CN117180266B - New use of carnosol and/or rosmanol in medicine - Google Patents

Abstract

The invention discloses a new pharmaceutical use of carnosol and/or rosmarinol, which belongs to the field of biomedicine. The use includes any of the following uses: preparing an activator for calcium-activated chloride ion channel ANO1; preparing an activator for improving intestinal motility deficiency. Medicines; preparation of medicines for treating xerostomia; preparation of medicines for preventing and treating acute lung injury. The present invention discovered for the first time that carnosol and its analogue rosmarinol are ANO1 activators. Experiments have proven that both carnosol and rosmarinol can enhance the contractile tension and frequency of guinea pig ileum by activating ANO1, that is, Carnosol and/or rosmarinol are effective drugs for treating intestinal motility insufficiency; it is also found that carnosol and/or rosmarinol can also be used to treat xerostomia and acute lung injury; the present invention finds that The new pharmacological mechanism of carnosol and rosmarinol provides new ideas and effective ways for the treatment of diseases that target ANO1, such as intestinal insufficiency, xerostomia and acute lung injury.

Description

New pharmaceutical uses of carnosol and/or rosmarinol

Technical field

The present invention relates to the field of biomedicine, in particular to new pharmaceutical uses of carnosol and/or rosmarinol.

Background technique

The calcium-activated chloride channel ANO1 is highly expressed in human intestinal pacemaker cells Cajal. Activating the ANO1 channel will lead to changes in intracellular chloride ion concentration. Activating Cajal cells by changing the cell membrane potential causes intestinal contraction, so ANO1 activators are screened. Promoting intestinal contraction and enhancing intestinal peristalsis by activating ANO1 in Cajal cells is one of the effective ways to improve digestion capacity. ANO1 ion channels are also highly expressed in salivary glands and bronchial mucosa submandibular gland acini. Oral administration of ANO1 activators can in principle activate the ANO1 channels here, thereby increasing the amount of saliva secreted by the salivary glands and increasing mucus secreted by the respiratory tract, which can effectively relieve xerostomia. , cystic fibrosis and acute lung injury. However, there are currently very few reports on the screening of drugs that are effective in treating intestinal insufficiency, xerostomia, and acute lung injury. To this end, the present invention specifically provides the use of a medicine in treating intestinal insufficiency, xerostomia and acute lung injury.

Contents of the invention

The purpose of the present invention is to provide new pharmaceutical uses of carnosol and/or rosmarinol to solve the problems existing in the above-mentioned prior art. The present invention confirms that carnosol and its analog rosmarinol can significantly activate ANO1, discovered the new pharmacological mechanism of carnosol and rosmarinol, providing new ideas and effective ways for the treatment of diseases targeting ANO1, such as intestinal motility deficiency, xerostomia and acute lung injury.

In order to achieve the above objects, the present invention provides the following solutions:

The present invention provides uses of carnosol and/or rosmarinol, and the uses include any of the following uses:

(1) Prepare an activator of calcium-activated chloride channel ANO1;

(2) Preparing drugs for improving intestinal motility deficiency;

(3) Preparing medicines for treating xerostomia;

(4) Prepare drugs for preventing and treating acute lung injury.

Furthermore, the carnosol and/or rosmarinol exert a medicinal effect of improving intestinal motility by promoting intestinal contraction.

Further, the carnosol and/or rosmarinol exert the effect of treating xerostomia by promoting saliva secretion.

Further, the carnosol and/or rosmarinol prevent and treat acute lung injury by inhibiting the content of white blood cells and inflammatory cytokines in the body.

Further, the inflammatory cytokines include TNF-α and IL-6.

The present invention also provides a medicine for preventing and treating ANO1 dysfunction diseases, the medicine contains carnosol and/or rosmarinol; the ANO1 dysfunction diseases include intestinal motility deficiency, acute lung injury, cystic fibrosis and One or more of Sjogren's syndrome.

