CN115813983A - The preparation method and application of the effective extract of Coptidis Magnolia officinalis and its microemulsion - Google Patents

Abstract

The invention discloses a preparation method and application of a coptis chinensis magnolia officinalis high-efficiency extract and microemulsion thereof, wherein the preparation method of the coptis chinensis magnolia officinalis high-efficiency extract comprises the following steps: reflux-extracting Coptidis rhizoma decoction pieces and cortex Magnolia officinalis decoction pieces with water or mixture of water and ethanol as extraction solvent, and concentrating to obtain Coptidis rhizoma cortex Magnolia officinalis extract; when the extraction solvent is a mixture of water and ethanol, the mass percent of the ethanol is 25-75%; wherein the mass ratio of the coptis root decoction pieces to the magnolia bark decoction pieces is 1:1, the ratio of material to liquid is 1: (8-12), the extraction frequency is 1-3, and the extraction time is 30-60 min each time. The method has simple process, and the prepared coptis and magnolia bark extract has high content of effective components; the prepared microemulsion can further improve the solubility of the coptis and magnolia bark extract, improve the stability of the extract and promote the absorption of the medicament; the coptis and magnolia bark extract and the microemulsion thereof have obvious effect on treating ulcerative colitis.

Description

The preparation method and application of the effective extract of Coptidis Magnolia officinalis and its microemulsion

technical field

The invention belongs to the technical field of traditional Chinese medicine, and in particular relates to the preparation method and application of the high-efficiency extract of coptis magnolia bark and microemulsion thereof.

Background technique

Coptis chinensis and Magnolia officinalis soup is mainly composed of Coptidis chinensis and Magnolia officinalis, which is derived from "Deshengtang Prescription" quoted in Volume 1, 3, and 3 of "Puji Fang". The components in Coptis Rhizoma mainly include Berberine, Coptisine, Epiberberine, Palmatine, Worenine, Hydrogenated Berberine ((R) -canadine) and other alkaloid components. Berberine can play a protective role against ulcerative colitis (UC) by improving the intestinal mucosal barrier function, maintaining the balance of intestinal flora, inhibiting inflammatory response and oxidative stress. Modern studies have found that berberine can reduce the expression of inflammatory factors (such as TNF-α) in serum and Toll-like receptor protein in colon tissue, up-regulate the expression level of tight junction protein in colon tissue, and inhibit the apoptosis of intestinal epithelial cells to exert its anti-inflammatory effect. The therapeutic role of UC. At the same time, berberine can also improve UC metabolic disorders and play an anti-inflammatory role by regulating the metabolism of microbiome and the level of nitric oxide (NO) in serum. There are 8 potential active ingredients in magnolia bark, such as magnolol, honokiol, honokiol, neohesperidin, etc. Magnolol can treat UC by immunomodulating phagocytosis, activating the mitogen-activated protein kinase (MAPK) signaling pathway, and reducing the levels of TNF-α, IL-1β, and IL-12 in colon tissue.

The active ingredients in this formula are mainly alkaloids and phenolic compounds, which have extremely poor water solubility and low bioavailability in the body. It is difficult to reach the corresponding concentration in the body after oral administration, which is not conducive to the active ingredients to play a role in inflammatory bowel disease. therapeutic effect. Microemulsion is a thermodynamically stable homogeneous phase system composed of water phase, oil phase, emulsifier and auxiliary emulsifier, with a particle size between 10 and 100 nm. It has the advantages of improving drug solubility, improving stability, and promoting drug absorption, and its water phase dissolves water-soluble components, and its oil phase dissolves fat-soluble components, which is suitable for the simultaneous inclusion of complex and multi-component Chinese medicine.

Contents of the invention

In order to solve the defects in the prescriptions mentioned in the background technology, the present invention provides the high-efficiency extract of Coptidis Magnolia officinalis, its microemulsion, preparation method and application of microemulsion, adopting microemulsion as the dosage form of encapsulating the Coptidis Magnolia officinalis extract, Can further improve its anti-UC effect.

The present invention adopts following technical scheme:

The preparation method of the high-efficiency extract of Coptidis Magnolia officinalis, using water or a mixture of water and ethanol as the extraction solvent, carries out reflux extraction on the decoction pieces of Coptidis Rhizoma and Magnolia officinalis, and concentrates to obtain the extract of Coptidis Magnolia officinalis; when the extraction solvent is water and ethanol During the mixture, the mass percent of ethanol is 25%～75%; Wherein, the mass ratio of Coptidis Rhizoma decoction pieces and Magnolia officinalis officinalis slices is 1:1, the solid-liquid ratio is 1:(8～12), and the number of extractions is 1～3, Each extraction time is 30min～60min.

