Cynanchum paniculatum total phenol extract as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicines, and relates to a paniculate swallowwort root total phenol extract, a preparation method thereof and application of the paniculate swallowwort root total phenol extract in preparation of a medicine for protecting helicobacter pylori infection, and the paniculate swallowwort root total phenol extract is prepared by the following method: adding a paniculate swallowwort root medicinal material and sodium chloride into water together, adjusting the pH value to be alkaline, and soaking; carrying out steam distillation at 250-270 DEG C, and carrying out redistillation on the collected fraction to obtain a distillate; storing the distillate at 0-4 DEG C for 12-48 hours, and then carrying out suction filtration, collection and drying to obtain a paniculate swallowwort root total phenol extract which is a needle-like white crystal; the mass percent content of paeonol in the paniculate swallowwort root total phenol extract is 90% or above. The cynanchum paniculatum total phenol extract containing specific functional components is prepared through process treatment of cynanchum paniculatum, and the cynanchum paniculatum total phenol extract has an excellent helicobacter pylori inhibition effect and an obvious elimination effect on gastrointestinal inflammation caused by pylorus infection.

Description

Technical Field

[0001] The invention relates to the field of medicines, and in particular to a Cynanchum cynanchum total phenol extract and a preparation method and application thereof. Background Art

[0002] Cynanchum chinense is a common Chinese medicinal material with warm nature and pungent taste. It has the effects of dispelling wind, removing dampness, relieving pain and relieving itching. It is often used for rheumatic pain, stomach pain, toothache, back pain, pain from falls, urticaria, eczema and other diseases. Modern pharmacological studies have shown that paeonol can inhibit the production of intracellular O2-free radicals, and has sedative, analgesic, antibacterial, anti-inflammatory, detoxifying, antiviral, anti-tumor, anti-allergic, lipid-lowering and other effects. It is the main active ingredient of Cynanchum chinense.

[0003] Patent CN117530930A discloses a Cynanchum chinense extract and a preparation method thereof, a Cynanchum chinense extract and a preparation method thereof, which collects paeonol by steam distillation and cooling crystallization, has a stable process and a high transfer rate of paeonol. This method can reduce the sublimation and volatilization of paeonol, improve the extraction efficiency of paeonol, and thereby increase the paeonol content of the Cynanchum chinense formula granules.

[0004] Patent CN110227091A discloses the extraction of the medicinal ingredients of Cynanchum chinense, its formula granules and preparation method. The water distillation method is used to simultaneously prepare the inclusion compound of the volatile oil, which retains the volatile oil components to the maximum extent. In addition, the paeonol in the prepared medicinal ingredients is highly stable and not easily destroyed, thereby improving the quality of the finished formula granules.

[0005] Cynomorium root mainly exerts its effects through its effective components. Effective components with different contents have different effects. Different processing processes will obtain Cynomorium root total phenol extracts containing different effective components. However, there is little research on its effective components in the prior art. Therefore, it is often difficult to obtain Cynomorium root total phenol extracts with excellent application effects. Since decoction (including water extract) is still the main form of administration in clinical application of traditional Chinese medicine, there are few reports on the anti-inflammatory effect of Cynomorium root extracts. It is very meaningful to study the effect of Cynomorium root total phenol extracts on gastrointestinal mucosal damage caused by Helicobacter pylori infection. Summary of the invention

[0006] The purpose of the present invention is to solve the above problems and to provide a Cynanchum chinense total phenol extract and a preparation method thereof, as well as the application of the extract in the preparation of drugs for protecting Helicobacter pylori infection.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for preparing a Cynanchum chinense total phenolic extract, which is prepared by the following method:

[0009] Add the Radix Cynanchum wilfordii and sodium chloride into water, adjust the pH to alkaline, and soak;

[0010] Perform steam distillation at 250-270°C, and redistill the collected fraction to obtain a distillate;

[0011] The distillate is stored at 0-4°C for 12-48 hours, then filtered, collected, and dried to obtain a needle-shaped white crystal total phenol extract of Cynanchum chinense;

[0012] The mass percentage content of paeonol in the Cynanchum chinense total phenol extract is above 90%.

[0013] As a preferred technical solution of the present invention, the added amounts of the Cynanchum chinense medicinal material, sodium chloride and distilled water are 180-220 g: 35-40 g: 2500-3500 mL.

