Polygonum multiflorum in-situ gel as well as preparation method and application thereof

Abstract

The invention discloses rubus idaeus in-situ gel as well as a preparation process and application thereof, and belongs to the technical field of pharmaceutical preparations. The rubus taiwanianus in-situ gel is prepared from the following components in percentage by mass: 16 to 20 percent of poloxamer 407, 1 to 5 percent of poloxamer 188, 7.5 to 20 percent of rubus taiwanianus extract liquid medicine, 0.2 percent of sodium phytate and 54.8 to 75.3 percent of distilled water. The rubus taiwanianus in-situ gel is prepared by mixing rubus taiwanianus extract liquid medicine, poloxamer, sodium phytate and distilled water according to optimal process parameters provided by the invention. The prepared rubus multibracteatus in-situ gel has a good forming effect and also has a slow release effect, and a reference is provided for further research of a novel traditional Chinese medicine preparation taking rubus multibracteatus as a main medicine.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical preparations, and in particular to a large black bubble in-situ gel and a preparation process and application thereof. Background Art

[0002] Periodontal disease is one of the most common and oldest diseases in human beings. At present, periodontal disease has become the leading cause of tooth loss in adults in my country. Periodontal disease is a chronic disease with no pain in the early stage and is easily ignored by patients. The cause of periodontal disease is called gingivitis, which only invades the gums in the early stage. The symptoms are bleeding and redness of the gums. If it is not treated in time to control the inflammation, it will cause secondary symptoms such as tooth sensitivity and pain. When the inflammation develops to the deeper periodontal tissue, it will cause suppuration. According to the results of the fourth national oral health epidemiological survey, the proportion of adults in my country with unhealthy periodontal disease, caries and tooth loss continues to rise. Among them, 80% to 97% of adults have varying degrees of periodontal disease; in the age group of 35 to 44 years old, the periodontal health rate is only 14.2%, and in the age group of 65 to 77 years old, the periodontal health rate is as low as 13.6%; in addition, children, as a special population, have a very high incidence of caries and serious oral health risks. Therefore, there is an urgent need for a safe and long-lasting functional oral care product.

[0003] Dawupao is a commonly used medicine of the Miao people in our province. It is a plant of the genus Rubus (Rubus multibracteatus Levl. & Vant.) of the Rosaceae family. It is also known as Wupao, June bubble, inverted root, and cow hair bubble thorn. It is a deciduous shrub of the genus Rubus (Rubus L.) of the Rosaceae family. It tastes slightly bitter and cool in nature. It enters the liver and spleen meridians. It is mainly produced in Guizhou, Yunnan, Sichuan and other places. Dawupao is rich in nutrients and contains 17 kinds of amino acids. The roots, stems, leaves and fruits can all be used as medicine. It has the effects of dispelling wind and dampness, clearing away heat and detoxifying, setting bones, cooling blood and stopping bleeding. It is often used by the local Miao people to treat colds, fever, enteritis, dysentery, nosebleeds and other diseases. Dawupao is also an important main drug in the traditional Chinese medicine preparation Jianchi Ning lozenges produced by Guizhou Kaidi Chengtian Pharmaceutical Co., Ltd. It is mainly used to treat periodontitis, gingivitis, caries pain, etc. Related studies have shown that the water extract of Psoralea corylifolia has good anti-inflammatory, analgesic and hemostatic effects on mice; in addition, the Psoralea corylifolia extract has a strong inhibitory effect on Lactobacillus that causes dental caries, Staphylococcus aureus that causes oral inflammation, and Helicobacter pylori that is related to bad breath and dental plaque formation, and has broad prospects for development in the oral market.

[0004] Insitu gel is a new type of drug release system. Among them, the temperature-sensitive insitu gel is in solution at room temperature, and can be quickly converted from liquid to semi-solid gel at body temperature (37°C) after administration. It can be used not only for skin administration, but also for oral, nasal, eye, rectum, vagina, intramuscular and other parts, and is widely used.

[0005] At present, there is no relevant research report on the in-situ gel of Dawupao. Based on this, the present invention provides a temperature-sensitive Dawupao in-situ gel and a preparation method thereof, aiming to provide a scientific basis for the clinical application of Dawupao and provide a reference for the development and application of new oral care products and new preparations with Dawupao as the main drug. Summary of the invention

[0006] The purpose of the present invention is to provide a kind of large black bubble in-situ gel.

[0007] Another object of the present invention is to provide a preparation process and application of the above-mentioned large black bubble in-situ gel.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] The in-situ gel of the present invention comprises the following components in percentage by mass: 16-20% of poloxamer 407, 1-5% of poloxamer 188, 7.5-20% of a medicinal solution of a poloxamer extract, 0.2% of sodium phytate, and 54.8-75.3% of distilled water.

[0010] Preferably, the in-situ gel of the present invention comprises the following components in mass percentage: 16-18% of poloxamer 407, 2-4% of poloxamer 188, 12.5-17.5% of the medicinal solution of the extract of the Psoralea corylifolia, 0.2% of sodium phytate, and 60.3-69.3% of distilled water.

[0011] Further preferably, the in-situ gel of the present invention comprises the following components in percentage by mass: 17% of poloxamer 407, 3% of poloxamer 188, 15% of a drug solution of a poloxamer extract, 0.2% of sodium phytate, and 64.8% of distilled water.

[0012] Among the components of the in-situ gel of the present invention, the concentration of the medicinal solution of the extract of the Bupleurum truncatum is 0.4 g / mL, that is, each mL of the medicinal solution contains 0.4 g of the Bupleurum truncatum extract.

[0013] The method for preparing the large black bubble in-situ gel of the present invention comprises the following steps:

[0014] S1 Preparation of the extract of Bupleurum truncatum:

[0015] Take an appropriate amount of the extract of the Chinese prickly ash, add 70% ethanol, add activated carbon for decolorization, filter, wash the filter paper with 70% ethanol, combine the filtrate, recover the ethanol under reduced pressure, and concentrate the filtrate to obtain a Chinese prickly ash extract solution;

[0016] S2 Preparation of gel solution:

[0017] Weigh poloxamer 407 and poloxamer 188, place them in a conical flask, gradually add the above-prepared radix eryngii extract solution and distilled water dissolved with 0.2% sodium phytate, seal, and refrigerate until they are completely dissolved and clear.

