and once a day.

Drop pills

Grinding cortex Dictamni Radicis 10.5g into fine powder, heating and melting 24.8g polyethylene glycol 4000, adding cortex Dictamni Radicis antiviral active fraction, vibration milling for 20min, mixing, and making into dripping pill. Each pill weighs 0.35g, and is orally administered 1 granule each time, three times a day.

Injection preparation

Taking 10.5g of the antiviral active part of the cortex dictamni, grinding into fine powder, adding β cyclodextrin 4.5g, sucrose 500g and a proper amount of pure water (about 390g), stirring and heating to dissolve, boiling for 10 minutes, slightly cooling, adding 10% (W/V) ethyl hydroxybenzoate ethanol solution, stirring uniformly, adjusting to 100ml with water, filtering, subpackaging and sterilizing to obtain the oral preparation, wherein each 10ml is taken orally, 1 pill is taken every time, and the oral preparation is taken three times a day.

The pharmaceutical composition containing the effective part of cortex dictamni can be properly selected according to the common auxiliary materials in the pharmaceutical field and according to the dosage form and the actual situation, for example, the common auxiliary materials include starch, low-substituted hydroxypropyl cellulose, aerosil, magnesium stearate, starch slurry, sucrose, dextrin, sodium carboxymethyl starch, talcum powder, polysorbate, polyethylene glycol, soybean phospholipid for injection, glycerol for injection and the like; when various dosage forms of the desired drug are prepared, they can be prepared according to conventional methods of pharmacy, such as mixing the compound with one or more carriers and then making the corresponding dosage forms. Preferably, the dosage form of the pharmaceutical preparation includes injection, tablet, suppository, ointment, gel, pill, tablet, granule, capsule, mixture and the like.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. An antiviral active fraction of cortex Dictamni Radicis is characterized by that the total content of limonin in the active fraction is more than 50%, and the active fraction contains limonin, phellodendron ketone, fraxinellone and procambricin.

2. The active site of claim 1 having a total limonin content of 80% or more and comprising limonin, obacunone, fraxinellone and procambardione.

3. Use of an active site according to any one of claims 1-2 in the preparation of an antiviral medicament.

4. The active site of claim 3, wherein the active site is for use in the preparation of an antiviral medicament.

5. The method for preparing an antiviral active site of cortex dictamni as claimed in any one of claims 1-2, comprising the steps of:

6. The method of claim 5, wherein the macroporous resin column type number in the removing step is DH101, DM-130, AB-8.

7. A method for measuring the content of an antiviral active site in cortex dictamni, which is characterized by comprising the following steps: (1) preparing a test sample: precisely weighing 10.0mg of antiviral active site in cortex Dictamni Radicis, dissolving with chromatographic pure methanol, diluting to a constant volume of 10ml volumetric flask, and filtering with 0.45 μm microporous membrane to obtain cortex Dictamni Radicis antiviral active site sample;