[0014] As a preferred technical solution of the present invention, the water is distilled water;

[0015] The amount of the Cynanchum chinense medicinal material, sodium chloride and distilled water added is 195-205g: 35-37g: 2900-3100mL;

[0016] Preferably, the added amount of the Cynanchum chinense medicinal material, sodium chloride and distilled water is 200g:36g:3000mL.

[0017] As a preferred technical solution of the present invention, the pH is adjusted to 8-9 by adding an alkaline substance, preferably, the pH is adjusted to 7.8-8.2, and most preferably, the pH is adjusted to 8;

[0018] And / or, the alkaline substance includes sodium hydroxide and potassium hydroxide.

[0019] As a preferred technical solution of the present invention, the soaking time is 1 to 2 hours, preferably 1.5 hours.

[0020] As a preferred technical solution of the present invention, steam distillation is carried out at 260° C. for 3 to 7 hours until no turbid distillate drips out.

[0021] As a preferred technical solution of the present invention, the distillate is stored at 4°C for 36 hours and then filtered. After collection and drying, the needle-shaped white crystals are obtained and stored in a vacuum dryer. The mass percentage content of paeonol in the Cynanchum wilfordii total phenol extract is above 93%.

[0022] A Cynanchum chinense total phenolic extract is prepared by using the above-mentioned preparation method of the Cynanchum chinense total phenolic extract.

[0023] Application of a Cynanchum chinense total phenol extract in preparing a drug for protecting against Helicobacter pylori infection.

[0024] The Cynanchum chinense total phenol extract is used as an active component and is added with auxiliary materials to prepare corresponding Chinese medicine solid preparations, including tablets and granules. The finished product is easy to prepare, has little toxic and side effects, and is safe and efficient.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The Cynanchum chinense total phenol extract of the present invention is a Chinese medicine extract, which has less toxic side effects and is safe and efficient compared with traditional Western medicine, and has obvious therapeutic effect on treating gastric inflammation caused by Helicobacter pylori infection. The present invention adopts a steam distillation and weighted distillation extraction method, and the Cynanchum chinense total phenol extract obtained has high purity. At the same time, the method has low energy consumption and high yield, is simple to operate and is easy to convert to industrial production, which promotes the application of the Cynanchum chinense total phenol extract in drugs for protecting Helicobacter pylori infection.

[0027] The present invention has been verified through experiments that the prepared Cynanchum chinense total phenol extract has obvious repairing effect on gastrointestinal mucosal damage caused by Helicobacter pylori infection, obvious alleviating effect on gastrointestinal inflammation caused by Helicobacter pylori infection, and obvious effect of inhibiting gastrointestinal Helicobacter pylori. It has obvious improvement effect on the pathological structure of intestinal mucosa of zebrafish caused by Helicobacter pylori infection and gastrointestinal mucosal damage, and the efficacy is positively correlated with the dosage. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a typical image of the gastrointestinal area of ​​zebrafish after treatment with Cynanchum quinata total phenols (the yellow dotted area is the analyzed part of the zebrafish gastrointestinal tract);

[0029] Figure 2 is the gastrointestinal tract area of ​​zebrafish after treatment with Cynanchum quinata total phenols;

[0030] Figure 3 This is a typical image of neutrophils in the gastrointestinal tract of zebrafish after treatment with Cynanchum cyrtonema total phenols (the yellow dotted area is the zebrafish intestinal analysis area, and the green fluorescent particles are neutrophils);

[0031] Figure 4 is the number of neutrophils in the gastrointestinal tract of zebrafish after treatment with Cynanchum quinata total phenols;

[0032] Figure 5 This is a typical fluorescence image of Helicobacter pylori in the gastrointestinal tract of zebrafish after treatment with total phenols from Cynanchum chinense (the yellow dotted box is the analyzed part of the zebrafish intestine);

[0033] Figure 6 is the fluorescence intensity of Helicobacter pylori in the gastrointestinal tract of zebrafish after treatment with total phenols from Cynanchum paniculatum;

[0034] Figure 7 This is a typical image of zebrafish intestinal pathological section after treatment with Cynanchum paniculatum total phenols (the red dotted area is the observation area of ​​zebrafish intestine). DETAILED DESCRIPTION

[0035] The present invention is described in detail below with reference to specific embodiments.

