CN117815319B - Compound isatis root preparation, preparation method thereof and application thereof in resisting influenza - Google Patents

Abstract

The invention relates to a preparation for improving the anti-influenza efficacy of a compound isatis root preparation and a preparation method thereof. The method adopts medium-low concentration ethanol (35%-48%) to heat and reflux to extract isatis indigotica, adopts a water extraction and alcohol precipitation method to extract isatis indigotica, combines the two and concentrates to obtain a compound isatis root granule improved new drug paste, which can be used to prepare the compound isatis root granule improved new drug preparation, maximizes the utilization and efficacy strength of active ingredients in isatis root and isatis indigotica, and significantly improves the anti-influenza efficacy of the compound isatis root granule compared with that before the improvement. The method is simple and easy to operate, has high safety, and has good clinical value and application prospect.

Description

A compound Radix Isatidis preparation and its preparation method and application in anti-influenza

Technical Field

The invention relates to the field of traditional Chinese medicine preparations, and in particular to a compound Radix Isatidis preparation and a preparation method thereof and application thereof in anti-influenza.

Background Art

Compound Isatis Root Granules are included in the drug standards issued by the Ministry of Health (Volume 12 of Chinese Medicine Formula Preparations, Standard No.: WS3-B-2377-97). They are made of the classic heat-clearing and detoxifying Chinese medicine Isatis Root and Folium Isatidis in a ratio of 2:3 through water extraction, alcohol precipitation, concentration, granulation and other processes. They have the effects of clearing heat and detoxifying, cooling blood, and are widely used in the prevention and treatment of upper respiratory tract diseases such as wind-heat colds and sore throats. They are also used in diseases such as chronic hepatitis B and flat warts, with definite efficacy. Modern research shows that Compound Isatis Root Granules have a diverse chemical composition, mainly containing alkaloids, amino acids, organic acids, nucleosides, polysaccharides, flavonoids and other ingredients, and have multiple pharmacological effects such as antiviral, antibacterial, immunomodulatory, antipyretic, and anti-inflammatory.

However, the current standard of compound isatis root granules still has bottleneck problems such as the transfer utilization rate of active ingredients, quality control level, and the need to expand and improve the efficacy.

In the prior art, Jiang Bin et al. developed an improved method for the production process of compound isatis root granules, which includes extracting isatis leaf with 70% ethanol under reduced pressure, concentrating to obtain an extract, and then extracting the residue with isatis root, concentrating and granulating. This method can increase the content of indigo and indigotin in the product (Jiang Bin, Zhang Xiaohong, Zhou Fujiong. Improvement of the production process of compound isatis root granules [J]. Chinese Journal of Hospital Pharmacy, 2000(06):52.); Zhang Qianzhen et al. proposed a method of using 7 times the amount of 80% ethanol to reflux extract isatis root and isatis leaf twice, each time for 80 minutes, to prepare a compound isatis root preparation, which can significantly increase the indigo content (Zhang Qianzhen, Li Dongqin, Chen Guoliang. Research on the extraction process of indigo in compound isatis root preparation [J]. Journal of Anhui Health Vocational and Technical College, 2020, 19(06):93-95.).

In the prior art, high-concentration ethanol greater than 70% is usually used for heating and reflux extraction of isatis leaf or isatis root and isatis leaf, which has the disadvantage of high extraction cost, especially the need to be produced in an explosion-proof workshop, involving certain safety risks that cannot be ignored. According to the current relevant records, it is usually and only aimed at improving the content of indigo red and indigo components, and the improvement of clinical efficacy is not shown, or the alcohol precipitation process is not adopted, resulting in a high paste rate, which is not conducive to the subsequent preparation of granules. For example, the prior art "Research on the Extraction Process of Indigo Red in Compound Isatis Root Preparation" records the process route of using isatis root and isatis leaf to extract with ethanol, and the process route uses a high concentration of ethanol as a solvent. For another example, the prior art "Improvement of the Production Process of Compound Isatis Root Granules" records the process route of using isatis leaf alcohol extraction, and then extracting the drug residue with isatis root together with water, and the process also uses a high concentration of ethanol solvent. The purpose of its process improvement is only to increase the content of indigo and indigo red, and does not involve improving the efficacy strength.

The inventors found in their research that different extraction methods have a significant impact on the active ingredients of the compound isatis root preparation, especially on one of the effects of the compound isatis root preparation - the activity of treating influenza.

Summary of the invention

Based on this, the purpose of the present invention is to provide a compound Radix Isatidis preparation with improved anti-influenza efficacy and a preparation method thereof, as well as the application of the compound Radix Isatidis preparation in anti-influenza.

The first aspect of the present invention is to provide a method for preparing a compound isatis root preparation with improved anti-influenza efficacy. The preparation method uses medium- and low-concentration ethanol heating reflux to extract isatis indigotica, and uses water extraction and alcohol precipitation to extract isatis root. The two are combined and concentrated to obtain an improved new drug paste for the compound isatis root preparation, which can be used to prepare an improved new drug preparation for the compound isatis root preparation, thereby maximizing the utilization and efficacy of the active ingredients in isatis root and isatis indigotica.

A method for preparing a compound Radix Isatidis preparation, wherein the active ingredient raw materials of the compound Radix Isatidis preparation are Radix Isatidis and Folium Isatidis in a mass ratio of 2:3, and the preparation method comprises the following steps:

SA. Take the isatis root, add 6-10 times water, heat to reflux or boil and extract 2-3 times, each time for 30-90 minutes, filter, and concentrate the filtrate to a volume of 0.5mL/g-1mL/g crude drug, add 1.5-4 times the volume of ethanol to the liquid, let stand for 12-24h, filter, and take the filtrate to obtain an isatis root extract;

SB. Take the isatis leaf, add 6-10 times of 35%-48% ethanol aqueous solution, heat and reflux to extract 2-4 times, each time for 60-120 minutes, filter, and take the filtrate to obtain an isatis leaf extract;

SC. The above Radix Isatidis extract and Folium Isatidis extract are combined and concentrated to obtain a fluid paste, and the fluid paste is mixed with pharmaceutically acceptable excipients to prepare a preparation.

