阅读说明：本技术 一种蒙药肋柱花提取物的应用 (Application of Mongolian medicine extract of lomatogonium carinthiacum ) 是由 付明海 阿丽沙 于 2021-06-29 设计创作，主要内容包括：本发明提供了一种蒙药肋柱花提取物的应用；所述蒙药肋柱花提取物在制备调节肠胃的药物中的应用。本发明所涉及的肋柱花提取物经化学成分检测结果,选择含量最高的獐牙菜苦苷,进而证明肋柱花提取物和獐牙菜苦苷对小鼠胃排空和小肠推进的影响,再用DA和5-HT建立胃肠动力障碍模型,证明蒙药肋柱花提取物对神经递质紊乱性胃肠动力障碍疾病具有一定的影响。(The invention provides an application of a Mongolian medicine extract of costaria lemaneiformis; the application of the Mongolian medicine ribwort extract in preparing the medicine for regulating the intestines and stomach. According to the invention, the stigmata extract is subjected to chemical component detection, the highest-content swertiamarin is selected, the influence of the stigmata extract and the swertiamarin on the gastric emptying and small intestine propulsion of a mouse is further proved, and a gastrointestinal motility disorder model is established by using DA and 5-HT, so that the Mongolian medicine stigmata extract has certain influence on neurotransmitter disorder gastrointestinal motility disorder diseases.)

1. An application of a Mongolian medicine ribcage flower extract is characterized in that the Mongolian medicine ribcage flower extract is applied to preparing medicines for regulating intestines and stomach.

2. The use of the extract of the Mongolian medicine ribbing flower as claimed in claim 1, wherein the preparation method of the extract of the Mongolian medicine ribbing flower comprises the following steps:

step 1, taking the lomatogonis, crushing, sieving, adding dichloromethane, performing reflux extraction, and filtering;

step 2, treating the filtrate, recovering dichloromethane, adding ethanol into filter residue, performing reflux extraction, filtering, treating the filtrate, and recovering ethanol to obtain an extract;

and 3, drying the extract to obtain the finished product.

3. The use of the Mongolian medicine extract of costaphyllum as claimed in claim 2, wherein in step 1, the ratio of the amount of costaphyllum to the amount of dichloromethane is 1: 10.

4. The use of the extract of the Mongolian medicine, ribwort, as claimed in claim 2, wherein in step 1, the reflux extraction is performed 2 times each for 4 hours.

5. The use of the extract of the Mongolian medicine Ribes burejense of claim 2, wherein in the step 2, the recovered apparatus is a rotary evaporator RE-2010.

6. The use of the Mongolian medicine ribwort extract as claimed in claim 2, wherein in step 2, the dosage ratio of the filter residue to the ethanol is 1: 10.

7. The use of the extract of the Mongolian medicine, ribwort, as claimed in claim 2, wherein in step 2, the reflux extraction is performed 2 times, each time 6 hours.

8. The use of the extract of the Mongolian medicine Ribes nivalis as claimed in claim 2, wherein the concentration of the ethanol in step 2 is 95 wt%.

9. The use of the Mongolian medicine extract of Ribis scholaris in claim 2, wherein in step 3, the drying apparatus is a freeze dryer FD-1A-50.

10. The use of the extract of the Mongolian medicine, ribwort, as claimed in claim 2, wherein in step 3, the drying time is (3-5h) and the drying temperature is (-50 ℃).

Technical Field

The invention belongs to the field of Mongolian medicine ribwort extract; in particular to an application of a Mongolian medicine ribwort extract.

Background

Costal column flower is a Mongolian medicine characteristic medicinal material. Has extremely bitter taste, has the effects of calming, invigorating stomach, healing wound and clearing heat, and is commonly used for treating diseases such as heat in the morning, typhoid fever, jaundice, influenza, heatstroke and fever, plague, liver and gallbladder heat, injury heat, stomach in the morning and evening. Costcolumn flower is also the main traditional medicine for treating liver and gall diseases by Mongolian medicine. It has been shown in previous studies to have therapeutic effects on dyspepsia, gallbladder swelling, jaundice, and inflammation of the liver and gallbladder. Later, the flavonoids of the Mongolian medicine ribwort flower are found to have the functions of reducing blood fat and treating obesity. In addition, researches report that swertia pseudochinensis of Gentianaceae is a traditional medicine for treating gastrointestinal diseases such as nausea, gastroparesis, gastric retardation and the like. However, the costal column flower as the Mongolian medicine has less experimental research on the gastrointestinal function and mechanism.

In recent years, with the improvement of living standard of people, the incidence rate of gastrointestinal motility disorder diseases in China is higher and higher, and the disease becomes a research hotspot of a digestion clinic. Gastrointestinal motility is an extremely complex neuromuscular activity that can ensure a person's normal digestive function, and its gastrointestinal motility disorders may lead to a variety of gastrointestinal motility disorder diseases. However, with the development of modern medicine, more and more researches show that the factors for regulating the gastrointestinal motility are diversified, mainly comprising gastrointestinal hormones, intestinal flora, neurotransmitters, some proteins and ion channels, and besides bitter taste and receptors thereof are also involved in the regulation of the gastrointestinal motility. Researches report that the bitter substance bendinylamine can inhibit gastric emptying and reduce food intake, and quinine can inhibit gastrointestinal motility and influence the release of motilin and ghrelin.

