CN113694144A

CN113694144A - Pseudo-ginseng and bletilla striata pH responsive in-situ gel and preparation method and application thereof

Info

Publication number: CN113694144A
Application number: CN202111162375.4A
Authority: CN
Inventors: 狄留庆; 王令充; 张雯; 周冰倩
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2021-11-26
Anticipated expiration: 2041-09-30
Also published as: CN113694144B

Abstract

The invention discloses a traditional Chinese medicine composition for treating digestive tract diseases, a pH responsive in-situ gel and a preparation method thereof. The traditional Chinese medicine composition consists of pseudo-ginseng, rhizoma bletillae and dendrobium officinale. The invention adopts animal experiment screening to determine the composition and dosage of the traditional Chinese medicine compound; according to the invention, the characteristic that the low acetyl gellan gum and the sodium alginate can form gel through phase change under the pH value of gastric acid is found through screening, the pH responsive in-situ gel is prepared with the traditional Chinese medicine composition, the gastric emptying of the effective component of the traditional Chinese medicine composition can be delayed, the action time of the medicine in the stomach is prolonged, and the experimental result shows that the pH responsive in-situ gel can obviously improve the gastric mucosa damage caused by ethanol, and has good gastric mucosa protection effect in the aspects of gastric tissue morphology, histopathological section, gastric ulcer index and inhibition rate, and biochemical indexes (SOD and VEGF-A, EGF) related to gastric mucosa protection.

Description

Pseudo-ginseng and bletilla striata pH responsive in-situ gel and preparation method and application thereof

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition for treating digestive diseases, a pH responsive in-situ gel and a preparation method thereof.

Background

Peptic Ulcer (PUD) is one of the most common diseases worldwide, affecting about four million people worldwide, with high morbidity and mortality throughout the year, characterized by mucosal denudation with defects extending into the submucosa or muscularis propria. About two-thirds of peptic ulcer patients are asymptomatic, and among symptomatic patients, the common symptoms are epigastric pain, dyspepsia, abdominal distension, nausea. Gastric Ulcer (GU) is the most common one of peptic ulcers and is one of the major gastrointestinal diseases with increasing incidence and prevalence worldwide. The pathogenesis of peptic ulcer is various, and the factors mainly comprise the following factors: (1) infection with helicobacter pylori: developing countries are more common and are the main factor causing gastric and duodenal ulcers; (2) the medicine has the following functions: the use of non-steroidal anti-inflammatory drugs (NSAIDs) is the most prominent factor, especially the use of aspirin. Other drugs include corticosteroids and bisphosphonate combinations with non-steroidal anti-inflammatory drugs; the use of sirolimus, 5-hydroxytryptamine reuptake inhibitors (SSRIs), 5-fluorouracil (5-FU) also increases the incidence of peptic ulcers; (3) gastric juice digestion: including the erosion effect of gastric acid and pepsin on the stomach wall; (4) genetic factors: the incidence of the population with G43G genotype and O-type blood is higher; (5) weakening of mucosal defense repair barrier function: including gastric mucus HCO₃ ^-Damage to the barrier and epithelial barrier, insufficient blood flow of gastric mucosa, activation of immune cell-inflammatory reaction, and insufficient repair and reconstruction factors; (6) a stress factor; (7) bad life habits: including smoking, alcohol abuse, especially alcohol damage to gastric mucosa; (8) complications due to other diseases: tumor, chronic disease, liver cirrhosisPatients with advanced kidney diseases are more likely to suffer from peptic ulcer. (9) Age: the incidence of peptic ulcer increases with age. Among these, H.pylori infection and the use of NSAIDs are high risk factors for PUD.

The diagnosis means of peptic ulcer mainly adopts upper gastrointestinal endoscope to make diagnosis. The judgment of whether peptic ulcer caused by helicobacter pylori infection is mainly made by urea breath test, fecal antigen test, rapid urease test or gastric biopsy histology and serology test at gastroscopy. The western medicine treatment means for treating peptic ulcer are almost all synthetic drug therapy, and the used drugs comprise the following classes: (1) proton Pump Inhibitor (PPI): h⁺-K⁺An ATPase inhibitor, which is the first choice drug for treating peptic ulcer and can inhibit the final process of gastric acid secretion inside parietal cells, and common drugs include omeprazole and lansoprazole; (2) h2 receptor blockers: is a main medicine for treating peptic ulcer related gastrointestinal hemorrhage, can selectively block H2 receptor on a parietal cell membrane, reduce gastric acid secretion, and inhibit secretion of basic gastric acid, wherein common medicines comprise ranitidine, cimetidine and famotidine; (3) gastric mucosa protective agent: comprises gastrointestinal hormone drugs (roxaprost and misoprostol), sulfur hydrogen bond drugs (sucralfate), bismuth agent, columnar cell stabilizer, etc. In addition, the medicine also comprises common anticholinergic medicine, antacid medicine and helicobacter pylori carrying medicine. However, synthetic drug therapy is accompanied by serious side effects, and although the symptoms can be alleviated, ulcers cannot be cured, so that the characteristics of traditional Chinese medicine treatment of PUD are highlighted.

In the field of traditional Chinese medicine, the traditional Chinese medicine names of peptic ulcer are stomachache, epigastric upset and gastrohelcosis, the peptic ulcer belongs to epigastric pain, and is mostly caused by stasis syndrome, and dyspepsia, exogenous pathogenic factors, dysfunction of liver and spleen, and emotional internal injury are common causes of epigastric pain. The diagnosis and treatment of epigastric pain with traditional Chinese medicine has a long history, and can be traced back to the internal classic of yellow emperor at the earliest time. Aiming at the occurrence mechanism of peptic ulcer, the traditional Chinese medicine treatment mainly takes strengthening spleen and stomach, removing blood stasis and relieving pain as the main principle. The treatment means mainly comprise: acupuncture therapy, diet conditioning and traditional Chinese medicine therapy. The traditional Chinese medicine treatment is used for integrally preventing and treating the diseases through the action characteristics of multiple components, multiple targets and multiple ways, and has the advantages of obvious curative effect, high safety and low recurrence rate compared with a plurality of drug-induced adverse reactions brought by synthetic drugs although the effect is slow.

In order to improve the medicinal performance of the active ingredients of the traditional Chinese medicine and enable the active ingredients to enter clinical application, the material basis, the action target and the pharmaceutics of the active ingredients need to be researched, and the active ingredients are prepared into safe and effective preparations by applying the current advanced scientific and technical means, so that the development and the application of the active ingredients of the traditional Chinese medicine in the field of pharmacy are expanded.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a traditional Chinese medicine composition with good peptic ulcer prevention and treatment effects and a pH-responsive in-situ gel of the traditional Chinese medicine composition.

The technical scheme is as follows: in order to realize the purpose, the invention adopts the technical scheme that:

a Chinese medicinal composition for treating digestive tract diseases comprises Notoginseng radix, rhizoma Bletillae, and herba Dendrobii.

As a preferred scheme, the traditional Chinese medicine composition for treating the digestive tract diseases comprises 3-9 parts of pseudo-ginseng, 3-9 parts of rhizoma bletillae and 3-9 parts of dendrobium officinale.

As a preferred scheme, the traditional Chinese medicine composition for treating the digestive tract diseases comprises 3 parts of pseudo-ginseng, 3 parts of rhizoma bletillae and 3 parts of dendrobium officinale.

The preparation method of the traditional Chinese medicine composition for treating the digestive tract diseases comprises the following steps,

the preparation method comprises the steps of taking pseudo-ginseng, bletilla striata and dendrobium officinale according to the weight parts, adding 6-15 times of water, soaking for 30-45 min, heating, decocting, extracting for 30-90 min, extracting for 1-3 times, combining extracting solutions, and concentrating under normal pressure until the relative density is 1.04-1.2 and the concentration is 0.5-1 g/mL.

A pH-responsive in-situ gel containing radix Notoginseng and rhizoma Bletillae is characterized by comprising radix Notoginseng, rhizoma Bletillae, herba Dendrobii traditional Chinese medicine extract, low acetyl gellan gum and sodium alginate.

As a preferred scheme, the panax notoginseng and bletilla striata pH-responsive in-situ gel comprises panax notoginseng, bletilla striata and dendrobium officinale traditional Chinese medicine extract concentrated solution, low acetyl gellan gum with the mass concentration of 0.1% and sodium alginate with the mass concentration of 0.5%.

The preparation method of the pseudo-ginseng and pH responsive in-situ gel comprises the following steps:

(1) taking pseudo-ginseng, bletilla striata and dendrobium officinale according to the weight parts, adding 6-15 times of water, soaking for 30-45 min, heating, decocting and extracting for 30-90 min, extracting for 1-3 times, combining extracting solutions, and concentrating under normal pressure to obtain an extracting concentrated solution with the relative density of 1.04-1.2 and the concentration of 0.5-1 g/mL;

(2) and (2) adding the low acetyl gellan gum and the sodium alginate into deionized water to fully swell to form an in-situ gel matrix solution, slowly pouring the extraction concentrated solution obtained in the step (1), heating, uniformly stirring, and stirring and cooling to room temperature to obtain the gel.