In some embodiments, in step SB.: take the isatis indigo leaf, add 8-10 times of 40%-48% ethanol aqueous solution, heat and reflux to extract 2-3 times, each time for 80-100 minutes, filter, take the filtrate, and obtain the isatis indigo leaf extract.

In some of the more preferred embodiments, in step SB.: take the isatis indigo leaf, add 8-10 times of 43%-46% ethanol aqueous solution, heat and reflux to extract twice, each time for 80-100 minutes, filter, take the filtrate, and obtain the isatis indigo leaf extract.

In some embodiments, in step SA.: take the isatis root, add 6-8 times of water, heat reflux or decoction for extraction 2-3 times, each time for 60-90 minutes, filter, take the filtrate and concentrate it to a volume of 0.67mL/g-1mL/g of crude drug, add 2.33-4 times the volume of ethanol to the drug solution, let it stand for 12-18h, filter, take the filtrate, and obtain the isatis root extract.

In some of the more preferred embodiments, in step SA.: take the isatis root, add 6-7 times of water, heat reflux or decoct for extraction 3 times, each time for 60-70 minutes, filter, take the filtrate and concentrate it to a volume of 1 mL/g crude drug, add 3.5-4 times the volume of ethanol to the drug solution, let it stand for 12-15 hours, filter, take the filtrate, and obtain the isatis root extract.

The second aspect of the present invention provides a compound Radix Isatidis preparation obtained by the above-mentioned preparation method.

In some of the embodiments, when preparing the compound Isatis Radix preparation, the extraction rate of isovitexin in the Folium Isatidis extract is not less than 1.10 mg/g of Folium Isatidis original medicinal material, and more preferably, the extraction rate of isovitexin is not less than 1.20 mg/g of Folium Isatidis original medicinal material, and even more preferably, the extraction rate of isovitexin is not less than 1.30 mg/g of Folium Isatidis original medicinal material.

In some embodiments, the dosage form of the preparation is granules. In addition, the dosage form of the preparation can also be a common dosage form in the medical field, such as capsules, ointments, or tablets or other acceptable dosage forms in medicine.

The third aspect of the present invention provides the use of the above-mentioned compound Radix Isatidis preparation in the preparation of prevention and treatment of influenza.

In some of the embodiments, the application includes reducing or preventing the patient's condition from developing into severe influenza after influenza virus infection, that is, the application of treating influenza includes reducing the occurrence of severe influenza.

In some embodiments, the use includes reducing the degree of lung tissue damage and the up-regulated level of lung inflammatory factors caused by influenza virus infection.

In some embodiments, the influenza virus (influenza virus) is type A (A), B (B), or C (C), preferably type A influenza virus.

The present invention adopts a preparation method different from that of the prior art, aims to improve the anti-influenza efficacy, establishes a simple and easy compound isatis root preparation for improving the anti-influenza efficacy and a preparation method thereof, and provides a compound isatis root preparation with significantly improved anti-influenza efficacy.

The preparation method of the present invention adopts medium and low concentration ethanol (35%-48%) to heat and reflux to extract the isatis leaf, and adopts water extraction and alcohol precipitation method to extract the isatis root, and the two are combined and concentrated to obtain the improved new drug paste of the compound isatis root preparation, which can be used to prepare the improved new drug preparation of the compound isatis root preparation, and maximizes the utilization and efficacy strength of the active ingredients in the isatis root and the isatis leaf. Compared with the compound isatis root preparation before the improvement, the anti-influenza efficacy of the compound isatis root preparation is significantly improved, especially the selectivity coefficient (SI) against influenza virus is significantly increased, showing the efficacy strength of high efficiency and low toxicity; reducing the lung damage caused by influenza virus and the up-regulation of lung inflammatory factors is significantly stronger, and showing a significant anti-influenza death protection effect, reducing the occurrence of severe influenza. The preparation method of the present invention is simple and easy to implement, and is also more suitable for industrial production, with good clinical value and application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the process route of sample 1 in Example 1.

Figure 2 shows the process routes of samples 2 and 3 in Example 1.

FIG3 is a technical route of the present invention.

FIG. 4 is a chromatogram of isovitexin detected in sample 1 and sample 4 by HPLC in Example 1 (1: reference substance, 2: sample 4, 3: sample 1).

FIG5(A) is a response surface plot of the relationship between the extraction rate of isovitexin (R1) and the ethanol concentration (A).

FIG5(B) is a response surface plot of the relationship between the extraction rate of isovitexin (R1) and the amount of solvent used (B).

FIG5(C) is a response surface plot of the relationship between the extraction rate of isovitexin (R1) and the extraction time (C).

Figure 6 is the main effect diagram of the orthogonal experiment mean of Isatis indigotica water extraction process.

Figure 7 is the main effect diagram of the orthogonal experiment mean of Isatis indigotica alcohol precipitation process.

Figure 8 shows the effects of the improved new drug sample of Compound Isatis Root Granules and Compound Isatis Root Granules on the lung index of mice (#: P < 0.05 compared with the blank control group; *: P < 0.05 compared with the virus-infected group).

Figure 9 shows the effects of the improved new drug sample of Compound Isatis Root Granules and Compound Isatis Root Granules on inflammatory cytokines in mice (#: P < 0.05 compared with the blank control group; *: P < 0.05 compared with the virus-infected group).

Figure 10 shows the effects of the improved new drug sample of Compound Isatis Root Granules and Compound Isatis Root Granules on HE pathological sections of mouse lungs.

DETAILED DESCRIPTION

In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and/or" used in the present invention includes any and all combinations of one or more of the related listed items.

The invention provides a method for preparing a compound isatis root preparation capable of improving the anti-influenza efficacy. The method adopts medium- and low-concentration ethanol heating and reflux to extract active ingredients in isatis indigotica, wherein flavonoid active ingredients such as isovitexin are mainly represented, and a water extraction and alcohol precipitation method is adopted to extract active ingredients in the isatis root, wherein (R, S)-goyichun and other ingredients are represented, and the two are combined and concentrated to obtain an improved new drug paste for the compound isatis root preparation, which can be used to prepare the improved new drug preparation for the compound isatis root preparation, maximizes the utilization and efficacy strength of the active ingredients in the isatis root and the isatis indigotica, and significantly improves the anti-influenza efficacy of the compound isatis root preparation before the improvement, and the method is simple and easy to implement, reduces the cost of producing and using reagents such as ethanol and the operational safety risk, and has good clinical value and application prospects.