Furthermore, the medicine also includes pharmaceutically acceptable excipients.

Further, the auxiliary materials include one or more of diluents, fillers, excipients, binders, wetting agents, disintegrating agents, absorption accelerators, surfactants, adsorption carriers, lubricants and flavors. kind.

Further, the dosage forms of the drug include tablets, capsules, granules, injections, nasal drops or sprays.

Further, the method of taking the medicine is oral administration, nasal administration or parenteral administration.

The invention discloses the following technical effects:

The present invention discovered for the first time that carnosol and its analogue rosmarinol are ANO1 activators. Experiments have proven that both carnosol and rosmarinol can enhance the contractile tension and frequency of guinea pig ileum by activating ANO1, that is, Carnosol and/or rosmarinol are effective drugs for the treatment of intestinal insufficiency. At the same time, it was also found that carnosol and/or rosmarinol can promote saliva secretion in mice, inhibit the increase of white blood cells and inflammatory cytokines in acute lung injury models, and play a protective role against acute lung injury in mice, that is, Carnosol and/or rosmarinol may also be used to treat xerostomia and acute lung injury. The present invention discovers a new pharmacological mechanism of carnosol and rosmarinol, and provides new ideas and effective ways for the treatment of diseases targeting ANO1, such as intestinal motility deficiency, xerostomia and acute lung injury.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the drawings of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exerting creative efforts.

Figure 1 shows the molecular formula of carnosol;

Figure 2 is the molecular formula of rosmarinol;

Figure 3 shows the typical current and statistical diagram of whole cells activated by carnosol;

Figure 4 is a typical current and statistical diagram of whole cells activated by rosmarinol;

Figure 5 shows the typical current and statistical diagram of ANO1 whole cells activated by the combined administration of carnosol and rosmarinol;

Figure 6 shows that carnosol enhances the contractility of guinea pig ileum;

Figure 7 shows that rosmarinol enhances the contractility of guinea pig ileum;

Figure 8 shows that the combined administration of carnosol and rosmarinol enhances the contractility of the ileum of guinea pigs;

Figure 9 shows that different concentrations of carnosol enhance saliva secretion in mice;

Figure 10 shows that different concentrations of rosmarinol enhance saliva secretion in mice;

Figure 11 shows that the combined administration of carnosol and rosmarinol enhances saliva secretion in mice;

Figure 12 shows the number of cells in mouse bronchoalveolar lavage fluid;

Figure 13 shows the content of inflammatory cytokines in mouse bronchoalveolar lavage fluid.

Detailed ways

Various exemplary embodiments of the invention will now be described in detail. This detailed description should not be construed as limitations of the invention, but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It should be understood that the terms used in the present invention are only used to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that every intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or value intermediate within a stated range, and any other stated value or value intermediate within a stated range, is also included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents relate. In the event of conflict with any incorporated document, the contents of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to the skilled person from the description of the invention. The specification and examples of the present invention are exemplary only.

The words "includes", "includes", "has", "contains", etc. used in this article are all open terms, which mean including but not limited to.

Example 1 Verification of the activation effect of carnosol and rosmarinol on ANO1 channel

In this embodiment, patch clamp technology is used to record the current of ANO1 in whole-cell mode. The instrument used was an EPC 10 amplifier (HEKA, Germany) and data recording was performed using PatchMaster software (HEKA). CHO cells were purchased from the Cell Bank of the Chinese Academy of Sciences, and ANO1 lentivirus was purchased from Shanghai Jikai Gene Medical Technology Co., Ltd. Method for constructing CHO cells stably transducing the ANO1 channel: Construct the ANO1 gene lentivirus system, and express it in CHO1 cells through exogenous transfection. After 3 days, 2 μg/mL puromycin is added for screening, and then the medium is changed every 2 days. After continuous screening for two weeks, the expression of ANO1 in CHO cells was identified through fluorescence microscopy observation, and the selected CHO cells stably transduced with ANO1 were expanded, cultured and cryopreserved.