[0018] In the preparation method of the in-situ gel of Bupleurum ovale of the present invention, step S1 is specifically as follows: 20 g of Bupleurum ovale extract is put into a 200 mL conical flask, 200 mL of 70% ethanol is added, 0.5-3% activated carbon is added, and the mixture is decolorized for 20-120 min at a temperature of 30-80° C., the mixture is filtered, the filter paper is washed with 70% ethanol, the filtrate is combined, the ethanol is recovered under reduced pressure, and the filtrate is concentrated to 50 mL to obtain a Bupleurum ovale extract solution with a concentration of 0.4 g / mL.

[0019] Preferably, in the preparation method of the in-situ gel of the Bupleurum ovale of the present invention, step S1 is specifically as follows: take 20 g of the Bupleurum ovale extract into a 200 mL conical flask, add 200 mL of 70% ethanol, add 1.0-2.5% activated carbon, decolorize for 60-120 min at a temperature of 40-60°C, filter, wash the filter paper with 70% ethanol, combine the filtrate, recover the ethanol under reduced pressure, and concentrate the filtrate to 50 mL to obtain a Bupleurum ovale extract solution with a concentration of 0.4 g / mL.

[0020] Further preferably, in the preparation method of the in-situ gel of the Bupleurum ovale of the present invention, step S1 is specifically as follows: take 20 g of the Bupleurum ovale extract into a 200 mL conical flask, add 200 mL of 70% ethanol, add 2% activated carbon, decolorize for 80 min at 50°C, filter, wash the filter paper with 70% ethanol, combine the filtrate, recover the ethanol under reduced pressure, and concentrate the filtrate to 50 mL to obtain a Bupleurum ovale extract solution with a concentration of 0.4 g / mL.

[0021] In the method for preparing the large black bubble in situ gel of the present invention, the refrigeration conditions in step S2 are: 4° C., refrigeration for 24 hours.

[0022] The invention discloses an application of the large black bubble in-situ gel in preparing a drug for treating oral inflammation.

[0023] Beneficial effects of the present invention:

[0024] 1. The present invention uses an ultraviolet-visible spectrophotometer to establish a method for determining the content of total flavonoids in Psoralea corylifolia and its extracts. The establishment of this detection method has laid a solid foundation for the sustainable development of the effective ingredients of Psoralea corylifolia.

[0025] 2. The present invention adopts the previous research method to obtain the extract of the big black bubble. Since the drug color is dark, it is not conducive to the molding effect of the final product. The inventors have conducted a large number of investigations and tests on the factors affecting the decolorization effect by measuring the decolorization rate and the total flavonoids retention rate, and determined that the optimal decolorization process conditions are: the amount of activated carbon added is 2%, the decolorization temperature is 50°C, and the decolorization time is 80min. Under the above decolorization process conditions, the removal efficiency of the pigment can reach (35.11±2.29)%, and the retention rate of flavonoids is as high as (80.11±1.96)%. This process not only significantly improves the color quality of the total flavonoids of the big black bubble, but also effectively improves the purity of the total flavonoids of the big black bubble, providing valuable data support and research basis for the preparation of the in-situ gel of the big black bubble.

[0026] 3. The inventor conducted single factor investigation and orthogonal test investigation on the proportion of poloxamer 407, the proportion of poloxamer 188, and the proportion of drug content, and determined the optimal preparation process parameters: the addition amount of poloxamer 407 is 17%, the addition amount of poloxamer 188 is 3%, the proportion of the drug solution of the large black bubble extract is 15%, and the proportion of sodium phytate is 0.2%. The gel prepared under the above process parameters has a good molding effect, the gelation temperature is (36.1±0.44)℃, the gelation time is 95s, and the gelation dehydration is 0cm. The gel is liquid at room temperature and maintains the fluidity of the liquid drug. When the phase transition temperature is reached, it can be transformed into a semi-solid state, which increases the contact area between the affected area and the drug, and facilitates the continuous release of the drug in the lesion area in the oral cavity.

[0027] 4. According to the in vitro release determination method of sustained-release preparations in the 2020 edition of the Chinese Pharmacopoeia, the release rate of the Dawupao in-situ gel was investigated under the condition of simulating artificial saliva in vitro. The results showed that the product prepared under the above-mentioned optimal preparation process can slowly release the active ingredient (total flavonoids of Dawupao) at a non-constant rate for 9 hours, and the cumulative release rate is more than 90%. This shows that the Dawupao in-situ gel has significant characteristics of a sustained-release preparation.

[0028] 5. The present invention adopts modern preparation technology for the first time, combines the effective ingredient extract of Miao medicine Dawupao with in-situ gel technology to develop a new type of dental care product - Dawupao in-situ gel. The gel is liquid at room temperature. After entering the oral cavity (37°C), it undergoes a phase change due to the increase in temperature and becomes a gel state, and can adhere to the affected area for a long time. Compared with the existing market gel, the Dawupao gel provided by the present invention has the following significant advantages: ① Simple preparation process; ② After administration, it quickly forms a gel in the body, can be retained in the administration site for a long time, and has good drug sustained release characteristics; ③ It has good tissue compatibility and is biodegradable. There is no foreign body sensation after use, and the patient compliance is good; ④ Good fluidity, which can make the drug fully contact with the affected area; ⑤ Common excipients for temperature-sensitive gels, such as poloxamer, polyethylene glycol, etc., are safe and non-toxic, and can be used for in vivo administration with high safety. The present invention provides a scientific basis for the clinical application of Dawupao, and provides a reference for further research on new Chinese medicine preparations and oral care products with Dawupao as the main drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the linear regression curve of rutin;

[0030] Figure 2 The effect of different activated carbon addition amounts on the decolorization effect;

[0031] Figure 3 The effect of different decolorization time on decolorization effect;

[0032] Figure 4 The influence of different decolorization temperatures on the decolorization effect;

[0033] Figure 5 It is the liquid medicine of the extract of the giant radix scabra which has been decolorized by using the best decolorization process;

[0034] Figure 6 The effect of different amounts of poloxamer 407 on gel formation;

[0035] Figure 7 The effect of different amounts of poloxamer 188 on gel formation;

[0036] Figure 8 The effect of different proportions of liquid medicine on gelation;

[0037] Fig. 9 This is the state diagram of the large black bubble after in-situ gel phase transition (gelation);

[0038] Fig.10 This is the in vitro release curve of the thermosensitive oral Dawupao in-situ gel;

[0039] Fig.11 This is a product picture of the temperature-sensitive oral Dawubu in-situ gel. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is described in detail below in conjunction with specific embodiments. The following embodiments are only used for explanation and illustration, and do not constitute a limitation on the technical solution of the present invention.