The preparation method of the present invention adopts medium and low concentration ethanol heating reflux to extract the active ingredients in the isatis indigotica leaf, which reduces the production cost and safety risk, and adopts the water extraction and alcohol precipitation method to extract the active ingredients in the isatis root, and the two are combined and concentrated to obtain the improved new drug paste of compound isatis root granules, which can be used to prepare the improved new drug preparation of compound isatis root granules, maximizes the utilization and efficacy of the active ingredients in the isatis root and the isatis indigotica leaf, and has significantly improved the anti-influenza efficacy of the compound isatis root granules compared with before the improvement, and can prevent the occurrence of severe influenza (based on the conventional clinical diagnostic standards), and the method is simple and easy to implement, and has good clinical value and application prospects.

The present invention is further described in detail below with reference to specific embodiments.

Example 1: Process route for preparing compound Radix Isatidis preparation

1. Materials and Methods

1.1 Preparation of samples from different process routes

1.1.1 Main experimental materials

The medicinal materials of Isatis root and Isatis indigotica were provided by Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd., purchased from Guangzhou Caizhilin Pharmaceutical Co., Ltd., and produced in Zhangye City, Gansu Province.

1.1.2 Experimental methods

(1) Sample 1: This sample adopts the standard compound isatis root granule extraction process, as shown in Figure 1. Take 60g of isatis root medicinal material and 90g of isatis indigotica medicinal material, add 8 times the volume of water and boil twice, each time for 1 hour, filter, combine the filtrate, concentrate to an appropriate amount, add three times the amount of ethanol, stir well, let stand for 24 hours, filter, recover ethanol from the filtrate, and concentrate to a thick paste.

(2) Sample 2: As shown in FIG2 , 60 g of Radix Isatidis and 90 g of Folium Isatidis were taken, and 8 times the volume of 40% low-concentration ethanol was added and heated under reflux for extraction twice, each time for 1 hour, and filtered. The filtrates were combined and concentrated to a thick paste without alcohol taste.

(3) Sample 3: As shown in FIG2 , 60 g of Radix Isatidis and 90 g of Folium Isatidis were taken, and 8 times the volume of 70% high concentration ethanol was added, and the mixture was heated under reflux and extracted twice, each time for 1 hour, and filtered, and the filtrates were combined and concentrated to a thick paste without alcohol taste.

(4) Sample 4: As shown in Figure 3, take 60g of Radix Isatidis, add 8 times the volume of water and boil twice, each time for 1 hour, filter, combine the filtrate, concentrate to an appropriate amount, add three times the volume of ethanol, stir well, let stand for 24 hours, and filter; take 90g of Folium Isatidis, add 8 times the volume of 40% low-concentration ethanol solution and extract twice, each time for 1 hour, filter, and combine the filtrate; combine the extracts of Radix Isatidis and Folium Isatidis, and concentrate to a thick paste without alcohol taste.

(5) Sample 5: As shown in Figure 3, take 60g of Radix Isatidis, add 8 times the volume of water and boil twice, each time for 1 hour, filter, combine the filtrate, concentrate to an appropriate amount, add three times the volume of ethanol, stir well, let stand for 24 hours, and filter; take 90g of Folium Isatidis, add 8 times the volume of 70% high concentration ethanol solution and extract twice, each time for 1 hour, filter, and combine the filtrate; combine the extracts of Radix Isatidis and Folium Isatidis, concentrate to a thick paste without alcohol taste, and obtain.

1.2 Anti-influenza A virus efficacy screening

1.2.1 Main experimental materials

MDCK cells were stored in the virus laboratory of Guangzhou Institute of Respiratory Health; A/PR/8/34 (H1N1) influenza A virus, with a titer of TCID50 = 105/100 μL, was stored at -80°C in the virus laboratory of Guangzhou Institute of Respiratory Health. This experiment used a virus titer of 100 TCID50. Oseltamivir was purchased from Sigma-Aldrich, USA, and was used as the positive control drug for this experiment.

1.2.2 Experimental methods

(1) Wash the 96-well plate monolayer cells once with PBS solution;

(2) Add 100 TCID50 virus solution, 100 μL/well, and incubate at 37°C for 1 hour;

(3) Discard the virus incubation solution and add 100 μL/well of virus culture solution containing a final concentration of 1.5 μg/mL. Instead of adding virus culture medium to the left 6 duplicate wells in row A, add 200 μL/well of virus culture medium dissolved with drugs. Then, perform gradient dilution from top to bottom using a gun. Observe the results after 2 days.

(4) After observing and recording the CPE results, add 20 μL of MTT solution (5 mg/mL) to each well and continue incubating in a 37°C, 5% CO2 incubator for 4 hours. Aspirate and discard the supernatant, then add 100 μL of 2-methyl sulfoxide (DMSO) to each well and shake at a low speed for 10 minutes to fully dissolve the crystals. Select a wavelength of 490 nm and measure the light absorbance of each well on an ELISA reader.

(5) The half inhibitory concentration (IC50) was calculated by the Reed-Muench method and expressed as the selection index SI (SI = TC50/IC50, the TC50 of each test drug was detected by the MTT method before the experiment). SI>2 indicates low toxicity and high efficiency, SI=1-2 indicates high toxicity and low efficiency, and SI<1 indicates ineffectiveness.

As shown in Table 1, sample 4 showed significantly better influenza virus inhibition activity compared to samples from other processes, and had the characteristics of low toxicity and high efficiency. The process route of "low concentration ethanol (35%-48%) extraction of isatis indigotica and water extraction and alcohol precipitation extraction of isatis root" of this sample can significantly improve the anti-influenza activity of the existing compound isatis root preparation. The above results also show that the method route adopted by the present invention is superior to the existing standard or technology for preparing compound isatis root granules in terms of anti-influenza efficacy.