Of the digestive outpatients, about 40% of patients are diagnosed with gastrointestinal motility disorders, which seriously affect the physical health and quality of life of the patients. Although many drugs for treating such diseases have been developed in the market, such as domperidone, cisapride and the like which are clinically and frequently used at present, cardiovascular disorders are caused by long-term use of such drugs. Researches show that the natural botanical drug has good curative effect on gastrointestinal diseases, has small side effect and low cost, and has been deeply researched by many researchers. The pathogenesis factors inducing the diseases are reported to be many, and the bitter taste and the receptor thereof are found to be involved in the gastrointestinal function and regulate the gastrointestinal motility in recent years.

The bitter Mongolian medicine costcolumn flower also has a certain effect on regulating gastrointestinal motility as a traditional botanical medicine for treating liver and gall diseases. However, the mechanism of action has not been reported.

Bitter compounds modulate gut hormone secretion and gastrointestinal motility through T2Rs located in the tongue taste buds and in enteroendocrine cells. Vida Bitarafan et al, by clinical trials, mixed quinine in the diet of healthy men, observed blood glucose response, gastric emptying and subsequent energy intake, found that intragastric administration of quinine reduced postprandial blood glucose, stimulated insulin and GLP-1, but did not slow gastric emptying or reduce energy intake. David Wicks et al, in order to verify that functional dyspepsia is nausea and vomiting is related to gastric emptying, fed healthy women with a slimming stick with improved bitter taste or a stick with a pleasant strawberry taste, and observed the gastric emptying time and the antral movement time noninvasively by electrical impedance tomography and a bioelectricity graph, found that the gastric emptying time of the bitter-taste fed group is significantly prolonged compared with that of the pleasure stick, and it is considered that unpleasant bitter taste delays gastric emptying without significantly impairing antral motility.

Disclosure of Invention

The invention aims to provide application of a Mongolian medicine ribwort extract.

The invention is realized by the following technical scheme:

the invention relates to application of a Mongolian medicine ribwort extract in preparing a medicine for regulating intestines and stomach.

Preferably, the preparation of the extract of the Mongolian medicine ribwort comprises the following steps:

step 1, taking the lomatogonis, crushing, sieving, adding dichloromethane, performing reflux extraction, and filtering;

step 2, treating the filtrate, recovering dichloromethane, adding ethanol into filter residue, performing reflux extraction, filtering, treating the filtrate, and recovering ethanol to obtain an extract;

and 3, drying the extract to obtain the finished product.

Preferably, in step 1, the ratio of the amount of the ribbing flower to the amount of the dichloromethane is 1: 10.

Preferably, in step 1, the reflux extraction is performed for 2 times, and each time is 4 hours.

Preferably, in step 2, the recovered apparatus is a rotary evaporator RE-2010.

Preferably, in the step 2, the dosage ratio of the filter residue to the ethanol is 1: 10.

Preferably, in step 2, the reflux extraction is performed for 2 times, and each time is 6 hours.

Preferably, in step 2, the concentration of ethanol is 95 wt%.

Preferably, in step 3, the drying apparatus is a freeze dryer FD-1A-50.

Preferably, in step 3, the drying time is (3-5h), and the drying temperature is (-50 ℃).

The principle of the invention is as follows: the regulating effect of the bitter Mongolian medicine ribbing flower on gastrointestinal motility is verified by adopting gastric emptying and small intestine propulsion experiments. In order to verify the influence of the ribbing extract on the gastrointestinal function, the highest-content swertiamarin is selected according to the detection result of chemical components of the ribbing extract, the influence of the ribbing extract and the swertiamarin on the gastric emptying and the small intestine propulsion of a mouse is proved, a gastrointestinal motility disorder model is established by DA and 5-HT, and the certain influence of Mongolian medicine ribbing on neurotransmitter disorder gastrointestinal motility disorder diseases is proved.

The invention has the following advantages:

(1) the invention utilizes Ach and CaCl₂After the isolated ileocecal smooth muscle is induced to cause spastic contraction, the extract of the lomatogonium carinthiacum and the main chemical components of hesperetin and coumarin thereof can obviously inhibit the sudden spastic contraction, so that the contraction action tends to be normal. Therefore, the extract of the lomatogonium carinthiacum is suspected to have potential treatment effect on neurotransmitter disturbance gastrointestinal motility disorder diseases, and the effective components are primarily considered to be hesperetin and coumarin.

(2) According to the detection result of the chemical components of the costal column flower extract, the highest content of swertiamarin is selected, the influence of the costal column flower extract and the swertiamarin on the gastric emptying and small intestine propulsion of a mouse is proved, and a gastrointestinal motility disorder model is established by using DA and 5-HT, so that the Mongolian medicine costal column flower has certain influence on neurotransmitter turbulent gastrointestinal motility disorder diseases.

Drawings

FIG. 1 is a graph showing the effect of swertiamarin in an extract of costaphylus asiaticus on mice; wherein A is an influence graph of swertiamarin on gastric emptying of mice; b is a graph of the influence of swertiamarin on the small intestine propulsion of the mouse;

FIG. 2 is an image of extract of Crotalaria ribacterica on DA-injected mice; wherein a is a graph of the effect on gastric emptying in mice; b is a graph of the effect on intestinal transit in mice;

FIG. 3 is a photograph of extract of ribcage versus 5-HT injected mice; wherein a is a graph of the effect on gastric emptying in mice; b is a graph of the effect on intestinal transit in mice;

FIG. 4 is a schematic diagram of isolated small intestine smooth muscle contraction activities at different parts of a mouse;

FIG. 5 is a graph of the effect of extract of ribbonfil on ex vivo ileocecal smooth muscle in mice; wherein A is an influence graph of contraction amplitude; b is an influence graph of frequency;