Further, the ratio of solid to liquid is preferably 1:8.

Further, the number of times of extraction is preferably 2.

Further, each extraction time is preferably 60 minutes.

Further, when the extraction solvent is a mixture of water and ethanol, the mass percentage of ethanol is 25%-50%.

Further, the mass percentage of ethanol is preferably 50%.

The preparation method of the microemulsion of the above-mentioned coptis magnolia bark superior extract comprises:

The prepared Coptidis Magnolia officinalis extract is prepared into freeze-dried powder;

Mix the lyophilized powder with the oil phase, surfactant and co-surfactant, place it in a centrifuge tube with stopper and vortex; then place it in a constant temperature oscillator at 37°C and shake it in the dark to reach the dissolution equilibrium. After centrifugation, take the Serum is microemulsion;

Wherein, the mass percentage of the oil phase is 10%-60%, the mass percentage of the surfactant is 40%-70%, and the mass percentage of the co-surfactant is 10%-60%.

Further, the oil phase is preferably oleic acid, the surfactant is preferably EL-40, and the co-surfactant is preferably PEG400.

The prepared coptis magnolia superior extract can be applied to the preparation of medicines for treating ulcerative colitis.

The microemulsion prepared above can be applied to the preparation of medicine for treating ulcerative colitis.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

(1) The process is simple, and the prepared Coptidis Magnolia officinalis extract has high active ingredient content.

(2) The prepared microemulsion can further improve the solubility of Coptidis Magnolia officinalis extract, improve its stability, and promote drug absorption.

(3) Coptidis magnolia extract and its microemulsion have a significant effect on the treatment of ulcerative colitis.

Description of drawings

Figure 1 is the pictures of mouse colons treated with extracts of different ethanol concentrations for UC, wherein, figure (a) is the blank group, figure (b) is the model group, and figures (c)-(g) are the ethanol concentration 0%, respectively. 25%, 50%, 75%, 90% of the therapeutic effect of the extract;

Fig. 2 is the colon length of treating UC with extracts of different ethanol concentrations;

Figure 3 is the body weight change of UC treated with extracts of different ethanol concentrations;

Figure 4 is the survival rate of UC treated with extracts of different ethanol concentrations;

Figure 5 is the DAI score of UC treated with extracts of different ethanol concentrations;

Fig. 6 is the colon tissue section of UC treated with extracts of different ethanol concentrations, wherein, Fig. (a) is the blank group, Fig. (b) is the model group, Fig. (c) ~ (g) are the ethanol concentration 0%, 25% respectively %, 50%, 75%, 90% extracts of colon tissue sections;

Figure 7 shows the levels of inflammatory factors in the colon tissue of UC treated with extracts of different ethanol concentrations, where Figures (a) to (d) are the levels of TNF-α, IL-1β, IL-6, and IL-10 inflammatory factors, respectively;

Figure 8 is a ternary phase diagram of the microemulsion.

Detailed ways

The specific implementations and technical effects of the present invention are provided below, wherein the specific implementations are only for illustrative purposes, but are not intended to limit the implementation scope of the present invention, and there are still many other applicable places in the present invention.

The specific process of this embodiment is as follows:

1. Preparation and characterization of coptis magnolia bark extract

1.1. Extract Coptidis Magnolia Extract

Weigh 37.3g each of Coptidis Rhizoma and Magnolia officinalis, put them in a 1000mL round-bottomed flask, add an appropriate amount of solution for reflux extraction, and concentrate the extract to 50mL to obtain Coptidis Magnolia officinalis extract.

1.2. Preparation of samples

Take 1 mL of Coptidis Magnolia officinalis extract, add methanol to make up to 8 mL, and filter through a 0.22 μm microporous membrane to obtain a sample.

1.3. Preparation of reference substances

Take an appropriate amount of epiberberine, coptisine, palmatine, and berberine reference substance, weigh them accurately, add methanol to make each 1mL contain epiberberine 1.49mg/mL, coptisine 1.955mg/mL, and palmatine 1.4075mg/mL, berberine 1.435mg/mL Coptidis mixed reference substance. Take an appropriate amount of honokiol and magnolol, weigh them accurately, add methanol to make a magnolol reference substance containing 1.36 mg/mL of honokiol and 1.77 mg/mL of magnolol solution per 1 mL.