Table 1 The efficacy of compound Radix Isatidis samples prepared by different processes in inhibiting influenza virus

1.3 Content determination

Based on the above experimental results, isovitexin is a representative anti-inflammatory and antioxidant flavonoid active ingredient in Folium Isatidis. The HPLC method was used to further detect the content of isovitexin in the fluid paste of Sample 4 and Sample 1. The results are shown in Figure 4. The peak area of isovitexin in Sample 1 prepared by the current marketed compound isatis root granule standard is extremely small, which is below the quantitative range of the detection method and cannot be detected; while the content of isovitexin in Sample 4 using the process route of the present invention is relatively high, and the calculated extraction rate is greater than 1.31 mg/g Folium Isatidis original medicinal material, which is significantly higher than Sample 1. This result also suggests that the extraction rate of isovitexin may be closely related to the anti-influenza efficacy.

Example 2: Optimization of process route for preparation of compound Radix Isatidis preparation

Flavonoid components such as isovitexin in Folium Isatidis are representative active ingredients of the low-concentration ethanol extraction part of Folium Isatidis, and components such as (R, S)-gauyichun in Radix Isatidis are representative active ingredients of the water extraction part of Radix Isatidis. In this experiment, HPLC was used to detect the contents of isovitexin and (R, S)-gauyichun in the extract, the extraction rate/transfer rate was calculated, and the extraction process parameters of the compound Radix Isatidis preparation were evaluated and optimized.

1. Optimization of Isatis indigotica Process

1.1 Investigation of extraction times

Take 50g of Isatidis Leaf medicinal material, extract it three times under the conditions of 50% ethanol, 10 times solvent, and 75min, take the first, second, and third extracts respectively, concentrate and fix the volume, and detect the content of isovitexin in the extract and the rate of paste, and the results are shown in Table 2. Considering the extraction effect and cost, the effect of the third extraction is obviously poor, so it is selected to extract twice.

Table 2 Effects of different extraction times on the extraction rate and paste yield of flavonoid components

sample Isovitexoside(mg/g) Cream yield (%) First extraction 0.757359606 13.75862069 Second extraction 0.228169458 5.43546798 The third extraction 0 2.086699507

1.2 Investigation of ethanol concentration, solvent dosage, and extraction time

Take 50g of Folium Isatidis, fix the extraction times to 2, use the Box-Behnken designed process parameters to carry out the Folium Isatidis extraction experiment, detect the content of flavonoid components in the extract and the rate of paste, and calculate the extraction rate. The experimental design and test results are shown in Tables 3 and 4.

Table 3 Box-Behnken design factor table

Table 4 Box-Behnken design and test results

The design expert software was used to analyze the experimental results, and the extraction rate of the representative flavonoid component isovitexin was used as the evaluation index to optimize the response surface method. First, a mathematical model between the extraction rate of isovitexin and the extraction conditions was established, among which the quadratic equation model had the best fitting effect, and the fitting equation was: extraction rate of isovitexin (R1)

=1.46849-0.044348A+0.11977B+0.078774C+0.06563BC-0.196606A2-0.161846B2-0.0748457C2, the model is significant and the fitting effect is good. The response surface plots of the relationship between the extraction rate of isovitexin (R1) and ethanol concentration (A), solvent dosage (B), and extraction time (C) are shown in Figures 5 (A) to (C).

Using design expert software, with the goal of maximizing the extraction rate of isovitexin (R1), an optimal solution was calculated to be 38% ethanol concentration, about 12 times the solvent dosage, and about 105 minutes of extraction time. Under this solution, the predicted extraction rate of isovitexin was 1.527 mg/g; considering the actual production, the solvent dosage and extraction time should not be too large, so with the goal of maximizing the extraction rate of isovitexin (R1) and minimizing the solvent dosage (B), a solution of 45% ethanol concentration, 9 times the solvent dosage, and 90 minutes of extraction time was adopted after comprehensive consideration. Under this solution, the predicted extraction rate of isovitexin was 1.4429 mg/g. The extraction rate of isovitexin and process parameters were both within an acceptable range, so this parameter was selected as the optimal extraction condition.

1.3 Verification of the extraction process of Isatis indigofera

Take about 100g of Folium Isatidis, extract twice with 45% ethanol concentration, 9 times the amount of solvent, and 90min extraction time, filter, combine, and obtain Folium Isatidis extract, and measure the volume. The above extraction experiment is repeated 3 times. Take an appropriate amount of extract for the detection of flavonoid components and the determination of the yield of the paste. The experimental results are shown in Table 5. The average extraction rate of isovitexin in the three batches of experiments is 1.4016mg/g, and the predicted accuracy reaches 97.14%, reaching the expected goal.

Table 5. Results of the validation experiment of the extract of Folium Isatidis

serial number Extraction rate of isovitexin (mg/g) 1 1.3856 2 1.4376 3 1.3817

2. Optimization of Isatis Radix Water Extraction Process

2.1 Orthogonal experiment

The amount of water added, extraction time and number of extractions were taken as the investigation factors, and the extraction rate/transfer rate and paste yield of (R, S)-galactopyran in Radix Isatidis were taken as the investigation indicators. The L9(34) orthogonal table was used for experimental design. The factor levels, experimental design and results are shown in Tables 6 and 7.

Take 25g of Radix Isatidis, extract according to the experimental design, filter the extract, combine, concentrate, add water to make the volume to 100mL, and save it for detecting the (R, S)-gaoichun content and the paste yield in the extract.

Taking into account the importance of the transfer of indicator components, the comprehensive score was calculated according to the weight ratio, comprehensive score = paste yield * 20% + (R, S) - transfer rate of gaoichun * 80%, and the orthogonal test results were analyzed.

Table 6 Factor levels of orthogonal experiment for water extraction of Radix Isatidis

level Water addition/times (A) Extraction time/min(B) Number of extractions/times (C) 1 6 30 1 2 8 60 2 3 10 90 3

Table 7 Orthogonal experimental design table of Isatis indigotica water extraction process

Minitab software was used to perform orthogonal (Taguchi) design analysis, and the results are shown in Table 8 and Figure 6.

Table 8 Mean response table of orthogonal test of water extraction process of Radix Isatidis

level A B C 1 43.38 45.47 40.51 2 50.23 54.07 35.08 3 49.07 43.14 67.10 Delta 6.85 10.92 32.02 Ranking 3 2 1

Minitab software was used for regression and variance analysis, and the results are shown in Table 9.