[0036]

Preparation of total phenolic extract of Cynanchum chinense

[0037] The total phenolic extract of Cynanchum chinense was prepared according to the following processes.

[0038] 1. Particle size of medicinal pieces

[0039] The Cynanchum chinense slices (2205119, Sichuan Xinhehua Chinese Medicine Slices Co., Ltd.) were crushed and passed through the No. 2, No. 3, No. 4 and other standard pharmacopoeia sieves, and about 40g of powder was weighed and placed in a 1000mL round-bottom flask, and 150g / kg of NaCl of the crude drug was added, and 600mL of distilled water (pH 6) was used as the extraction solvent. After appropriate soaking, steam distillation was performed at 240°C, and distillation was stopped when no turbid distillate dripped out; the distillate was redistilled, and the distillate was collected. After standing at 4°C for 24h, the crystals were collected by suction filtration; after vacuum drying at 40°C, the total phenol extract of Cynanchum chinense was obtained. The results are shown in Table 1.

[0040] Table 1 Investigation results of particle size of decoction pieces (n=3)

[0041]

[0042] Experiments show that the extraction results are better when the material is crushed and then passed through a No. 2 sieve.

[0043] 2.NaCl dosage

[0044] Weigh 40g of powder (pass through No. 2 sieve, 2205119, Sichuan Xinhehua Chinese Medicine Piece Co., Ltd.), place in a 1000mL round-bottom flask, add 600mL of solution (pH 6) containing NaCl (120g / kg, 150g / kg, 180g / kg crude drug), soak appropriately, perform steam distillation at 240°C, and stop distillation when no more turbid distillate drips out; redistillate the distillate, collect the distillate, refrigerate and stand at 4°C for 24h, collect the crystals by suction filtration, dry in vacuum at 40°C, and weigh to obtain the total phenol extract of Cynanchum cyrtonema. The results are shown in Table 2.

[0045] Table 2 Investigation results of NaCl amount (n=3)

[0046]

[0047] Experiments show that the extraction results are better when the NaCl dosage is 180g / kg crude drug.

[0048] 3. Extraction solvent pH

[0049] Weigh 40g of powder (pass through No. 2 sieve), place in a 1000mL round-bottom flask, add 600mL of NaCl solution (pH 5, 6, 7, 8) containing 180g / kg crude drug, soak appropriately, perform steam distillation at 240°C, and stop distillation when no more turbid distillate drips out; redistillate the distillate, collect the distillate, refrigerate and stand at 4°C for 24h, and collect the crystals by suction filtration; after vacuum drying at 40°C, weigh, and obtain the total phenolic extract of Cynanchum cyrtonema. The results are shown in Table 3.

[0050] Table 3 Soaking pH value investigation results (n=3)

[0051]

[0052] Experiments show that the results are better when the solvent pH is 8.

[0053] 4. Extraction temperature

[0054] Weigh 40g of powder (pass through No. 2 sieve, 2205119, Sichuan Xinhehua Chinese Medicine Piece Co., Ltd.), put it in a 1000mL round-bottom flask, add 600mL (pH 8) of NaCl solution containing 180g / kg crude drug, soak it appropriately, and steam distill it at 200, 240, 260, and 280°C, and stop distilling when no more turbid distillate drips out; redistillate the distillate, collect the distillate, refrigerate and stand it at 4°C for 24h, and collect the crystals by suction filtration; after vacuum drying at 40°C, weigh it, and the total phenol extract of Cynanchum chinense is obtained. The results are shown in Table 4.

[0055] Table 4 Extraction temperature investigation results (n=3)

[0056]

[0057] Experiments show that the results are better when the extraction temperature is 260℃.

[0058] 5. Crystallization temperature

[0059] Weigh 40g of coarse powder (passed through No. 2 sieve, 2205119, Sichuan Xinhehua Chinese Medicine Pieces Co., Ltd.), put it in a 1000mL round-bottom flask, add 600mL of NaCl solution (pH 8) containing 180g / kg crude drug, soak it appropriately, and steam distill it at 260°C until no more turbid distillate drips out, stop distillation; redistillate the distillate, collect the distillate, let it stand for 24h at 4°C and 25°C, respectively, and collect the crystals by suction filtration; after vacuum drying at 40°C, weigh it, and the total phenol extract of Cynanchum cyrtonema was obtained. The results are shown in Table 5.

