Application of Lemon Balm in the Preparation of Drugs for the Prevention or Treatment of Heatstroke

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of lemon balm glycosides in the preparation of drugs for the prevention or treatment of heatstroke. By constructing a mouse model of heatstroke-induced myocardial injury and observing indicators such as rectal temperature, blood routine tests, blood biochemistry, oxidative stress, and cardiac function, this invention found that lemon balm glycosides can significantly reduce the increase in rectal temperature caused by heatstroke in mice, increase the content of red blood cells and albumin in plasma, and reduce the level of oxidative stress in myocardial cells. Therefore, the application of lemon balm glycosides in the preparation of drugs for the prevention or treatment of heatstroke is proposed.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of lemon balm glycoside in the preparation of drugs for the prevention or treatment of heatstroke. Background Technology

[0002] Heatstroke is a severe form of heat exhaustion. Its core physiological and pathological process involves a severe imbalance between heat production and dissipation in a hot and humid environment, leading to a rapid increase in core body temperature (usually exceeding 40°C). This triggers a severe systemic inflammatory response, disseminated intravascular coagulation, and multiple organ dysfunction syndrome. Regarding myocardial damage, extreme heat and the inflammatory storm can directly damage myocardial cells, causing heat-toxic myocardial injury. Simultaneously, the heart needs to rapidly increase its output load to compensate for heat loss through vasodilation in the skin. This high-load state easily induces myocardial ischemia, arrhythmias, and even heart failure, which is one of the key factors contributing to the rapid deterioration and death of heatstroke. Therefore, early intervention and treatment of heatstroke are of great importance. Currently, clinical treatment for heatstroke often involves a comprehensive approach, including physical cooling, fluid resuscitation, replacement therapy, and antiarrhythmic or vasoactive drugs.

[0003] Lemon glycoside, a natural flavonoid glycoside, primarily exhibits anti-tumor, neuroprotective, hepatoprotective, and anti-inflammatory effects. Studies have shown that it can effectively inhibit cancer cell growth through multiple pathways, including inducing apoptosis and inhibiting proliferation and metastasis. Simultaneously, lemon glycoside possesses significant neuroprotective activity, improving cognitive impairment in models such as Alzheimer's disease, and demonstrating good protective effects against alcohol- or drug-induced liver damage. However, its role in heatstroke has not yet been reported in existing technologies. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of lemon balm glycoside in the preparation of drugs for the prevention or treatment of heatstroke.

[0005] Application of lemon balm glycosides in the preparation of drugs for the prevention or treatment of heatstroke.

[0006] This invention constructs a mouse model of heatstroke myocardial injury and measures the rectal temperature of mice. Compared with the control group, the rectal temperature of the heatstroke group was significantly higher. The rectal temperature of the experimental groups given low, medium and high doses decreased after modeling. Therefore, the application of lemon balm glycoside in the preparation of drugs for the prevention or treatment of heatstroke is proposed.

[0007] Preferably, the drug is used to suppress the increase in anal temperature caused by heatstroke.

[0008] Preferably, the drug is used to improve cardiac dysfunction caused by heatstroke.

[0009] Preferably, the drug is an oral or injectable formulation.

[0010] Preferably, the drug is prepared from lemon balm glycoside and pharmaceutically acceptable excipients.

[0011] Preferably, the pharmaceutically acceptable excipients include any one of solubilizers and diluents.

[0012] Preferably, the diluent comprises DMSO.

[0013] Preferably, when the drug is an injectable formulation, the concentration of lemon balm glycoside is 10 μM to 80 μM.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] This invention, by constructing a mouse model of heatstroke-induced myocardial injury, observed indicators such as body weight, rectal temperature, complete blood count, blood biochemistry, oxidative stress, and cardiac function. The results showed that lemon balm glycoside can significantly reduce the increase in rectal temperature caused by heatstroke in mice and increase the levels of white blood cells (WBC), lymphocytes (LYM), and red blood cells (RBC) in the blood.

[0016] Lemon balm glycosides can also significantly improve cardiac function decline caused by heatstroke. Significant improvements are seen in cardiac function indicators including ejection fraction (EF), short-axis systolic rate (FS), left ventricular end-systolic volume (LVESV), and left ventricular posterior wall systolic thickness (LVPWs). This effectively enhances cardiac pumping function and alleviates organic damage to the heart.

[0017] This invention, through the construction of an HL-1 cardiomyocyte heatstroke model, and by detecting indicators such as cell viability, JC-1 staining, and ROS staining, found that lemon balm glycoside can significantly improve the cell viability and reduce cell death rate of HL-1 cardiomyocytes after heatstroke injury. Simultaneously, lemon balm glycoside can reduce the generation of intracellular reactive oxygen species after injury and maintain mitochondrial membrane potential.