1.4. Chromatographic detection

Because the chromatographic mobile phase requirements for the alkaloid components of Coptidis rhizome are complex, and the mobile phase required by the active components of Magnolia officinalis is quite different, so the method of separate determination is used for content determination. With reference to the 2020 edition of "Chinese Pharmacopoeia", the chromatographic conditions and system suitability test of Coptidis rhizome are as follows: use octadecylsilane bonded silica gel as filler; use acetonitrile-0.05mol/L potassium dihydrogen phosphate solution (50:50) (per Add 0.4 g of sodium lauryl sulfate to 100 mL, then adjust the pH value to 4.0 with phosphoric acid) as the mobile phase, and the detection wavelength is 345 nm. The number of theoretical plates should not be less than 5000 based on the berberine hydrochloride peak. Magnolia officinalis chromatographic conditions and system suitability test are as follows: chromatographic conditions and system suitability test use octadecylsilane bonded silica gel as filler; use methanol-water (78:22) as mobile phase; detection wavelength is 294nm, theoretical The plate number should not be less than 3800 based on the magnolol peak.

5. Optimization of extraction process

The extraction solvent, solid-liquid ratio, extraction time and extraction times were selected as the investigation factors, and each factor took three levels. The factor levels are shown in Table 1. Orthogonal experiments were designed, as shown in Table 2. Content of index components (mg/g) in Table 2 = total content of index components (mg)/amount of Coptidis Magnolia officinalis decoction pieces (g).

Table 1 Orthogonal test factor level table

level extraction solvent solid to liquid ratio extraction time Extraction times 1 water 1:8 30 1 2 25% ethanol 1:10 45 2 3 50% ethanol 1:12 60 3

Table 2 Orthogonal test table

According to the analysis of the results of the orthogonal test, the primary and secondary relationship of each factor affecting the extraction rate of active ingredients is as follows: extraction times, extraction solvent, extraction time, and solid-liquid ratio. The best extraction process is: the solvent is 50% ethanol, the extraction times are 2 times, the extraction time is 60min, and the ratio of solid to liquid is 1:10. The extract of Coptidis Magnolia officinalis was extracted by the best extraction process, and prepared in parallel three times. After the content determination by HPLC, the total content of berberine, coptisine, palmatine, berberine, magnolol and honokiol The content is an index component, and the average content of the extract index component is 58.83mg/g.

2. Components and efficacy testing of extracts with different ethanol concentrations

According to the aforementioned orthogonal experiment, the most significant effect on the extraction rate of active ingredients is the extraction solvent. As the concentration of ethanol increases, the extraction rate of active ingredients increases. However, the effect of the active ingredient content on anti-UC efficacy is still unclear, so the concentration of ethanol was further refined, and the samples were extracted with 0%, 25%, 50%, 75%, and 90% ethanol as the extraction solution, and the content was determined. As well as preliminary experiments on the efficacy of anti-UC drugs.

2.1 Sample preparation

Weigh 37.3g each of Coptidis Rhizome and Magnolia officinalis, put them in a 1000mL round bottom flask, add 0%, 25%, 50%, 75%, 90% ethanol solution for reflux extraction, and add 10 times of extraction each time Solvent, extracted twice, each extraction time 60min, the extract was concentrated to 50mL to obtain a sample.

2.2 Content determination

According to the chromatographic conditions in 1.4, the content of the sample was determined, and the results are shown in Table 3. It can be seen from Table 3 that the extraction rate of the effective components of Coptidis Rhizoma and Magnolia officinalis is different when extracted with different ethanol concentrations, especially the content of the index components of Magnolia officinalis. The addition of ethanol greatly improves the content of Magnolia officinalis index components, which is greater than the aqueous solution in each ethanol solution. When extracted with 50% ethanol, the active ingredients of Coptidis Rhizome and Magnolia officinalis have the highest content.

Table 3 Contents of each sample index component

2.3 Drug efficacy evaluation

(1) Animal modeling and administration

Male ICR mice weighing 20-25 g were divided into 6 groups, 12 in each group. Divided into blank, model, 0% ethanol extract, 25% ethanol extract, 50% ethanol extract, 75% ethanol extract, 90% ethanol extract. After one week of adaptive feeding, the mice were fed with 3% DSS freely to make a UC model. After 3 days, the blank and model groups were fed with normal saline, and the drug group was fed with the corresponding extract (crude drug amount 2g/kg). Rats were fasted without food and water for 24 hours after the last administration, and the materials were collected.

(2) Drug efficacy index

(a) Observation indicators: After the modeling started, observe and record the changes in the body, spirit, activity status, hair luster, stool properties and blood in the stool of mice in each group every day, and record the DAI score and death situation. For the DAI score standard, see Table 4.