[0060] Table 5 Crystallization temperature investigation results (n=3)

[0061]

[0062] Experiments show that the results are better when the crystallization temperature is 4°C.

[0063] 6. Crystallization time

[0064] Weigh 40g of coarse powder (passed through No. 2 sieve, 2205119, Sichuan Xinhehua Chinese Medicine Pieces Co., Ltd.), put it in a 100mL round-bottom flask, add 600mL (pH 8) of NaCl solution containing 180g / kg crude drug, soak it appropriately, and steam distill it at 260°C until no more turbid distillate drips out, stop distillation; redistillate the distillate, collect the distillate, let it stand at 4°C for 12h, 24h and 36h, respectively, and collect the crystals by suction filtration; after vacuum drying at 40°C, weigh it, and the total phenol extract of Cynanchum cyrtonema was obtained. The results are shown in Table 6.

[0065] Table 6 Crystallization time investigation results (n=3)

[0066]

[0067] Experiments show that the results are better when the crystallization time is 36h.

[0068] 7. Small test verification

[0069] Weigh 40g of powder (pass through No. 2 sieve, 20221119, Shanghai Yanghetang Chinese Medicine Piece Co., Ltd.), put it in a 1000mL round-bottom flask, add 600mL (pH 8) of NaCl solution containing 180g / kg crude drug, soak it appropriately, and steam distill it at 260°C until no more turbid distillate drips out, stop distillation; redistillate the distillate, collect the distillate and let it stand for 36h at 4°C, and collect the crystals by suction filtration; after vacuum drying at 40°C, weigh it, and get the total phenol extract of Cynanchum chinense. The results are shown in Table 7.

[0070] Table 7 Results of three batches of small tests (n=3)

[0071]

[0072] Note: The "content" in Tables 1 to 7 refers to the total phenol content of Cynanchum chinense in the same batch of medicinal pieces tested under certain process conditions, and the average value of three parallel experiments is taken.

[0073] [Example]

[0074] The total phenol extract of Cynanchum cyrtonema was prepared according to the optimal process conditions determined above

[0075] Weigh 200g of Radix Cynanchii and 36g of NaCl, mix, add 3000mL of distilled water (adjusted to pH 8 with sodium hydroxide), soak for 1.5h, and steam distill (about 5h) at 260°C until no turbid distillate drips out. The collected fractions are then redistilled, and the distillate obtained is stored at 4°C for 36h and then filtered. The obtained product is collected, dried, and stored in a vacuum dryer at 40°C. The obtained product is needle-shaped white crystals, i.e., Radix Cynanchii total phenolic extract (CP-VE), and its paeonol content is 93.29wt% after testing.

[0076]

Efficacy test of total phenols in Cynanchum chinense

[0077] 1. Test materials

[0078] 1.1. Sample preparation information

[0079] Administration: The Cynanchum chinense total phenolic extract (CP-VE) prepared in the example is needle-shaped white crystals and is stored in a vacuum desiccator at room temperature. It is prepared into a 100 mM mother solution with DMSO and stored at -20°C.

[0080] Positive control: Clarithromycin, white powder, batch number E1529013, Shanghai Aladdin Biochemical Technology Co., Ltd., stored at 4°C. Prepared into 16.7 mM stock solution with DMSO and stored at -20°C.

[0081] 1.2. Experimental animals and bacteria

[0082] Zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / LCaCO3), the experimental animal use license number was: SYXK(Zhejiang)2022-0004, and the breeding management met the requirements of the international AAALAC certification (certification number: 001458).

[0083] Transgenic neutrophil green fluorescent zebrafish (MPX) were bred by natural pair mating. Zebrafish aged 3 days post fertilization (3dpf) were used to evaluate the efficacy of Cynanchum chinense total phenol in alleviating gastrointestinal inflammation in Pylori infection.

[0084] Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish aged 3 dpf were used to determine the maximum detectable concentration (MTC) of total phenols from Cynanchum chinense, the repair of gastrointestinal mucosal damage caused by Helicobacter pylori infection, the efficacy of inhibiting gastrointestinal Helicobacter pylori, and gastrointestinal histopathological evaluation.