The CHO cells stably transduced to the ANO1 channel were spread on the cell slide. The composition of the cell bath solution was 145mmol/L NaCl, 5mmol/L KCl, 1mmol/L MgCl ₂ , 2mmol/L CaCl ₂ , 10mmol/L HEPES, and 100 μmol/L. Carnosol and/or 100 μmol/L rosmarinol (the molecular formulas of carnosol and rosmarinol are shown in Figures 1 and 2). The components of the cell bath solution in the control group did not contain carnosol and/or rosmarinol, and the remaining components were the same as above. Osmotic pressure is controlled at 300-310mOsm/L. The components of the liquid inside the electrode are 130mmol/L KCl, 2mmol/L mol/MgCl ₂ , 10mmol/L EGTA and 10mmol/L HEPES (pH 7.4, adjusted with NaOH), and the osmotic pressure is controlled at 290-300mOsm/L.

The patch clamp recording program has a single stimulation duration of 1.15 seconds. The membrane voltage is first clamped at 0mV for 100ms, and then different step voltages from -80 to +80mV are given to the cells (each step is 20mV). Each duration is 750ms, the final membrane voltage is -80mV, the duration is 300ms, repeat 9 times, activate the ANO1 channel, and count the current data at +80mV to obtain the current value. The results are shown in Figures 3, 4 and 5. Figure 3 shows the current size of ANO1 under 100 μmol/L carnosol. Figure 4 shows the current size of ANO1 under 100 μmol/L rosmarinol. Figure 5 shows the current size of ANO1 under 100 μmol/L rosmarinol. The current magnitude of ANO1 after combined administration of L carnosol and 100 μmol/L rosmarinol. It can be seen that both 100 μmol/L carnosol and/or 100 μmol/L rosmanol can significantly activate the ANO1 current, and the ANO1 Channels are activating. It is speculated that carnosol and rosmarinol have the potential to treat diseases that target ANO1, such as intestinal insufficiency, acute lung injury, cystic fibrosis, Sjogren's syndrome, etc.

Example 2 Biological experiments verify the medicinal uses of carnosol and rosmarinol

1Carnosol and rosmarinol enhance intestinal motility in guinea pigs

Take a Hatley guinea pig, kill it and immediately dissect the abdominal cavity, take out a 10cm section of ileum, and place it in an oxygen-saturated benchtop culture dish. Remove the mesentery along the intestinal wall, and then cut the ileum into 3-8 small sections with a length of 1-1.5cm. Use a 5mL syringe to absorb desktop liquid to rinse the intestinal contents, and replace with fresh desktop liquid for later use.

Take one of the above-mentioned spare intestinal segments and place it in a petri dish filled with desktop liquid. Thread the diagonal walls at both ends with suture needles and tie them. Be careful to keep the intestines open and not to close them. One end of the intestinal tube was tied to the fixed hook of the bath, and then placed into the 37°C Maxwell bath. Then tie the other end of the intestinal tube to the cantilever beam of the tension transducer, and adjust the preload to 1g. After the isolated ileum has stabilized for 30 minutes, record the normal contraction curve at one end, then drop carnosol solution into the McBurney bath, observe and record the contraction curve, and the results are shown in Figure 5: First, acetylcholine was added to cause the ileum to contract. contraction, and then adding atropine inhibited the contraction, thus verifying that the ileal tissue used in the experiment had good activity. Subsequently, 100 μmol/L carnosol was added, as shown in Figure 6, which showed that it caused the contraction of the ileum. Finally, the ANO1 inhibitor T16Ainh-A01 was used to inhibit the contraction of the ileum. In the same way, as shown in Figure 7, using 100 μmol/L rosmarinol can also promote the contraction of the ileum. It was proved that both carnosol and rosmarinol can enhance intestinal motility deficiency in guinea pigs. As shown in Figure 8, the combined administration of 100 μmol/L carnosol and 100 μmol/L rosmarinol can also enhance intestinal motility dysfunction in guinea pigs, and combined administration enhances contraction efficiency more efficiently.

