CN113171412A

CN113171412A - Callicarpa nudiflora traditional Chinese medicine composition and preparation method thereof

Info

Publication number: CN113171412A
Application number: CN202110551593.0A
Authority: CN
Inventors: 刘洋洋; 唐菲
Original assignee: Hainan Chongyuan Drug Development Co ltd
Current assignee: Hainan Chongyuan Drug Development Co ltd
Priority date: 2021-05-20
Filing date: 2021-05-20
Publication date: 2021-07-27

Abstract

The invention discloses a preparation method of a callicarpa-nauclea traditional Chinese medicine composition, which comprises the steps of mixing callicarpa nudiflora and nauclea traditional Chinese medicine raw materials to form a traditional Chinese medicine mixture, adding water with the mass being 20 times of that of the traditional Chinese medicine mixture into the traditional Chinese medicine mixture, heating, refluxing and extracting for 3 times, wherein the heating, refluxing and extracting time is 0.5h each time, combining 3 times of traditional Chinese medicine extracting solutions, filtering the traditional Chinese medicine extracting solutions, heating, concentrating and drying to obtain callicarpa nauclea traditional Chinese medicine extract powder; mixing the obtained traditional Chinese medicine extract powder of the callicarpa bodinieri and auxiliary materials according to the mass ratio of 1:1, and filling to obtain the traditional Chinese medicine composition of the callicarpa bodinieri. The preparation method of the callicarpa bodinieri traditional Chinese medicine composition provided by the invention is simple in preparation process, the prepared capsule is stable in quality, and the obtained callicarpa bodinieri traditional Chinese medicine composition has the effects of diminishing inflammation, detoxifying, astringing and stopping bleeding. Can be used for treating respiratory tract inflammation, acute infectious hepatitis, and hemorrhage of respiratory tract and digestive tract.

Description

Callicarpa nudiflora traditional Chinese medicine composition and preparation method thereof

Technical Field

The invention relates to the technical field of medicine preparation, and particularly relates to a callicarpa-cholangiopsis traditional Chinese medicine composition and a preparation method thereof.

Background

The Callicarpa nudiflora medicinal material is dried Callicarpa nudiflora (Callicarpa) of Callicarpa of Verbenaceae, also named as caulis seu radix Nothopanacis Delavayi, red nodulation and the like, and is distributed in Guangdong, Hainan and overseas Malaysia of China and the like. According to the record of 'Chinese pharmacopoeia' 2020 edition, the medicinal material Gymnothorax kidaku has the effects of diminishing inflammation, relieving swelling and toxin, eliminating dampness and turbidity, stopping bleeding and the like. The Chinese medicinal composition is clinically used for treating inflammatory pyogenic infections, acute infectious hepatitis and internal and external injury bleeding caused by bacterial infection. Research reports record that the chemical components mainly contained in the callicarpa nudiflora are flavonoids, phenylpropanoids and terpenoids. In addition, the Chinese patent medicine nakedflower beautyberry tablet extracted and processed by taking nakedflower beautyberry as a main raw material has been clinically applied for many years, is used for treating inflammation, acute infectious hepatitis, respiratory tract and digestive tract bleeding and the like caused by bacterial infection, and has good effect.

Nauclea officinalis (Nauclea officinalis Pierre ex Pitard.) is a plant of India madder, is also called as a bear gall tree, a yellow gall wood, a bear gall, a tree phellodendron bark and the like in China, is mainly produced in places such as Guangdong, Guangxi, Hainan and the like in China, is only accepted by the medicinal material standard of Hainan province at present, has a long medicinal history among Li nationalities, can be used for treating various diseases, has particularly obvious effects of resisting bacteria, diminishing inflammation and the like, is widely developed into various Chinese patent medicines such as a gall-wood extract tablet and the like, has a name of 'green antibiotic', and has wide research on chemical components, pharmacological actions and the like of leaves, stems, barks, roots and the like of the gall-wood in recent years due to the obvious medicinal value.

At present, the best extraction process of the two traditional Chinese medicine raw materials and the effect of the capsule prepared by the extract of the callicarpa bodinieri are not reported.

Disclosure of Invention

Therefore, the invention provides a preparation method of a callicarpa-cholangiopsis traditional Chinese medicine composition.

In order to achieve the above purpose, the invention provides the following technical scheme:

the embodiment of the invention provides a preparation method of a callicarpa bodinieri traditional Chinese medicine composition, which comprises the following steps

Mixing traditional Chinese medicine raw materials of beautyberry and nauclea officinalis to form a traditional Chinese medicine mixture, adding water with the mass being 20 times of that of the traditional Chinese medicine mixture into the traditional Chinese medicine mixture, performing reflux extraction for 3 times, wherein the reflux time is 0.5h each time, combining 3 times of traditional Chinese medicine extracting solutions, filtering the traditional Chinese medicine extracting solutions, heating, concentrating and drying to obtain traditional Chinese medicine extract powder of beautyberry and nauclea officinalis;

mixing the obtained traditional Chinese medicine extract powder of the callicarpa bodinieri and auxiliary materials according to the mass ratio of 1:1, and filling to obtain the traditional Chinese medicine composition of the callicarpa bodinieri.