FIG. 6 is a graph of the effect of extract of ribbonfil on ex vivo ileocecal smooth muscle in mice; wherein A is an influence graph of contraction amplitude; b is an influence graph of frequency;

FIG. 7 is a diagram showing that the normal contraction state can be recovered by repeated washing;

FIG. 8 is a graph of the effect of extract of ribbonfil on Ach-induced ex vivo ileal smooth muscle in mice; wherein A is an influence graph of contraction amplitude; b is an influence graph of frequency;

FIG. 9 is the extract of Crotalaria costata vs. CaCl₂Graph of the effect of induced mice ex vivo ileal smooth muscle; wherein A is an influence graph of contraction amplitude; b is an influence graph of frequency;

FIG. 10 is a graph of the effect of HC and HP on Ach-induced ex vivo ileo-smooth muscle in mice; wherein A is an influence graph of contraction amplitude; b is an influence graph of frequency;

FIG. 11 is HC and HP vs CaCl₂Graph of the effect of induced mice ex vivo ileal smooth muscle; wherein A is an influence graph of contraction amplitude; b is an influence graph of frequency;

FIG. 12 is a graph of the effect of intercostal flower extract (LR) on galectin-induced ex vivo ileal smooth muscle in mice; wherein A is an influence graph of contraction amplitude; and B is an influence graph of frequency.

Detailed Description

The present invention will be described in detail with reference to specific examples. It should be noted that the following examples are only illustrative of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

This example relates to the use of extract of the Mongolian drug, ribbing, on gastric emptying and small intestinal transit in mice.

1. Effects on gastric emptying and intestinal transit in Normal mice

Taking 18 mice, randomly dividing the mice into 3 groups, and each group comprises 6 mice;

respectively: normal control group, low dose group (1g kg) of extract solution of costaphyllum^-1) Hippocampus costata extract high dose group (4g kg)^-1)。

The method comprises the following specific steps: the corresponding drug solution is administered in the dosage groups in a gavage manner with the dosage of 0.1 mL-10 g^-1The normal control group was given an equal volume of CMC-Na solution. The groups were fasted before administration without water deprivation for 16 h. And 5min after administration on the next day, perfusing the blue semi-solid nutrient paste into the stomach, wherein the lavage volume is 0.5mL for each, after 10min, removing the neck, killing the mice of each group, rapidly cutting open the abdomen, taking out the stomach, wiping the stomach with filter paper, weighing the full weight of the stomach, then cutting the stomach body along the greater curvature of the stomach, flushing the content in the stomach, wiping the stomach, and weighing the clean weight of the stomach. Simultaneously, the small intestine is immediately taken out, is gently stripped and is directly placed on an operating table cloth, and the distance from the pylorus to the blue semisolid nutrient paste and the total length of the small intestine are measured. The evacuation rate of the gastric blue semi-solid nutritional paste and the small intestine propulsion rate were calculated according to the following formulas. The emptying rate (%) of the stomach blue semisolid nutrient paste is 1- (stomach full weight-stomach net weight/stomach full weight) multiplied by 100%; the small intestine propulsion rate (%) -, the distance from pylome to blue semisolid nutrient paste/small intestine full length × 100%.

As a result: results of the extract of the ribbing flower on the gastric emptying rate and the small intestine propulsion rate of normal mice show that compared with the normal group, the low dose of the extract of the ribbing flower can promote the gastric emptying rate (P < 0.001), and the high dose group can slow the gastric emptying rate and reduce the small intestine propulsion rate (P < 0.0001); swertiamarin also significantly reduced gastric emptying in mice (P <0.01) without significant effect on small intestine propulsion rate (P > 0.05). The effect of swertiamarin on gastric emptying and intestinal propulsion in mice is shown in figure 1, with values: mean ± standard error, six mice per group. Note: p <0.01, P <0.0001 (n-4-6) compared to normal group. Wherein A is an influence graph of swertiamarin on gastric emptying of mice; b Effect of swertiamarin on small intestine propulsion of mice.

2. Influence on gastric emptying and intestinal propulsion inhibition of mice caused by dopamine hydrochloride (DA)

24 mice were randomly divided into 4 groups, namely a normal control group and a dopamine model group (1 mg. kg)^-1) Low dose of extract of ribbonfil + DA group, high dose of extract of ribbonfil + DA group. Each administration group was administered by intragastric administration with 0.1 mL. multidot.10 g of the corresponding drug solution^-1Blank groups were given equal volumes of CMC-Na solution. The groups were fasted before administration without water deprivation for 16 h. After 5min of administration next day, except for normal control group, each group was injected with DA intraperitoneally, after 5min, each group was administered with blue semi-solid nutritional paste, the intragastric administration volume was 0.5mL each, and after 10min, the neck was removed to kill the mice, and the gastric blue liquid diet evacuation rate and small intestine propulsion rate were calculated.

As a result: the results of the extract of ribbonfil on gastric emptying and small intestine propulsion of mice with gastrointestinal motility disorder model induced by intraperitoneal injection of DA are shown in FIG. 2, and the values are: mean ± standard error, six mice per group. Note: model group compared to normal group, # P < 0.05; p <0.05, P <0.01 in the administration group compared to the model group. The gastric emptying rate and the small intestine propulsion rate of the mice in the dopamine model group were significantly reduced compared to the normal group (P < 0.05). Compared with the DA model group, the extract of the lomatogonium carinthiacum has a remarkable promotion effect on the DA-induced emptying delay of the mouse gastric blue semisolid nutrition paste (P <0.05 and P <0.01 respectively).