[0041] Example 1

[0042] The formula of the in-situ gel of Psoralea corylifolia comprises the following components in percentage by weight: 17% of poloxamer 407, 3% of poloxamer 188, 15% of Psoralea corylifolia extract liquid, 0.2% of sodium phytate, and 64.8% of distilled water.

[0043] The preparation process of the above-mentioned large black bubble in-situ gel:

[0044] S1 Preparation of the extract of the giant aralia

[0045] Take 20g of the extract of the Radix Achyranthis Bidensii in a 200mL conical flask, add 200mL of 70% ethanol, add 2% activated carbon, decolorize at 50°C for 80min, filter, wash the filter paper with 70% ethanol, combine the filtrate, recover the ethanol under reduced pressure, and concentrate the filtrate to 50mL to obtain a Radix Achyranthis Bidensii extract solution with a concentration of 0.4g / mL.

[0046] S2 Preparation of gel solution

[0047] Weigh poloxamer 407 and poloxamer 188, place them in a conical flask, gradually add the above-prepared phytate solution and distilled water dissolved with 0.2% sodium phytate, seal the flask, and refrigerate at 4°C for 24 hours until the solution is completely dissolved and clear.

[0048] Example 2

[0049] The formula of the in-situ gel of the Herba Lysimachiae includes the following components in percentage by weight: 16% of poloxamer 407, 5% of poloxamer 188, 7.5% of Herba Lysimachiae extract liquid, 0.2% of sodium phytate, and 71.3% of distilled water.

[0050] The preparation process of the above-mentioned large black bubble in-situ gel:

[0051] S1 Preparation of the extract of the giant aralia

[0052] Take 20g of the extract of the Herba Lycopodiellae in a 200mL conical flask, add 200mL of 70% ethanol, add 0.5% activated carbon, decolorize for 120min at 80°C, filter, wash the filter paper with 70% ethanol, combine the filtrate, recover the ethanol under reduced pressure, and concentrate the filtrate to 50mL to obtain a Herba Lycopodiellae extract solution with a concentration of 0.4g / mL.

[0053] S2 Preparation of gel solution

[0054] Weigh poloxamer 407 and poloxamer 188, place them in a conical flask, gradually add the above-prepared phytate solution and distilled water dissolved with 0.2% sodium phytate, seal the flask, and refrigerate at 4°C for 24 hours until the solution is completely dissolved and clear.

[0055] Example 3

[0056] The formula of the in-situ gel of the Herba Lysimachiae includes the following components in percentage by weight: 20% of poloxamer 407, 1% of poloxamer 188, 20% of Herba Lysimachiae extract liquid, 0.2% of sodium phytate, and 58.8% of distilled water.

[0057] The preparation process of the above-mentioned large black bubble in-situ gel:

[0058] S1 Preparation of the extract of the giant aralia

[0059] Take 20g of the extract of the Herba Cynoglossifolia in a 200mL conical flask, add 200mL of 70% ethanol, add 3% activated carbon, decolorize at 80°C for 20min, filter, wash the filter paper with 70% ethanol, combine the filtrate, recover the ethanol under reduced pressure, and concentrate the filtrate to 50mL to obtain a Herba Cynoglossifolia extract solution with a concentration of 0.4g / mL.

[0060] S2 Preparation of gel solution

[0061] Weigh poloxamer 407 and poloxamer 188, place them in a conical flask, gradually add the above-prepared phytate solution and distilled water dissolved with 0.2% sodium phytate, seal the flask, and refrigerate at 4°C for 24 hours until the solution is completely dissolved and clear.

[0062] Example 4

[0063] The formula of the in-situ gel of the Herba Lysimachiae includes the following components in percentage by weight: 17% of poloxamer 407, 3% of poloxamer 188, 15% of Herba Lysimachiae extract liquid, 0.2% of sodium phytate, and 64.8% of distilled water.

[0064] The preparation process of the above-mentioned large black bubble in-situ gel:

[0065] S1 Preparation of the extract of the giant aralia

[0066] Take 20g of the extract of the Radix Achyranthis Bidensii in a 200mL conical flask, add 200mL of 70% ethanol, add 3% activated carbon, decolorize for 120min at 30°C, filter, wash the filter paper with 70% ethanol, combine the filtrate, recover the ethanol under reduced pressure, and concentrate the filtrate to 50mL to obtain a Radix Achyranthis Bidensii extract solution with a concentration of 0.4g / mL.

[0067] S2 Preparation of gel solution

[0068] Weigh poloxamer 407 and poloxamer 188, place them in a conical flask, gradually add the above-prepared phytate solution and distilled water dissolved with 0.2% sodium phytate, seal the flask, and refrigerate at 4°C for 24 hours until the solution is completely dissolved and clear.

[0069] Example 5

[0070] The above-mentioned large black bubble in-situ gel is used for preparing oral inflammation therapeutic drugs.

[0071] In order to further verify the reliability of the present invention and screen out the best solution, the inventor conducted a series of experiments, as follows:

[0072] 1. Preparation of the extract of Radix Achyranthis Bidentatae

[0073] Refer to "Study on the screening of anti-inflammatory drug effect sites of Dawupao and determination of its effective ingredients" [1] The method is used to prepare the extract of Dawupao, which is as follows: take 1kg of Dawupao and use 10 times the amount of 70% ethanol to reflux and extract three times, and the extraction time is 3h, 3h and 2h respectively; combine the filtrate, recover the ethanol until it is tasteless, and concentrate to 1g / mL to obtain the ethanol extract of Dawupao. Take an appropriate amount of the above-mentioned ethanol extract of Dawupao, extract it three times with 4 times the amount of ethyl acetate and n-butanol respectively, collect the extract, recover the solvent, and vacuum dry to obtain the dry extract of ethyl acetate and n-butanol extraction parts of Dawupao. Take the dry extracts of ethyl acetate and n-butanol parts and mix them evenly to obtain the effective ingredient extract of Dawupao.