Preferably, the preparation method of notoginseng and pH-responsive in situ gel comprises the following steps:

(1) taking 3 parts of pseudo-ginseng, 3 parts of bletilla striata and 3 parts of dendrobium officinale according to the parts by weight, adding 12 times of water, soaking for 45min, heating, decocting and extracting for 90min, extracting for 2 times, combining extracting solutions, and concentrating under normal pressure to obtain an extraction concentrated solution with the relative density of 1.04 and the concentration of 0.5 g/mL;

(2) and (2) adding deionized water into the low acetyl gellan gum and the sodium alginate for fully swelling to form an in-situ gel matrix solution, slowly pouring the extract concentrated solution obtained in the step (1), heating at 90 ℃, uniformly stirring at 150rpm, stirring at 170rpm, and cooling to room temperature to obtain the gel.

Preferably, in the preparation method of pseudo-ginseng and pH-responsive in-situ gel, the mass concentration of the low acetyl gellan gum is 0.1% and the mass concentration of the sodium alginate is 0.5%.

Has the advantages that: the invention provides pseudo-ginseng bletilla striata pH responsive in situ gel and a preparation method thereof;

1. the invention adopts animal experiment screening to determine the composition and dosage of the traditional Chinese medicine compound; according to the invention, the characteristic that the low acetyl gellan gum and the sodium alginate can be subjected to phase change to form gel under the pH value of gastric acid is found through screening, and the pH responsive in-situ gel is prepared by the low acetyl gellan gum and the sodium alginate and an aqueous extract of a Chinese herbal compound (pseudo-ginseng, bletilla striata and dendrobium officinale).

According to the invention, the preparation process of the pH responsive in-situ gel (PBG) is designed by considering the optimal extraction and concentration process of the traditional Chinese medicine compound water extract (PGR); the PBG preparation is preliminarily inspected on fluidity, in-vitro and in-vivo in-situ gel characteristics, rheological characteristics, in-vitro drug release characteristics and bioadhesion, and the gastric mucosa protection effect of the PBG preparation is further verified.

The invention firstly carries out the efficacy evaluation before the prescription, adopts an ethanol-induced acute gastric mucosa injury model, takes the gastric tissue morphology, histopathological sections, the gastric ulcer index and inhibition rate and biochemical indexes (SOD, MDA, IL-6, TNF-alpha, PGE2, EGF and VEGF-A) related to the gastric mucosa protection as evaluation indexes, verifies the efficacy of PGR, and compares the efficacy difference between the traditional notoginseng common bletilla pseudobulb formula and the PGR. The results show that the PGR low, medium and high dose groups have certain protection effect on acute gastric mucosa injury caused by ethanol; the efficacy of the pseudo-ginseng and rhizoma bletillae groups is slightly stronger than that of the low-dose groups and lower than that of the medium-dose and high-dose groups. Compared with the radix notoginseng and rhizoma bletillae groups, the dosage of the PGR is lower, and the gastric mucosa protection effect is stronger. The mechanism by which PGRs exert a protective effect on the gastric mucosa may be related to antioxidant, anti-inflammatory, and ulcer healing promotion. The medium and high doses do not show significant difference in most indexes, so the dose after conversion of the medium dose group is determined as the recommended dose, and the prescription composition of the recommended dose is as follows: 3g of pseudo-ginseng, 3g of rhizoma bletillae and 3g of dendrobium officinale.

Then, the extraction and concentration processes of PGR and the preparation process of PBG were studied. In PGR extraction process investigation, taking the comprehensive score of the total content of index components and the paste yield as an evaluation index, carrying out single-factor investigation and L9(34) orthogonal experiment on the soaking time, the decoction time, the water adding quantity and the decoction times, carrying out single-factor investigation on the extraction times, and determining the extraction process parameters as follows: 1 prescription dose of the medicinal materials, adding 12 times of water, soaking for 45min, boiling the water, extracting for 90min, and extracting for 2 times. In PGR concentration process investigation, the concentration degree and the concentration mode are successively investigated, the comprehensive scores of the state of the concentrated solution, the content of index components and the paste yield are respectively taken as evaluation indexes, and finally, the concentration process parameters are determined as follows: concentrating under normal pressure to 10 times, with relative density of 1.04 and concentration of about 0.5 g/mL. In the preparation process research, the normalized values of the apparent viscosity and the pH response incremental viscosity of the PBG are used as evaluation indexes, and the dosage of the low acetyl gellan gum (Kel) in the PBG is further optimized to be 0.1% w/v and the dosage of the Sodium Alginate (SA) is 0.5% w/v by a star point design-effect surface method. The influence of the process factors (heating temperature, stirring speed during heating and stirring speed during cooling) on the PBG is examined by taking the normalized values of the PBG form, the apparent viscosity and the pH response increment viscosity as evaluation indexes. The result shows that the technological factors have little influence on the apparent viscosity and pH response increment viscosity of the PBG and have certain influence on the preparation form. The process for finally determining the prescription of the preparation comprises the following steps: fully swelling Kel (0.1% w/v) and SA (0.5% w/v) in deionized water to form an in-situ gel matrix solution, slowly pouring the concentrated solution, heating at 90 ℃, uniformly stirring at 150rpm, stirring at 170rpm, and cooling to room temperature to obtain the gel.

Formulation characterization of PBG was then performed. The PBG has good in vitro fluidity, is easy to pour, has moderate sample viscosity, and is beneficial to oral administration. The gel was formed in a short time in either simulated gastric fluid or rat stomach, indicating that PBG had good in situ gel properties. The rheological measurement result shows that the viscosity of PBG is reduced along with the increase of the shear rate, the PBG is in the phenomenon of shear thinning, the PBG is a pseudoplastic fluid, the trend of a rheological characteristic curve is consistent with that of a blank gel matrix, and the gelling property of the gel matrix is not changed by adding PGR; meanwhile, the addition of PGR enhances the water binding capacity of the gel matrix, improves the stability and mechanical property of the gel matrix, improves the syneresis phenomenon of the gel matrix after forming gel, and the PBG has stable property within the range of 25-45 ℃. In vitro release studies show that the release of the active ingredient Militarine has low pH dependence, the cumulative release rate of 12h in water and pH 6.8 media is only about 80%, the cumulative release rate of 12h in pH 1.2 media can reach 100%, and the release of the drug is more consistent with the first order kinetic equation. The PBG has better in-vitro gastric adhesion performance, and the examination experiment on the in-vivo gastric retention time and the residual quantity of active ingredients shows that the PBG can be retained in the stomach of a rat for a longer time compared with the extracting solution, and related pharmacodynamic ingredients can be still detected in the gastric contents after administration for 240min, which indicates that the PBG can delay the gastric emptying of the active ingredients after forming gel in the stomach and prolong the action time of the medicament in the stomach.

Finally, whether the PBG improves the drug effect of the PGR is verified. An acute gastric mucosa injury model made of ethanol is adopted, and the pharmacological effects of PBG are evaluated by taking the gastric tissue morphology, histopathological sections, the index and inhibition rate of gastric ulcer and biochemical indexes (SOD, MDA, IL-6, TNF-alpha, PGE2, EGF and VEGF-A) related to gastric mucosa protection as evaluation indexes. The result shows that PBG can obviously improve the gastric mucosa injury caused by ethanol, and shows stronger gastric mucosa protection drug effect than PGR in the aspects of gastric tissue morphology, histopathological section, gastric ulcer index and inhibition rate, and biochemical indexes (SOD and VEGF-A, EGF) related to gastric mucosa protection.

Drawings

Figure 1 is the gastric ulcer index (n-6) in rats;^*P<0.05vs MC，^**P<0.01vs MC；^aP<0.05vs PC，^aaP<0.01vs PC；

^ΔP<0.05vs PB，^ΔΔP<0.01vs PB。

FIG. 2 shows the results of pathological section (magnification 200 ×) (A) blank; (B) a model group; (C) a group of positive drugs; (D) a low dose group; (E) a medium dose group; (F) a high dose group; (G) notoginseng and bletilla striata.

FIG. 3 shows the results of measurement of SOD and MDA in serum (n is 6),^#P<0.05vs NC，^##P<0.01vs NC；^*P<0.05vs MC，^**P<0.01vs MC；^aP<0.05vs PC，^aaP<0.01vs PC；

^ΔP<0.05vs PB，^ΔΔP<0.01vs PB。

FIG. 4 shows the results of measuring the activities of SOD and MDA in stomach tissue,^#P<0.05vs NC，^##P<0.01vs NC；^*P<0.05vs MC，^**P<0.01vs MC；^aP<0.05vs PC，^aaP<0.01vs PC；

^ΔP<0.05vs PB，^ΔΔP<0.01vs PB。

FIG. 5 shows the results of measuring the IL-6 and TNF-alpha contents in stomach tissues,^#P<0.05vs NC，^##P<0.01vs NC；^*P<0.05vs MC，^**P<0.01vs MC；^aP<0.05vs PC，^aaP<0.01vs PC；

^ΔP<0.05vs PB，^ΔΔP<0.01vs PB。

FIG. 6 shows the results of measuring the contents of PGE2, EGF and VEGF-A in stomach tissues,^#P<0.05vs NC，^##P<0.01vs NC；^*P<0.05vs MC，^**P<0.01vs MC；^aP<0.05vs PC，^aaP<0.01vs PC；

^ΔP<0.05vs PB，^ΔΔP<0.01vs PB

FIG. 7 shows the range of gellan gum concentrations (A: apparent viscosity; B: pH response incremental viscosity; C: OD).