3. Influence on gastric emptying and intestinal hyperfunction of mice caused by serotonin (5-HT)

24 mice were randomly divided into 4 groups, namely a normal control group and a 5-HT model group (4 mg. kg)^-1) Low dose of extract of ribbonfil + group DA, high dose of extract of ribbonfil + group DA. Each administration group was intragastrically administered with 0.1 mL. multidot.10 g of the corresponding drug solution^-1Blank groups were given equal volumes of CMC-Na solution. The groups were fasted before administration without water deprivation for 16 h. After 5min of administration the next day,except for a normal control group, each group is injected with 5-HT in the abdominal cavity, after 10min, each group is given blue semisolid nutrient paste, the intragastric administration volume is 0.5mL of each, after 10min, the neck is removed, the mice are killed, and the gastric blue liquid diet evacuation rate and the small intestine propulsion rate are calculated.

As a result: the results of the extract of ribbonfil on gastric emptying and intestinal transit in mice model of gastrointestinal motility disorder caused by intraperitoneal injection of 5-HT are shown in FIG. 3, and the values are: mean ± standard error, six mice per group. Note: model group compared to normal group, # P < 0.05; dose group and model group comparison, P <0.05, P <0.001, P < 0.0001. The 5-HT model group showed a significant increase in gastric emptying rate (P <0.05) compared to the normal group. Compared with the 5-HT model group, the high and low doses of the extract of the ribbing flowers have obvious reduction effects on the emptying rate and the small intestine propulsion rate of the blue semisolid nutrient paste in the stomach of the mouse (P is less than 0.05, P is less than 0.001 and P is less than 0.0001).

In the embodiment, a gastrointestinal motility disorder model mouse is established by using neurotransmitters DA and 5-HT, wherein DA is a precursor of epinephrine and norepinephrine and is also a neurotransmitter which plays a role through a specific receptor, and has the effects of relaxing stomach smooth muscle, relaxing intestinal canal, expanding mesentery, inhibiting the propulsion of a stomach body and a gastric antrum, shrinking pylorus, delaying gastric emptying and the like. DA is an important gastrointestinal neurotransmitter due to its wide distribution in the gastrointestinal tract, and is also one of the protective factors of the gastrointestinal mucosa. According to the pharmacology, DA mainly activates alpha, beta adrenaline and peripheral dopamine receptors, inhibits the propulsion of the stomach body and the antrum of the stomach and slows the gastric emptying. 5-HT is also one of important neurotransmitters, is mainly distributed in chromaffin cells of intestinal mucosa, and has the effects of promoting gastrointestinal wall contraction and gastrointestinal peristalsis. An increase in such neurotransmitters in the human body also causes many gastrointestinal motility disorders. The test proves that: the extract of the costal columna can promote the delayed gastric emptying and the small intestine propulsion of mice caused by DA; and has inhibitory effect on gastric emptying and small intestine propulsion caused by 5-HT. Therefore, the Mongolian medicine costaria flower can reduce the symptoms of gastrointestinal motility disorder caused by 5-HT increase.

According to the invention, the extract of the lomatogonium carinthiacum with different concentrations is adopted to induce the gastric emptying rate and the small intestine propulsion rate of the mouse, and the surprising discovery shows that the 1g/kg dose of the extract of the lomatogonium carinthiacum can obviously promote the gastric emptying of the mouse, and the 4g/kg dose of the extract of the lomatogonium carinthiacum slows the gastric emptying of the mouse and inhibits the small intestine propulsion, so that the extract of the lomatogonium carinthiacum is preliminarily considered to be capable of regulating the gastric emptying in two directions. The invention also discovers that the 0.2g/kg dosage of the swertiamarin obviously delays gastric emptying after the main bitter component of the costal flower extract is induced by the swertiamarin.

In combination with the above, the Mongolian medicine costaphyllum can bidirectionally regulate the gastric emptying of mice, namely the low dose promotes the gastric emptying, the high dose delays the gastric emptying, and the effective component of the inhibition effect of the Mongolian medicine costaphyllum is likely to be swertiamarin. And is synergistic with DA; can inhibit gastrointestinal diseases caused by 5-HT increase, and its pathogenesis needs further research. The experiment provides experimental basis for clinically applying Mongolian medicine costellus to improve gastrointestinal motility disorder diseases.

Example 2

The embodiment relates to application of a Mongolian medicine ribbing flower extract, and the effect of the Mongolian medicine ribbing flower extract on isolated small intestine smooth muscle is researched.

The method comprises the steps of firstly screening various fragments of mouse small intestines by using an Organ bath technology, and selecting small intestine fragments with higher contractile activity. And selecting five chemical components with high content, such as Swertiamarin (ST), Swertiamarin (SO), Geniposide (GA), Hesperetin (HP), Hydroxycoumarin (HC) and the like according to the detection results of the chemical components in the chapter II, and verifying the effect of the Mongolian medicine ribbing flower extract and the five chemical components thereof on isolated ileocecal smooth muscle. Using Ach and CaCl₂Pretreatment of the ilex latus muscle resulted in spastic contraction, and the effect of the extract of the pileus costatus and the effective components HP and HC on the spastic contraction is clarified. Bitter taste is one of six, and is sensed by 25T 2Rs in the body, thereby regulating various physiological functions. In addition, studies have demonstrated the regulatory effect of bitter tasting substances on small intestine smooth muscle contraction. Therefore, in the experiment, the bitter taste mechanism of the lomatogonium carinthiacum is verified by using the bitter taste inhibitor galenin.