2Carnosol and rosmarinol improve xerostomia

BALB/c mice, ordinary grade, body weight 18-22g, were randomly divided into 7 groups, 5 mice in each group; the blank control group was physiological saline, and experimental group 1 to experimental group 3 were administered carnosol, and the dosages were in order The doses were 5 mg/kg, 10 mg/kg and 20 mg/kg; experimental group 4 to 6 were administered rosmarinol, and the dosages were 5 mg/kg, 10 mg/kg and 20 mg/kg; experimental group 7 was 10 mg/kg. Carnosol and 10 mg/kg rosmarinol were administered simultaneously. The drug was administered intragastrically once a day for a total of 6 weeks, and the salivary flow rate of the mice was measured weekly starting from the third week. Fast for 1 hour before the measurement and water is not allowed. The dry cotton ball method was used for measurement: Place sterile absorbent cotton into the cheek of the experimental mouse, take it out after 3 minutes, and weigh the wet weight. The results are shown in Figure 9, Figure 10 and Figure 11. It can be seen from Figure 9, Figure 10 and Figure 11 that carnosol and rosmarinol can promote the secretion of saliva in mice, and as the dosage increases, the amount of saliva secretion also increases. Combined administration The drug effect is better than the same dose administered alone, indicating that carnosol and rosmarinol have the function of promoting saliva secretion and can be used to prepare drugs for the treatment of xerostomia.

3Carnosol and rosmarinol improve acute lung injury in mice

Establishment of mouse acute lung injury model: Balb/c mice, male, weighing 20±1g, were randomly divided into 7 groups, 5 mice in each group; LPS: physiological saline with a concentration of 0.001% LPS; carnosol and methionine The dosage of senol is 20 mg/kg. Administration was administered by intraperitoneal injection. 1 hour after administration, intranasal instillation was performed to create a model. After 12 hours of intranasal instillation, the mice were sacrificed to collect bronchoalveolar lavage fluid and lung tissue. The corresponding indicators were detected. Each group was administered intraperitoneal injection and The drugs administered for nasal drip modeling are shown in Table 1. The experimental results are shown in Figures 12 and 13. Both carnosol and rosmarinol can inhibit the increase in leukocytes and inflammatory cytokines in the body, play a protective role against acute lung injury in mice, and the combined administration is more effective than either alone. The medicine is more effective.

Table 1 Drugs administered by intraperitoneal injection and nasal drip modeling in each group

Based on the above, the present invention discovered for the first time that carnosol and its analog rosmarinol are ANO1 activators. Experiments have proven that both carnosol and rosmarinol can enhance the contractile tension and frequency of guinea pig ileum by activating ANO1. , that is, carnosol and/or rosmarinol are effective drugs for the treatment of intestinal insufficiency. At the same time, it was also found that carnosol and/or rosmarinol can promote saliva secretion in mice, inhibit the increase of white blood cells and inflammatory cytokines in acute lung injury models, and play a protective role against acute lung injury in mice, that is, Carnosol and/or rosmarinol may also be used to treat xerostomia and acute lung injury.

The above-described embodiments only describe the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. All deformations and improvements shall fall within the protection scope determined by the claims of the present invention.

Claims (5)

1. Use of carnosol in the preparation of drugs for improving intestinal motility deficiency. 2. The use according to claim 1, characterized in that the carnosol exerts the medicinal effect of improving intestinal motility by promoting intestinal contraction. 3. The use according to claim 1, characterized in that the medicine further includes pharmaceutically acceptable excipients. 4. The use according to claim 1, characterized in that the dosage form of the drug is tablets, capsules, granules or injections. 5. The use according to claim 1, characterized in that the method of taking the drug is oral or parenteral administration.