Table 4DAI score

scoring weight loss Stool properties bloody stool condition 0 ≤1 normal Negative 1 ≤5 soft, shaped Occult blood (+) 2 5-10 slimy, formed Occult blood (++) 3 10-15 Soft, mucous, shaped stools Occult blood (+++) 4 ≥15 Soft, mucus-filled, shapeless stools Gross bloody stool

(b) Colon length: After the last administration, the rats were anesthetized, and the abdomen of the rat was cut along the linea alba. The colon was removed and rinsed with ice-cold saline, and the length of the colon was measured with a ruler.

(c) Tissue section: colon tissue was fixed in 4% paraformaldehyde for 48 hours, dehydrated, routinely embedded in paraffin, sectioned (thickness 5 μm), and attached to a high-adhesion glass slide, numbered, overnight at 37°C, HE staining was performed separately. The changes of colonic tissue damage were observed under a microscope.

(d) Elisa cytokine assay: After the mouse colon was homogenized, according to the kit instructions, the amount of tissue protein was first measured, and then the tissue IL-1β, TNF-α and IL-6; IL-10, Arg-1 were measured. content.

2.4 Drug efficacy test results

(1) Observation indicators

The pharmacological effect of Coptidis Magnolia officinalis extracts with different ethanol concentrations on DSS-induced ulcerative colitis in mice was preliminarily evaluated by daily recording of mouse body weight, DAI score, survival status and colon length. Refer to the index observation results in Figures 1 to 5. The results of the colon length show that: DSS free diet can significantly reduce the colon length, and Magnolia Coptidis Rhizoma Coptidis in each group can improve this change, among which Coptis Magnolia 50% and 90% ethanol extracts can Significantly improved; body weight results showed that the weight of the mice in the normal group continued to increase, while the body weight of the model group decreased significantly after 7 days after modeling, and each administration group could be relieved to a certain extent; on the 10th day, 50% ethanol extract There were significant differences between the mice in the model group and the mice in the model group; the DAI score results showed that, compared with the model group, the DAI scores of each administration group decreased. The survival curve showed that the survival rate of blank group and 50% ethanol extract was 100%, and all other groups died. According to the comprehensive analysis, the 50% ethanol extract of Coptidis Magnolia has the best medicinal effect.

(2) Pathological morphology of colon tissue and expression of inflammatory factors

Referring to Figure 6, from the H&E slices of colon tissue and the levels of inflammatory factors, the degree of colon inflammation in mice in each experimental group was compared. The results of H&E slices showed that the colonic mucosal crypt structure of the mice in the model group was severely damaged, the colonic epithelium was thickened, and the mucosa and submucosa were edematous and accompanied by a large number of inflammatory cell infiltration. Each administration group can be relieved to a certain extent, and the 100 μm is the inflammatory site of the mouse colon tissue of each group. It can be clearly seen that the crypt structure of the inflammatory area of 0% ethanol extract is destroyed, and the inflammatory cells infiltrate, but concentrated In one area; 25% ethanol extract showed obvious edema at the inflammatory site, the crypt structure was partially destroyed, and inflammatory cells infiltrated the submucosa; 50% ethanol extract colon tissue crypts were intact, and inflammatory cells were rarely seen; 75% ethanol extract The inflammatory site of the extract was in the mucosal layer, with inflammatory cell infiltration; the inflammatory site of the 90% ethanol extract was scattered, with inflammatory cell infiltration in the mucosa and submucosa.

See Figure 7, the results of cellular inflammatory factors showed that compared with the blank group, the model group significantly increased the pro-inflammatory factors TNF-α, IL-1β, IL-6, and decreased the level of the anti-inflammatory factor IL-10. Compared with the model group, each medication group could down-regulate the levels of TNF-α, IL-1β, and IL-6, and up-regulate the level of anti-inflammatory factor IL-10. Among them, 0%, 50%, 75% ethanol extracts can significantly down-regulate the level of TNF-α; 0%, 25%, 59%, 75% ethanol extracts also have significant differences in the down-regulation of IL-1β; The drug group can significantly down-regulate the level of IL-6. Combining 2.2 Content Determination and 2.4.1 Observation Index, Coptis Magnolia officinalis 50% ethanol extract has the best efficacy, which is consistent with the content determination index.

Through the content determination of different ethanol concentrations of Coptidis Magnolia and the preliminary comparative study of drug efficacy, it was found that the 50% ethanol extract of Coptidis Magnolia had the highest index content and the best drug effect.