[0074] 2. Decolorization of the extract of Dawupao

[0075] 1. Instruments and Materials

[0076] 1.1 Experimental Instruments

[0077] The experimental instruments are shown in Table 1.

[0078] Table 1 Experimental instruments

[0079]

[0080] 1.2 Experimental Reagents

[0081] The experimental reagents are shown in Table 2.

[0082] Table 2 Experimental reagents

[0083]

[0084] 2. Establishment of the determination method of total flavonoids content in Rhizoma Cibotii

[0085] 2.1 Preparation of test solution

[0086] The research group has previously studied the extraction process of total flavonoids from the anti-inflammatory part of the radix pyrifolia, so the radix pyrifolia extract was obtained using the previous research method. Since the drug is dark in color, it is not conducive to the molding effect of the final product, so the extract was decolorized. Take 2g of the radix pyrifolia extract powder, accurately weigh it, add 50mL of 70% ethanol, weigh it, ultrasonically dissolve it for 50min, the dissolution temperature is 60℃, the dissolution frequency is 53kHz, put it to room temperature, weigh it, make up the lost weight with 70% ethanol, shake it well, filter it, and you are done.

[0087] 2.2 Preparation of reference solution

[0088] Accurately weigh 9.91 mg of rutin reference substance, place it in a 25 mL volumetric flask, add 70% ethanol and shake to dissolve, dilute to the scale, shake well, and obtain a reference substance solution containing 0.3964 mg of rutin per 1 mL.

[0089] 2.3 Determination of UV detection conditions

[0090] 2.3.1 Determination of color developer

[0091] Weigh 2.413g of aluminum chloride hexahydrate and dissolve it in 100mL of distilled water to obtain 0.1mol / L aluminum chloride solution; take a certain amount of 0.2mol / L sodium acetate solution and add 0.2mol / L acetic acid solution, adjust the pH to 5.2 to obtain acetic acid-sodium acetate buffer solution; draw 0.5mL of 0.3964mg / mL rutin reference solution into a 10mL volumetric flask, accurately add 0.1mol / L aluminum chloride solution and 0.5mL sodium acetate-acetic acid buffer solution 2.0mL, dilute to the scale with 70% ethanol, shake well, and let it stand for 15min as the reference solution.

[0092] 2.3.2 Determination of color wavelength

[0093] Accurately pipette 50μL of the test solution prepared under "2.1" into a 10mL volumetric flask, add the color developer to obtain the test solution, and measure it at 200-600nm using a UV-visible spectrophotometer. The results show that the test sample has good absorption at 284nm, and the maximum absorption wavelength of the rutin reference substance is 273nm. The maximum absorption wavelengths of the two are relatively close, and the test solution after color development is light yellow, clear, and has no precipitation. Therefore, this method can be used for color development, and 273nm is selected as the detection wavelength, and the color development time is 15min.

[0094] 2.4 Methodological Review

[0095] 2.4.1 Drawing of Rutin Standard Curve

[0096] Accurately measure 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, and 0.80 mL of 0.3964 mg / mL rutin reference solution in a 10 mL volumetric flask, develop color according to the conditions in "2.3", develop color in the same way as a blank, and measure the absorbance. Draw a standard curve with rutin concentration as the horizontal axis and absorbance as the vertical axis. The linear regression equation is y=25.17x-0.0197, R 2 =0.9992, indicating that the reference solution showed a good linear relationship in the range of 0.0079 to 0.0316 mg / mL. The linear regression curve of rutin is shown in Figure 1 .

[0097] 2.4.2 Precision experiment

[0098] Prepare the test solution according to "2.1", accurately pipette 50 μL of the test solution, transfer it to a 10 mL volumetric flask, develop the color according to the method under "2.3", determine the total flavonoid content at a wavelength of 273 nm, and measure 6 times continuously. The results are shown in Table 3. From the results, it can be seen that the RSD is 0.92, indicating that the method has good precision.

[0099] Table 3 Precision experiment

[0100]

[0101] 2.4.3 Repeatability Experiment

[0102] Take 6 portions of the powder of the extract of the big wu bubble, prepare the test solution according to "2.1", use a pipette to draw 50 μL of the test solution into a 10 mL volumetric flask, develop the color according to the method under "2.3", measure the absorbance and calculate the total flavonoid content, and the results are shown in Table 4. As can be seen from Table 4, the flavonoid content of the 6 portions of the big wu bubble extract is relatively stable, with an RSD of 1.82. From the repeatability results, it can be seen that the flavonoid content of the big wu bubble extract is (155.29±2.82) mg / g.

[0103] Table 4 Repeatability experiment

[0104]

[0105] 2.4.4 Stability test

[0106] Take 1 portion of the powder of the extract of the Radix Achyranthis Bidentatae, prepare the test solution according to the method in "2.1", place it in a 10 mL volumetric flask, develop color according to the method in "2.3", measure the absorbance at 0, 2, 4, 8, 12, and 24 hours after color development, and calculate the total flavonoid content. The results are shown in Table 5. It can be seen from the results that this method has good stability within 24 hours, with an RSD of 0.84.

[0107] Table 5 Stability test

[0108]

[0109]

[0110] 2.4.5 Sample recovery experiment

[0111] Weigh 15.83 mg of rutin reference substance accurately, place it in a 100 mL volumetric flask, and dilute to volume with 70% ethanol to obtain a reference substance solution containing 0.1583 mg of rutin in 1 mL of solution. Weigh 6 portions of Dawupao extract powder accurately, 10 mg each, add 10 mL of 0.1583 mg / mL rutin reference substance solution, weigh the weight, ultrasonicate for 50 minutes, bring to room temperature, weigh, make up the lost weight with 70% ethanol, shake well, filter, and obtain. Take 1 mL and develop color according to the method under "2.3". The results are shown in Table 6. From the results, it can be seen that the average recovery rate of 6 Dawupao samples is 101.45%, and the RSD is 4.14, indicating that the sample recovery rate results are good.