[0018] Based on the above findings, this invention provides the application of lemon balm glycoside in the preparation of drugs for the treatment or prevention of heatstroke, particularly for the treatment of cardiac damage caused by heatstroke. The drug is an oral administration formulation, and the dosage is 2.5 mg / kg, 5 mg / kg, or 10 mg / kg body weight. Attached Figure Description

[0019] Figure 1 Statistical graphs of rectal temperature in mice after treatment with different concentrations of lemon balm. Results are expressed as mean ± standard deviation, n=10, * P<0.05, ** P<0.005, *** P<0.0005, **** P<0.00005.

[0020] Figure 2 The graph shows the statistical changes in blood routine tests in mice after treatment with different concentrations of lemon balm glycoside. Approximately 50 μL of blood was collected from the eyeballs after drug treatment and model establishment and analyzed using a blood routine instrument. In the graph, a represents white blood cells; b represents lymphocytes; and c represents red blood cells. The results are expressed as mean ± standard deviation. n=10. * P<0.05, ** P<0.005, *** P<0.0005, **** P<0.00005.

[0021] Figure 3 Statistical and example graphs of small animal cardiac ultrasound examinations in mice treated with different concentrations of lemon balm are shown. In the figure, a represents ejection fraction; b represents left ventricular short-axis systolic rate; c represents left ventricular posterior wall systolic thickness; and d represents left ventricular posterior wall diastolic thickness. Results are expressed as mean ± standard deviation, n=10, * P<0.05, ** P<0.005, *** P<0.0005, **** P<0.00005.

[0022] Figure 4 This is a typical M-mode image of the long-axis section of the left ventricle beside the sternum.

[0023] Figure 5 The graphs and statistical results show the cell viability of HL-1 cardiomyocytes after treatment with different concentrations of lemon balm. The statistical results are expressed as mean ± standard deviation. * P<0.05, ** P<0.005, *** P<0.0005, **** P<0.00005.

[0024] Figure 6 These are microscopic images of the cells from each experimental group.

[0025] Figure 7 Fluorescence patterns and statistical results of reactive oxygen species (ROS) generation in HL-1 cardiomyocytes after treatment with different concentrations of lemon balm glycosides. Green fluorescence represents ROS. Statistical results are expressed as mean ± standard deviation. * P < 0.05, ** P < 0.005, *** P < 0.0005, **** P < 0.00005.

[0026] Figure 8 The image shows the JC-1 staining results of HL-1 cardiomyocytes after treatment with different concentrations of lemon balm glycoside. Red fluorescence indicates healthy cells with high mitochondrial membrane potential, while green fluorescence indicates apoptotic or damaged cells with low mitochondrial membrane potential.

[0027] Figure 9 The statistical results for staining are expressed as mean ± standard deviation, n=10, * P<0.05, ** P<0.005, *** P<0.0005, **** P<0.00005. Detailed Implementation

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0029] The mouse constant temperature and humidity exposure model is a classic model of heatstroke. The method involves placing mice in an environment with precisely controlled temperature and humidity (39.5°C, 60%) for continuous exposure until their core body temperature reaches approximately 42.0°C to induce a heatstroke model. This invention uses this constant temperature and humidity exposure animal model as the research subject. According to existing technology, this animal model can induce myocardial damage caused by heatstroke.

[0030] The animals used in these examples were purchased from the Experimental Animal Center of the Air Force Medical University, the cells were purchased from the ATCC cell bank, and the reagents were commercially available. Unless otherwise specified, the experimental methods or related detection methods used in the following examples are those known in the art.

[0031] The structural formula of lemon balm glycoside is: .

[0032] Example 1: Lemon balm glycosides can reduce the increase in core body temperature in mice caused by heatstroke.

[0033] Mice were administered low, medium, and high doses of lemon balm glycoside (dissolved in 1 g / μL DMSO solution, with dosages calculated based on body weight of 2.5 mg / kg, 5 mg / kg, and 10 mg / kg, respectively) every other day for 6 days. After administration, mice were continuously exposed to a constant temperature and humidity (39.5℃, 60%) incubator until their core body temperature reached 42℃ (approximately 2 hours). This established an individual-level model of heatstroke and heatstroke-related myocardial injury.

[0034] BALB / c mice were used as the research subjects. Following the study design, mice were randomly grouped using a random number table. After drug administration, the mice were modeled using the aforementioned constant temperature and humidity protocol to replicate heatstroke and myocardial injury models. The specific experimental steps are as follows:

[0035] (1) Grouping: BALB / c mice were divided into control group, heatstroke group, and low (2.5 mg / kg), medium (5 mg / kg), and high (10 mg / kg) lemon glycoside experimental groups, with 10 mice in each group.