[0085] Helicobacter pylori, Helicobacter pylori culture medium (7 mL of sterile defibrinated sheep blood and 1 tube of Helicobacter pylori additive per 100 mL), microaerophilic, 37°C, 150 rpm shaking culture.

[0086] 1.3. Instruments, consumables and reagents

[0087] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); electric focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan); slicer (KD2258, Jinhua Kedi Medical Instrument Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); 6-well plate (Nest Biotech, China), constant temperature oscillator (HZ-9211K, Taicang Hualida Experimental Equipment Co., Ltd., China); vertical laminar flow clean bench (CA-1390-1, Shanghai Shangjing Purification Equipment Co., Ltd., China); desktop large-capacity low-speed centrifuge (TD5A, Changsha Yingtai Instrument Co., Ltd., China).

[0088] Trinitrobenzenesulfonic acid (TNBS, batch number SLCK4178, Sigma, USA), methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, batch number 20171016, Sinopharm Chemical Reagent Co., Ltd., China); 4% tissue cell fixative (batch number 20210828, Beijing Solebold Technology Co., Ltd., China); xylene (batch number 20201113, Sinopharm Chemical Reagent Co., Ltd., China); PBS buffer (batch number 70115000, Biosharp, China); Mayer's hematoxylin staining solution (batch number 20210506, Shanghai Yihe Biotechnology Co., Ltd., China); eosin staining solution (batch number 20210506, Shanghai Yihe Biotechnology Co., Ltd., China). He Biotechnology Co., Ltd., China); neutral gum (batch number 330A021, Solarbio, China); Huabao high-efficiency section paraffin (batch number 202101102, Shanghai Huayong Paraffin Co., Ltd., China); anhydrous ethanol (batch number 20210529, Sinopharm Chemical Reagent Co., Ltd., China); Helicobacter pylori culture medium (liquid) (batch number 20200507, Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd., China); defibrinated sheep blood (batch number 20211026, Haibo Biotechnology Co., Ltd., China); Helicobacter pylori additive (batch number 20211104, Haibo Biotechnology Co., Ltd., China); CM-DiI (batch number 2335589, Thermo Fisher Scientific (China) Co., Ltd., China).

[0089] 2. Detection Method

[0090] 2.1. MTC determination

[0091] 3dpf wild-type AB strain zebrafish were randomly selected in 6-well plates, and 30 zebrafish were treated in each well (experimental group). Total phenols of Cynanchum indica were given in water (concentrations shown in Table 8). A normal control group and a model control group were set up at the same time, and the volume of each well was 3mL. Except for the normal control group, the other experimental groups were first given TNBS in water. After treatment at 28℃ for 48h, TNBS was removed, and 5×10 7 The gastrointestinal mucosal injury model of Helicobacter pylori infection in zebrafish was established by using CFU / mL Helicobacter pylori. After treatment at 28℃ for 24h, the MTC of Cynanchum chinense total phenols on the model zebrafish was determined.

[0092] 2.2. Evaluation of the efficacy of repairing gastrointestinal mucosal damage caused by Pylori infection

[0093] 3dpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). Total phenols of Cynanchum indica were given in water (concentrations are shown in Table 9), and clarithromycin was 167μM in the positive control group. A normal control group and a model control group were also set up. The volume of each well was 3mL. Except for the normal control group, TNBS was first given in water to the other experimental groups. After 48h of treatment at 28℃, TNBS was removed, and 5×10 7 The gastrointestinal mucosal injury model of Helicobacter pylori infection in zebrafish was established by using CFU / mL Helicobacter pylori. After treatment at 28℃ for 24h, 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope and the pictures were saved. The data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the gastrointestinal area of ​​zebrafish. The statistical analysis results of this indicator were used to evaluate the repair effect of Cynanchum chinense total phenols on gastrointestinal mucosal injury caused by Helicobacter pylori infection in zebrafish. The statistical analysis results were expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0094] 2.3. Evaluation of efficacy in alleviating gastrointestinal inflammation caused by pylori infection