The Organ bath is an advanced instrument which has novel design, simple structure, convenient use and easy maintenance and can measure the tension of any in-vitro tissue. Currently, the Organ bath technology is widely applied to in vitro dynamic research, such as in vitro smooth muscle, arterial blood vessel ring, uterine tissue, vas deferens and the like. Ach is an excitatory neurotransmitter released by cholinergic nerves, which binds specifically to M receptors on smooth muscle cells of the gastrointestinal tract in vivo, thereby regulating gastrointestinal motility and promoting contraction of smooth muscles of the gastrointestinal tract. Calcium ion is an important second messenger that regulates cell movement, secretion, metabolism and differentiation, thereby regulating the contraction and relaxation functions of the smooth muscles of the stomach and intestine. In addition, studies have shown that high concentrations of calcium ions contract smooth muscle and low concentrations relax smooth muscle.

The method comprises the following specific steps:

first, experimental materials and methods

Healthy male Kunming mice, SPF grade, weight 28-32g, purchased from Liaoning Biotechnology Limited company, laboratory animals using license number SCXK (Liao) 2015-0001, breeding and SPF grade laboratory animals, temperature 20 + -5 deg.C, humidity 40% -50%, freely taking food and drinking water, pre-experiment adaptive breeding for at least 7 days, mouse feeding provided by Liaoning Biotechnology Limited company. All experimental mice were treated according to the "experimental animal ethics committee regulation" revised by the national science and technology committee of the people's republic of china.

1. Experimental medicine and instrument equipment

See table 1 for details.

TABLE 1

2. Preparation of the solution

Preparation of krebs solution: 6.2g NaCl, 0.315g KCI, 0.535g MgSO₄，0.145g KH₂PO₄，1.87g NaHCO₃1.8g of glucose and 0.011 CaCl₂Dissolve in 1L distilled water is mixed evenly and is used as the product.

Preparing the medicine: all experimental drugs were dissolved in dimethyl sulfoxide (DMSO), and the drugs were first dissolved in DMSO to 100mmoL/L and diluted to 10mmoL/L with distilled water before use.

3. Preparation and contractility determination of isolated intestinal smooth muscle strips

Healthy mice were taken and fasted without water deprivation for 16 h. Cervical dislocation, cutting the abdomen along the midline of the abdomen, taking out the small intestine, and adding krebs-containing solution (95% O in the tube)₂And 5% CO₂Mixed gas), cutting off adipose tissues adhered to an intestinal canal, cleaning intestinal contents, and cutting into intestinal segments with length of 1-1.5cm for later use. Ex vivo smooth muscle tone recording: RM6240E multichannel physiological signal acquisition and processing system (Chengdu instrument factory) is opened and connected with JZ100 tension transducer (Kyoto Xinghangxing Kogyo Co., Ltd., Beijing) and zeroed. KWB-05 constant temperature circulating water bath (Beijing, gold, Hongtai technology Co., Ltd.) distilled water was added to keep the water temperature at 37.5 ℃.10 mL of krebs solution was added to the bath and 95% O was continuously added₂And 5% CO₂And (4) mixing the gases. After the instrument is stabilized, taking the intestinal canal, threading and ligating the two ends of the intestinal canal by an operation needle at the diagonal wall, fixing one end of the intestinal canal on a bath fixer, connecting the other end of the intestinal canal to an L-shaped tension transducer, putting the intestinal canal into the bath, properly adjusting the position and the height of the transducer to enable the isolated intestinal segment to be vertically suspended in the center of the bath, avoiding the isolated intestinal segment from contacting the inner side of the canal wall to influence the experimental result, and giving a load of about 1g to the intestinal canal for balancing for 30 min. Adding medicine after the intestinal canal has stable contraction activity, recording the change condition of intestinal canal contraction activity, calculating the contraction amplitude inhibition rate and contraction frequency inhibition rate [12 ]]. The contraction amplitude suppression ratio (%) -1-average contraction amplitude after administration/average contraction amplitude before administration × 100%; the contraction frequency inhibition ratio (%) -1-average contraction frequency after administration/average contraction frequency before administration × 100%.

3.1 mouse Ex vivo intestinal sampling site investigation

Respectively taking separated small intestine, namely stomach pylorus, duodenum, upper end of small intestine, lower segment of small intestine and ileum, from different parts of mouse, suspending 1-1.5cm in bath containing 10mL krebs solutionContinuously introducing 95% of O₂And 5% CO₂Mixing gas, applying 1g load, after the intestinal canal is contracted stably, beginning to record the contraction amplitude and frequency of each intestinal section, comparing the contraction activity difference of the intestinal sections at different positions, and screening out the intestinal section with stronger contraction amplitude, better activity and stable frequency. Results figure 4, a schematic diagram of the isolated intestinal smooth muscle contraction activity in different sites of the mouse.

3.2 influence of extract of Crotalaria costata and its main chemical components on contractility of isolated ileum of mouse

Based on the results of the chemical composition of the extract of costal Style costatum, the effects of LR, ST, SO, GA, HP, HC (in DMSO), etc. on the in vitro ileocecal smooth muscle were observed. The tube was suspended vertically in a 10mL bath of krebs solution and 95% O was continuously added₂And 5% CO₂The gas was mixed and a load of 1g was applied and stabilized for 30 min. After the isolated intestinal canal has stable contraction activity, the extract of costaphyllum costatum (final concentrations of 0.1mg/mL, 0.3mg/mL, 0.6mg/mL, 1mg/mL, 3mg/mL and 6mg/mL, respectively) and five chemical components (final concentrations of 10uM, 30uM, 100uM, 300uM, 1mM and 3mM, respectively) are added in a micro-cumulative manner, the dosing intervals are 5min, blank groups are set at the same time, and the experiment is carried out by only using DMSO solution with the same concentration. And (5) counting the change of the contraction curves 1min before and 1min after dosing, and calculating the contraction amplitude and the frequency change rate.