3. Preparation of Coptis Magnolia Extract Microemulsion

The dried and freeze-dried powder of Coptidis Magnolia officinalis 50% ethanol extract is used as a drug. The main components of magnolia bark, magnolol and honokiol, also have the problems of low solubility in gastrointestinal fluid and high metabolism in the liver and intestines, which is not conducive to the drug's efficacy. Microemulsion is a thermodynamically stable homogeneous phase system composed of water phase, oil phase, emulsifier and auxiliary emulsifier, with a particle size between 10 and 100nm. It has the advantages of improving drug solubility, improving stability, and promoting drug absorption, and its water phase dissolves water-soluble components, and its oil phase dissolves fat-soluble components, which is suitable for the simultaneous inclusion of complex and multi-component Chinese medicine. Therefore, the present invention further selects the microemulsion as the formulation for encapsulating the extract of Magnolia officinalis Coptidis Rhizoma Magnoliae, in order to further improve its anti-UC effect.

3.1 Drug solubility test

Precisely weigh 1g of Coptidis Magnolia officinalis 50% ethanol extract freeze-dried powder, mix with 5mL oil phase, surfactant and co-surfactant respectively, place in a centrifuge tube with stopper, vortex for 30min, and place at 37°C for constant temperature oscillation Shake in the dark for 48 hours to reach the dissolution equilibrium, centrifuge at 10000r/min for 15 minutes, take 1mL of supernatant, add methanol to make up to 4mL, mix well, pass through a 0.22μm filter membrane, and take the subsequent filtrate to obtain the final product. Perform detection according to the chromatographic conditions under item 1.3, inject 10 μL of sample, and detect the main active ingredients of Magnolia officinalis and Coptidis Rhizoma.

The oil phase can be castor oil, glyceryl caprylate, olive oil, isopropyl palmitate, ethyl oleate, oleic acid, isopropyl myristate, soybean oil, macrogol glycerol oleate, mono Glyceryl Linoleate. Surfactant can adopt alkylphenol polyoxyethylene ether, castor oil polyoxyethylene 40, castor oil polyoxyethylene 30, Tween-80, caprylic acid macrogol glyceride, propylene glycol monocaprylate. Co-surfactants can be polyethylene glycol 400, 1,2-propylene glycol, absolute ethanol, glycerol, and 1,3-butanediol.

3.2 Solubility test results

From the solubility results of each material to Coptidis Magnolia officinalis 50% ethanol extract: in the oil phase, the solubility of oleic acid and oleic acid macrogol glyceride is higher than other oil phases; It is generally larger than the oil phase, and the solubility of Span 80 and castor oil polyoxyethylene 40 (EL-40) is higher than that of other surfactants; among co-surfactants, their solubility to drugs is significantly increased, and polyethylene glycol 400, 1,2-propanediol, glycerol, and 1,3-butanediol all have greater solubility to drugs. Therefore, the excipients with higher solubility were selected for further optimization.

3.3 Preliminary screening of microemulsion formulations

(1) Oil phase screening

With EL-40 as the surfactant and PEG400 as the co-surfactant, the mass ratio Km of the surfactant and the co-surfactant is determined to be 2:1 as the mixed surfactant, and the different oil phases and the mixed surfactant are respectively ( Oleic acid, oleic acid macrogol glyceride) according to the ratio of 1:9, 2:8, 3:7, 4:6, 5:5, stir while adding appropriate amount of water, and make microemulsion after high-pressure homogenization. Fixed as an oil phase, the total amount of surfactant and co-surfactant is 5g, and the amount of water added is 15g, and an appropriate oil phase is selected according to its appearance and particle size distribution.

(2) Screening of surfactants

With the best oil phase screened, PEG400 is a co-surfactant, and the Km value is determined to be 2. The mixed surface activity of the oil phase and different surfactants (Span 80, castor oil polyoxyethylene 40 (EL-40)) According to the ratio of 1:9, 2:8, 3:7, 4:6, 5:5, add appropriate amount of water while stirring, and make microemulsion after high-pressure homogenization. Fixed as an oil phase, the total amount of surfactant and co-surfactant is 5g, the amount of water added is 15g, and a suitable surfactant is selected according to its appearance and particle size distribution.

(3) Co-surfactant screening

The best oil phase, the best surfactant, surfactants and different co-surfactants (PEG400, 1,2-propanediol) were screened to prepare mixed surfactants according to Km as 2, oil phase and mixed surfactants According to the ratio of 1:9, 2:8, 3:7, 4:6, 5:5, add appropriate amount of water while stirring, and make microemulsion after high-pressure homogenization. Fixed as the oil phase, the total amount of surfactant and co-surfactant is 5g, the amount of water added is 15g, and a suitable co-surfactant is selected according to its appearance and particle size distribution.