In one embodiment of the invention, the mass ratio of the callicarpa nudiflora and the nauclea officinalis is 4: 1.

In one embodiment of the invention, the nauclea officinalis extracting solution at least comprises the following active ingredients:

in the extracting solution, active ingredients derived from callicarpa nudiflora: phenylethanoid glycosides, flavonoids, iridoids;

in the extracting solution, active ingredients derived from the nauclea officinalis: phenolic acid compounds, indole alkaloids and quinolinone alkaloids.

In one embodiment of the invention, the phenylethanoid glycosides compounds include forsythoside E and cistanoside F.

In an embodiment of the present invention, the iridoid compound includes geniposide.

In one embodiment of the invention, the extract yield is 32.2%.

In one embodiment of the invention, in the callicarpa bodinieri traditional Chinese medicine composition, verbascoside is not less than 7.6mg, and strictosamide is not less than 0.47 mg.

In one embodiment of the invention, each traditional Chinese medicine composition of callicarpa bodinieri contains 0.39g of traditional Chinese medicine extract powder of callicarpa bodinieri.

In one embodiment of the invention, the callicarpa bodinieri traditional Chinese medicine extract powder is mixed with auxiliary materials, passes through a 80-mesh sieve and is filled into capsules.

In one embodiment of the invention, the humidity of the process environment for preparing the callicarpa bodinieri traditional Chinese medicine composition is less than 70%.

The invention has the following advantages:

the invention provides a preparation method of a callicarpa-cholangiopsis traditional Chinese medicine composition, the preparation process is simple, the quality of the prepared capsule is stable, and the obtained callicarpa-cholangiopsis traditional Chinese medicine composition has the effects of diminishing inflammation, detoxifying, astringing and stopping bleeding. Can be used for treating respiratory tract inflammation, acute infectious hepatitis, and hemorrhage of respiratory tract and digestive tract.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

FIG. 1 is a graph illustrating the results of a critical RH test provided by an embodiment of the present invention;

FIG. 2 is a photograph showing the content of the Chinese medicinal extract powder of the nauclea officinalis Chinese medicinal composition provided by the embodiment of the present invention;

FIG. 3 is a pathological observation diagram of H & E stained lung tissue of a mouse provided by an embodiment of the present invention, wherein A is a blank control; b is a model group; c is a control group; d is an administration group;

fig. 4 is a graph of the difference in edema of lung tissues measured by the wet/dry weight method of mice provided by the example of the present invention, wherein the difference is significant (P <0.05) and the difference is very significant (P <0.01) compared with the blank; compared with the model group, the # difference is obvious (P is less than 0.05), and the # difference is extremely obvious (P is less than 0.01), and the difference of the pulmonary tissue edema is detected by a mouse wet/dry weight method;

FIGS. 5-7 are graphs of the detection of inflammatory factors in mice provided by the present invention;

FIG. 8 shows a negative ion mode TIC of a sample of an aqueous extract of a nauclea officinalis traditional Chinese medicine composition provided by the embodiment of the present invention;

fig. 9 shows a sample positive ion mode TIC of an aqueous extract of a callicarpa bodinieri traditional Chinese medicine composition provided by an embodiment of the invention;

FIG. 10 is a forsythoside E secondary mass spectrum provided in the examples herein;

FIG. 11 is a cistanche deserticola F secondary mass spectrum provided by an embodiment of the present invention;

FIG. 12 is a second-order mass spectrum of genipin nucleotide provided by the embodiment of the invention.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation Process of Artocarpus communis traditional Chinese medicine extract powder

The embodiment provides a preparation method of a callicarpa bodinieri traditional Chinese medicine composition, which comprises the following steps:

in the embodiment, a water reflux extraction method is selected, water addition amount (A), extraction time (B) and extraction times (C) are taken as investigation factors, each factor is selected to be 3 levels, the yield of the traditional Chinese medicine extract is taken as an investigation index, the extraction method of the callicarpa bodinieri extract is optimized, and the factor levels are shown in table 1.