3.3 Pachyrhizus extract vs. Ach and CaCl₂Effect of induced Ex vivo ileal smooth muscle contractility in mice

After the intestinal canal contraction curve is stabilized, 0.1uM acetylcholine (Ach) and 1mM CaCl are respectively added to the intestinal canal₂Acting for 20min, adding the extract of Crotalaria ribacterica with different concentrations to final concentrations of 0.1mg/mL, 0.3mg/mL, 0.6mg/mL, 1mg/mL, 3mg/mL, and 6mg/mL respectively, adding medicine at intervals of 5min each time, observing the Papilaria ribacterica extract to Ach and CaCl₂And (3) inducing the action of spastic contraction, recording the contraction amplitude and frequency 1min before and 1min after dosing, and calculating the contraction amplitude and frequency change rate.

3.4HC and HP vs Ach and CaCl₂Induced mice in vitro ileocecal smooth muscleInfluence of the contractile force

After the intestinal canal contraction curve is stabilized, 0.1uM acetylcholine (Ach) and 1mM CaCl are respectively added to the intestinal canal₂Acting for 20min, adding HC and HP with different concentrations to make final concentrations reach 10uM, 30uM, 100uM, 300uM, 1mM, and 3mM respectively, each time adding the medicine at an interval of 5min, observing HC and HP to Ach and CaCl₂And (3) inducing the action of spastic contraction, recording the contraction amplitude and frequency 1min before and 1min after dosing, and calculating the contraction amplitude and frequency change rate.

3.5 Effect of extract of Crotalaria on the bitter inhibitor galectin-induced isolated ileum of mice

After the intestinal contraction curve is stabilized, a G protein beta gamma subunit inhibitor galenin (the final concentration is 75uM) is added to block a T2Rs signal channel, after 20min is blocked, the extract of the costalius costalilis with different concentrations is cumulatively added to enable the final concentration to respectively reach 0.1mg/mL, 0.3mg/mL, 0.6mg/mL, 1mg/mL, 3mg/mL and 6mg/mL, the interval of each dosing is 5min, the influence of the extraction of the costalius costalilis on the contraction force of the mouse ileum smooth muscle induced by the galenin is observed, the contraction amplitude and frequency of 1min before and 1min after the dosing are recorded, and the contraction amplitude and frequency change rate are calculated.

This example uses Graphpad prism 8.0.2 statistical analysis software to perform statistical analysis processing of data. Experimental data are presented as mean plus minus standard error (X ± SEM). Two groups of data in the experiment are compared and analyzed by adopting t test, a plurality of groups of data are compared and analyzed by adopting One-way ANOVA (One-way ANOVA), pairwise comparison is carried out by adopting Least Significant Difference (LSD) test, and P <0.05 is the difference, so that the statistical significance is realized.

Second, result in

1. Preparation and contractility determination of isolated intestinal smooth muscle strips

The spontaneous contraction activity results of the smooth muscles of different parts of the mouse excised small intestine show that the contraction amplitude of the ileum section is obviously larger than that of the upper and lower sections of the stomach pylorus, duodenum and small intestine; the frequency was also relatively stable (see table 2 and fig. 4).

TABLE 2

Section of intestine	Amplitude (g)	Frequency (times/min)
			Pylorus part	0.156±0.027	27.00±1.140
Duodenum	0.181±0.155	24.33±3.445
			Upper part of small intestine	0.138±0.096	22.50±1.643
Lower part of small intestine	0.171±0.093	20.50±2.345
			Ileum	0.319±0.225	17.50±1.378

2. Influence of extract of ribbonfil and its main chemical components on contractility of isolated ileum of mouse

2.1 Effect of extract of Crotalaria costata on contractility of isolated ileum of mice

The effect of the extract of ribbonfil on ex vivo ileum contractility is shown in figure 5. Compared with the extract before administration, the extract of the lomatogonium carinthiacum has bidirectional regulation effect on the contractility of the isolated ileum of the mouse, the intestinal canal is stimulated to shrink (P is less than 0.001) at low concentration, and the shrinkage rate can reach 45.10% when the shrinkage rate is 0.3 mgmL; the intestinal canal contraction is inhibited (P is less than 0.001 and P is less than 0.0001) at high concentration, and the inhibition rate can reach 83.21% at 6 mg/mL; the extract of the lomatogonium carinthiacum has no obvious influence on the contraction frequency of an intestinal canal (P is more than 0.05) at low concentration, the inhibition effect is obviously enhanced along with the increase of the drug concentration (P is less than 0.05, P is less than 0.0001), and the inhibition rate can reach 94.1% at 6 mg/mL.

2.2 Effect of the major chemical Components of the extract of Crotalaria on contractility of isolated ileum of mice

The effect of 5 chemical components in the extract of ribbonfil on the contractile force of isolated ileum of mice is shown in fig. 6. Compared with DMSO group, ST, SO and GA have no significant difference on the contraction amplitude and frequency of the isolated ileocecal smooth muscle of the mice (P is more than 0.05); HC and HP can inhibit spontaneous contraction amplitude of isolated ileum of mice (P is less than 0.05, P is less than 0.01, P is less than 0.001, and P is less than 0.0001) in a dose-dependent manner, and the inhibition effect on the contraction frequency of the intestinal canal is enhanced along with the increase of the concentration of the drug, the inhibition effect is strongest at 3mM, and can reach 85.60% and 72.73% respectively; compared with DMSO group, HC and HP low concentration has no obvious effect on contraction frequency (P > 0.05), but the inhibition effect is enhanced along with the increase of concentration (P <0.01, P <0.0001), and the contraction frequency inhibition rate of HC for completely inhibiting the contraction frequency HP at the final concentration of 3mM can reach 72.71% at 3 mM; but can be restored to the normal contraction state after repeated washing (as shown in figure 7).