(4) Km value screening

The best oil phase, the best surfactant, and the best co-surfactant are mixed according to the Km value of 4:1, 3:1, 2:1, 1:1, and 1:2 to obtain the mixed surface activity. Agent, oil phase and mixed surfactant according to the ratio of 1:9, 2:8, 3:7, 4:6, 5:5, stir while adding appropriate amount of water, and make microemulsion after high-pressure homogenization. Fixed as the oil phase, the total amount of surfactant and co-surfactant is 5g, the amount of water added is 15g, and the appropriate Km value is selected according to its appearance and particle size distribution.

(5) Selection of drug concentration

Get the best oil phase, surfactant and co-surfactant, add 10, 20, 40, 80, 160, 320, 640 mg of Coptis Magnolia 50% ethanol extract respectively, dissolve in oil phase, surfactant and In the mixture of co-surfactants, the remaining amount is supplemented with water, and the microemulsion is prepared after high-pressure homogenization. Take 10mL of microemulsion and centrifuge at 10000r/min for 15min, take an appropriate amount of 0.5mL of the supernatant into a brown measuring bottle, add methanol to dilute to 4mL, and measure the content of Coptis magnolia bark index components in each sample. Select the appropriate drug concentration according to its appearance, particle size distribution and drug loading.

3.4 Preliminary Screening Results of Prescription Optimization

(1) Oil phase screening results

EL-40 was used as surfactant, PEG400 was used as co-surfactant, the mass ratio Km of surfactant and co-surfactant was determined to be 2:1 as mixed surfactant, glycerol monolinoleate and oleic acid Prepared for the oil phase, see Table 5-6, can form a blank microemulsion with slightly bluish light, and the microemulsion becomes more and more opaque as the proportion of the oil phase increases. However, the microemulsion formed by glycerol monolinoleate cannot present a single particle size peak, and the particle size is generally >100nm. However, the microemulsion prepared with oleic acid has a single peak, and the particle size is less than 100nm. Therefore, the oil phase was selected as oleic acid for the next test.

Table 5 The appearance and particle size of glyceryl monolinoleate-EL40-PEG 400-water

Table 6 The appearance and particle size of oleic acid-EL 40-PEG 400-water

(2) Surfactant screening results

Use oleic acid as the oil phase, PEG400 as the co-surfactant, determine the mass ratio Km of surfactant and co-surfactant as 2:1 as the mixed surfactant, and use Span 80 and EL-40 as the surfactant respectively , high-pressure homogeneous preparation of microemulsion. It was found that the microemulsion could not be prepared with Span 80 as the surfactant, and the mixed solution was stratified and oily matter floated, so it was directly eliminated. The microemulsion prepared with EL-40 as the surfactant is like the microemulsion of oleic acid-EL 40-PEG 400-water in 4.4.1, so the surfactant is finally determined to be EL-40.

(3) Co-surfactant screening results

Use oleic acid as the oil phase, EL-40 as the surfactant, determine the mass ratio Km of the surfactant and co-surfactant as 2:1 as the mixed surfactant, and use PEG400 and 1,2-propylene glycol as the co-surfactant respectively The active agent is prepared as a microemulsion by high pressure homogenization. It was found that both PEG400 and 1,2-propanediol could form microemulsions with a particle size of <100nm. In consideration of safety, PEG400 was finally selected as the final co-surfactant.

Table 7 Appearance and particle size of oleic acid-EL 40-1,2 propylene glycol-water

(4) Km value screening

Using oleic acid as the oil phase, EL-40 as the surfactant, and PEG400 as the co-surfactant, the microemulsion was prepared after mixing with the oil phase at different Km ratios, and a ternary phase diagram was drawn, as shown in Figure 8. The results show that the black area in the figure is a sample with a particle size of <100nm. It can be seen that when the amount of oleic acid is 10% to 60%, the amount of L40 is 40% to 70%, and the amount of polyethylene glycol 400 is 10% to 60%. 60% of them can form microemulsion with particle size less than 100nm.

(5) Screening of dosage

Using oleic acid as the oil phase, EL-40 as the surfactant, and PEG400 as the co-surfactant, the mass ratio Km of the surfactant and the co-surfactant is determined to be 2:1 as the mixed surfactant, and the thickness of Coptis chinensis of different quality The 50% extract of Pakuri is dissolved in the mixture of oil phase, surfactant and co-surfactant, and 30mL of water is used as the water phase, after mixing, the microemulsion is prepared by high-pressure homogeneity. The results showed that with the increase of the dosage, the particle size tended to become larger, but the encapsulation efficiency decreased continuously. When the dosage was >320mg, the encapsulation efficiency was <80%.

The screening of dosage of table 8

Through single factor investigation, oleic acid was preliminarily screened out as the oil phase, EL-40 as the surfactant, PEG400 as the co-surfactant, and the dosage of each excipient was oleic acid: 10%-60%, EL40: 40%-70%, Polyethylene glycol 400: 10% to 60%. Microemulsions can be formed when the drug loading is less than 320mg.