TABLE 1

Weighing callicarpa nudiflora powder which is sieved by a No. 4 sieve, accurately weighing 5g and 9 parts in total according to a ratio of 4:1, numbering test groups No. 1-9 in sequence, heating, refluxing, extracting and filtering according to conditions specified in Table 1, combining filtrates, heating and concentrating, placing a concentrated solution into an evaporation dish which is dried to constant weight, drying the concentrated solution on a water bath, placing the dried solution into an oven at 105 ℃ for drying for 3h, quickly transferring the dried solution into a dryer for cooling for 0.5h to obtain callicarpa nudiflora traditional Chinese medicine extract powder, quickly and accurately weighing the mass until the weight is constant, and calculating the yield of the traditional Chinese medicine extract. The formula for calculating the extract yield is shown in formula (1).

Extract yield ═ extract weight (g))/(crude drug (g)) × 100% (1)

The Chinese medicine extractum rate of the callicarpa bodinieri is shown in the orthogonal experimental design and results of the extraction process in the table 2 and the variance analysis results in the table 3.

TABLE 2

TABLE 3

By comparing the mean deviation of the factors in table 2, the factor C (the number of times of extraction) has the greatest influence on the test results, and the factor a (the amount of the water added) and the factor B (the extraction time) are the next factor, so that the influence of the factors on the extract extraction yield is ranked as C > a > B. As shown in the results of the ANOVA in Table 3, the factor C has significant difference (p is less than 0.05), and the factors A and B have no obvious influence on the test results, so that the optimal process for extracting the water of the callicarpa bodinieri extractum is A3B1C3, namely the water adding amount of the medicinal materials is 20 times, and the extraction is carried out for 3 times, and each time is 0.5 hour, according to the mean value of the factors and the aspects of practical production and production time saving.

Example 2 preparation of a Callicarpa Nudiflora traditional Chinese medicine composition

1. Taking 3 parts of callicarpa nudiflora and nauclea officinalis, weighing 10g of callicarpa bodinieri and nauclea officinalis in a ratio of 4:1, verifying the water extraction process of the medicinal materials according to the optimal process conditions determined in the example 1, and determining results are shown in an orthogonal test verification experiment result of a traditional Chinese medicine extracting solution in a table 4. The result shows that the yield of the callicarpa bodinieri extractum extracted by the determined optimal scheme is higher, and the scheme is more stable.

TABLE 4

As can be seen from Table 4, the best method determined in example 1 has a high yield of Callicarpa nudiflora-Nauclea officinalis extractum, the method is stable, and the process is reasonable and feasible.

2. Method for extracting nauclea nudiflora extract

Taking Callicarpa nudiflora and Nauclea officinalis powder which are sieved by a No. 4 sieve, accurately weighing 1180g of medicinal material powder according to a ratio of 4:1, extracting according to an optimal extraction process preferably selected in an orthogonal experiment shown in Table 2 of example 1, filtering an extracting solution, combining filtrates, heating and concentrating, placing a concentrated solution into an evaporation dish which is dried to constant weight, drying the concentrated solution in a drying oven at 105 ℃ for 3 hours after drying by distillation on a water bath, quickly transferring the dried solution into a dryer for cooling for 0.5 hour, and quickly and accurately weighing the mass until the constant weight is achieved to obtain the traditional Chinese medicine extract of the Callicarpa nudiflora.

3. Preparation of traditional Chinese medicine composition of callicarpa bodinieri and nauclea officinalis

Pulverizing dry traditional Chinese medicine extract of nauclea nudiflora, sieving dry traditional Chinese medicine extract powder of nauclea nudiflora, mixing with starch at a ratio of 1:1, and manually granulating with 400-granule capsule filling plate.

4. Selection of formulation of nakedflower beautyberry and nakedflower gall traditional Chinese medicine composition

The folk proved recipe of the callicarpa bodinieri decoction has the disadvantages of troublesome decoction, low efficiency, inconvenient carrying and the like, and limits the development of the callicarpa bodinieri decoction to a certain extent. Compared with granules, tablets and the like, the capsule has the advantages of less auxiliary material consumption, convenient carrying, convenient taking, high bioavailability and the like. Although the callicarpa bodinieri traditional Chinese medicine extract is easy to absorb moisture, the problem can be solved by adding a proper amount of auxiliary materials for filling, so that the preparation form of the embodiment is finally determined to be a capsule.

5. Determination and performance investigation of traditional Chinese medicine composition auxiliary materials of callicarpa nudiflora

Determination of auxiliary materials: the traditional Chinese medicine extract has strong hygroscopicity, so that the investigation on the hygroscopicity resistance and the flowability of the medicinal powder is particularly critical when the capsule is prepared.

Determination of auxiliary materials: when the callicarpa bodinieri extract powder is mixed with the auxiliary material starch in a ratio of 1:1, the mixture can be completely sieved, is loose and free of lumps and is suitable for being filled into capsules.