FIG. 8 shows the range of sodium alginate concentration (A: apparent viscosity; B: pH response incremental viscosity; C: OD value).

Fig. 9 is a three-dimensional view of the effect plane and a contour map.

Fig. 10 shows the results of measuring the activity of SOD and the content of MDA in serum (n ═ 6).^aP<0.05vs NC，^aaP<0.01vs NC；^*P<0.05vs MC，^**P<0.01vs MC；^#P<0.05vs PC，^##P<0.01vs PC；

^ΔP<0.05vs PGR，^ΔΔP<0.01vs PGR。

FIG. 11 shows the results of measuring SOD activity and MDA content in stomach tissue,^aP<0.05vs NC，^aaP<0.01vs NC；^*P<0.05vs MC，^**P<0.01vs MC；^#P<0.05vs PC，^##P<0.01vs PC；

^ΔP<0.05vs PGR，^ΔΔP<0.01vs PGR。

FIG. 12 shows the results of measuring the IL-6 and TNF-alpha contents in the stomach tissue,^aP<0.05vs NC，^aaP<0.01vs NC；^*P<0.05vs MC，^**P<0.01vs MC；^#P<0.05vs PC，^##P<0.01vs PC；

^ΔP<0.05vs PGR，^ΔΔP<0.01vs PGR。

FIG. 13 measurement results of PGE2, EGF and VEGF-A contents in stomach tissue,^aP<0.05vs NC，^aaP<0.01vs NC；^*P<0.05vs MC，^**P<0.01vs MC；^#P<0.05vs PC，^##P<0.01vs PC；

^ΔP<0.05vs PGR，^ΔΔP<0.01vs PGR。

Detailed Description

The present invention is further illustrated below with reference to specific examples, which should not be construed as limiting the invention.

Example 1 pharmacodynamic study of the protective action of Notoginseng radix and rhizoma Bletillae on stomach and gastric mucosa

1 instruments and materials

1.1 instruments

TGL-16M desk-top high-speed refrigerated centrifuge (shanghai luxiang instrument centrifuge, ltd); Milli-Q Synthesis108 ultrapure water meter (Millipore, USA); CPA225D electronic analytical balance (sydows scientific instruments beijing ltd); VORTEX-5 VORTEX mixer (Haiman, its Linebel instruments, Inc.); a Tecan Infinite 200PRO microplate reader (Tecan, Switzerland); digital display calipers (shanghai constant measuring tool, ltd); HH-4 digital display constant temperature water bath (national electric appliance Co., Ltd.); MB100-2A microplate constant temperature oscillator (Thermo Fisher, Germany); GL-802B micro bench vacuum pump (Haiman, Leibel instruments, Inc.); retsch MM400 high speed ball mill homogenizer (Retsch company, germany); IMS-20 Ice machine (snow appliances, Inc., Utility City).

1.2 reagents and reagents

Omeprazole (batch No. 19121801, Jiangsu Changjiang river pharmaceutical Co., Ltd.); absolute ethanol (AR, lot No. 20200601, national chemical group reagents, Inc.); SOD reagent box (batch number: 20200623, Nanjing institute of bioengineering); MDA kit (batch number: 20200624, Nanjing institute of bioengineering); BCA kit (batch number: 20200629, Nanjing institute of bioengineering); TNF-alpha, IL-6, VEGF-A, PGE2, EGF kit (batch No. 202006003, Nanjing institute of bioengineering); sodium chloride (batch No. 20191001, Jiangsu Qiangsheng functional chemistry Co., Ltd.).

1.3 herbs

The decoction pieces are purchased from Wansheng pharmaceutical products Co., Ltd, and are detected to accord with the standard of the pharmacopoeia of 2020 edition. Notoginseng radix (batch number: D20180910); bletilla striata (batch number: 190601); dendrobium officinale (batch number: D20180912).

1.4 animals

SD rat, male, 180-220 g, provided by Nanjing university of traditional Chinese medicine laboratory animal center, license number SCXK (Zhe) 2019-.

2 methods and results

2.1 animal groups

42 SD rats, male, with the body mass of 180-220 g, the breeding environment temperature of 23-25 ℃, free drinking and eating, random grouping after 1 week of adaptive feeding, and weighing. The experiments were divided into 7 groups: 1. blank group (Normal control, NC), 2 Model group (Model control, MC), 3 Positive drug group (PC), 4 Low dose group (Low dose group, LG), 5 medium dose group (Middle dose group, MG), 6 High dose group (High dose group, HG), 7 Panax notoginseng rhizoma bletillae group (Panax notoginseng & Bletilla striata, PB).

2.2 administration and dosage

The blank and model groups were drenched with saline.

The positive drug group is used for feeding omeprazole, the dose is converted into rat equivalent dose, namely 80mg/kg, according to the clinical dosage of human, the omeprazole is used for gastric lavage according to 10mL/kg, and the concentration of omeprazole aqueous suspension to be prepared is 8 mg/mL.

The extraction method of the traditional Chinese medicine composition consisting of the pseudo-ginseng, the bletilla striata and the dendrobium officinale comprises the following steps: adding 12 times of water into Notoginseng radix, rhizoma Bletillae and iron sheet at a weight ratio of 1:1:1, soaking for 45min, heating, decocting for 90min, extracting for 2 times, mixing extractive solutions, concentrating, and making into low, medium and high dosage groups with administration dosage of 0.315g/kg, 0.945g/kg and 2.845g/kg respectively. The medicine is administrated by stomach irrigation according to 10mL/kg, and the concentration of the extracting solution is respectively as follows: 31.5mg/mL, 94.5mg/mL, 283.5 mg/mL. The extraction method comprises the steps of adding 12 times of water into 1:1 by weight of the pseudo-ginseng and the bletilla striata, soaking for 45min, heating, decocting, extracting for 90min, extracting for 2 times, combining the extracting solutions, concentrating to 189mg/mL to prepare the finished product with the administration dose of 1.89g/kg, and intragastrically irrigating each group according to the volume of 10mL/kg for 1 time/1 d.

2.3 Molding

Fasting for 24h before the last administration, full fasting, emptying of bedding and faeces. After 2h after the last administration, except for the blank group, all the tested animals were gavaged with 1mL of absolute ethyl alcohol, and 1h after anesthesia and material drawing observation.

2.4 sample Collection

After 1h of modeling, the rats are anesthetized, 7mL of abdominal aorta blood is collected in an anticoagulant-free vacuum blood collection tube, after standing for 1h, the abdominal aorta blood is centrifuged for 15min at 4 ℃ and 3500rpm, serum is separated, and the serum is respectively filled into a0.5 mL centrifuge tube and stored at the temperature of minus 20 ℃ in a 150 mu L tube and taken at any time. Taking the stomach, splitting along the greater curvature of the stomach, washing the contents of the stomach with precooled normal saline, observing the gastric mucosa of the rat by naked eyes, measuring the ulcer length with a vernier caliper, recording the ulcer index, taking the stomach tissue of the glandular stomach 1cm × 1cm after recording, quickly fixing the stomach tissue in 4% paraformaldehyde fixing solution, and making a tissue pathological section. Adding 9 times of ice PBS buffer solution into the rest stomach tissue according to the weight-volume ratio, fully homogenizing on ice to prepare 10% tissue homogenate, centrifuging for 10min at 5000Xg, and taking supernatant to store at-20 ℃.

2.5 morphological Observation of rat stomach tissue

After the stomach is cut open, the damage degree of the stomach tissue of each group of rats is directly observed by naked eyes and photographed and recorded.

2.6 gastric mucosal Ulcer Index (UI) and ulcer inhibition ratio determination

The degree and morphology of gastric mucosal lesions were visually observed, and the length and width (mm) of bleeding points or bands were measured with a vernier caliper and scored. The Ulcer Index (UI) of rat gastric mucosa is shown in formula 1-1, and its scoring standard is shown in

In Table 1, the ulcer inhibition ratio is calculated by the formula 1-2.

Ulcer Index (UI) ═ bleeding point score + length score + (width score × 2)	Equation 1-1
		I(％)＝(Mo-Me)/Mo×100％	Equations 1-2

Wherein I (%) is ulcer inhibition rate, Mo is ulcer index of model group, and Me is ulcer index of administration group.