[0036] Dosage group: Ten adult mice of appropriate weight were randomly selected and injected intraperitoneally with the corresponding dose of lemon balm on days 1, 3 and 5. After administration on day 6, the mice were placed in a constant temperature and humidity (39.5℃, 60%) incubator to establish the model until the core body temperature of the mice reached 42℃ (about 2 hours) and then the samples were collected.

[0037] Control group: Ten mice with similar weight to the dosage group were selected and collected after being raised in a suitable environment for 6 days.

[0038] Heatstroke group: Ten mice with similar weight to the dosage group were selected and kept in a suitable environment for 6 days. Then, they were placed in a constant temperature and humidity (39.5℃, 60%) incubator to model the heatstroke. The mice were collected after their core body temperature reached 42℃ (about 2 hours).

[0039] (2) Immediately after the modeling was completed, the rectal temperature of mice in the control group, heatstroke group and dosage group was measured. The mice were fixed and the thermometer probe was inserted into the rectum about 2 cm. The rectal temperature was recorded after the reading of the rectal thermometer stabilized.

[0040] result:

[0041] After the isothermal and humidity modeling process is completed, the rectal temperature of the mice is measured. Figure 1 As shown, compared with the control group, the rectal temperature of the heatstroke group was significantly increased (P<0.05). The rectal temperature of the experimental groups given low, medium and high doses (2.5mg / kg, 5mg / kg and 10mg / kg, respectively) decreased after modeling, with the 10mg / kg dose group showing the most significant decrease (P<0.05).

[0042] Example 2: Lemon balm glycosides can improve blood routine changes caused by heatstroke.

[0043] The above scheme was used to construct a heatstroke myocardial injury model at the individual level. Low, medium and high doses (2.5 mg / kg, 5 mg / kg and 10 mg / kg respectively) of lemon balm were used for preprotective treatment, and the specific steps were the same as in Example 1.

[0044] Changes in various blood routine indicators in mice after heatstroke modeling were detected: Blood was collected in anticoagulant tubes (approximately 0.05 mL) using the enucleation method, and then analyzed using a fully automated blood routine instrument.

[0045] result:

[0046] After the mouse modeling process was completed under constant temperature and humidity, changes in various blood routine indicators were detected: such as... Figure 2As shown, heatstroke causes changes in routine blood parameters in mice, such as a decrease in white blood cells (WBC), lymphocytes (LYM), and red blood cells (RBC) (P<0.05). Administration of different doses of lemon balm glycosides improved these parameters to varying degrees (P<0.05), indicating that the damage caused by heatstroke was repaired to some extent.

[0047] Example 3: Lemon balm glycosides can improve cardiac dysfunction caused by heatstroke.

[0048] The above scheme was used to construct a heatstroke myocardial injury model at the individual level. Low, medium and high doses (2.5 mg / kg, 5 mg / kg and 10 mg / kg respectively) of lemon balm were used for preprotective treatment, and the specific steps were the same as in Example 1.

[0049] Small animal ultrasound examination of cardiac function in mice after constant temperature and humidity modeling: The day before the ultrasound examination, the mice were shaved of their chest fur. On the day of the ultrasound, the mice were anesthetized by inhaling 3% isoflurane (air flow rate of 1 L / min). After the limb withdrawal reflex disappeared, the mice were fixed on a 37°C constant temperature plate and inhaled 1.5% isoflurane (air flow rate of 1 L / min) to completely cover the left thoracic cavity with coupling agent. Using a 30 MHz probe, standard parasternal left ventricular long axis section and standard left ventricular papillary muscle short axis section were selected to record M-mode cardiac ultrasound images.

[0050] Measurement metrics include: EF, FS, LVESV, and LVPWs.

[0051] During the testing process, the following details should be noted as they may affect the test results: First, the anesthesia should not be too deep, otherwise it will affect the mouse's heart rate and contractile function; second, the mouse's body position should be correct, and its limbs should not be fixed too long or too tight, otherwise it will compress the mouse's heart and ultimately affect the accuracy of the cardiac function test; third, the area of ​​the mouse near the heart should be shaved at least one day in advance. Shaving the hair too early will cause new hair to grow during the test, generating artifacts during imaging and affecting the ultrasound results, while shaving the hair too late will put the mouse in a state of stress and interfere with the cardiac function results.