(1) Powder flowability investigation

Mixing the traditional Chinese medicine extract powder of the callicarpa bodinieri and the auxiliary material starch, sieving by a 80-mesh sieve, inspecting the repose angle of the medicine powder by a fixed funnel method, and measuring for 3 times on average. When the angle of repose alpha is less than 30 degrees, the flow state of the medicinal powder is good; the flow state of the medicine powder is poor when the angle of repose alpha is more than 40 degrees; when the angle of repose is 30 degrees < alpha <40 degrees, the flow state of the medicinal powder is better. As shown in Table 5, the results of the angle of repose test are calculated to show that the three angles of repose are all less than 30 degrees, which proves that the powder has good fluidity.

TABLE 5

(2) Investigation of critical relative humidity

Making a moisture absorption curve: weighing 1.5g of each of 3 parts of the callicarpa bodinieri traditional Chinese medicine composition, respectively spreading the weighed materials in flat weighing bottles which are dried to constant weight, opening bottle caps, placing the flat weighing bottles in a glass dryer containing saturated sodium chloride solution (with the relative humidity of 75%), storing the flat weighing bottles in a constant temperature environment at 25 ℃, taking out the flat weighing bottles after 2h, 4h, 8h, 12h, 24h, 30h, 36h, 48h, 60h and 72h respectively, precisely weighing, calculating the moisture absorption percentage, drawing by taking time as an abscissa and moisture absorption rate as an ordinate to obtain a moisture absorption curve, and finding out the corresponding time (h) when the moisture absorption is balanced. The results of the moisture absorption rate test are shown in Table 6. The moisture absorption balance time of the callicarpa bodinieri traditional Chinese medicine composition is 60 hours, and the callicarpa bodinieri traditional Chinese medicine composition is placed for 72 hours in order to ensure a better moisture absorption balance effect.

TABLE 6

(3) Investigation of determination of Critical Relative Humidity (CRH)

Weighing 7 parts of the content of the callicarpa bodinieri traditional Chinese medicine composition, wherein each part is 1.5g, placing the content in glass dryers in different humidity environments respectively, storing the content in a constant temperature environment of 25 ℃ for three days, and calculating the moisture absorption percentage by taking the moisture absorption percentage as a Y axis. Taking different humidity as X axis as hygroscopic balance curve, making two tangent lines at inflection point, and obtaining CRH as the corresponding abscissa of tangent line focus. As shown in fig. 1, the critical relative humidity of the callicarpa bodinieri traditional Chinese medicine composition is 73%, so the relative humidity of the environment should be controlled below 70% in the preparation process.

6. Preparation result of traditional Chinese medicine composition of callicarpa bodinieri and nauclea officinalis

Mixing with appropriate amount of starch and dry extract powder of nakedflower beautyberry and Nakedflower lignum according to a ratio of 1:1, and filling the mixture with 400 granules of artificial capsules to obtain the nakedflower beautyberry and Nakedflower lignum traditional Chinese medicine composition. As shown in fig. 2, the content of the traditional Chinese medicine extract powder of the callicarpa bodinieri traditional Chinese medicine composition prepared in this embodiment is shown.

Example 3 preparation of Callicarpa Nudiflora traditional Chinese medicine capsules

1. Taking 472g of beautyberry medicinal material and 118g of nauclea officinalis medicinal material which are screened by a No. 4 screen, adding 20 times of water, extracting for 3 times, 0.5 hour each time, combining extracting solutions, filtering, heating, concentrating and drying to obtain 195g of beautyberry and nauclea officinalis dry extract. Pulverizing the above dry extract, sieving, and making into capsule.

The callicarpa bodinieri traditional Chinese medicine capsule is a hard capsule, and the content is tan powder, and the capsule is slightly fragrant in smell, slightly sweet in taste and slightly bitter in taste.

2. Taking 0.1g of the content of the callicarpa bodinieri and nauclea officinalis traditional Chinese medicine capsule, putting the content into a 1ml volumetric flask, adding 65% methanol to the volume of 1ml, and carrying out ultrasonic treatment for 30 min. Cool down and make up with 65% methanol. Shaking, filtering, and collecting the filtrate. Taking verbascoside and strictosamide reference substances, preparing reference substance solutions according to the method, respectively dropping on the same 0.5% NaOH silica gel G thin layer plate, adding chloroform: ethyl acetate: methanol: developing with acetic acid (9:8.6:3:0.8) as developing agent, taking out, air drying, spraying with 3% aluminum trichloride ethanol solution, heating at 105 deg.C for 5min, and inspecting under ultraviolet light (365 nm). The test chromatogram shows fluorescent spots of the same color at the positions corresponding to those of the control chromatogram. The callicarpa bodinieri traditional Chinese medicine capsule prepared in the embodiment meets all relevant regulations under the capsule item (general rule 0103).