TABLE 1 ulcer index score criteria

Degree of damage	1 minute (1)	2 is divided into	3 points of	4 is divided into
					Bleeding point	1 is provided with	-	-	-
Length of bleeding band/mm	1-5	6-10	10-15	>15
					Width/mm of hemorrhage zone	1-2	>2

2.7 section observation of pathological tissue of gastric mucosa

The stomach tissue fixed in 4% paraformaldehyde solution was dehydrated, embedded, and sectioned according to a conventional method, stained with hematoxylin-eosin (HE), and observed under a microscope.

2.8 determination of Biochemical indicators (SOD, MDA) in serum

The SOD activity in rat serum is measured by xanthine oxidase method (hydroxylamine method), and the MDA content in rat serum is measured by thiobarbituric acid method (TBA method).

2.9 determination of relevant indices in stomach tissue

2.9.1 measurement of SOD and MDA in stomach tissue

The SOD activity in rat stomach tissue is measured by xanthine oxidase method (hydroxylamine method), and the MDA content in rat stomach tissue is measured by thiobarbituric acid method (TBA method).

2.9.2 measurement of inflammatory factors in stomach tissue

The ELISA kit method is adopted to determine the contents of IL-6 and TNF-alpha in the stomach tissue of the rat.

2.9.3 determination of factors associated with gastric mucosal protection in gastric tissue

The contents of PGE2, EGF and VEGF-A in stomach tissues were determined by ELISA kit method.

2.10 statistical processing and data representation

Statistical analysis was performed by t-test using SPSS 22.0 statistical software and results were expressed in x ± s, with significant differences expressed as P <0.05 and very significant differences expressed as P < 0.01. All data processing herein is performed in this manner, if not specifically indicated.

3 results and analysis

3.1 morphological Observation of rat stomach tissue

The gastric mucosa of the rats in the blank group is smooth and has no damage, the gastric mucosa of the rats in the model group has serious damage, bleeding zones with wide surface distribution and swelling and congestion of the mucosa. Each of the remaining groups had different degrees of congestion, swelling, bleeding, but were reduced compared to the model group. The gastric mucosa of the rats in the positive drug group, the medium and high dose groups is basically intact, and the damage is light. Compared with the low-dose group, the panax notoginseng and rhizoma bletillae group has the advantages that the density of bleeding points is reduced, and the injury degree is higher than that of the medium-dose and high-dose groups.

3.2 gastric mucosal Ulcer Index (UI) and ulcer inhibition rate measurement result

The results of the ulcer index and inhibition rate determination are shown in fig. 1 and table 2, and the results show that compared with the model group, the ulcer indexes of the positive drug group, the medium-dose group, the high-dose group and the pseudo-ginseng bletilla striata group are obviously smaller than that of the model group (P <0.01), and the ulcer index of the low-dose group is smaller than that of the model group but has no significant difference (P > 0.05). Compared with the low dose group, the ulcer index of the medium dose group and the high dose group was significantly reduced (P <0.05), and there was no significant difference between the medium dose group and the high dose group (P > 0.05). Compared with the low-dose group, the pseudo-ginseng bletilla striata group has no obvious difference, and the improvement condition of the medium-dose and high-dose groups is obviously better than that of the pseudo-ginseng bletilla striata group (P < 0.05).

The ulcer inhibition rates of the low-dose group, the pseudo-ginseng and rhizoma bletillae group are 23.05 percent and 25.93 percent respectively, and the ulcer inhibition rates of the medium-dose group and the high-dose group exceed 50.62 percent of the positive medicine group and respectively reach 51.85 percent and 52.68 percent.

Table 2 ulcer index and ulcer inhibition results (n ═ 6)

^*P<0.05vs MC，^**P<0.01vs MC；^aP<0.05vs PC，^aaP<0.01vs PC；

^ΔP<0.05vs PB，^ΔΔP<0.01vs PB

3.3 section observation of pathological tissues of gastric mucosa

The pathological section results are shown in figure 2, and the tissue structure of the gastric mucosa of the rats in the blank group is complete and has a normal glandular structure of the gastric mucosa. The model group rats suffered from severe damage to gastric mucosa and were characterized by extensive mucosal epithelial loss of the tissue, necrosis of part of the mucosal epithelium and glands (black arrows), visible congestion of capillaries (green arrows), and more mononuclear cell infiltration (yellow arrows). The remaining groups had different degrees of damage, but were all reduced compared to the model group. The damage of the positive drug group is obviously improved compared with the model group, and local epithelial cell shedding (yellow arrow) and a small amount of glandular epithelial cell nucleus consolidation (red arrow) can be seen, and bleeding and mucosal layer edema do not exist. The low dose group had a small number of bleeding bands and edema, and extensive mucosal epithelial detachment of the tissue was observed (black arrows); partial mucosal epithelial cell detachment (yellow and black arrows) was visible in the medium dose group, partial mucosal epithelial detachment (black arrows) and cytoplasmic eosinophilic enhancement (green arrows) was visible in the high dose group; pseudo-ginseng and bletilla striata groups were seen with little proliferation of connective tissue, necrosis of local mucosal upper layers of the tissue (black arrows), congestion and bleeding of capillaries (green arrows), and necrosis of part of glandular epithelial cells (blue arrows). Compared with the low-dose group and the pseudo-ginseng and bletilla striata group, the medium-high dose group has lower damage degree.

3.4 determination of SOD and MDA in serum

The contents of SOD and MDA are related indexes of oxidative stress and are related to the antioxidant activity of the medicine.

As can be seen from FIG. 3, the SOD activity in the serum of the model group rat is significantly reduced (P is less than 0.01) compared with that of the blank group, which indicates that the modeling is successful; SOD activity in the serum of rats in each administration group is obviously improved compared with that in the model group (P is less than 0.01); the SOD content of the medium and high dose groups is obviously increased compared with that of the low dose group (P is less than 0.01, P is less than 0.05); the SOD activity of the high-dose group is improved compared with that of the medium-dose group (P is more than 0.05); the SOD activity of the rhizoma bletillae is higher than that of the low-dose group and lower than that of the medium-dose and high-dose groups (P is more than 0.05).

The MDA content in the serum of the model rat is obviously increased (P is less than 0.01) compared with that of the blank group, which indicates that the model is successfully made; the MDA content in the blood serum of the positive medicine rat is reduced (P is less than 0.05) compared with that of the model group, and the MDA content in the blood serum of the middle and high dose groups, the blood serum of the pseudo-ginseng and bletilla striata groups is obviously reduced (P is less than 0.01) compared with that of the model group; compared with the low-dose group, the MDA content of the medium-dose group and the high-dose group is remarkably reduced (P is less than 0.01); the MDA content in the serum of the high-dose group is reduced compared with that of the medium-dose group, but no significant difference exists. The content of MDA in the blood serum of the rhizoma bletillae group is lower than that of the low-dose group (P is more than 0.05), and is obviously lower than that of the medium-dose group and the high-dose group (P is less than 0.01).

3.5 determination of relevant indices in stomach tissue

3.5.1 measurement of SOD and MDA in stomach tissue

SOD and MDA in stomach tissue are measured as shown in figure 4. The results show that after the ethanol molding, the activity of SOD in rat stomach tissue is reduced (P is less than 0.05), and the MDA content is increased (P is less than 0.05), which indicates the success of molding.

In the index SOD activity, the positive medicine group and the medium-dose group are obviously increased (P is less than 0.01) compared with the model group, and the high-dose group, the pseudo-ginseng and the bletilla striata group are increased (P is less than 0.05) compared with the model group; SOD activity of low, medium and high dose groups is increased in sequence (P is more than 0.05); the panax notoginseng and bletilla striata groups have high lower dose groups and low higher and middle dose groups (P is more than 0.05).

In the index MDA content result, the positive drug group, the medium-dose group and the high-dose group are reduced compared with the model group (P is less than 0.05), wherein the high-dose group is obviously reduced compared with the model group (P is less than 0.01). The content of the pseudo-ginseng and bletilla striata MDA in the group is higher than that in the low, medium and high dose groups (P is more than 0.05).

3.5.2 measurement of inflammatory factors in stomach tissue

IL-6 and TNF-alpha are cytokines reflecting inflammatory reactions of the body, and are closely related to inflammatory reactions. IL-6 stimulates neutrophils, macrophages and lymphocytes at the site of inflammation, producing various harmful products, reactive oxygen radicals and lysosomal enzymes that cause damage to gastric ulcer tissue. TNF-alpha stimulates neutrophil infiltration and epithelial apoptosis, reduces gastric microcirculation around ulcers, and delays gastric ulcer healing. The results of the determination of IL-6, TNF- α levels in stomach tissue are shown in FIG. 5.

The results show that after ethanol molding, the levels of inflammatory factors IL-6 and TNF-alpha in rat gastric tissues are both obviously increased (P is less than 0.01), and the levels of the positive drug group and the model group are both obviously reduced, which indicates that the molding is successful.