3. Extract of costaria costata for Ach and CaCl₂Effect of induced Ex vivo ileal smooth muscle contractility in mice

3.1 the effect of different concentrations of extract of ribbonfil on the contractile activity of ex vivo ileum of mice after Ach pretreatment is shown in figure 8.

As can be seen from FIG. 8, after 0.1uM of the tool drug Ach is added for the pretreatment, the baseline of the isolated ileocereus muscle of the mouse has an ascending trend, and compared with the baseline before the drug is added, the change of the contraction amplitude (P is less than 0.001) of the ileocereus muscle is obvious, has obvious difference and has no influence on the contraction frequency; compared with an Ach control group, the extract of the Stylosanthes guianensis can obviously inhibit the amplitude and frequency of contraction caused by Ach after being induced by different concentrations of the extract, the inhibition effect is enhanced along with the increase of the concentration (P is less than 0.05, P is less than 0.001, and P is less than 0.0001), the inhibition effect on the amplitude and frequency of the contraction is strongest when the final concentration is 6mg/mL, the inhibition rate of the amplitude of the contraction is 88.27%, and the inhibition rate of the frequency of the contraction is 94.92%.

3.2 different concentrations of Pachylomis costata extract Pair CaCI₂The effect of ex vivo ileal contractile activity in the mice after pretreatment is shown in figure 9.

As can be seen in FIG. 9, the pre-treatment with 1mM CaCI₂After induction, the baseline of the isolated ileocereus muscle of the mouse rapidly rises, and compared with the condition before dosing, the change of the contraction amplitude (P is less than 0.0001) and the frequency (P is less than 0.0001) of the ileocereus muscle is obvious and has obvious difference; and CaCI₂The control group added with different concentrations of extract of Crotalaria costata to control CaCI in low concentration range₂The spasmodic contraction has no significant effect, but the CaCI can be significantly inhibited when the concentration is proper₂The resulting amplitude and frequency of contraction and the inhibition with increasing concentration were increased (P <0.01, P <0.0001), with the most potent inhibition of amplitude and frequency of contraction at a final concentration of 6mg/mL, with an inhibition of amplitude of contraction of 88.90% and an inhibition of frequency of contraction of 55.19%.

HC and HP vs Ach and CaCl₂Effect of induced Ex vivo ileal smooth muscle contractility in mice

4.1 effects of different concentrations of HC and HP on isolated ileal contractile activity in mice after Ach pretreatment are shown in FIG. 10.

As can be seen from FIG. 10, after 0.1uM of the tool drug Ach is added for the pretreatment, the baseline of the isolated ileocereus muscle of the mouse has an ascending trend, and compared with the baseline before the drug addition, the change of the contraction amplitude (P is less than 0.01) of the ileocereus muscle is obvious and has obvious difference, but no influence is caused on the contraction frequency; compared with an Ach control group, the amplitude and frequency of contraction caused by Ach can be obviously inhibited after the induction by HC and HP with different concentrations, the inhibition effect is enhanced along with the increase of the concentrations (P is less than 0.01, P is less than 0.001, and P is less than 0.0001), the inhibition effect on the amplitude and frequency of the contraction is strongest when the final concentration is 3mM, the inhibition rate of the amplitude of the contraction is 93.23%, and the inhibition rate of the frequency of the contraction is 90.48%.

4.2 different concentrations of HC and HP vs. CaCI₂The effect of ex vivo ileal contractile activity in the mice after pretreatment is shown in figure 11.

As can be seen in FIG. 11, the pre-treatment with 1mM CaCI₂After induction, the baseline of the isolated ileocereus muscle of the mouse rapidly rises, and compared with the condition before dosing, the change of the contraction amplitude (P is less than 0.0001) and the frequency (P is less than 0.0001) of the ileocereus muscle is obvious and has obvious difference; and CaCI₂Control group compared with control group, after adding different concentrations of HC and HP, the concentration range is low for CaCI₂The spasmodic contraction has no significant effect, but the CaCI can be significantly inhibited when the concentration is proper₂The resulting amplitude and frequency of contraction, and the inhibition increased with increasing concentration (P <0.01, P <0.0001), with the final concentration of 3mM showing the strongest inhibition of amplitude and frequency of contraction, 90.96% and 67.20%, respectively, while HC and HP showed less inhibition of frequency of contraction, with the inhibition of frequency of contraction being 67.15% and 65.40%, respectively, only at the highest concentration of 3 mM.

5. Effect of extract of Crotalaria on isolated ileum of mice induced by the bitter taste inhibitor galectin

5.1 the effect of extracts of ribcage from different concentrations on the contractile activity of the isolated ileum of mice after blocking the bitter taste receptor of the smooth muscles of the ileum by the G protein β γ subunit inhibitor galectin is shown in FIG. 12.