3. Measuring by high performance liquid chromatography (0512).

Chromatographic conditions and system applicability test, using octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A, and 0.1% formic acid solution as mobile phase B, and performing gradient elution according to the specification in the following table; the column temperature was 28 ℃; the detection wavelength was 258 nm. The number of theoretical plates should not be less than 5000, calculated as acteoside peak and vincoside lactam.

Preparation of a reference solution, preparation of a verbascoside reference solution: precisely weighing herba Verbasci Thapsi glycoside reference substance, adding 50% ethanol to obtain standard solution containing 1.135mg of herba Verbasci Thapsi glycoside per 1 ml.

Preparation of strictosamide reference solution: accurately weighing strictosamide reference substance, adding 50% ethanol to obtain strictosamide standard solution containing 0.438mg of strictosamide per 1 ml.

Preparation of a test solution: grinding the contents of the product with different contents, precisely weighing about 0.1g, placing in a 50ml volumetric flask, adding 50% ethanol to constant volume to 50ml, weighing, and performing ultrasonic treatment for 30 min. Cooling, weighing, and adding 50% ethanol. Shaking, filtering, and collecting the filtrate to obtain the sample solution.

Measuring by accurately sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

Each traditional Chinese medicine capsule containing callicarpa nudiflora prepared by the implementation method is not less than 7.6mg calculated by verbascoside (C29H36O15) and not less than 0.47mg calculated by strictosamide (C26H30N2O 8).

The prepared beautyberry and nauclea officinalis traditional Chinese medicine capsule has the functions and main treatment: diminish inflammation, remove toxicity, astringe and stop bleeding. Can be used for treating airway inflammation, acute infectious hepatitis, and hemorrhage of airway and digestive tract.

The usage and dosage of the callicarpa bodinieri traditional Chinese medicine capsule prepared in the embodiment are as follows: is administered orally. 2-3 granules at a time, 3-4 times a day. Specification: each capsule contains 0.39g (each capsule contains 0.39g of dry extract). And (3) storage: drying and sealing.

Experimental example, anti-inflammatory action of beautyberry and nauclea officinalis traditional Chinese medicine composition

1. Experimental animals: healthy male Kunming mice, 4-5 weeks old, with a weight of 18-20g, were purchased from the center of the laboratory animals of the Hubei pharmaceutical institute and kept in a clean laboratory. After adaptive feeding for 3-5d, random grouping administration experiments are carried out.

2. Experimental materials and reagents: LPS (batch number: L-2880) Sigma, USA; dexamethasone (batch No. D4902-25MG) Sigma USA; chloral hydrate (batch No. 302-17-0), Shanghai-derived PhylloBiotech Co., Ltd; ELISA kit (batch number: H417) was built into bioengineering institute; methanol (batch No. 32058), science of west longe; anhydrous ethanol (batch No. 32061) science of West Longsu; xylene (batch number: 2020112223), Guangzhou chemical industries; 4% paraformaldehyde (batch No. P0099-500ml), Biyuntian Biotech Co., Ltd; physiological saline (batch: R21479), Shanghai-derived leaf Biotech Co., Ltd.; hematoxylin stain (G1004-100), Wuhan Severe Biotech, Inc.; eosin dye liquor (G1001-100), Wuhan Severe Biotech, Inc.; the callicarpa bodinieri traditional Chinese medicine composition prepared in example 1.

3. Experimental method, mice random grouping and administration

40 mice were randomly divided into 4 groups, which were a blank control group (normal saline), a model control group (normal saline), a positive control group (dexamethasone 3mg/kg), and the nauclea officinalis traditional Chinese medicine composition group of example 2 (400mg/kg), each group containing 10 mice. The composition group of the callicarpa bodinieri and the nauclea officinalis is administrated for 1 time/d and 20ml/kg by gastric gavage, the positive control group (dexamethasone) is administrated for 1 time/d and 10ml/kg by intraperitoneal injection and is continuously administrated for 7d, and the normal control group and the model group are administrated with corresponding amount of physiological saline by gastric gavage. After 1h of the last administration, the mice except the blank control group are respectively dripped with 10mg/kg of nasal LPS and 25 mul/mouse, and the mice are induced with acute lung injury. After 2h of nasal drip LPS modeling, the eyeballs are respectively picked and blood is taken, then the mice are sacrificed, the right upper lung is taken, the redundant tissues are removed, and the mice are placed in 4 percent paraformaldehyde solution for pathological section observation; the wet/dry weight ratio of the lung was measured by taking the lower right lung tissue.