[0095] 3dpf transgenic neutrophil green fluorescent MPX strain zebrafish were randomly selected in 6-well plates, and 30 zebrafish were treated in each well (experimental group). Total phenols of Cynanchum indica were given in water (concentrations are shown in Table 10), clarithromycin concentration of 167μM was given to the positive control group, and a normal control group and a model control group were set up at the same time. The volume of each well was 3mL. Except for the normal control group, the other experimental groups were first given TNBS in water, and after 48h treatment at 28℃, TNBS was removed, and 5×10 7 The gastrointestinal mucosal injury model of Helicobacter pylori infection in zebrafish was established by using 1000 CFU / mL Helicobacter pylori. After 24 h of treatment at 28°C, 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope and the pictures were saved. The data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the number of zebrafish intestinal neutrophils. The statistical analysis results of this indicator were used to evaluate the efficacy of Cynanchum chinense total phenols in relieving gastrointestinal inflammation in zebrafish Helicobacter pylori infection. The statistical analysis results were expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0096] 2.4. Evaluation of efficacy in inhibiting gastrointestinal Helicobacter pylori

[0097] 3dpf wild-type AB strain zebrafish were randomly selected in 6-well plates, and 30 zebrafish were treated in each well. Total phenols of Cynanchum indica were given in water (concentrations are shown in Table 11), clarithromycin concentration of 167μM was given to the positive control group, and a normal control group and a model control group were set up at the same time, with a volume of 3mL per well. Except for the normal control group, TNBS was first given in water to the other experimental groups. After treatment at 28℃ for 48h, TNBS was removed, and 5×10 7 The gastrointestinal mucosal injury model of Helicobacter pylori infection in zebrafish was established by using the CFU / mL Helicobacter pylori. After 24 h of treatment at 28°C, 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope and the pictures were saved. The data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the fluorescence intensity of the zebrafish gastrointestinal tract. The statistical results of this indicator were used to evaluate the inhibitory effect of Cynanchum chinense total phenols on Helicobacter pylori. The statistical processing results were expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0098] 2.5. Gastrointestinal histopathological evaluation

[0099] 3dpf wild-type AB strain zebrafish were randomly selected in 6-well plates, and 30 zebrafish were treated in each well (experimental group). Total phenols of Cynanchum indica were given in water (concentrations are shown in Table 11), and clarithromycin was 167μM in the positive control group. A normal control group and a model control group were also set up. The volume of each well was 3mL. Except for the normal control group, TNBS was first given in water in the other experimental groups. After 48h of treatment at 28℃, TNBS was removed, and 5×10 7 CFU / mL Helicobacter pylori was used to establish a gastrointestinal mucosal injury model of zebrafish Helicobacter pylori infection. After treatment at 28℃ for 24h, the zebrafish in each group were fixed, dehydrated, embedded, sectioned, and stained with H&E for histopathological analysis and observation.

[0100] 3. Test results

[0101] 3.1.MTC

[0102] Under the experimental conditions, the maximum detection concentration (MTC) of Cynanchum chinense total phenols on the gastrointestinal mucosal injury model zebrafish infected with Helicobacter pylori was 125 μM. See Table 8 for details.

[0103] Table 8 Experimental results of the concentration of Cynanchum chinense total phenol in repairing gastrointestinal mucosal damage in Pylorus infection (n=30)

[0104]

[0105] 3.2. Repair of gastrointestinal mucosal damage caused by Pylori infection

[0106] Under the experimental conditions, total phenols from Cynanchum chinense have a significant repairing effect on gastrointestinal mucosal damage caused by Helicobacter pylori infection. See Table 9 for details. Figure 1 , 2 .

[0107] Table 9 Experimental results of evaluating the efficacy of Cynanchum chinense total phenols in repairing gastrointestinal mucosal damage caused by Helicobacter pylori infection (n=10)

[0108]

[0109] Compared with the model control group, *p<0.05, ***p<0.001

[0110] 3.3. Efficacy of reducing gastrointestinal inflammation caused by Pylori infection

[0111] Under the experimental conditions, total phenols from Cynanchum chinense have a significant effect on relieving gastrointestinal inflammation caused by Helicobacter pylori infection. See Table 10 for details. Figure 3 and 4 .