[0052] result:

[0053] The cardiac function of mice in each group after constant temperature and humidity modeling was detected by small animal ultrasound. The results are as follows: Figure 3 and Figure 4(Left ventricular long-axis ultrasound section) is shown. Data analysis revealed that compared to the control group, after heatstroke injury, ejection fraction (EF), short-axis contractile rate (FS), and left ventricular posterior wall systolic thickness (LVPWs) were significantly decreased (P<0.05), while left ventricular end-systolic volume (LVESV) was significantly increased (P<0.05). In the experimental groups given low, medium, and high doses (2.5 mg / kg, 5 mg / kg, and 10 mg / kg, respectively) of lemon balm glycoside, EF, FS, and LVPWs were significantly increased (P<0.05), while LVESV was significantly decreased (P<0.05). This suggests that administration of lemon balm glycoside significantly improved cardiac function in mice. The 10 mg / kg group showed the most significant effect.

[0054] Example 4: Lemon balm glycosides can improve the decreased survival rate of HL-1 cardiomyocytes caused by heatstroke.

[0055] (1) Basic culture conditions: Mouse HL-1 cardiomyocytes were used as the research object. The cells were cultured in a 37°C, 5% CO2 incubator with complete medium containing 10% FBS. The cells adhered to the wall and grew. The medium was changed every other day. When the cells adhered to the wall for 80%-90%, they were digested and passaged with 0.25% trypsin. Cardiomyocytes in good growth status during the logarithmic growth phase were used for experiments.

[0056] (2) Grouping: HL-1 cells were divided into control group, heatstroke (HS) group, 10μM Didymin +HS group, 20μM Didymin +HS group, 40μM Didymin +HS group, and 80μM Didymin +HS group. Among them:

[0057] Control group: HL-1 cells were cultured in a basal culture (cells were cultured in a 37°C, 5% CO2 incubator with complete medium containing 10% FBS, adherent growth, and medium was changed every other day) for 24 hours.

[0058] HS group: Based on the basic culture conditions, HL-1 cells were placed in a carbon dioxide incubator and cultured at 42.5℃ for 9 hours.

[0059] Dosage groups: Based on the basic culture conditions, the samples were cultured for 3 hours in complete medium containing 10% FBS with lemon balm glycoside concentrations of 10, 20, 40, and 80 μM, respectively, and then placed in a carbon dioxide incubator at 42.5℃ for 9 hours.

[0060] After culture, the supernatant was collected, washed with PBS, digested with trypsin, and the cell suspension was collected. After centrifugation, the reagent was added according to the Muse Count & Viability apoptosis kit (purchased from Merck) and mixed well. The mixture was reacted at room temperature in the dark for 15 min, and the cells were detected by flow cytometry within 30 min.

[0061] result:

[0062] Cell vitality such as Figure 5 As shown, the morphology under the cell microscope is as follows: Figure 6 As shown, compared with the control group, the cell survival rate in the heatstroke group was significantly decreased (P<0.05); after administration of different doses of lemon balm, the cell survival rate was significantly increased (P<0.05).

[0063] Example 5: Lemon balm glycosides reduce the production of reactive oxygen species in HL-1 cardiomyocytes caused by heatstroke damage:

[0064] (1) Basic culture conditions: Mouse HL-1 cardiomyocytes were used as the research object. The cells were cultured in a 37°C, 5% CO2 incubator with complete medium containing 10% FBS. The cells adhered to the wall and grew. The medium was changed every other day. When the cells adhered to the wall for 80%-90%, they were digested and passaged with 0.25% trypsin. Cardiomyocytes in good growth status during the logarithmic growth phase were used for experiments.

[0065] (2) Grouping: HL-1 cells were divided into control group, heatstroke (HS) group, 10μM Didymin +HS group, 20μM Didymin +HS group, 40μM Didymin +HS group, and 80μM Didymin +HS group. Among them:

[0066] Control group: HL-1 cells were cultured in a basal culture (cells were cultured in a 37°C, 5% CO2 incubator with complete medium containing 10% FBS, adherent growth, and medium was changed every other day) for 24 hours.

[0067] HS group: Based on the basic culture conditions, HL-1 cells were placed in a carbon dioxide incubator and cultured at 42.5℃ for 9 hours.

[0068] Dosage groups: Based on the basic culture conditions, the samples were cultured for 3 hours in complete medium containing 10% FBS with lemon balm glycoside concentrations of 10, 20, 40, and 80 μM, respectively, and then placed in a carbon dioxide incubator at 42.5℃ for 9 hours.