4. Pathological observation of H & E stained lung tissue in mice

Taking lung tissue in 4% formaldehyde solution, trimming the tissue, placing in an embedding box, washing the stationary liquid with running water, respectively placing in ethanol with different concentrations for dehydration, placing the tissue block in xylene solvent after dehydration, preparing lung tissue paraffin block, slicing to 5 μm, HE staining, and sealing. The sections were observed under a microscope and photographed to record lung morphology and inflammatory lesions. As shown in FIG. 3, the pathological observation of H & E stained lung tissue in mice can reflect the severity of acute lung injury induced by LPS and the anti-inflammatory effect of drugs on the acute lung injury. Observation under a light microscope shows that the lung tissue structure of the blank control group is normal, the alveolar cavities are clear, no obvious inflammatory cells and red blood cells infiltrate into the cavities, and the alveolar spaces are fine and have complete structures; compared with a blank control group, the pulmonary alveolus shape of the model group is obviously changed, partial pulmonary alveolus is broken, the pulmonary alveolus structure is damaged, the alveolar wall is thickened, and obvious inflammatory cell infiltration is achieved, so that the establishment of the acute pneumonia mouse model is proved to be successful. Compared with the model group, the lung interval of the control group is obviously thinned, inflammatory cells and red blood cells in the alveolar space and the pulmonary interstitium are obviously reduced, and the alveolar structure is complete. The results of the experimental group are consistent with those of the control group, the pathological changes are obviously reduced compared with the model group, the infiltration of inflammatory cells is reduced, the lung interval is obviously thinned, and the alveoli are relatively complete, which shows that the callicarpa bodinieri traditional Chinese medicine composition can improve acute mouse lung injury induced by LPS.

5. Method for detecting pulmonary edema difference by mouse wet/dry weight method

Taking the right lower lung tissue of the mouse, removing dry surface water, placing the mouse in a clean glass ware, accurately weighing the mouse by an electronic balance, placing the mouse in a constant-temperature oven at 80 ℃ for drying for 24 hours, then weighing the mouse, and calculating the ratio of the right lower lung tissue to the dry lower lung tissue, namely the lung wet/dry weight ratio (W/D). As shown in FIG. 4, the difference of edema of lung tissue was measured by the wet/dry weight method of mice, and W/D of the model group mice was significantly increased (P <0.05) compared to the blank group. Compared with a model group, the W/D of a mouse is remarkably reduced (P is less than 0.05) by administrating the corresponding dose of the callicarpa bodinieri traditional Chinese medicine composition through intragastric administration, and the result shows that the pulmonis edema caused by acute lung injury can be remarkably relieved by administrating the callicarpa bodinieri traditional Chinese medicine composition through intragastric administration, which shows that the callicarpa bodinieri traditional Chinese medicine composition has a certain protection effect on the lung tissue of the mouse and is consistent with the result of a dexamethasone group.

6. Mouse inflammatory factor detection

Serum stored after centrifugation (3000r/10min) is respectively subjected to ELISA kit detection according to the method of the kit specification, the levels of TNF-alpha, IL-1 beta and IL-6 are detected, the OD value is measured by an enzyme-linked immunosorbent assay, and the level of inflammatory cytokines is calculated according to the specification. As shown in FIGS. 5-7, the data obtained from the mouse inflammatory factor test were averaged over 5 replicates, the intra-group comparisons were performed using one-way anova, the comparisons between the two groups were performed using t-test, and differences of P <0.05 or P <0.01 were statistically significant and plotted using Origin software. As shown in fig. 5-7, the difference was significant (P <0.05), and the difference was very significant (P <0.01), compared to the blank; compared with the model group, the # difference is significant (P <0.05), and the # difference is extremely significant (P < 0.01). Compared with the blank group, the mass concentrations of TNF-alpha, IL-1 beta and IL-6 in the model group are all obviously higher than that in the blank group, and LPS can obviously increase the activities of proinflammatory factors of TNF-alpha, IL-1 beta and IL-6(P is less than 0.01). Compared with a model group, the callicarpa bodinieri traditional Chinese medicine composition group and the dexamethasone group can obviously reduce the expression of TNF-alpha (P <0.01), IL-1 beta (P <0.01) and IL-6(P <0.05), which shows that the callicarpa bodinieri traditional Chinese medicine composition has a certain anti-inflammatory effect on acute lung injury of mice induced by LPS. The research results show that the callicarpa bodinieri traditional Chinese medicine composition can improve the inflammatory reaction of lung tissues by regulating the expression of proinflammatory factors and anti-inflammatory factors, and the results are consistent with the results of a dexamethasone group.