[0112] Table 10 Experimental results of evaluating the efficacy of Cynanchum chinense total phenols on the relieving of gastrointestinal inflammation caused by pylori infection (n=10)

[0113]

[0114]

[0115] Compared with the model control group, ***p<0.001

[0116] 3.4. Efficacy in inhibiting gastrointestinal Helicobacter pylori

[0117] Under the experimental conditions, total phenols of Cynanchum chinense have a significant inhibitory effect on Helicobacter pylori in the gastrointestinal tract. See Table 11 for details. Figure 5 and 6 .

[0118] Table 11 Experimental results of evaluating the efficacy of Cynanchum chinense total phenols in inhibiting gastrointestinal Helicobacter pylori (n=10)

[0119]

[0120] Compared with the model control group, **p<0.01, ***p<0.001

[0121] 3.5. Gastrointestinal histopathology

[0122] Intestinal pathology results ( Figure 7) showed that the number of intestinal folds of zebrafish in the normal control group was large and obvious, and the intestinal epithelial cells were tightly connected to the cilia and mucosa. The number of intestinal folds of zebrafish in the model control group was reduced, the intestinal cavity was significantly enlarged (the red double-headed arrows show the intestinal cavity diameter), and the mucosal tissue was destroyed. The intestinal expansion of zebrafish in the Cynanchum cylindrica total phenol group was significantly restored, and the morphology of intestinal tissue cells was similar to that of the normal control group. This shows that Cynanchum cylindrica total phenol has a significant effect on improving the pathological structure of the intestinal mucosa of zebrafish with gastrointestinal mucosal damage caused by Helicobacter pylori infection. For details, see Figure 7 .

[0123]

MIC concentration test of drugs against Helicobacter pylori under different pH environments

[0124] 1. Experimental Materials

[0125] 1.1 Experimental equipment

[0126] Table 12 Main equipment involved in the experiment

[0127]

[0128] 1.2 Main experimental reagents

[0129] Table 13 Main reagents involved in the experiment

[0130]

[0131] 1.3 Experimental strains

[0132] Table 14 Experimental strains

[0133]

[0134] 2. Experimental Procedure

[0135] 1) Preparation of drug stock solution:

[0136] CP-VE: Weigh an appropriate amount of CP-VE prepared in Example 1, dissolve it in anhydrous ethanol to prepare a 64 mg / mL stock solution, and store it at 4°C for later use.

[0137] 2) Preparation of pH 5.3 BHI defibrinated sheep blood solid drug-containing culture medium: weigh 12.25 g BHIB culture medium powder and 7.5 g agar, dissolve in 500 mL distilled water with heating and stirring, adjust the pH to 3.0, dispense into Erlenmeyer flasks, sterilize at 121° C. for 15 minutes, cool to about 50° C., dispense into 50 mL centrifuge tubes, add different volumes of CP-VE drug stock solution in proportion, and add defibrinated sheep blood at a ratio of 5%. Mix well, shake well, and pour into a 12-well plate to cool for use.

[0138] 3) Preparation of pH 7.0 BHI defibrinated sheep blood solid drug-containing culture medium: weigh 12.25 g BHIB culture medium powder and 7.5 g agar, dissolve in 500 mL distilled water with heating and stirring, adjust the pH to 7.0, dispense into Erlenmeyer flasks, sterilize at 121°C for 15 minutes, cool to about 50°C, dispense into 50 mL centrifuge tubes, add different volumes of CP-VE drug stock solution in proportion, add defibrinated sheep blood at a ratio of 5%, mix well, shake well, pour into 12-well plates, cool and set aside.

[0139] 4) Preparation of pH 9.0 BHI defibrinated sheep blood solid drug-containing culture medium: weigh 12.25 g BHIB culture medium powder and 7.5 g agar, dissolve in 500 mL distilled water with heating and stirring, adjust the pH to 9.0, dispense into Erlenmeyer flasks, sterilize at 121°C for 15 minutes, cool to about 50°C, dispense into 50 mL centrifuge tubes, add different volumes of CP-VE drug stock solution in proportion, and add defibrinated sheep blood at a ratio of 5%. Mix well, shake well, and pour into a 12-well plate to cool for use.

[0140] 5) NC membrane pretreatment: Cut the NC membrane into 1 cm×1 cm size, sterilize it by high pressure, and spread it into each well of the drug-containing culture medium for later use.