Table 9 Variance analysis of orthogonal test of water extraction technology of Radix Isatidis

From the intuitive analysis of the above results, it can be seen that test number 5 has the highest score, and the conditions are: extraction 3 times, adding 8 times of water, and extraction for 60 minutes. From the results of variance analysis, it can be seen that the factors affecting the comprehensive score (extraction rate and (R, S)-co-isocyanate transfer rate) are extraction times> extraction time> water addition. The extraction times have a greater impact on the extraction rate and (R, S)-co-isocyanate transfer rate of Radix Isatidis, and 3 extractions are significantly better.

The results of variance analysis show that within the range of water addition set in the experiment, the water addition had no significant effect on the comprehensive score of Isatis root extraction. Considering the production cost and production efficiency, the water addition was selected as 6 times; within the range of extraction time set in the experiment, the extraction time had no significant effect on the comprehensive score of Isatis root extraction, but the (R, S)-coitrin transfer rate was low under too short or too long extraction time. Considering the production cost, production efficiency and (R, S)-coitrin transfer rate, the extraction time was selected as 60 minutes; within the range of extraction times set in the experiment, the extraction times had no significant effect on the comprehensive score of Isatis root extraction, but there was a trend of the greatest influence, and 3 extractions were significantly better, so 3 extractions were selected.

Based on the above experimental results and comprehensive actual situation, the final extraction process of Isatis root was determined as follows: Isatis root was heated and refluxed for extraction 3 times, with 6 times the amount of water added each time, and the extraction lasted for 1 hour.

2.2 Validation experiment of water extraction of Radix Isatidis

Take 250g of Isatis root, heat and reflux and extract 3 times, add 6 times water each time, extract for 1 hour, combine the filtrates of the 3 extractions, concentrate and make up to volume with water to obtain the Isatis root extract, which is used to detect the transfer rate and paste yield of (R, S)-gaoichun, and repeat the verification 3 times.

Table 10 Verification of Isatis indigotica water extraction process results

3. Optimization of alcohol precipitation process of Radix Isatidis

3.1 Orthogonal experiment

The concentration ratio of medicinal solution (raw drug mass: extract volume, g/mL), alcohol precipitation concentration (volume percentage of ethanol added, %), and standing time (h) were used as investigation factors, and the extraction rate/transfer rate and paste yield of (R, S)-galactopyran of Isatis root were used as investigation indicators. The L9(34) orthogonal table was used for experimental design. The factor levels, experimental design and results are shown in Tables 11 and 12.

Taking into account the importance of the transfer of indicator components, the comprehensive score was calculated according to the weight ratio, comprehensive score = paste yield * 20% + (R, S) - transfer rate of gaoichun * 80%, and the orthogonal test results were analyzed.

Table 11 Factor levels of orthogonal experiment of alcohol precipitation process of Radix Isatidis

Table 12 Orthogonal experimental design table of alcohol precipitation process of Radix Isatidis

Minitab software was used to perform orthogonal (Taguchi) design analysis, and the results are shown in Table 13 and Figure 7.

Table 13 Mean response table of orthogonal experiment of alcohol precipitation process of Radix Isatidis

level A B C 1 41.34 15.58 22.33 2 20.81 24.43 24.84 3 11.54 33.68 26.53 Delta 29.80 18.10 4.20 Ranking 1 2 3

Regression and variance analysis were performed using Minitab software, and the results are shown in Table 14.

Table 14 Variance analysis of orthogonal test of alcohol precipitation process of Radix Isatidis

source Degrees of Freedom Adj SS Adj MS F-number P-value A 2 1395.71 697.86 18.56 0.051 B 2 491.38 245.69 6.53 0.133 C 2 26.76 13.38 0.36 0.737 error 2 75.19 37.60 total 8 1989.05

From the intuitive analysis of the above results, it can be seen that test number 3 has the highest score, and the conditions are: the liquid concentration ratio is 1:1, ethanol is added to a volume ratio of 80%, and the alcohol precipitation is left to stand for 24 hours. From the results of variance analysis, it can be seen that the factors affecting the comprehensive score (paste yield and (R, S)-co-isoquinoline transfer rate) are mainly liquid concentration ratio> ethanol concentration> standing time. The liquid concentration ratio has a greater impact on the paste yield and (R, S)-co-isoquinoline transfer rate of Isatis indigotica alcohol precipitation, and the concentration to 1:1 is significantly better.

From the results of variance analysis, it can be seen that within the range of drug solution concentration ratio set in the experiment, the drug solution concentration ratio has no significant effect on the comprehensive score of Isatis root alcohol precipitation, but the trend is obvious, and the drug solution concentration ratio is selected as 1; within the range of ethanol concentration set in the experiment, the extraction time has no significant effect on the comprehensive score of Isatis root alcohol precipitation, but the trend is obvious, and the ethanol concentration (added volume fraction) is selected as 80%; within the range of alcohol precipitation time set in the experiment, the alcohol precipitation time has no significant effect on the comprehensive score of Isatis root alcohol precipitation, and the trend is also the smallest. Considering the cost factor, the alcohol precipitation time is selected as 12h.

Based on the above experimental results and comprehensive actual considerations, the final alcohol precipitation process for Radix Isatidis was determined as follows: the water extract of Radix Isatidis was concentrated to a medicinal solution: medicinal material ratio of 1mL:1g, ethanol was added to make the ethanol volume fraction 80%, and the alcohol precipitation was performed for 12 hours.

3.2 Validation experiment of alcohol precipitation of Radix Isatidis

Take 100g of Radix Isatidis, treat it three times according to the above optimal water extraction and alcohol precipitation process, take the Radix Isatidis extract, and detect the paste rate and (R,S)-gaoichun content.