[0069] Intracellular total reactive oxygen species (ROS) levels were detected using Beyotime's reactive oxygen species (ROS) detection kit (DCFH-DA). DCFH-DA itself is non-fluorescent and can freely cross the cell membrane to enter the cell, where it is hydrolyzed by intracellular esterases to generate DCFH. DCFH cannot penetrate the cell membrane and is thus retained inside the cell. When ROS are present intracellularly, DCFH is oxidized to generate DCF, which exhibits strong green fluorescence. The detected fluorescence intensity is directly proportional to the intracellular ROS level. The specific method is as follows:

[0070] 1. Dilute DCFH-DA with serum-free culture medium to prepare working solution, replace the original culture medium of cardiomyocytes, add probe working solution, and incubate in a cell culture incubator at 37°C in the dark for 20-60 minutes.

[0071] 2. After incubation, wash the cells 2-3 times with preheated PBS or serum-free culture medium to thoroughly remove probes that have not been taken up by the cells, thereby reducing background fluorescence.

[0072] 3. After washing, perform the test. Observe under a fluorescence microscope using the FITC (green) channel.

[0073] result:

[0074] Staining results as follows Figure 7 As shown: Compared with the Control group, the heatstroke (HS) group showed increased ROS generation, which was reflected in a significant increase in green fluorescence intensity; compared with the HS group, the 10, 20, 40, and 80 μM lemon balm glycoside groups showed significantly decreased ROS generation, which was reflected in a decrease in green fluorescence intensity.

[0075] Example 6: Lemon balm glycosides improve mitochondrial membrane potential imbalance in HL-1 cardiomyocytes caused by heatstroke.

[0076] (1) Basic culture conditions: Mouse HL-1 cardiomyocytes were used as the research object. The cells were cultured in a 37°C, 5% CO2 incubator with complete medium containing 10% FBS. The cells adhered to the wall and grew. The medium was changed every other day. When the cells adhered to the wall for 80%-90%, they were digested and passaged with 0.25% trypsin. Cardiomyocytes in good growth status during the logarithmic growth phase were used for experiments.

[0077] (2) Grouping: HL-1 cells were divided into control group, heatstroke (HS) group, 10μM Didymin +HS group, 20μM Didymin +HS group, 40μM Didymin +HS group, and 80μM Didymin +HS group. Among them:

[0078] Control group: HL-1 cells were cultured in a basal culture (cells were cultured in a 37°C, 5% CO2 incubator with complete medium containing 10% FBS, adherent growth, and medium was changed every other day) for 24 hours.

[0079] HS group: Based on the basic culture conditions, HL-1 cells were placed in a carbon dioxide incubator and cultured at 42.5℃ for 9 hours.

[0080] Dosage groups: Based on the basic culture conditions, the samples were cultured for 3 hours in complete medium containing 10% FBS with lemon balm glycoside concentrations of 10, 20, 40, and 80 μM, respectively, and then placed in a carbon dioxide incubator at 42.5℃ for 9 hours.

[0081] Resuspend cells from each group in 0.5 ml of cell culture medium containing serum and phenol red, add 0.5 ml of JC-1 staining working solution, invert several times to mix, and incubate at 37°C for 20 min in a cell culture incubator. Then centrifuge at 600 g for 3-4 min to pellet the cells and discard the supernatant. Wash twice with JC-1 staining buffer: resuspend cells in 1 ml of JC-1 staining buffer, centrifuge at 600 g for 3-4 min to pellet the cells and discard the supernatant; resuspend cells again in 1 ml of JC-1 staining buffer, centrifuge at 600 g for 3-4 min to pellet the cells and discard the supernatant. After resuspending with JC-1 staining buffer again, observe under a fluorescence microscope or laser confocal microscope.

[0082] result:

[0083] JC-1 staining results are as follows Figure 8 and Figure 9 As shown: Compared with the Control group, the red fluorescence of the heatstroke (HS) group was significantly weakened and the green fluorescence was significantly enhanced; after treatment with different doses of lemon balm, compared with the HS group, the red fluorescence of each dose group was significantly enhanced and the green fluorescence was significantly weakened.

[0084] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0085] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0086] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of lemon balm glycoside in the preparation of drugs for the prevention of heatstroke, characterized in that, The drug is used to inhibit the increase in rectal temperature caused by heatstroke; the drug is used to improve cardiac dysfunction caused by heatstroke.

2. The application according to claim 1, characterized in that, The drug is an oral or injectable formulation.

3. The application according to claim 2, characterized in that, The drug is prepared from lemon balm glycoside and pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, Pharmaceutically acceptable excipients include any one of solubilizers and diluents.

5. The application according to claim 4, characterized in that, The diluent includes DMSO.

6. The application according to claim 2, characterized in that, When the drug is an injectable formulation, the concentration of lemon balm glycoside is 10 μM to 80 μM.

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