In the embodiment, a mouse model of acute lung injury is prepared after nasal cavity instillation of LPS, and the anti-inflammatory effect of the callicarpa bodinieri traditional Chinese medicine composition is researched. In the acute lung injury research of mice, IL-6 in the lung has obvious proinflammatory characteristics, TNF-alpha and IL-1 beta are considered to be one of the most important proinflammatory cytokines for causing ALI, so the proinflammatory cytokines TNF-alpha, IL-1 beta and IL-6 are selected as research indexes in the experiment. The research result shows that the stimulation of LPS obviously increases the expression of proinflammatory factors TNF-alpha, IL-1 beta and IL-6, meanwhile, the H & E stained lung tissue of a mouse is pathologically observed that the shape of alveolus is changed, part of alveolus is broken, the structure of the alveolus is destroyed, the alveolar wall is thickened, and obvious inflammatory cell infiltration is realized; the W/D of the mouse lung is obviously lower, and the expression content of proinflammatory factors such as TNF-alpha, IL-1 beta, IL-6 and the like is obviously lower than that of an LPS model group. Therefore, the experimental result shows that the callicarpa bodinieri traditional Chinese medicine composition can play an anti-inflammatory role by reducing the expression of proinflammatory factors such as TNF-alpha, IL-1 beta, IL-6 and the like induced by LPS, relieving lung edema induced by LPS, relieving lung injury, improving pathological changes of lung tissues and the like, and inhibiting ALI lung tissue injury and inflammatory reaction induced by LPS.

Test example 2 analysis of chemical Components of an extract solution of a composition of a Michelia purpurea and Nauclea officinalis

1. Preparation of callicarpa nudiflora extract

Pulverizing naturally air dried folium Callicarpae Formosanae, sieving with No. 4 sieve, accurately weighing 5g medicinal powder, adding 75ml water, heating and refluxing for 2 hr, shaking, filtering, heating and concentrating the filtrate, and oven drying to obtain dry folium Callicarpae Formosanae extract. Taking about 0.1g of Callicarpa nudiflora extract, precisely weighing, placing in a 50ml measuring flask, adding 10% methanol solution to constant volume to 50ml, sealing, weighing, ultrasonically treating for 30min, cooling, and supplementing with 10% methanol. Shaking, filtering, and collecting the filtrate as Callicarpa nudiflora extractive solution.

2. Preparation of Nauclea officinalis extract

Pulverizing air-dried lignum naucleae, sieving with No. 4 sieve, accurately weighing 1g medicinal powder, adding 100ml water, heating and refluxing for 2 hr, shaking, filtering, heating and concentrating the filtrate, and oven drying to obtain dry extract. Taking about 0.1g of the extract, precisely weighing, placing in a 50ml measuring flask, adding 10% methanol solution to reach a constant volume of 50ml, sealing, weighing, ultrasonically treating for 30min, cooling, and supplementing with 10% methanol. Shaking, filtering, and collecting the filtrate as sample solution.

3. Preparation of callicarpa bodinieri extract

Precisely weighing 0.1g of the content of the callicarpa bodinieri traditional Chinese medicine composition, placing the content in a 50ml measuring flask, adding 10% methanol solution to fix the volume to 50ml, sealing, weighing, ultrasonically treating for 30min, cooling, and supplementing with 10% methanol. Shaking, filtering, and collecting the filtrate as folium Callicarpae Formosanae extractive solution.

4. UPLC-Q-TOF-MS detection conditions

Chromatographic conditions are as follows: using an ACQUITY UPLC BEH C18(2.1 mm. times.50 mm, 2.2 μm) chromatographic column at a flow rate of 0.25 ml/min^-1The sample introduction amount is 5 mu l, and the column temperature is 30 ℃; the mobile phase was 0.1% formic acid solution (a) -acetonitrile solution (B), gradient elution: 0-6 min, 8% -15% A; 6-16 min, 15% -25% A; 16-17 min, 25% -28%; 17-22 min, 28-35%.

Mass spectrum conditions: an electrospray ionization source ESI is adopted to scan in two modes of positive ions and negative ions respectively, the scanning range is 50-1500 m/z, the scanning time is 1.5s, the detection time is 22min, the low-energy collision voltage is 6V, the high-energy collision voltage is 20-60V, and the atomizing gas and the drying gas are both nitrogen.

Compound analysis and assignment method: and identifying the chemical components according to retention time, ultraviolet absorption characteristics, molecular ion peaks and corresponding secondary fragment ions of each chemical component in the callicarpa nudiflora water extract, the nauclea officinalis extract and the callicarpa bodinieri traditional Chinese medicine composition extract and by combining related literature data.

As shown in fig. 8 and 9, the total ion flow of the aqueous extract of the composition of nauclea purpurea swingle is analyzed in the positive and negative ion mode, respectively, to obtain the total ion flow chart (TIC) in the positive and negative ion mode. According to the obtained quasi-molecular ion peak and secondary mass spectrum fragment ions, 59 compounds in the callicarpa bodinieri traditional Chinese medicine composition are speculated and identified by analyzing the components of the compounds and comparing the results with the results of single medicinal materials, wherein 37 compounds come from callicarpa nudiflora and 22 compounds come from nauclea officinalis, and all the components conform to the chemical components of the single-component extracting solution analyzed from callicarpa nudiflora and nauclea officinalis.