Table 15 Isatis root alcohol precipitation verification test results

4. Research Summary and Discussion

Based on the above experimental results, the process route of the compound isatis root preparation extraction process, the ethanol concentration and extraction times used in the extraction of isatis leaf, the extraction times used in the water extraction and alcohol precipitation of isatis root, the concentration ratio before alcohol precipitation, etc. will significantly affect the content of active ingredients and the efficacy of the compound isatis root preparation. The optimal process route proposed in this experiment is:

Step 1: According to the weight ratio of the compound isatis root granules, take 2 parts of isatis root, add 6 times of water, heat to reflux or decoct for extraction 3 times, each time for 60 minutes, filter, take the filtrate and concentrate it to a volume of 1mL/g crude drug, add 4 times the volume of ethanol (i.e., alcohol precipitation concentration of 80%) to the liquid, let it stand for 12 hours, filter, take the filtrate, and obtain the isatis root extract.

Step 2: according to the weight ratio of the compound isatis root granules, take 3 parts of isatis indigotica, add 9 times of 45% ethanol aqueous solution, heat and reflux to extract twice, each time for 90 minutes, filter, take the filtrate, and obtain the isatis indigotica extract.

Step 3: Combine and concentrate the above-mentioned Radix Isatidis extract and Folium Isatidis extract to obtain an improved new drug paste of compound Radix Isatidis preparation.

Example 3: Anti-influenza animal experiment

1. Main materials and instruments

1.1 Drugs

Compound Isatis Root Paste 1 was provided by Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd. The preparation method refers to the current standard method for Compound Isatis Root Granules, namely: taking Isatis Root and Folium Isatidis in a mass ratio of 2:3, adding water and decocting twice, each time for 1 hour, filtering, combining the filtrate, concentrating to an appropriate amount, adding three times the amount of ethanol, stirring, standing for 24 hours, filtering, recovering ethanol from the filtrate, and concentrating to a thick paste. Compound Isatis Root Paste 2 was provided by Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd. The preparation method refers to the preparation method of sample (5) in Example 1. The improved compound isatis root paste is provided by Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd. The preparation method refers to the optimal process parameters determined in Example 2, that is: take the isatis root and the isatis leaf medicinal materials with a mass ratio of 2:3, add 9 times the amount of 45% ethanol to the isatis leaf and heat it under reflux for extraction twice, each time for 90 minutes, and combine the filtrates to obtain the isatis leaf extract; add 6 times the amount of water to heat it under reflux for extraction 3 times, each time for 60 minutes, combine the filtrates, concentrate to a medicinal liquid: medicinal material ratio of 1mL:1g, add ethanol to make the ethanol volume fraction 80% (i.e. +4 times the amount), precipitate with alcohol for 12 hours, and filter to obtain the isatis root extract. Combine the isatis root and the isatis leaf extract, concentrate until there is no alcohol taste and it is in a thick paste.

1.2 Cell lines and virus strains

MDCK cells were purchased from ATCC and passaged according to standard cell culture methods. Influenza A virus A/Aichi/2/68 (H3N2) strain was screened by the virus laboratory of Guangzhou Institute of Respiratory Health, Guangzhou Medical University to obtain a mouse lung-adapted strain, and was amplified and preserved according to standard virus culture methods.

1.3 Animals

SPF BALB/c male mice, 8 weeks old, weighing 21±2g, were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. (License No.: SCXK(Zhejiang)2019-0001). All animal experiments were conducted in the Development Zone Laboratory of Guangzhou Analysis and Testing Center, China (negative pressure laboratory, license No.: SYXK(Guangdong)2019-0201), and all operations strictly followed the guidelines of the International Laboratory Animal Assessment and Accreditation Committee. The animal room maintained a temperature of 20-28°C, a relative humidity of 40-70%, and a light of 12h/d. IVC cages, bedding, and feed were sterilized. Mice were adaptively fed for 1 week before the experiment.

1.4 Main reagents and consumables

Picric acid (Sigma-Aldrich, USA), isoflurane (Shenzhen Reward Life Science Co., Ltd.), PBS, DMEM/F-12, trypsin-EDTA (0.05%) (Gibco, USA), etc.

1.5 Main instruments

Cryo-grinding apparatus (JXFSTPRP-CLN-48, Shanghai Jingxin Industrial Development Co., Ltd.), Class II biological safety cabinet (BSC-1000-II-B1, Suzhou Huayu Purification Equipment Co., Ltd.), etc.

2. Main experimental methods

2.1 Animal experiments

Sixty SPF BALB/c male mice were randomly divided into 6 groups, 10 mice in each group, and were adaptively fed for 1 week after picric acid labeling. Starting from 2 days before infection modeling, mice in the blank control group and virus infection model group were gavaged with 200 mL of PBS every day; mice in the compound isatis root paste group 1 were gavaged with 6 mg crude drug/g of compound isatis root paste sample 1; mice in the compound isatis root paste group 2 were gavaged with 6 mg crude drug/g of modified compound isatis root paste sample 2 every day; mice in the high, medium, and low doses of modified compound isatis root paste groups were gavaged with 3 mg crude drug/g, 1.5 mg crude drug/g, and 0.75 mg crude drug/g of modified compound isatis root paste every day. On the day of infection modeling, mice were anesthetized with isoflurane in a biological safety cabinet, and the blank group was given 50 μL of PBS intranasally, and the other groups were infected with 50 μL of 1LD ₅₀ virus solution intranasally. The general condition of the mice was observed every day, and the body weight and survival were recorded. Observe continuously until the 5th day after infection. 5 days after infection, weigh each group of mice and draw blood from the eyeballs. After the blood is drained, the mice are killed by cervical dislocation, the chest cavity of the mice is exposed, the complete lungs of the mice are taken out, and the residual blood is blotted on sterile gauze. The lungs of 4 mice in each group are fixed in 5 volumes of formalin at room temperature for 25 hours. The lungs of 6 mice are weighed, and the lung index is obtained as lung weight/body weight × 100%, and then stored at -80℃ for future use. The dissection process strictly follows the principle of sterility.

2.2 Detection of cytokine expression in mouse lungs (ELISA method)

The supernatant of rat lung homogenate was taken out from -80℃ and thawed on ice. According to the instructions of BCA protein quantitative analysis kit (Cat: NCI3225CH), the corresponding concentration standards were prepared: 2000, 1500, 1000, 750, 500, 250, 125, 25, 0μg/mL, and the determination working solution was prepared according to A solution: B solution = 50:1. The standard and the sample to be tested were added to the 96-well plate at 10μL per well, 2 replicate wells were set, and 200μL of working solution was added to each well, and incubated at 37℃ in the dark for 30min. After taking out the 96-well plate, the absorbance of each well at a wavelength of 562nm was detected with a full-wavelength microplate reader. The standard curve was obtained according to the OD value and concentration of the standard, and the protein concentration of the sample to be tested was calculated.