In the index IL-6, compared with the model group, the contents of the medium and high dose groups and the contents of the pseudo-ginseng and rhizoma bletillae groups are all obviously reduced (P is less than 0.01); compared with the low-dose group, the content of the medium-high dose group is obviously reduced (P is less than 0.05, P is less than 0.01), and no obvious difference exists between the medium-high dose group and the high-high dose group; compared with the pseudo-ginseng and bletilla striata groups, the low-dose group is higher, and the medium-high dose group is lower.

In the index TNF-alpha, compared with a model group, the contents of a medium-high dose group, a pseudo-ginseng and a bletilla striata group are obviously reduced (P is less than 0.05 or P is less than 0.01); compared with the low-dose group, the content of the medium-dose group is reduced, the content of the high-dose group is obviously reduced (P is less than 0.01), and no obvious difference exists between the medium-dose group and the high-dose group; compared with the pseudo-ginseng and bletilla striata groups, the low-dose group is higher, and the medium-high dose group is lower.

3.5.3 determination of factors related to gastric mucosa protection in gastric tissue

PGE2 is an important cytoprotective factor that, as one of the major metabolites of arachidonic acid, repairs damaged gastric mucosa by inhibiting gastric acid secretion, increasing blood flow to the gastric mucosa to maintain cellular integrity, increasing mucus secretion, and promoting protein and cellular synthesis, enhancing resistance of gastric mucosal cells to stimuli. The result of the measurement of the content of PGE2 in the stomach tissue is shown in FIG. 6, and the result shows that compared with the blank control group, the content of PGE2 in the homogenate of the rat stomach tissue in the model group is obviously reduced (P is less than 0.01), and the content of the positive drug group is obviously increased (P is less than 0.01) compared with the model group. Compared with the model group, the content of the low-dose group is increased, and the content of the medium-dose group and the high-dose group is obviously increased (P is less than 0.05 and P is less than 0.01); the content of the high-dose group is obviously increased compared with that of the low-dose group (P is less than 0.01); compared with the medium-dose group, the content of the high-dose group is obviously increased (P is less than 0.05); compared with the radix notoginseng and rhizoma bletillae groups, the low-dose group has lower content, the medium-dose group is equivalent to the radix notoginseng and rhizoma bletillae groups, and the high-dose group is higher.

EGF can enhance immunity by inducing a JAK/STAT signal channel and a phosphatidylinositol channel, the concentration of EGF in the stomach of a large number of patients with gastric ulcer is remarkably reduced, and EGF can promote cell repair and migration, promote epithelial cell proliferation of gastrointestinal mucosa, improve blood circulation of gastric mucosa, enhance mucosal barrier, improve serum EGF level of patients with peptic ulcer and effectively promote ulcer healing. The EGF content in the stomach tissue is shown in figure 6, and the results show that compared with a blank control group, the EGF content in the rat stomach tissue homogenate of the model group is obviously reduced (P is less than 0.01), and the content of the positive drug is obviously increased (P is less than 0.01) compared with the model group. In the content of EGF in stomach tissues, the content of EGF in the low, medium and high dose groups is remarkably increased compared with that in the model group (P <0.01), and the content of EGF is increased along with the increase of the administration dose, but no statistical difference exists among the low, medium and high dose groups. Compared with the pseudo-ginseng and rhizoma bletillae groups, the low, medium and high dose groups are all improved.

VEGF-A plays an important role in promoting ulcer healing and angiogenesis and protecting the integrity of gastric mucosa, and the higher the content of VEGF-A is, the more beneficial to the healing of gastric mucosa injury is. The result of measuring the VEGF-A content in the stomach tissue is shown in figure 6, and the result shows that in the VEGF-A content in the stomach tissue, compared with a blank control group, the VEGF-A content in the gastric tissue homogenate of a rat model group is obviously reduced (P is less than 0.01), and the VEGF-A content in the positive drug is obviously increased (P is less than 0.01) compared with the model group. The low dose group has higher content compared with the model group, but has no statistical difference; the content of the medium-dose group is increased (P is less than 0.05) compared with that of the model group, and the content of the high-dose group is obviously increased (P is less than 0.01) compared with that of the model group; the content of the high-dose group is obviously increased compared with that of the low-dose group (P is less than 0.05); no significant difference between the medium and high dose groups; compared with the pseudo-ginseng and rhizoma bletillae groups, the low, medium and high dose groups are all improved.

Example 2 formulation Process Studies of Panax notoginseng and pH-responsive in situ gel

1 materials and instruments

1.1 instruments

Model JJ-1A digital display electric stirrer (yokoku instruments ltd); ZNHW type digital display electrocaloric jacket (Zhengzhou Biochemical instruments Co., Ltd.); model ZG-8020 electronic thermometer (ningbo zhao electrical appliances limited); PHS-3C type pH meter (Shanghai apparatus electric science Co., Ltd.); sartorius CPA225D electronic balance (sydows scientific instruments beijing ltd); Milli-Q Synthesis108 ultrapure water meter (Millipore, USA); NDJ-1B rotational viscometer (Shanghai Changji geological instruments Co., Ltd.).

1.2 reagents and reagents

Sodium alginate (SA, batch number: S11053, Shanghai-sourced leaf organism Co., Ltd.); low acyl gellan gum (Kel, lot number: G821481, Shanghai Michelin Biochemical technology Ltd.); hydrochloric acid (batch No. 20200107, analytical grade, national pharmaceutical group chemical Co., Ltd.); pepsin (batch: WXBC2086V, from porcine gastric mucosa, Shanghai Michelin Biotechnology Ltd.); sodium chloride (batch No. 20191001, analytical purity, Jiangsu Qiangsheng functional chemistry Co., Ltd.);

a concentrated solution of a traditional Chinese medicine composition (3 parts of pseudo-ginseng, 3 parts of rhizoma bletillae and 3 parts of dendrobium officinale are taken according to the parts by weight, 12 times of water is added, the mixture is soaked for 45min, then heated, decocted and extracted for 90min and extracted for 2 times, extracting solutions are combined, and the concentrated solution is concentrated to an extraction concentrated solution with the relative density of 1.04 and the concentration of 0.5g/mL at normal pressure).

2 methods and results

2.1 Studies on the amount of Panax notoginseng and pH-responsive in situ gel (PBG) prescription

2.1.1 Simulated Gastric Fluid (SGF) formulation

Taking a proper amount of concentrated hydrochloric acid, sodium chloride and pepsin, adding deionized water to prepare a solution containing 0.7% of hydrochloric acid, 0.02% of sodium chloride and 0.32% of pepsin, and adjusting the pH to 1.2.

2.1.2 preparation of PBG

Accurately weighing a certain amount of SA and Kel respectively, fully swelling in deionized water, slowly adding PGR concentrated solution, stirring uniformly at 80 ℃ by using an electric stirrer at the rotating speed of 150rpm, stirring at the rotating speed of 150rpm, and cooling to room temperature to obtain the compound.

2.1.3 in situ gel matrix concentration Studies

The experimental target prepares PBG with good in-vitro fluidity, small apparent viscosity, high speed of forming gel in gastric acid environment and large pH response increment viscosity. In the preliminary experiment process, the finding shows that when the gellan gum is used as the gel matrix alone, the speed of forming gel in gastric juice by the PBG is slow, which is not beneficial to forming gel instantaneously; sodium alginate is used as a gel matrix independently, and the prepared PBG has small gel viscosity in gastric juice and is not beneficial to exerting the function of gastric mucosa adhesion, so that two gel matrixes of Kel and SA are selected in the experiment to prepare the PBG.

Taking a proper amount of Kel and SA, respectively and fully swelling in deionized water to prepare an in-situ gel sample, observing the state of the in-situ gel, and inspecting the upper limit and the lower limit of the dosage of the in-situ gel matrix.

The results show that gelation occurs naturally with no flowability when the Kel concentration is higher than 0.2% w/v. At SA concentrations above 1% w/v, PBG agglomerated in SGF and were difficult to disperse. When the concentrations of Kel and SA are below 0.01% w/v and 0.05% w/v, respectively, the concentrations are too low and the prepared sample does not form a gel in the gastric acid environment. The concentration range for the single factor investigation was thus determined: kel 0.01-0.2% w/v, SA 0.05-1% w/v.

2.1.4 Effect of prescription factors on PBG Properties

2.1.4.1 evaluation index

Taking 15mL of in-situ gel, adding 15mL of deionized water, and uniformly mixing to obtain the viscosity, namely the equivalent water dilution viscosity. Taking 15mL of in-situ gel, adding 15mL of SGF, and uniformly mixing to obtain the viscosity, namely the equivalent gastric juice dilution viscosity. The difference value of the equivalent gastric juice diluted viscosity and the equivalent water diluted viscosity is the pH response incremental viscosity, the calculation formula is shown as 1-5, the calculation formula is shown as 1-6 when the pH response incremental viscosity d1 is larger, the calculation formula is shown as 1-7 when the apparent viscosity d2 is smaller, the Hassan method is adopted, the comprehensive score is converted into a normalization value of 0-1, the geometric average value is calculated by using the normalization values of all indexes, and the calculation formula is shown as 1-8 when the normalization value (OD) of the pH response incremental viscosity and the apparent viscosity is used as an evaluation index.