3.5 Box-Benhnken Response Surface Method to Optimize Microemulsion Prescription

The types of excipients in the microemulsion system were preliminarily confirmed through single factor investigation, but the optimal ratio of each phase was not clear. Determine the dosage range of each phase according to the pseudo-ternary phase diagram; use the Box-Behnken design-response surface method to optimize the prescription ratio, and arrange the experiments in accordance with Tables 9-10 to weigh the corresponding mass fractions of oil phase, surfactant and Co-surfactants were mixed, stirred under the condition of 35°C, and the optimal concentration of Lianbo extract was added slowly, the remaining amount was supplemented with water, and after high-pressure homogenization, a microemulsion sample was obtained. Take an appropriate amount of 1 g in a brown measuring bottle, add methanol to dilute to 8 mL, and measure the drug loading in each group. Another 1 mL was diluted 100 times with water, and the particle size was measured by Zetasizer laser particle size analyzer. The mass fractions of A (oil phase), B (surfactant: co-surfactant Km value) and C (dosage amount) are independent variables, and Y1 (particle size) and Y2 (total content of index components) are used as response values. Design Expert 8.0.5 software was used to optimize the design and determine the optimal ratio of the prescription.

Table 9 Factor Level Arrangement Table

variable test A(%) B(%) C (mg) -1 20 1:2 160 0 30 1:1 320 1 40 2:1 640

Table 10 Test arrangements and results

serial number A oil phase B Km value C dosage Y1 particle size (nm) Y2 encapsulation rate (%) 1 0 1 1 62.70 90.70 2 1 0 -1 68.41 104.21 3 0 -1 1 75.36 87.70 4 0 0 0 75.51 93.17 5 0 1 -1 69.97 95.05 6 -1 1 0 59.74 87.47 7 0 0 0 72.19 91.95 8 0 0 0 75.46 92.93 9 0 0 0 70.82 89.80 10 1 0 1 68.16 91.35 11 -1 0 1 58.28 94.63 12 1 -1 0 74.62 89.93 13 0 -1 -1 63.86 93.34 14 -1 -1 0 65.48 83.75 15 -1 0 -1 59.35 95.07 16 1 1 0 69.02 87.53 17 0 0 0 70.67 92.96

3.6 Analysis of variance and model fitting

From the variance analysis table of the particle size, it can be known that the influence of the three factors on the particle size is A﹥B﹥C, the factor A, that is, the mass fraction of the oil phase, has the most significant effect on the particle size (P﹤0.05), and the factor B, the surface activity The km value of surfactant and co-surfactant also had a very significant effect on the particle size (P﹤0.05), but the factor C drug loading had no significant effect on the particle size (P﹤0.05).

It can be seen from the variance analysis table of the encapsulation rate that the influence of the three factors on the encapsulation rate of the drug is C﹥A﹥B respectively, that is, the dosage of factor C has the most significant effect on the encapsulation rate of the drug (P﹤0.05), and the factor A That is, the mass fraction of the oil phase also has a significant effect on the drug encapsulation efficiency (P﹤0.05), factor B, namely the Km value of the surfactant and the co-surfactant, has no significant effect on the drug encapsulation efficiency, which is determined by AB, AC, and BC. The P value of the item shows that the interaction between factors AB and AC has a significant impact on the drug loading.

According to the analysis of the experimental data by Design Expert 8.0.6 software, it is predicted that the best preparation method of microemulsion is: Weigh 5.754g of oleic acid, 4.9515g of EL40, 4.2945g of PEG400, and then add 160mg of Coptidis Magnolia Bark 50% ethanol extract and mix Evenly, add 30g of purified water and stir for 10min. Homogenize for 20 times at an ultra-high pressure of 850 rpm to obtain Coptidis Magnolia Microemulsion. The appearance properties were evaluated, and an appropriate amount was taken to measure the particle size and encapsulation efficiency respectively. Results The three microemulsions were all yellow solutions with light halo and fluidity like water samples; the measured particle diameters were 58.45nm, 52.28nm and 61.94nm respectively, the average particle diameter was 57.56nm, and the predicted particle diameter was 58.815nm. The encapsulation efficiency was 98.68%, 96.03%, 99.23%, the average content was 97.98%, the predicted encapsulation rate was 102.89%, the measured values of each index were close to the predicted values, and the measured values of particle size and ligustilide content were in line with those of The relative deviations of the predicted values are 1.255nm and 4.91%, respectively, indicating that the model has good predictability and the process is stable and feasible.