[0112] Table 6 Sample recovery experiment

[0113]

[0114] 3. Research on the process of removing pigments from Dawupao activated carbon

[0115] 3.1 Calculation formula

[0116] The extract solution of the radix pyriferum (without color developer and buffer solution) was subjected to full wavelength scanning of the UV-visible spectrum in the wavelength range of 200-600nm, and the optimal detection wavelength was selected. The results showed that the total flavonoids solution of the radix pyriferum had the largest absorption peak at 224nm. The decolorization absorbance was measured at this wavelength, and the pigment removal rate was calculated according to the following formula.

[0117]

[0118] Determine the absorbance of total flavonoids of Radix Achyranthis Bidentatae before (after) decolorization, combine it with the regression equation of the rutin standard curve, and then calculate the retention rate of total flavonoids according to the following formula.

[0119]

[0120] Results The comprehensive weighted scoring method was used to measure the pigment removal rate and flavonoid retention rate respectively, which were used as evaluation indicators. The comprehensive score was calculated according to the following formula.

[0121]

[0122] 3.2 Single factor investigation

[0123] To explore the amount of activated carbon added (M / V), decolorization time (T 1 ), decolorization temperature (t1 ) on the decolorization effect of the extract of the big black bubble, and a single factor experiment was conducted with the flavonoid retention rate and the pigment removal rate as evaluation indicators. Prepare the test solution according to the method under "2.1", take 5mL in a test tube, weigh it, treat it according to the corresponding single factor conditions, filter it, make up the weight, take 50μL of the filtrate in a 10mL volumetric flask with 70% ethanol and add it to the scale line, 70% ethanol is used as a blank, measure the pigment removal rate, and develop the color according to the color development method under "2.3", and measure the flavonoid retention rate. Perform a single factor experiment on activated carbon decolorization according to "Table 7", by first fixing two factors and changing the third factor; then investigate the influence of this factor on the experimental results, and select the best conditions. Each factor level is measured in parallel 3 times, and the data is processed using the comprehensive weighted scoring method.

[0124] Table 7 Activated carbon decolorization single factor experiment

[0125]

[0126] 3.2.1 Activated carbon addition amount

[0127] First, the decolorization temperature was fixed at 50°C and the decolorization time was 80 minutes. By setting different amounts of activated carbon addition (the test solution prepared under "2.1" was used as the calculation standard), the effect of different amounts of activated carbon addition on the pigment removal effect of the large black bubble extract solution was studied. The results of the effect of different amounts of activated carbon addition on the decolorization effect are shown in Table 8 and Figure 2 . The experimental results show that the amount of activated carbon added is positively correlated with the decolorization effect, that is, the more the amount of activated carbon added increases, the more significant the decolorization effect. However, when the amount of activated carbon added exceeds 2.0%, the decolorization effect is no longer significantly improved, and it shows a downward trend. In addition, with the increase in the amount of activated carbon added, the loss of flavonoids will also increase accordingly. When the amount of activated carbon added is set to 2.0%, the comprehensive score is the highest, which is 89.20 points. After weighing the decolorization effect and flavonoid loss, the amount of activated carbon added was finally determined to be 2.0%.

[0128] Table 8 Decolorization effect of different activated carbon addition amounts

[0129]

[0130]

[0131] 3.2.2 Decolorization time

[0132] First, the concentration of activated carbon was fixed at 2.0% and the decolorization temperature was 50°C, and the decolorization time was investigated to select the optimal decolorization time. The results of the effects of different decolorization times on the decolorization effect are shown in Table 9 and Figure 3. The experimental results show that as the decolorization time increases, the decolorization effect gradually increases. When the decolorization time is 80 minutes, the decolorization effect is most significant, with a comprehensive score of 90.83. At this time point, the decolorization effect reaches the best. At the same time, the experiment also found that there is a negative correlation between the retention rate of flavonoids and the decolorization time, that is, the longer the decolorization time, the lower the retention rate of flavonoids. After comprehensive consideration, 80 minutes was finally determined to be the best decolorization time.

[0133] Table 9 Decolorization effect of different decolorization time

[0134]

[0135] 3.2.3 Decolorization temperature

[0136] First, the decolorization temperature was investigated under the conditions of 2.0% activated carbon addition and 80 minutes of decolorization time. Through the analysis of experimental data, it was found that the activated carbon treatment temperature with the best decolorization effect was 50°C. Under this condition, the decolorization score reached the highest value, which was 88.43 points. The results of the influence of different decolorization temperatures on the decolorization effect are shown in Table 10 and Figure 4 The results showed that with the increase of decolorization temperature, the retention rate of flavonoids showed a downward trend, especially at 60℃ and 70℃, the flavonoid retention rate decreased significantly. In order to achieve the best decolorization effect while maintaining the flavonoid content, 50℃ was selected as the decolorization temperature.

[0137] Table 10 Decolorization effect at different decolorization temperatures

[0138]

[0139] 3.3 Verification Experiment

[0140] The optimal decolorization conditions of the extract of the Chinese magnolia were obtained by single factor screening, and three decolorization experiments were carried out in parallel. The results are shown in Table 11. Under the conditions of 2.0% activated carbon addition, 50°C decolorization temperature, and 80 min decolorization time, the flavonoid retention rate was (80.11±1.96)%, and the decolorization rate was (35.12±2.29)%.

[0141] Table 11 Decolorization verification experiment of flavonoid extract of Radix Achyranthis Bidentatae

[0142]

[0143] 5 Conclusion

[0144] The optimal decolorization process conditions were determined through a series of single-factor experiments. The experimental results showed that under the conditions of 2.0% activated carbon addition, 50°C decolorization temperature, and 80 minutes of decolorization time, the pigment removal efficiency can reach (35.11±2.29)%, while the flavonoid retention rate is as high as (80.11±1.96)%. Figure 5 This process not only significantly improved the color quality of flavonoids from Psoralea corylifolia, but also effectively improved the purity of total flavonoids from Psoralea corylifolia. The determination of this process provides valuable data support and research basis for the preparation of in-situ gel of Psoralea corylifolia.