η_pH＝η_SGF-η_DW	Equations 1 to 5
		d1＝(Y_n-Y_min)/(Y_max-Y_min)	Equations 1 to 6
d2＝(Y_max-Y_n)/(Y_max-Y_min)	Equations 1 to 7
		OD＝(d1×d2)^1/2	Equations 1 to 8

Wherein eta is_DWIs diluted with equal amount of water_SGFIs equivalent to the dilution viscosity of gastric juice_pHViscosity is increased for pH response. Y is_minTo determine the minimum value of viscosity, Y_maxThe maximum value of the measured viscosity is obtained.

2.1.4.2 Kel concentration Range examination

Weighing a certain amount of Kel, fully swelling in deionized water, slowly adding 30ml of the GR concentrated solution, heating while uniformly stirring by using an electric stirrer, evaporating the solution to be equal to the concentrated solution, stirring and cooling to room temperature to obtain the Kel-PGR in-situ gel. Kel concentrations were investigated separately: 0.01% w/v, 0.05% w/v, 0.1% w/v, 0.15% w/v, 0.2% w/v, and the effect of different concentrations of Kel on the apparent viscosity, pH-responsive delta viscosity, and OD values of the formulations was examined. Here, when Kel was used at 0.2% w/v, gelation occurred easily during cooling, so that this sample was subjected to viscosity measurement with stirring in real time to avoid coagulation, and the results are shown in FIG. 7.

The results show that the apparent viscosity gradually increases with increasing Kel concentration. At Kel concentrations below 0.1% w/v, the pH response delta viscosity is too low; kel at levels greater than 0.15% w/v increases dramatically and flow decreases. Therefore, in subsequent experiments, the Kel concentration is examined to be 0.1-0.15% w/v of the OD value which tends to increase.

2.1.4.3 SA dosage Range examination

Weighing a certain amount of SA, fully swelling in deionized water, slowly adding 30mL of PGR concentrated solution, heating while uniformly stirring by using an electric stirrer to evaporate the solution to be equal to the concentrated solution, stirring and cooling to room temperature to obtain the SA-PGR in-situ gel. The SA concentration ranges were examined separately: 0.05% w/v, 0.1% w/v, 0.5% w/v, 0.8% w/v, 1% w/v, and the effect of different amounts of SA on apparent viscosity, pH-responsive delta viscosity, and OD values were examined, and the results are shown in FIG. 8.

The results show that the apparent viscosity gradually increases with increasing concentration of SA. When the dosage of SA is less than 0.1% w/v, the pH response increment viscosity is too small; when the SA concentration is more than 0.5% w/v, the apparent viscosity increases sharply, which is disadvantageous for forming a cement having good fluidity. Therefore, in the subsequent experiments, the SA concentration is examined to be 0.1-0.5% w/v of the OD value which is in the rising trend.

2.1.5 star point design-effect surface method PBG prescription optimization process

2.1.5.1 star point design factor and level code table

The star point design factors and horizontal codes are shown in table 3.

TABLE 3 Star design factor and horizontal coding table

2.1.5.2 Star Point design-Effect surface experiment result

The apparent viscosity, pH response incremental viscosity and OD value were calculated in the same manner, and the results are shown in Table 4.

TABLE 4 Star design-response surface analysis combinations and test results

2.1.5.3 model fitting

The data obtained were analyzed experimentally using Design-Expert 11 software, and the results are shown in Table 5. And (3) carrying out binomial fitting on each level of each factor by using the total evaluation 'OD value' to obtain a multivariate nonlinear model equation with the total evaluation normalization value OD (Y) as a response value and 2 parameters as independent variables: y is 0.6320+0.0636A-0.0389B-0.2710A²-0.2885B²+0.1489A²B-0.1886AB²+0.3675A²B (r-0.9868, P-0.0012). Analysis of variance is shown in the following table: model item P<0.01, which shows that the model difference is statistically significant, and the mismatching term P is 0.7045, which indicates that the model has no outlier, and the residual error of the model may be generated by random error. Second order term A²、B²、A²B²Has a very significant influence on the model, AB²Has a significant effect on the model.

TABLE 5 analysis of variance

2.1.5.4 response surface analysis and process verification

And describing a three-dimensional effect surface according to a binomial expression, and selecting an experimental condition with a better overall evaluation of 'normalization value (OD)' from the effect surface, wherein the larger the OD value is, the better the experimental result is. The contour plot of the effect surface against the two independent variables A and B is shown in FIG. 9 with the total score "normalized" as the dependent variable. The prescription dose that maximizes the OD value predicted from the regression model is: kel dosage is 0.1% w/v, SA dosage is 0.5% w/v, the optimized prescription dosage is convenient for practical operation, and the predicted value of OD under the condition is 0.675.

Three portions of 30ml PLGR concentrated solution are taken, 3 portions of PBG are prepared in parallel according to the optimal prescription, the prescription dosage verification experiment is carried out according to the optimal optimized technological conditions, the deviation value is calculated, and the experimental result is shown in the table 6. The results show that the deviations (the deviation is (predicted value-measured value)/predicted value) are all less than 5.0%, which indicates that the established model is good in prediction.

Table 6 prescription dose verification experiment

2.2 Effect of Process factors on PBG Properties

2.2.1 preparation of PBG

The dosage of the prescription obtained by optimization according to the star point design-effect surface method is 0.1% w/v of Kel dosage and 0.5% w/v of SA dosage.

The preparation method comprises the following steps: taking 3 parts of pseudo-ginseng, 3 parts of bletilla striata and 3 parts of dendrobium officinale according to the parts by weight, adding 12 times of water, soaking for 45min, heating, decocting and extracting for 90min, extracting for 2 times, combining extracting solutions, and concentrating under normal pressure to obtain an extraction concentrated solution with the relative density of 1.04 and the concentration of 0.5 g/mL; and (2) adding deionized water into low acetyl gellan gum (Kel) and Sodium Alginate (SA) for fully swelling to form an in-situ gel matrix solution, slowly pouring the extraction concentrated solution obtained in the step (1), heating at 90 ℃, uniformly stirring at 150rpm, stirring at 170rpm, and cooling to room temperature to obtain the gel.

2.2.2 evaluation index

And (3) judging the advantages and disadvantages of the prescription process by using the form of the PBG, the normalized value (OD) of the apparent viscosity and the pH response increment viscosity as evaluation indexes and adopting an intuitive analysis and variance analysis method so as to select the optimal preparation process.

2.2.3 Effect of Process factors on PBG

2.2.3.1 heating temperature

The heating temperatures were 70 ℃, 80 ℃ and 90 ℃ respectively. Other conditions are as follows: PBG was prepared with a stirring speed of 150rpm for heating and 150rpm for cooling by an electric stirrer, and observed and measured, and the results are shown in Table 7.

The results show that the apparent viscosity and pH delta viscosity at 70 ℃ are both significantly lower by 90 ℃ (P <0.05), different heating temperatures have no significant effect on the OD values (P >0.05) and have no major effect on the appearance morphology of PBG. The low temperature will result in too slow evaporation rate, and the heating temperature is selected to be 90 ℃ in combination with the actual preparation process.

TABLE 7 influence of heating temperature

2.2.3.2 influence of stirring speed during heating

In the preliminary experiment process, when the stirring speed is more than 300rpm, the PBG is easy to generate thick bubbles, so that the apparent viscosity is increased, and the heating temperatures are respectively considered to be 100rpm, 200rpm and 300 rpm. Other conditions are as follows: PBG was prepared at a heating temperature of 90 ℃ and a stirring rotation speed of 150rpm at the time of cooling, and observed and measured, and the results are shown in Table 8.

The results show that the apparent viscosity at 100rpm is significantly lower (P <0.05), the apparent viscosity and pH delta viscosity are significantly higher (P <0.05) at 300rpm compared to 200rpm, and different stirring speeds have no significant effect on the OD value (P >0.05) but have a greater effect on the appearance morphology. The higher the rotation speed, the more likely the PBG is to generate dense bubbles inside, the more likely the upper layer is to generate foam, and the greater the apparent viscosity. The lower the rotation speed, the more uniform and fine the PBG, the slower the evaporation speed, and the stirring rotation speed is determined to be 150rpm in the heating process by combining the actual preparation process.

TABLE 8 influence of heating agitation speed

2.2.3.3 Effect of stirring speed during Cooling

In the preliminary experiment process, the stirring speed is higher than 300rpm when cooling, PBG is subjected to quick mechanical stirring, a large amount of gas is mixed in the PBG, and thick bubbles are easily generated, so that the apparent viscosity is increased, and the PBG needs to be kept stand for a long time for defoaming. Therefore, the stirring speeds in the cooling process were examined as 100rpm, 200rpm and 300rpm, respectively, and direct cooling without stirring was examined. Other conditions are as follows: the heating temperature is 90 ℃, and the stirring speed in the heating process is 150 rpm. PBG was prepared, observed and measured, and the results are shown in Table 9.