3.7 Parameter optimization of high pressure homogenization

Times of high-pressure homogenization: Mix the phases according to the optimal recipe obtained above, fix the homogenization speed at 850rpm, and measure the microemulsion particle size and encapsulation efficiency after homogenizing for 5, 10, 15, 20, and 25 times respectively.

High-pressure homogenization speed: The high-pressure homogenizer used in this test adjusts the pressure by the speed, and the higher the speed, the higher the pressure. The optimal recipe obtained from 4.6 is mixed with each phase, the number of homogenization is the optimal number of the above optimization, and the rotation speed is adjusted to 550, 650, 750, 850, 950rpm respectively, and the microemulsion particle size and encapsulation efficiency are measured after homogenization.

Judging from the particle size and encapsulation efficiency index, within a certain range, the more times of high-pressure homogenization, the smaller the microemulsion particle size. When it reaches 20 times, the microemulsion particle size is the smallest and the encapsulation efficiency is the highest. The homogeneous rotational speed has little effect on the particle size, but has a greater influence on the encapsulation efficiency. When the rotational speed is 750rpm, the drug loading is the highest.

Table 11 High pressure homogenization times

Homogenization times Particle size (nm) Encapsulation rate (%) 5 76.65 94.14 10 68.40 93.68 15 62.28 96.03 20 60.80 98.67 25 62.02 82.51

Table 13 High pressure homogeneous speed

Homogeneous speed(rpm) Particle size (nm) Encapsulation rate (%) 550 58.10 85.93 650 54.18 84.92 750 61.57 93.39 850 64.58 92.30 950 63.52 88.56

In summary, the preparation process of Coptis Magnolia Microemulsion is determined as follows: 5.754g of oleic acid, 4.9515g of EL40, 4.2945g of PEG, and then 160mg of Coptidis Magnolia 50% ethanol extract is mixed evenly, and 30g of purified water is added, after mixing well, Under the rotating speed of 750rpm, ultrahigh pressure homogenization was performed 20 times to obtain Coptidis Magnolia Microemulsion, with a particle size of 61.57nm and an encapsulation efficiency of 93.39%.

The above descriptions are only specific embodiments of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Accordingly, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims (10)

1. The preparation method of the coptis and magnolia bark effective extract is characterized by comprising the following steps:

reflux-extracting Coptidis rhizoma decoction pieces and cortex Magnolia officinalis decoction pieces with water or mixture of water and ethanol as extraction solvent, and concentrating to obtain Coptidis rhizoma cortex Magnolia officinalis extract; when the extraction solvent is a mixture of water and ethanol, the mass percent of the ethanol is 25-75%; wherein the mass ratio of the coptis root decoction pieces to the magnolia bark decoction pieces is 1:1, the ratio of material to liquid is 1: (8-12), the extraction frequency is 1-3, and the extraction time is 30-60 min each time.

2. The effective extract of the coptis and magnolia officinalis as claimed in claim 1, which is characterized in that:

the material-liquid ratio is 1:8.

3. the Magnolia officinalis superior effect extract of claim 1, wherein:

the number of extraction times is 2.

4. The Magnolia officinalis superior effect extract of claim 1, wherein:

the extraction time is 60min.

5. The effective extract of the coptis and magnolia officinalis as claimed in claim 1, which is characterized in that:

when the extraction solvent is a mixture of water and ethanol, the mass percent of the ethanol is 25-50%.

6. The effective extract of the coptis and magnolia officinalis as claimed in claim 5, which is characterized in that:

the mass percent of the ethanol is 50%.

7. The preparation method of the microemulsion of the coptis and magnolia officinalis optimal effect extract as the claim 1 is characterized in that:

preparing the coptis and magnolia bark extract prepared in the claim 1 into freeze-dried powder;

mixing the freeze-dried powder with an oil phase, a surfactant and a cosurfactant, and placing the mixture into a centrifuge tube with a plug for vortex; placing in a constant temperature oscillator at 37 deg.C, oscillating in dark to reach dissolving balance, centrifuging, and collecting supernatant to obtain microemulsion;

wherein, the oil phase accounts for 10 to 60 percent by mass, the surfactant accounts for 40 to 70 percent by mass, and the cosurfactant accounts for 10 to 60 percent by mass.

8. The method for preparing a microemulsion according to claim 7, wherein:

the oil phase is oleic acid, the surfactant is EL-40, and the co-surfactant is PEG400.

9. The use of the extract of the golden thread magnolia bark with good effect prepared in the claim 1 in the preparation of the medicament for treating ulcerative colitis.

10. Use of the microemulsion prepared according to claim 7 for the preparation of a medicament for the treatment of ulcerative colitis.

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