Dilute the lung supernatant sample to an appropriate concentration according to the concentration range of the standard. Take all reagents out of the refrigerator and return to room temperature. Prepare the washing buffer and standard. Add 50 μL of diluent to each well, then add the blank control, standard and sample to the corresponding wells, cover with a strip and gently shake for 1 minute to mix the liquid, and then incubate at room temperature for 2 hours. Aspirate the liquid in each well, wash each well with 400 μL of washing buffer in an automatic washer 5 times, aspirate the washing buffer in the well after the last time, turn the plate upside down, and wipe the liquid dry. Add 100 μL of coupling solution to each well, cover with a strip and incubate at room temperature for 2 hours. Repeat the washing 5 times, add 100 μL of substrate solution to each well after absorbing the washing solution, and incubate at room temperature for 30 minutes in the dark. Add 100 μL of stop solution to each well, gently shake the plate to mix the liquid thoroughly, and turn from blue to yellow. Measure the absorbance value at OD = 450nm on a full-wavelength fluorescence microplate reader. GraphPad Prism 8 software was used to establish a standard curve using the concentration and OD value of the standard, and the protein concentration of inflammatory factors in each sample was calculated based on the standard curve and the OD value of the sample.

2.3 Pathological sections and HE staining of mouse lung tissue

Fixation: After dissecting the mice and removing the lungs, they were fixed in formalin for 24 hours, and then the solution was changed and fixation continued for another 24 hours. Dehydration: The lung tissue was carefully removed with tweezers and placed in a dehydration box for dehydration with gradient alcohol, 50% alcohol for 90 minutes, 85% alcohol for 90 minutes, 95% alcohol for 90 minutes, 100% alcohol for 60 minutes, and 100% alcohol for 60 minutes. Transparency: The dehydrated lung tissue of the mouse was continued to be placed in a dehydration box and transparentized with gradient xylene, 50% alcohol + 50% xylene for 60 minutes, 100% xylene for 60 minutes, and 100% xylene for 60 minutes. Infiltration: The transparent lung tissue was infiltrated with gradient paraffin, 50% xylene + 50% paraffin for 90 minutes, paraffin for 120 minutes, and paraffin for 120 minutes at 62°C. Note that the paraffin penetration temperature in the last step should be lower than 65°C, and the time should be calculated after the paraffin is completely dissolved; embedding: embed the paraffin-penetrated lung tissue in melted wax, then quickly cool it at -20°C, and trim the wax block; sectioning: place the trimmed wax block on a slicer, cut out thin slices with a thickness of about 5 μm, float and flatten them in warm water at about 40°C, take out the thin slices with a slide, dry them at 60°C for 10 hours, and store them at room temperature; dewaxing and rehydration: dewax and rehydrate the blank slices in the order of xylene-alcohol-water, xylene 20 minutes, xylene 20 minutes, alcohol 10 minutes, alcohol 10 minutes, 95% alcohol 5 minutes, 90% alcohol 5 minutes, 80% alcohol 5 minutes, 70% alcohol 5 minutes, and wash with distilled water for 2 minutes; hematoxylin-eosin (HE) staining: stain with hematoxylin first, then with eosin. Hematoxylin for 5 min, distilled water for 10 s, 1% hydrochloric acid alcohol for 3 s, running water for blueing for 10 min, eosin for 2 min, distilled water for 20 s; dehydration and sealing: 75% ethanol for 5 min, 85% ethanol for 5 min, 95% ethanol for 5 min, anhydrous ethanol for 5 min, anhydrous ethanol for 5 min, xylene for 1 min, xylene for 1 min, dry, and seal with neutral gum; observe under a microscope and take pictures.

2.4 Data Processing

Data were statistically analyzed using GraphPad Prism 8. The test results of different groups were processed using Graphpad software and expressed as mean ± SEM, and the Tukey t test method was used to test the differences between groups.

3. Main results

3.1 Lung Index

After mice were infected with influenza virus and suffered pneumonia damage, although their weight decreased, their lung weight increased due to factors such as inflammatory congestion and edema, and substantial lesion damage. Therefore, the lung index can be used to evaluate the lung damage caused by influenza virus infection in mice. As shown in Figure 8, the lung index of mice in the virus infection model was significantly higher than that of the blank model group; after intervention with a high dose (3 mg crude drug/g) of the modified new drug flow paste of compound isatis root granules, the lung index of mice was significantly lower than that of the virus model group, indicating that the improved process of compound isatis root can improve the lung damage of mice caused by virus infection, and the lung index level is significantly lower than that of compound isatis root flow paste 1 and compound isatis root flow paste 2 groups (P < 0.05); however, the current compound isatis root granule flow paste sample and compound isatis root flow paste 2 sample had no significant improvement effect on the increase of the lung index of mice caused by virus infection at a dose of 6 mg crude drug/g.

3.2 Pulmonary inflammatory factors

As shown in Figure 9, during the process of viral infection-induced pneumonia, multiple cytokines (IL-6, IL-8, TNF-α) in the lungs of model mice were significantly upregulated; intervention with different doses of the modified new drug compound isatis granules paste had a significant inhibitory effect on the overexpression of IL-6, IL-8, and TNF-α in the lungs, indicating that the modified new drug compound isatis granules has a significant inhibitory effect on the excessive inflammatory response of the lungs in the influenza pneumonia model; in particular, the regulatory effect of the modified new drug compound isatis granules paste at a dose of 3 mg crude drug/kg on the three inflammatory cytokines was significantly better than that of the compound isatis paste 1 sample and the compound isatis paste 2 sample at a dose of 6 mg crude drug/kg, showing the advantage of the best efficacy strength.