As a result, the apparent viscosity at 300rpm was significantly high, the OD value decreased with the increase of the rotation speed, and the different rotation speeds had no significant influence on the OD value (P >0.05) and had a large influence on the appearance. The higher the rotation speed, the more likely the inside of the formulation is to generate dense bubbles, the more likely the upper layer is to generate foam, and the greater the apparent viscosity. The lower the rotating speed, the more uniform and fine the PBG, the slower the cooling speed, the more easy the PBG is to agglomerate, and the cooling stirring rotating speed is determined to be 170rpm by combining the actual preparation process.

TABLE 9 influence of stirring speed on Cooling

2.2.4 best Process verification experiment

Taking three parts of concentrated solution, preparing 3 parts of PBG in parallel according to the optimal formula, carrying out process verification, measuring apparent viscosity and pH response increment viscosity, calculating OD value, calculating RSD value of each index, observing PBG form, and obtaining the result shown in Table 10.

The verification test results show that the RSD value of each index is less than 3%, and the results show that the prepared 3 parts of PBG has stable process and good reproducibility, and the preparation process is stable and feasible.

Table 10 verification of the preparation Process

The notoginseng and the pH responsive in situ gel PBG consist of main medicaments (a traditional Chinese medicine composition extract PGR) and in situ gel matrixes (Kel and SA). The PBG preparation process is examined, and the influences of the prescription factors (Kel dosage and SA dosage) and the process factors (heating temperature, stirring speed during heating and stirring speed during cooling) on the PBG apparent viscosity, pH response increment viscosity and appearance form are examined. The single factor investigation result shows that slight changes of Kel and SA concentration can generate great influence on PBG viscosity, and too high concentration can cause too high apparent viscosity and poor fluidity of PBG; too low a concentration results in a pH response with an incremental viscosity that is too low to form a gel in SGF. The single factor examination Kel concentration range is 0.01-0.2% w/v, and the SA examination concentration range is 0.05-1% w/v.

PBG prepared by Kel and SA in combination shows a relatively obvious viscosity synergistic effect, the change of the concentration of PBG can influence the change of apparent viscosity and pH response increment viscosity, the change of apparent viscosity and pH response increment viscosity has interaction, and the optimal proportion exists in the dosage. Further optimizing the prescription of Kel and SA by adopting a star point design-effect surface method, wherein the dosage of the prescription obtained by final optimization is as follows: kel0.1% w/v and SA 0.5% w/v, and the PBG prepared by the optimized formula has good in-vitro flowability, high speed of forming gel in a gastric juice environment, large pH response increment viscosity and good dispersibility.

In the investigation of the preparation process, the influence of the heating temperature, the stirring speed during heating and the stirring speed during cooling on the PBG apparent viscosity, the pH response increment viscosity and the appearance form is investigated. The results show that the three selected process factors have no significant influence on the OD value, wherein the stirring speed has a large influence on the form of the preparation. The PBG is mixed with a large amount of bubbles due to the influence of mechanical stirring when the rotating speed is too high, and the PBG needs to be placed for a long time for defoaming; too low a rotational speed can result in solidification of the PBG without flowability. And finally determining the process factors of heating temperature of 90 ℃, stirring speed of 150rpm during heating and stirring speed of 170rpm during cooling by combining the single-factor investigation result. The prescription process is verified, and the 3 batches of PBG prepared by the method have stable process and better repeatability.

Example 3 evaluation of effectiveness of Notoginsen triterpenes and pH-responsive in situ gels

1 instruments and materials

1.1 instruments

1.2 reagents and reagents

Colloidal bismuth pectin (batch No. 191001, Renshi & pharmaceutical Co., Ltd.); absolute ethanol (AR, lot No. 20201230, national chemical group reagents, Inc.); SOD and MDA kit (batch number: 20210331, Nanjing institute of bioengineering); BCA kit (batch number: 20210406, Nanjing institute of bioengineering); TNF-alpha and IL-6 kit (batch No. 20210330002, Nanjing institute of bioengineering); VEGF-A, PGE2, EGF kit (batch No. 20210331003, Nanjing institute of bioengineering); sodium chloride (batch No. 20191001, Jiangsu Qiangsheng functional chemistry Co., Ltd.).

1.3 preparation of extract and preparation

The extraction method of the traditional Chinese medicine composition (PGR) comprises the following steps: taking 3 parts of pseudo-ginseng, 3 parts of bletilla striata and 3 parts of dendrobium officinale according to the parts by weight, adding 12 times of water, soaking for 45min, then heating, decocting and extracting for 90min, extracting for 2 times, combining extracting solutions, and concentrating under normal pressure to obtain an extraction concentrated solution with the relative density of 1.04 and the concentration of 0.5 g/mL.

The preparation method of the pseudo-ginseng and bletilla striata pH responsive in situ gel (PBG group) comprises the following steps:

1.4 animals

36 SD rats with male sex, 180-220 g, provided by Nanjing university of traditional Chinese medicine laboratory animal center, license number SCXK (Zhe) 2019-.

2 methods and results

2.1 animal groups

36 SD rats, male, with body mass of 180-220 g, feeding environment temperature of 23-25 deg.C, freely drinking water and eating, adaptively feeding for 1 week, randomly grouping, and weighing. The experiments were divided into 6 groups: 1. blank group (Normal control, NC), 2 Model group (Model control, MC), 3 Positive drug group (PC), 4 adjuvant group (Access groups, AG), 5 PGR group, 6 PBG group.

2.2 administration and dosage

The blank and model groups were drenched with distilled water.

The colloidal bismuth pectin is given to the positive drug group, the dosage is converted into rat equivalent dosage, namely 63mg/kg, according to the clinical dosage of human, the drug is administrated by gastric gavage according to 10mL/kg, and the concentration of the colloidal bismuth pectin aqueous suspension which needs to be prepared is 6.3 mg/mL.

The adjuvant group was gavaged with a gel matrix solution (Kel 0.1% w/v, SA 0.5% w/v); the PGR group is administered with notoginseng and bletilla striata stomach-protecting prescription by intragastric administration, and the PBG group is administered with notoginseng and bletilla striata pH responsive in situ gel by intragastric administration, with administration dosage of 0.945 g/kg.

The stomach was gavaged at a volume of 10mL/kg for 1 time/1 day for each group.

2.3 Molding

Fasting for 24h before the last administration, full fasting, emptying of bedding and faeces. After 2h after the last administration, except for the blank group, all the tested animals are gavaged and administered with 1 mL/animal of absolute ethyl alcohol to make an acute gastric mucosa injury model, and anesthesia and material drawing observation are carried out after 1 h.

2.4 sample Collection

After 1h of modeling, rats are anesthetized, 7mL of abdominal aorta blood is collected in an anticoagulant-free vacuum blood collection tube, after standing for 1h, the abdominal aorta blood is centrifuged for 15min at 4 ℃ and 3500rpm, serum is separated, and the serum is respectively filled in a0.5 mL centrifuge tube and stored at the temperature of minus 20 ℃ in a 150 mu L tube and taken at any time. Taking the stomach, splitting along the greater curvature of the stomach, washing the contents of the stomach with precooled normal saline, observing the gastric mucosa of the rat by naked eyes, measuring the ulcer length with a vernier caliper, recording the ulcer index, taking the stomach tissue of the glandular stomach 1cm × 1cm after recording, quickly fixing the stomach tissue in 4% paraformaldehyde fixing solution, and making a tissue pathological section. Adding 9 times of ice PBS buffer solution into the rest stomach tissue according to the weight-volume ratio, fully homogenizing on ice to prepare 10% tissue homogenate, centrifuging for 10min at 5000Xg, and taking supernatant to store at-20 ℃.

2.5 morphological Observation of rat stomach tissue

2.6 gastric mucosal Ulcer Index (UI) and ulcer inhibition ratio determination

The degree and morphology of gastric mucosal lesions were visually observed, and the length and width (mm) of bleeding points or bands were measured with a vernier caliper and scored. Ulcer Index (UI) scoring criteria are shown in table 11.

TABLE 11 ulcer index score criteria

2.7 section observation of pathological tissue of gastric mucosa

2.8 determination of Biochemical indicators (SOD, MDA) in serum

2.9 determination of relevant indices in stomach tissue

2.9.1 measurement of SOD and MDA in stomach tissue

2.9.2 measurement of inflammatory factors in stomach tissue

2.10 statistical processing and data representation

3 results and analysis

3.1 morphological Observation of rat stomach tissue

The gastric mucosa of the rats in the blank group is smooth and has no damage; the model group rat has serious gastric mucosa injury, wide bleeding zone on the surface, and serious mucosa swelling and congestion; the gastric mucosa of the rat in the positive medicine group is basically intact, and the injury is light; the degree of gastric mucosa injury of the auxiliary material group is lighter than that of the model group, and congestion, bleeding and wider bleeding zones still exist; both the rats in the PGR group and PBG group had less gastric mucosal damage, and the PBG group was slightly superior to the PGR group, as indicated by a smaller bleeding area and a narrower bleeding band.