[0145] 3. Preparation of Thermosensitive Oral Dawububble In-Situ Gel

[0146] 1. Instruments and Materials

[0147] 1.1 Experimental Instruments

[0148] The experimental instruments are shown in Table 12.

[0149] Table 12 Experimental instruments

[0150]

[0151] 1.2 Experimental Reagents

[0152] The experimental reagents are shown in Table 13.

[0153] Table 13 Experimental reagents

[0154]

[0155] 2. Methods and Results

[0156] 2.1 Preparation of the extract of Psoralea corylifolia

[0157] Take 20g of the extract of the Herba Lycopodii in a 200mL conical flask, add 200mL of 70% ethanol, decolorize according to the optimal decolorization conditions of the Herba Lycopodii decolorization part, filter, wash the filter paper with 70% ethanol, combine the filtrate, recover the ethanol under reduced pressure, and concentrate the filtrate to 50mL to obtain a high-concentration Herba Lycopodii extract solution with a concentration of 0.4g / mL.

[0158] 2.2 Preparation of gel solution

[0159] Weigh an appropriate amount of the drug solution of the Psoralea corylifolia extract, weigh a certain proportion of the matrix (Poloxamer 407 / Poloxamer 188) and place them in a conical flask, gradually add the drug solution and distilled water (dissolve 0.2% of sodium phytate), seal the bottle, and place it in a refrigerator at 4°C for 24 hours until it is completely dissolved and clear, and set aside.

[0160] 2.3 Evaluation indicators

[0161] 2.3.1 Determination of gelation temperature

[0162] Determination of gelation temperature (T 2 ) The test tube inversion method can be used: 2 mL of gel solution is placed in a sealed test tube and placed in a water bath with gradually increasing temperature. Every time the temperature rises by 0.1°C, the test tube is inverted and observed for 30 seconds. The temperature is recorded when the gel does not flow. The three measurements are consistent, and 5 points are deducted for every 1°C below 34°C or every 1°C above 37°C.

[0163] 2.3.2 Dehydration during gelation

[0164] Pour 5 mL of gel solution into a 10 mL test tube and place it in a constant temperature water bath at 37°C for 30 minutes. The dehydration during the gelation process (L) is an indicator of the water holding capacity of the in situ thermosensitive gel. The water holding capacity of the gel is evaluated by observing the amount of dehydration at 37°C for 30 minutes: less than 0.5 mL of dehydration is scored as 100 points, 0.5 to 1.5 mL of dehydration is scored as 50 points, and more than 1.5 mL of dehydration is scored as 0 points.

[0165] 2.3.3 Determination of gel time

[0166] The gelation time (t 2 ), put 2mL gel solution in a sealed test tube, put it in a water bath at 37℃, invert the test tube every 5s, and the time when the gel does not fall after the test tube is inverted is the gelation time. The time with the same result after three parallel measurements is recorded as the gelation time of the sample. The gelation time in the gelation process is scored by a stopwatch: 100 points for a fast gelation time (≤50s), and 5 points for every 10s over 50s.

[0167] 2.3.4 Evaluation index calculation formula

[0168] Results The comprehensive weighted scoring method was used to measure the gelation temperature, gelation time and gel dehydration, and use them as evaluation indicators to calculate the comprehensive score according to the following formula.

[0169] Comprehensive score = gelation temperature (T 2 )×50%+gelation time (t 2 )×30%+gel dehydration condition (L)×20%

[0170] 2.4 Single Factor Experiment

[0171] In order to investigate the effects of the ratio of poloxamer 407 (P407), poloxamer 188 (P188) and drug content on in situ gelation, the gelation temperature (T 2 ), gel time (t 2) and the dehydration condition during gelation (L) were used as evaluation indicators for single factor experiments. The corresponding volume of stock solution was taken, prepared according to the corresponding single factor conditions, and then distilled water was added to 10g. Each factor level was measured in parallel 3 times. The single factor experiment of in situ gel preparation process was carried out according to "Table 14", and the data were processed using the comprehensive weighted scoring method.

[0172] Table 14 Single factor experiment of in situ gel preparation process

[0173]

[0174] 2.4.1 Effect of Poloxamer 407 on Gel Formation

[0175] From the preliminary experiment, it can be seen that when the percentage of P407 is lower than 14%, the gelling effect is poor. When the percentage is greater than 23%, the gelling temperature is lower than room temperature. When the mass concentration of P407 is 17%, the gelling temperature is closest to human body temperature. The mass fraction of P188 is fixed at 3% and the drug loading is 15%. Different proportions of P407 are prepared and the experiment is carried out according to the method under "2.3". The results are shown in Table 15. Figure 6 The results show that the gelling temperature and gelling time both decrease with the increase of P407 proportion. When the percentage of P407 is 17%, the highest comprehensive score is 94.75 points. Considering all factors, the proportion of P407 is 17%.

[0176] Table 15 Effect of different proportions of P407 on gelling

[0177]

[0178] 2.4.2 Effect of Poloxamer 188 on Gel Formation

[0179] When P188 is used alone as a gelling material, it has gelling ability only when the dosage is higher than 50%, and the gelling temperature is too high to be suitable for human body. However, using it as an auxiliary gelling material can significantly improve the gelling temperature of gel. The mass fraction of P407 is fixed at 17%, the amount of liquid medicine is 15%, and different proportions of P188 are prepared. Experiments are carried out according to the method under "2.3". The results are shown in Table 16. Figure 7 The results show that as the proportion of P188 increases, the gelation temperature rises and the gelation time increases, and the gelation time is inversely proportional to the proportion of P188. When the percentage of P188 is 3%, the highest comprehensive score is 92.75 points. Considering the comprehensive selection of P188, the proportion is 3%.

[0180] Table 16 Effect of different proportions of P188 on gelling

[0181]

[0182] 2.4.3 Effect of drug loading on gelation

[0183] The mass fraction of P407 was fixed at 17% and that of P188 at 3%. Different proportions of the drug solutions were prepared and the experiment was carried out according to the method under "2.3". The results are shown in Table 17. Figure 8 The results show that the influence of drug proportion on gelation temperature and gelation time is not obvious. When the proportion of drug solution of Dawupao extract is 15%, the highest comprehensive score is 93.25. Considering comprehensively, the proportion of drug solution is 15.0%.