3.3 Pulmonary pathology

Pathological sections of lung tissue are important indicators for evaluating lung injury. As shown in Figure 10, after influenza virus infection, the alveoli of mice atrophied extensively, the alveolar walls thickened and expanded, and a large number of inflammatory cells infiltrated, the pulmonary interstitial blood vessels dilated and congested, and some alveolar cavities had inflammatory cell infiltration and tissue fluid infiltration, showing early manifestations of acute lung injury. Compared with the virus model group, the high-dose compound isatis root granule improved new drug flow paste group showed significant improvement in alveolar atrophy, a smaller range of inflammatory cell infiltration, a reduced degree, and a significant improvement in lung injury. Other dose groups also had a certain improvement effect; at the same time, the intervention of compound isatis root flow paste sample 1 and compound isatis root flow paste sample 2 at a dose of 6 mg crude drug/kg had a relatively weak effect on the pathological improvement of lung injury.

Influenza virus and other pathogens infection is one of the main causes of pneumonia and damage to lung tissue, which is specifically manifested in animal models as pulmonary congestion and edema, pathological changes in the lungs, excessive cytokine secretion and other characteristics. The experimental results of influenza virus-induced pneumonia animal model show that the improved new drug new paste of compound isatis root can significantly reduce pulmonary congestion and edema, inhibit alveolar shrinkage and atrophy and alveolar wall thickening and expansion, reduce inflammatory cell infiltration and cytokine expression in lung tissue, and its efficacy is significantly better than the existing process paste of compound isatis root preparation and the compound isatis root paste extracted from isatis indigotica with high concentration (70%) ethanol at a lower raw drug dose. In addition, our research group also found in the high-dose influenza virus-induced mouse death model experiment that the improved new drug flow paste of compound isatis root preparation has a significant death protection effect, while the current standard compound isatis root preparation and the compound isatis root flow paste extracted from isatis leaf with high concentration (70%) ethanol have no significant death protection effect. The above results show that the improved new drug flow paste of compound isatis root preparation prepared by the method of the present invention has a stronger anti-influenza effect than the current standard compound isatis root granules and some existing process compound isatis root flow pastes, which can significantly reduce excessive inflammation and tissue damage caused by influenza virus and reduce the probability of influenza aggravation. Although there are a large number of existing extraction methods for compound isatis preparations, we unexpectedly found that the extraction methods of isatis root and isatis leaf, including the process route, the appropriate changes in process parameters, have an effect on the content of effective ingredients in the prepared medicine, the rate of ointment and the intensity of influenza efficacy, and have found a more effective method for preventing and treating influenza, and preventing and treating the development of severe symptoms after influenza virus infection.

Example 4: New drug preparation prepared from improved compound Isatis indigotica ointment

1. Granules

According to the extraction method determined in Example 2, a compound isatis root granule improved new drug paste of a certain relative density is prepared; a spray dryer is used to dry to obtain a compound isatis root granule improved new drug spray-dried powder; magnesium stearate, silicon dioxide, and stevioside are added according to the formula amount, and dextrin is added to make up the formula amount, mixed for 20 minutes, and mixed to obtain a compound isatis root granule improved new drug total mixture; the compound isatis root granule improved new drug total mixture is taken, granulated by a dry granulator, the granules are sieved with 14 and 30 meshes, and the coarse particles and fine powders are re-granulated, and finally particles with a particle size in the range of 14-30 mesh and uniform color are obtained to obtain a compound isatis root granule improved new drug granule.

2. Tablets

According to the extraction method determined in Example 2, a compound isatis root granule improved new drug paste with a certain relative density is prepared; a spray dryer is used to dry the compound isatis root granule improved new drug spray-dried powder; the compound isatis root granule improved new drug spray-dried powder is taken, an appropriate amount of auxiliary materials are added, mixed, made into granules, pressed into tablets, and film-coated to obtain compound isatis root granule improved new drug tablets.

3. Capsules

According to the extraction method determined in Example 2, a compound isatis root granule improved new drug paste with a certain relative density is prepared; a spray dryer is used to dry the compound isatis root granule improved new drug spray-dried powder; the compound isatis root granule improved new drug spray-dried powder is taken, an appropriate amount of auxiliary materials is added, mixed, made into granules, and loaded into capsules to obtain compound isatis root granule improved new drug capsules.

4. Decoction

According to the extraction method determined in Example 2, a compound isatis root granule improved new drug paste with a certain relative density is prepared, and an appropriate amount of refined honey or invert sugar made from sucrose and other auxiliary materials are added to the paste, mixed, and continued to concentrate to a certain relative density to obtain a compound isatis root granule improved new drug decoction.

The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims (6)

1. A method for preparing a compound Radix Isatidis preparation, wherein the active ingredient raw materials of the compound Radix Isatidis preparation are Radix Isatidis and Folium Isatidis in a mass ratio of 2:3, characterized in that the method comprises the following steps: SA. Take the isatis root, add 6 times water, heat reflux or decoction extraction 3 times, each time for 60 minutes, filter, take the filtrate and concentrate it to a volume of 1mL / g crude drug, add 4 times the volume of ethanol to the liquid, let stand for 12h, filter, take the filtrate to obtain an isatis root extract; SB. Take the isatis leaf, add 9 times of 45% ethanol aqueous solution, heat and reflux extraction twice, each time for 90 minutes, filter, and take the filtrate to obtain an isatis leaf extract; SC. The above Radix Isatidis extract and Folium Isatidis extract are combined and concentrated to obtain a fluid paste, and the fluid paste is mixed with pharmaceutically acceptable excipients to prepare a preparation. 2. The compound Radix Isatidis preparation obtained by the preparation method according to claim 1, wherein the extraction rate of isovitexin in the Folium Isatidis extract during the preparation of the compound Radix Isatidis preparation is not less than 1.10 mg/g Folium Isatidis original medicinal material. 3. The compound Radix Isatidis preparation according to claim 2, characterized in that the preparation has a dosage form It is in the form of granules, capsules, paste or tablets. 4. Use of the compound Radix Isatidis preparation according to any one of claims 2 to 3 in the preparation of a medicament for preventing and treating influenza. 5. The use according to claim 4, characterized in that the use includes reducing or preventing the development of severe illness after influenza virus infection. 6. The use according to claim 5, characterized in that the use comprises reducing lung tissue damage and the expression level of lung inflammatory factors caused by influenza virus infection.