[0141] 6) Streak the test strain on a plate culture medium and culture at 37°C in a microaerophilic environment (6% O2, 7% CO2, 7% H2, 80% N2) for about 3 days until obvious bacterial plaques are visible.

[0142] 7) Pick a single colony, streak it on the plate medium again, and culture it at 37℃ microaerophilic (6% O2, 7% CO2, 7% H2, 80% N2) for about 3 days until obvious plaques are visible. After judging the colony morphology to be consistent with the naked eye, add 1 mL PBS to collect the plate colonies, adjust OD600 to 0.6, dilute 1000 times, take 20 μL and drop it on each NC membrane, culture it at 37℃ microaerophilic (6% O2, 7% CO2, 7% H2, 80% N2) for 14 days, and observe the growth of the colonies on the NC membrane.

[0143] 8) Interpretation of results: MIC (minimum inhibitory concentration) is the lowest inhibitory concentration that completely inhibits bacterial growth in a test tube or a small well in the dilution method of microbial identification.

[0144] 3. Experimental results

[0145] In this test, under pH 5.3, the MIC concentration of CP-VE against Helicobacter pylori ATCC26695 was 128 μg / mL; under pH 7, the MIC concentration of CP-VE against Helicobacter pylori ATCC26695 was 64 μg / mL; under pH 9, the MIC concentration of CP-VE against Helicobacter pylori ATCC26695 was 256 μg / mL;

[0146] Table 15 MIC concentration test results of CP-VE against Helicobacter pylori under different pH conditions

[0147]

[0148] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a total phenolic extract of Cynanchum chinense, characterized in that: Prepared by the following method: Add the Radix Cynanchum wilfordii and sodium chloride into water, adjust the pH to alkaline, and soak; Perform steam distillation at 250-270°C, and redistill the collected fraction to obtain a distillate; The distillate is stored at 0-4°C for 12-48 hours, then filtered, collected, and dried to obtain a needle-shaped white crystal total phenol extract of Cynanchum chinense; The mass percentage content of paeonol in the Cynanchum chinense total phenol extract is above 90%.

2. The method for preparing the Cynanchum chinense total phenolic extract according to claim 1, characterized in that: The added amounts of the Cynanchum chinense medicinal material, sodium chloride and distilled water are 180-220 g: 35-40 g: 2500-3500 mL.

3. The method for preparing the Cynanchum chinense total phenolic extract according to claim 2, characterized in that: The water is distilled water; The amount of the Cynanchum chinense medicinal material, sodium chloride and distilled water added is 195-205g: 35-37g: 2900-3100mL; Preferably, the added amount of the Cynanchum chinense medicinal material, sodium chloride and distilled water is 200g:36g:3000mL.

4. The method for preparing the Cynanchum chinense total phenolic extract according to claim 1, characterized in that: Adjust the pH to 8-9 by adding alkaline substances, preferably, adjust the pH to 7.8-8.2, and most preferably adjust the pH to 8; And / or, the alkaline substance includes sodium hydroxide and potassium hydroxide.

5. The method for preparing the Cynanchum chinense total phenolic extract according to claim 1, characterized in that: The soaking time is 1 to 2 hours, preferably 1.5 hours.

6. The method for preparing the Cynanchum chinense total phenolic extract according to claim 1, characterized in that: The distillation was carried out at 260°C for 3 to 7 hours until no turbid distillate dripped out.

7. The method for preparing the Cynanchum chinense total phenolic extract according to claim 1, characterized in that: The distillate is stored at 4° C. for 36 hours and then filtered. The needle-shaped white crystals are collected and dried and stored in a vacuum dryer. The mass percentage content of paeonol in the Cynanchum wilfordii total phenol extract is above 93%.

8. A Cynanchum chinense total phenolic extract, characterized in that The preparation is obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the Cynanchum chinense total phenolic extract as claimed in claim 8 in the preparation of a drug for protecting against Helicobacter pylori infection.

10. The use of a Cynanchum chinense total phenolic extract according to claim 9 in the preparation of a drug for protecting against Helicobacter pylori infection, characterized in that: The Cynanchum cyrtonema total phenol extract is used as an active component and is added with auxiliary materials to prepare corresponding traditional Chinese medicine solid preparations, including tablets and granules.

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