[0184] Table 17 Effect of different proportions of main drugs on gelation

[0185]

[0186] 2.5 Orthogonal experiment

[0187] According to the results of the single factor experiment, gelation temperature, gelation time, and gel dehydration were used as evaluation indicators, P188 ratio (A), P407 ratio (B) and liquid ratio (C) were used as factors, and each factor had three levels, an error sequence (D) was added, and L9 (3 4 ) table to carry out orthogonal experiment. The orthogonal experiment design table is shown in Table 18.

[0188] Table 18 Factor level table of preparation process

[0189]

[0190]

[0191] The results are shown in Tables 19 and 20. Through data analysis, the maximum R value of the P188 ratio is 33.3, the R value of the liquid medicine ratio is 30.2, and the R value of the P407 ratio is 25.2. It can be seen that the order of factors affecting the preparation process of the thermosensitive oral large black bubble in situ gel is: P188 ratio> liquid medicine ratio> P407 ratio, that is, the P188 ratio has the greatest impact on the comprehensive score, and the P407 ratio has the least impact on the preparation process of the thermosensitive oral large black bubble in situ gel. Therefore, the optimal process condition is A 2 B 2 C 2 , that is, P188 accounts for 3.0%, liquid medicine accounts for 15%, and P407 accounts for 17%.

[0192] Table 19 Orthogonal results of preparation process prescription

[0193]

[0194] Table 20 Analysis of variance

[0195]

[0196] 2.6 Verification Experiment

[0197] Three batches of samples were prepared in parallel according to the optimal process screened out, and the gelation temperature, gelation time and dehydration were investigated. The results are shown in Table 21. 2 ) is (36.1±0.44)℃, gelation time (t 2 ) was 95s, the gel dehydration condition (L) was 0cm, and the comprehensive scores were 92.75, 92.50, and 93.25 points, respectively, indicating that the preparation process was stable and reliable.

[0198] Table 21 Verification experiment

[0199]

[0200] 3. Conclusion

[0201] The optimal preparation process parameters were determined by the analysis of single-factor orthogonal experiments. The results showed that the optimal preparation process parameters were: P188 addition amount was 3%, P407 addition amount was 17%, drug proportion was 15%, sodium phytate proportion was 0.2%, gelling temperature (T 2 ) is (36.1±0.44)℃, gelation time (t 2 ) is 95s, and the gel dehydration condition (L) is 0cm. The thermosensitive in-situ gel prepared under this condition has a good phase change effect. It not only increases the contact area of ​​the drug, but also maintains the fluidity of the liquid drug. At the same time, it can be transformed into a semi-solid state at the phase change temperature (36.1±0.44)℃, and continuously releases the drug in the oral lesion area. The state of the large black bubble in-situ gel after phase change (gelation) is shown in the figure below. Fig. 9 shown.

[0202] 4. Quality inspection of thermosensitive oral Dawupao in-situ gel

[0203] 1. Instruments and Materials

[0204] 1.1 Experimental Instruments

[0205] RC-3 dissolution tester (Tianjin Guoming Pharmaceutical Co., Ltd.), and other instruments are the same as above.

[0206] 1.2 Experimental Reagents

[0207] The experimental reagents are shown in Table 22.

[0208] Table 22 Experimental reagents

[0209]

[0210] 2. Methods and Results

[0211] 2.1 Content determination

[0212] 2.1.1 Preparation of test solution

[0213] Three batches of temperature-sensitive oral large black bubble in situ gel were prepared, 50 μL (weighed on an analytical balance to be 0.055 g) was taken from each batch and added to a 10 mL volumetric flask. After adjusting the volume, 1 mL was taken from each batch and added to three 10 mL volumetric flasks. A color developer and a buffer were added, and the volume was adjusted to 10 mL with 70% ethanol. The color was developed for 15 min and the absorbance was measured.

[0214] 2.1.2 Preparation of blank gel

[0215] Add all gel components except the extract of D. occidentalis to prepare blank gel. Prepare 3 batches of blank gel solution according to "2.1.1" and measure the absorbance.

[0216] 2.1.3 Experimental Results

[0217] The absorbance of the reference solution and the blank gel solution was measured at 273 nm. The absorbance of the three batches of blank gel solutions was 0, indicating that the blank gel components of Dawupao had no effect on the absorbance. The results of measuring the three batches of test gel solutions are shown in Table 23. The total flavonoid content of the temperature-sensitive oral Dawupao gel was (19.39±0.44) mg / g.

[0218] Table 23 Total flavonoid content of gel

[0219]

[0220] 2.2 Release rate detection

[0221] 2.2.1 Preparation of artificial saliva

[0222] Weigh 0.8 g of sodium chloride, 1.590 g of calcium chloride, 0.8 g of potassium chloride, 2 g of urea, 1.56 g of sodium phosphate, and 0.031 g of sodium sulfide nonahydrate and dissolve them in 800 mL of deionized water preheated to an appropriate temperature to ensure that all solid substances are completely dissolved. Subsequently, filter the solution through filter paper to remove possible undissolved solids or impurities. After the solution is cooled to room temperature, continue to add deionized water to a total volume of 2000 mL. Use 1 mol / L sodium hydroxide solution to adjust the solution pH to 7, and store it in a refrigerator at 4°C for subsequent use.