As can be seen in FIG. 12, galectin had no effect on the mice isolated ileal smooth muscle contractility (P > 0.05); compared with the extract group of the lomatogonium carinthiacum, after the garlein is used for inducing the smooth muscles of the intestine, the intestine contracting effect of the lomatogonium carinthiacum extract in a low-concentration range disappears (P is less than 0.05 and P is less than 0.01 respectively), but the intestine contracting effect of the lomatogonium carinthiacum extract in a high-concentration range is not obviously influenced (P is more than 0.05); has no effect on the effect of the extract of the ribbonfil on inhibiting the contraction frequency of the smooth muscles of the ileum (P is more than 0.05).

The motility of the gastrointestinal smooth muscle is governed by the autonomic nervous system and the intramural plexus distributed in the wall of the gastrointestinal tract, and the isolated small intestine smooth muscle is still present despite the loss of the innervation of the external nerves, so that the isolated small intestine can independently regulate and control the gastrointestinal function to a certain extent.

The invention utilizes the Organ bath technology to firstly examine the spontaneous contraction of each segment of the small intestine of the mouse and finds that each segment of the small intestine of the mouse has rhythmic contraction activity, wherein the contraction amplitude of the ileum segment is the largest. After the isolated ileal smooth muscle is induced by the extract of the lomatogonium costatum, the surprisingly found that the low concentration range (0.1-0.3mg/mL) of the extract of the lomatogonium costatum promotes the contraction amplitude of the isolated ileal smooth muscle, and when the concentration reaches more than 0.4mg/mL, the contraction amplitude of the isolated ileal smooth muscle is gradually inhibited until the complete relaxation is realized. Therefore, the extract of ribbonfil is considered to have bidirectional regulation effect on the smooth muscles of the ileum and the bidirectional regulation result is consistent with the in vivo experiment result. This further verifies this interesting phenomenon. "bidirectional regulation" means that when the body is in an unbalanced state, a certain drug can transform the body from an hyperfunction state to a normal state, and can transform the body from a low state to a normal state, i.e., the diseased body tends to be normal, and finally the body reaches a normal balance state. It has been reported that Chinese medicines and prescriptions exhibit bidirectional regulation in the human body system such as digestion, urinary, circulation, respiration, nerves, immunity, and the like. For example, hawthorn can regulate gastrointestinal motility bidirectionally, promote gastrointestinal motility for constipation patients, and delay gastrointestinal motility for diarrhea patients. In vitro experiments are carried out by utilizing Organ bath technology, the wild ginseng herb and the like are found that the ginsenoside with different concentrations has a bidirectional regulating effect on isolated toad heart contraction movement, namely, under the condition of low-calcium anyhow liquid, the inhibited toad heart contraction is promoted, but the strong contraction caused by the induction of high-calcium anyhow liquid is induced, and the dose-dependent inhibition of heart contraction is realized. Therefore, the bidirectional regulation effect has extremely important significance for treating various diseases of organisms and improving clinical curative effect. Furthermore, Bert Avau et al induced antral smooth muscle with the bitter tasting substance denatonium benzoate found that denatonium benzoate bilaterally modulates antral smooth muscle, but the contractile function of denatonium benzoate bilaterally modulates antral smooth muscle is not further elucidated, presumably the contractile action may be associated with elevated calcium ions and the diastolic action may be associated with activation of the ROCK pathway. As is well known, studies have shown that bitter substances exert important physiological effects through T2Rs, and that T2Rs has higher expression in various segments of the intestinal tract. Therefore, in order to verify the action mechanism of bitter Mongolian medicine, namely the extract of the lomatogonium costatum, for regulating gastrointestinal motility, in the experiment, the bitter receptor inhibitor galenin is used for inducing the smooth muscle of the intestine to block a T2Rs passage, and the contraction effect of the extract of the lomatogonium costatum in a low concentration range is found to be obviously inhibited, so that the effect of the extract of the lomatogonium costatum on contracting the intestinal tract is suspected to be mediated through T2 Rs.

The generation and transmission of spontaneous contraction of smooth intestinal muscle are easily affected by bioactive substances and drugs such as neurotransmitter, gastrointestinal hormone, etc., such as acetylcholine (Ach), histamine and CaCl₂And the like. Ach is an excitatory neurotransmitter, and activates receptor-regulated calcium channels via M receptors to cause smooth muscle contraction and promote gastrointestinal motility. Calcium ions play an important role in gastrointestinal motility as a very important second messenger, and the influx of extracellular calcium ions and the release of intracellular calcium ions are the main sources of calcium ions in gastrointestinal smooth muscle cells. Research shows that the contraction and relaxation movement of gastrointestinal smooth muscle is affected by the change of free calcium ion inside cell, and when the calcium ion inside cell is raised to some extent, it will combine with creatin or creatinomodulin to cause the contraction of smooth muscle. The invention utilizes Ach and CaCl₂After the isolated ileocecal smooth muscle is induced to cause spastic contraction, the extract of the lomatogonium costatum and the main chemical components of hesperetin and cumicin thereof can obviously inhibit sudden spastic contraction, so that the contraction action tends to be normal. Therefore, the extract of the lomatogonium carinthiacum is suspected to have potential treatment effect on neurotransmitter disturbance gastrointestinal motility disorder diseases, and the effective components are primarily considered to be hesperetin and coumarin.

In combination, the extract of Crotalaria costata can regulate the mouse ileum bidirectionally, namely the low-concentration contracted intestinal canal and the high-concentration relaxed intestinal canal, and the action mechanism of the extract can be partially mediated by a T2Rs signal channel. In addition, the extract of the lomatogonium carinthiacum has potential therapeutic effect on gastrointestinal motility disorder diseases caused by neurotransmitter disorder.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.