The ulcer index and inhibition rate determination results are shown in table 12, and the results show that compared with the model group, the ulcer index of the auxiliary material group is obviously smaller than that of the model group (P is less than 0.05), and the ulcer indexes of the positive medicine group, the PGR group and the PBG group are obviously reduced (P is less than 0.01) compared with that of the model group. Compared with the adjuvant group, the ulcer indexes of the PGR group and the PBG group are obviously reduced (P is less than 0.01). Ulcer index was significantly lower in PBG group than in PGR group (P < 0.05).

The ulcer inhibition rate of the positive medicine is 14.91 percent, the ulcer inhibition rate of the auxiliary material group is 21.05 percent higher than that of the positive medicine, and the gel layer formed by the auxiliary materials in a gastric juice environment is supposed to be adhered to the gastric mucosa to play a physical protection role. The ulcer inhibition rates of the PGR group and PBG group were 43.86% and 56.58%, respectively.

TABLE 12 ulcer index and ulcer inhibition results (n ═ 6)

3.3 section observation of pathological tissues of gastric mucosa

Pathological section results show that the mucosa layer and the muscle layer can be seen under a blank group of glasses, the cell staining of the mucosa layer is deepened, and the structure of the muscle layer is complete and has no obvious abnormality. Muscular layer and submucosa can be seen under the model group lens, blood vessels of the submucosa are congested, and inflammatory cell infiltration can be seen around the blood vessels. The tissue structure of the muscle layer of the positive medicine group is compact, the cells are regularly arranged, and the loose performance can be seen locally. The auxiliary material group shows that mucous membrane layers are damaged, mucous membrane folds are damaged, the whole layer shows congestion performance, the muscular layer is thinned, and inflammatory cells can be occasionally seen in each layer. The PGR group muscularis cells are arranged regularly, compact and good in integrity, and the submucosa tissue structure is loose. Mucous membrane, gland and submucous muscular layer can be seen under the PBG group lens, inflammatory cells are occasionally seen, and other structures are not obviously abnormal.

3.4 determination of Biochemical indicators in serum

3.4.1 measurement of SOD and MDA in serum

The measurement results of SOD and MDA in serum are shown in FIG. 10. The results show that the SOD activity in the serum of the rat in the model group is obviously reduced (P is less than 0.01) compared with that in the blank group, and the SOD activity in the serum of the rat in the model group is obviously improved (P is less than 0.01) compared with that in the model group, which indicates that the model building is successful; SOD activity in serum of each administration group is increased compared with that of a model group, the auxiliary material group has significant difference (P is less than 0.05), and the PBG group has very significant difference (P is less than 0.01); SOD activity in PNG group serum is obviously higher than that in PGR group (P < 0.05).

The MDA content in the serum of the model group rat is obviously increased (P is less than 0.01) compared with that of the blank group, which indicates that the model building is successful; the content of each administration group is reduced compared with that of a model group, wherein the auxiliary material group has significant difference (P is less than 0.05), and the PGR group and the PBG group have very significant difference (P is less than 0.01); the MDA content in the serum of the PGR group and the PBG group is obviously lower than that of the positive medicine group (P is less than 0.01); the content of MDA in the PBG group is reduced compared with that in the PGR group (P is more than 0.05).

3.5 determination of relevant indices in stomach tissue

3.5.1 measurement of SOD and MDA in stomach tissue

SOD and MDA in stomach tissue are measured as shown in figure 11. The results show that after the ethanol molding, the activity of SOD in rat stomach tissue is reduced (P is less than 0.05), and the MDA content is increased (P is less than 0.01), which indicates the success of molding.

In the index SOD activity, the positive medicine group, the auxiliary material group and the PGR group are increased (P is less than 0.05) compared with the model group, and the PBG group is obviously increased (P is less than 0.01) compared with the model group; the SOD activity of the PBG group is obviously higher than that of the auxiliary material group (P is less than 0.05); SOD in stomach tissue of PGR group is higher than that of PBG group (P > 0.05).

In the index MDA content result, the auxiliary material group is reduced (P is less than 0.05) compared with the model group, and the positive medicine group, the PGR group and the PBG group are obviously reduced (P is less than 0.01) compared with the model group; the content of MDA in the gastric tissue of the PBG group is obviously lower than that of the blank group and the auxiliary material group (P is less than 0.01) and lower than that of the PGR group (P is more than 0.05).

3.5.2 measurement of inflammatory factors in stomach tissue

The results of the measurement of IL-6 and TNF-alpha contents in stomach tissues are shown in FIG. 12. The result shows that after ethanol molding, the water average of inflammatory factors IL-6 and TNF-alpha in rat stomach tissue is obviously increased (P is less than 0.01) compared with that of a blank group, which indicates that molding is successful.

In the index IL-6, compared with the model group, the content of the auxiliary material group is reduced (P is less than 0.05), and the content of the positive medicine group, the PGR group and the PBG group is obviously reduced (P is less than 0.01); the content of PBG group is lower than that of positive medicine group and that of PGR group (P > 0.05).

In the index TNF-alpha, compared with a model group, the contents of a positive medicine group, a PGR group and a PBG group are obviously reduced (P is less than 0.05); the content of PBG group is lower than that of positive medicine group and that of PGR group (P > 0.05).

The results of measuring the content of factors related to gastric mucosal protection (PGE2, VEGF-A, EGF) in stomach tissue are shown in FIG. 13.

In the content of the index PGE2, compared with a blank group, the content of the model group is obviously reduced (P is less than 0.05); compared with the model group, the positive medicine group and the auxiliary material group are increased (P is more than 0.05), and the PGR group and the PBG group are obviously increased (P is less than 0.01); the content of PGE2 in the PBG group is higher than that in the blank group, the positive medicine group and the auxiliary material group (P is less than 0.05); the PBG group content was higher than the PGR group, but there was no significant difference (P > 0.05).

In the index VEGF-A content, compared with a blank group, the content of a model group is obviously reduced (P is less than 0.05); compared with the model group, the content of the positive drug group is increased (P is less than 0.05), and the content of the PGR group and the PBG group is obviously increased (P is less than 0.01); the content of PBG group is obviously higher than that of blank group, positive medicine group and PGR group (P is less than 0.05).

In the index EGF content, the content of the model group is obviously reduced (P is less than 0.05) compared with that of the blank group; compared with a model group, the EGF content of each administration group is increased, wherein the auxiliary material group has obvious difference (P is less than 0.05), and the positive medicine group, the PGR group and the PBG group have extremely obvious difference (P is less than 0.01); the content of PBG group is higher than that of blank group (P is less than 0.05), and is obviously higher than that of auxiliary material group and PGR group (P is less than 0.01).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A Chinese medicinal composition for treating digestive tract diseases is characterized by comprising pseudo-ginseng, rhizoma bletillae and dendrobium officinale.

2. The traditional Chinese medicine composition for treating digestive tract diseases according to claim 1, which is prepared from 3-9 parts of pseudo-ginseng, 3-9 parts of bletilla striata and 3-9 parts of dendrobium officinale.

3. The traditional Chinese medicine composition for treating digestive tract diseases according to claim 2, which is prepared from 3 parts of pseudo-ginseng, 3 parts of bletilla striata and 3 parts of dendrobium officinale.

4. The preparation method of the Chinese medicinal composition for treating the digestive tract diseases as claimed in any one of claims 1 to 3, which comprises the following steps,

5. A pH-responsive in-situ gel containing radix Notoginseng and rhizoma Bletillae is characterized by comprising radix Notoginseng, rhizoma Bletillae, herba Dendrobii traditional Chinese medicine extract, low acetyl gellan gum and sodium alginate.

6. The pseudo-ginseng and bletilla striata pH-responsive in-situ gel as claimed in claim 5, wherein the gel comprises concentrated extracts of pseudo-ginseng, bletilla striata and dendrobium officinale, low acetyl gellan gum with a mass concentration of 0.1% and sodium alginate with a mass concentration of 0.5%.

7. The preparation method of pseudo-ginseng and pH responsive in situ gel according to claim 5 or 6, which is characterized by comprising the following steps:

8. The preparation method of pseudo-ginseng and pH responsive in situ gel according to claim 7, which comprises the following steps:

9. The method for preparing notoginseng and pH responsive in situ gel according to claim 8, wherein the mass concentration of low acetyl gellan gum is 0.1% and the mass concentration of sodium alginate is 0.5%.

10. Use of the Chinese medicinal composition of any one of claims 1 to 3 for the preparation of a medicament for the treatment of digestive tract diseases.

11. Use of the Chinese medicinal composition of any one of claims 1 to 3 for the preparation of a medicament for the treatment of gastrointestinal ulcer diseases.

12. Use of notoginseng and pH responsive in situ gel as claimed in claim 5 or 6 for the preparation of a medicament for the treatment of digestive tract diseases.

13. Use of notoginseng and pH responsive in situ gel as claimed in claim 5 or 6 for the preparation of a medicament for the treatment of digestive tract diseases.