[0223] 2.2.2 Study on the release rate of thermosensitive oral Dawupao in-situ gel

[0224] The release rate of the thermosensitive oral in-situ gel was determined by the slurry method. The in vitro release was carried out in the RC-3 dissolution tester. The medium was simulated artificial saliva with a volume of 500 mL, which met the conditions of the leak tank. The in-situ gel solution of the in-situ gel was added to the 3 mL vial, and the temperature was adjusted to about 37 ° C to make it a semi-solid gel. After preparing the fresh artificial saliva, its temperature was gradually raised to close to the human body temperature during gentle stirring, and the temperature was basically maintained at about 37 ° C, with an error of no more than 0.5 ° C, for subsequent use. Subsequently, the vial containing the drug was placed at the bottom of the 500 mL dissolution instrument cup. Then, artificial saliva was slowly poured along the inner wall of the dissolution instrument cup until the vial was completely filled with the medium. During this process, the stirring speed needs to be kept at 50 r / min, and the operating temperature of the entire system needs to be controlled at about 37 ° C, with an error of no more than 0.5 ° C. In order to evaluate the release of the drug, it is necessary to take samples at different time points. The specific operation is to take 3 mL of the dissolution solution and place it in a 10 mL volumetric flask at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 9 hours respectively (take 3 mL of blank artificial saliva to test whether the blank saliva has an effect on the absorbance), and add 3 mL of fresh constant temperature artificial saliva at the same time, determine the content according to the content determination method of the sample solution, and calculate the cumulative release. Determine the release characteristics and rate of the drug in artificial saliva.

[0225] 2.2.3 Experimental results

[0226] Artificial saliva and blank gel had no effect on the determination of total flavonoids in P. truncatula. Fig.10 It can be seen that the solubility of the thermosensitive oral Dawupao in-situ gel gradually increases with time and gradually dissolves. It can release the active ingredients slowly and at a non-constant rate, and can release continuously for 9 hours. The final solubility is higher than 90%, indicating that the Dawupao in-situ gel has significant characteristics of a sustained-release preparation.

[0227] Table 24 In vitro release of thermosensitive oral large black bubble in situ gel

[0228]

[0229]

[0230] 3. Conclusion

[0231] After the activated carbon decolorized Dawupao extract solution passed a series of single factor experiments, orthogonal experiments, and verification experiments, the optimal preparation process was obtained. The total flavonoid content of the active ingredient in the thermosensitive Dawupao in-situ gel prepared under the preparation process conditions was (19.39±0.44) mg / g. The release results showed that the release of the thermosensitive oral Dawupao in-situ gel was greater than 9h, and the release rate could reach more than 90%. The prepared Dawupao in-situ gel product is shown in the figure. Fig.11 The results showed that the product prepared under this process parameter had a sustained-release effect. It can provide a useful reference for the further research and development of new traditional Chinese medicine preparations with Dawupao as the main drug.

[0232] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0233] References:

[0234] [1] Tang Nian, Wang Qun, Zhang Meili, Wang Jiaojiao, et al. Screening of anti-inflammatory efficacy sites of Radix Aconiti Lateralis Preparata and determination of its active ingredient content[J]. World Latest Medical Information Abstracts. 2022, 22(69): 96-103.

Claims

1. A large black bubble in situ gel, characterized in that The invention comprises the following components in percentage by weight: 16-20% of poloxamer 407, 1-5% of poloxamer 188, 7.5-20% of phytic acid sodium, 0.2% of sodium phytate and 54.8-75.3% of distilled water.

2. The large black bubble in situ gel according to claim 1, characterized in that The invention comprises the following components in percentage by weight: 16-18% of poloxamer 407, 2-4% of poloxamer 188, 12.5-17.5% of phytic acid sodium and 0.2% of sodium phytate, and 60.3-69.3% of distilled water.

3. The large black bubble in situ gel according to claim 2, characterized in that The invention comprises the following components in percentage by weight: 17% of poloxamer 407, 3% of poloxamer 188, 15% of aconite extract liquid, 0.2% of sodium phytate and 64.8% of distilled water.

4. The large black bubble in situ gel according to any one of claims 1 to 3, characterized in that: The concentration of the extract of the Herba Eupatorii is 0.4 g / mL.

5. The process for preparing the large black bubble in-situ gel according to any one of claims 1 to 3, characterized in that: The steps include: S1 Preparation of the extract of Bupleurum truncatum: Take an appropriate amount of the extract of the Chinese prickly ash, add 70% ethanol, add activated carbon for decolorization, filter, wash the filter paper with 70% ethanol, combine the filtrate, recover the ethanol under reduced pressure, and concentrate the filtrate to obtain a Chinese prickly ash extract solution; S2 Preparation of gel solution: Weigh poloxamer 407 and poloxamer 188, place them in a conical flask, gradually add the above-prepared radix eryngii extract solution and distilled water dissolved with 0.2% sodium phytate, seal, and refrigerate until they are completely dissolved and clear.

6. The preparation process according to claim 5, characterized in that: Step S1 is specifically as follows: 20 g of the extract of the Herba Lycopodiellae is put into a 200 mL conical flask, 200 mL of 70% ethanol is added, 0.5-3% of activated carbon is added, and the mixture is decolorized for 20-120 min at a temperature of 30-80° C., filtered, the filter paper is washed with 70% ethanol, the filtrate is combined, the ethanol is recovered under reduced pressure, and the filtrate is concentrated to 50 mL to obtain a Herba Lycopodiellae extract solution with a concentration of 0.4 g / mL.

7. The preparation process according to claim 6, characterized in that: Step S1 is specifically as follows: 20 g of the extract of the Herba Lycopodiellae is put into a 200 mL conical flask, 200 mL of 70% ethanol is added, 1.0-2.5% of activated carbon is added, and the mixture is decolorized for 60-120 min at a temperature of 40-60° C., filtered, the filter paper is washed with 70% ethanol, the filtrate is combined, the ethanol is recovered under reduced pressure, and the filtrate is concentrated to 50 mL to obtain a Herba Lycopodiellae extract solution with a concentration of 0.4 g / mL.

8. The preparation process according to claim 7, characterized in that: Step S1 is specifically as follows: take 20 g of the extract of the Myrica chinensis leaf in a 200 mL conical flask, add 200 mL of 70% ethanol, add 2% activated carbon, decolorize for 80 minutes at a temperature of 50°C, filter, wash the filter paper with 70% ethanol, combine the filtrate, recover the ethanol under reduced pressure, and concentrate the filtrate to 50 mL to obtain a Myrica chinensis leaf extract solution with a concentration of 0.4 g / mL.

9. The preparation process according to claim 5, characterized in that: The refrigeration conditions in step S2 are: 4° C., refrigeration for 24 hours.

10. Use of the large black bubble in situ gel according to any one of claims 1 to 3 in preparing a drug for treating oral inflammation.

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