The secondary mass spectrum is shown in fig. 10,

peaks

5, 41 and 42 all have a similar mass spectrum cracking rule with verbascoside, peak 2 is an excimer ion peak M/z 461.1638[ M-H ] -, which indicates that the polarity is relatively large, the excimer ion peak of M/z 461.1638 has a neutral rhamnosyl group loss cracking reaction, and a fragment ion of M/z 315.1080, which is a parent nucleus fragment ion of dihydroxyphenylethanoid glycoside, is continuously cracked, and loses 1 molecule of glucose and water M/z180, so that a fragment ion of M/z 135.0432 is generated. And the compound contains main fragment ions with m/z of 205.0771 generated by benzene ring structure fracture on the basis of Rha loss, the compound contains rhamnosyl and dihydroxyphenylethanoid glycosides through a cracking way, and the compound is determined to be forsythin E after comparison with the literature.

As shown in FIG. 11, in the secondary mass spectrum, the molecular ion peak of peak 14 is M/z 487.1484[ M-H ] -, the fragment ion M/z 325.0930 generated after the caffeoyl group is lost can be seen in the secondary mass spectrum, the typical ion peak M/z 179.0333 of the caffeoyl group and the typical fragment ion M/z 135.0432 can also be seen in the secondary mass spectrum, and the compound is cistanoside F according to the literature report.

As shown in fig. 12, in the secondary mass spectrum, peak 2 forms an excimer ion peak M/z373.1129[ M-H ] -, and M/z 211.0594 mass spectrum ion fragments obtained by removing one molecule of glucose are visible in the secondary mass spectrum, on this basis, one molecule of carboxyl is removed to generate M/z 167.0699 fragment ions, one molecule of water is lost to generate 149.0578 fragment ions, and the compound is geniposide acid by comparing with the literature.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A preparation method of a callicarpa-nauclea officinalis traditional Chinese medicine composition is characterized by comprising the steps of mixing callicarpa nudiflora and nauclea officinalis traditional Chinese medicine raw materials to form a traditional Chinese medicine mixture, adding water with the mass being 20 times of that of the traditional Chinese medicine mixture into the traditional Chinese medicine mixture, heating, refluxing and extracting for 3 times, wherein the heating, refluxing and extracting time is 0.5h each time, combining 3 times of traditional Chinese medicine extracting solutions, filtering the traditional Chinese medicine extracting solutions, heating, concentrating and drying to obtain callicarpa nudiflora traditional Chinese medicine extract powder;

2. The method for preparing the callicarpa bodinieri traditional Chinese medicine composition as claimed in claim 1,

the mass ratio of the callicarpa nudiflora and the nauclea officinalis traditional Chinese medicine raw materials is 4: 1.

3. The method for preparing the callicarpa bodinieri traditional Chinese medicine composition as claimed in claim 1,

the callicarpa bodinieri extract at least comprises the following active ingredients:

4. The method for preparing the callicarpa bodinieri traditional Chinese medicine composition as claimed in claim 3,

the phenylethanoid glycosides compounds include forsythoside E and cistanoside F.

5. The method for preparing the callicarpa bodinieri traditional Chinese medicine composition as claimed in claim 3,

the iridoid compound comprises geniposide.

6. The method for preparing the callicarpa bodinieri traditional Chinese medicine composition as claimed in claim 1,

the extract yield is 32.2%.

7. The method for preparing the callicarpa bodinieri traditional Chinese medicine composition as claimed in claim 1,

in the traditional Chinese medicine composition of the callicarpa bodinieri, verbascoside in each capsule is not less than 7.6mg, and strictosamide is not less than 0.47 mg.

8. The method for preparing the callicarpa bodinieri traditional Chinese medicine composition as claimed in claim 1,

each traditional Chinese medicine composition of the callicarpa bodinieri contains 0.39g of traditional Chinese medicine extract powder of the callicarpa bodinieri.

9. The method for preparing the callicarpa bodinieri traditional Chinese medicine composition as claimed in claim 1,

mixing the traditional Chinese medicine extractum powder of the callicarpa bodinieri and auxiliary materials, sieving the mixture by a 80-mesh sieve, and filling the mixture into capsules.

10. The method for preparing the callicarpa bodinieri traditional Chinese medicine composition as claimed in claim 1,

the humidity of the process environment for preparing the callicarpa bodinieri traditional Chinese medicine composition is less than 70%.