Application of curcumin in the preparation of hemoglobin oxygen supply efficiency regulator

Abstract

The present invention discloses the application of curcumin in the preparation of a hemoglobin oxygen supply efficiency regulator. The present invention verifies through experiments that curcumin can bind to normal human hemoglobin (HbA) in a dose-effect manner, can increase hemoglobin oxygen affinity, and also enhance the Bohr effect of hemoglobin, so that hemoglobin can bind more oxygen in the lungs and can also promote hemoglobin to release more oxygen in peripheral tissues, that is, can enhance the oxygen supply efficiency of hemoglobin, indicating that curcumin can regulate the oxygen supply efficiency of hemoglobin, and thus can be used as a hemoglobin oxygen supply efficiency regulator for preventing and / or treating hypotonic hypoxia.

Description

Technical Field

[0001] The invention relates to the technical field of hypoxia drugs, and in particular to the application of curcumin in preparing a hemoglobin oxygen supply efficiency regulator. Background Art

[0002] The process of human body taking up oxygen includes inhaling air from the external environment, exchanging oxygen in the air with red blood cells in the alveoli, oxygen reaching peripheral tissues through arterial blood circulation, and releasing oxygen to cells, tissues, organs, etc. The atmospheric pressure of the air changes with the altitude, and the partial pressure of each gas component in the air (such as oxygen) also changes with the altitude. The oxygen content and oxygen partial pressure in the air in plateau areas decrease with the increase of altitude. The oxygen partial pressure in the air inhaled by people decreases, resulting in a decrease in the oxygen partial pressure in the human arterial blood, which causes insufficient oxygen supply to cells and tissues, resulting in hypoxia in the body. This hypoxia in plateau areas is called plateau hypoxia.

[0003] At present, the main drug used in China to prevent plateau hypoxia is Rhodiola rosea, but it is a traditional Chinese medicine and is defined as a health product or internal medicine. Its mechanism is unclear, and its effect has not been recognized by the US FDA. The United States uses acetazolamide and dexamethasone to prevent or treat hypoxia. Acetazolamide relieves hypoxia symptoms by increasing the discharge of bicarbonate, stimulating the respiratory tract, and increasing arterial oxygen partial pressure; dexamethasone is suitable for patients who are intolerant or allergic to acetazolamide. It may resist altitude sickness by reducing brain volume and inhibiting vascular endothelial growth factor and lipid peroxidation. Summary of the invention

[0004] The purpose of the present invention is to address the technical defects existing in the prior art. In a first aspect, the present invention provides the use of curcumin in the preparation of a hemoglobin oxygen supply efficiency regulator.

[0005] The concentration of curcumin is 9-30 μM.

[0006] The oxygen supply efficiency of hemoglobin includes the oxygen affinity of hemoglobin, expressed as P 50 The value indicates that the regulator can 50 Values ​​decreased by 6.5-21% (to 11.60-15.20 mmHg).

[0007] The oxygen supply efficiency of hemoglobin includes the Bohr effect of hemoglobin, which is characterized by the acid-base sensitive index (SI) of hemoglobin. The regulator can increase the SI by 86% (to 50%-80%).

[0008] The oxygen supply efficiency of hemoglobin also includes the theoretical ability of hemoglobin to bind to oxygen and release it to tissues and cells, which is characterized by the plain theoretical oxygen release capacity value and plateau theoretical oxygen release capacity value of hemoglobin. The regulator can increase the plain theoretical oxygen release capacity value by 38% (to 10.3%-11.3%) and the plateau theoretical oxygen release capacity value by 45% (to 17.3%-21.2%).

[0009] The binding rate constant of curcumin and hemoglobin Ka=1.92×10 3 mol -1 s -1 .

[0010] The dissociation rate constant of curcumin and hemoglobin is Kd=2.51×10 -2 s -1 .

[0011] The dissociation equilibrium constant of curcumin and hemoglobin KD=1.31×10 -5 mol.

[0012] In a second aspect, the present invention provides the use of curcumin in the preparation of a medicament for preventing and / or treating hypoxic injury.

[0013] The hypoxic injury is caused by acute high altitude hypoxia.

[0014] In a third aspect, the present invention provides a blood product comprising curcumin and stabilized hemoglobin, wherein the curcumin is bound to the hemoglobin.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention verifies through experiments that curcumin can present a dose-effect combination with normal human hemoglobin (HbA), can increase hemoglobin oxygen affinity, and can also enhance the Bohr effect of hemoglobin, so that hemoglobin can not only bind more oxygen in the lungs (i.e., take in more oxygen from the external environment), but also promote hemoglobin to release more oxygen in peripheral tissues, that is, enhance the oxygen supply efficiency of hemoglobin, indicating that curcumin can play a regulatory role in the oxygen supply efficiency of hemoglobin, so that it can be used as a hemoglobin oxygen supply efficiency regulator for preventing and / or treating hypotonic hypoxia. At the same time, experiments using acute high-altitude hypoxia animal models to evaluate the effect of curcumin on regulating the oxygen supply efficiency of hemoglobin show that curcumin can prevent and / or alleviate a series of pathological reactions caused by acute high-altitude hypoxia by regulating the oxygen supply efficiency of hemoglobin, indicating that curcumin can prevent and / or alleviate the damage caused by acute high-altitude hypoxia to the body by regulating the oxygen supply efficiency of hemoglobin, so as to be used as a drug for preventing and / or treating acute high-altitude hypoxia. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Shown is the binding curve of hemoglobin with curcumin solutions of different concentrations;

[0018] Figure 2 Shown is a graph showing changes in the proportion of oxygenated hemoglobin during deoxygenation after intervention with curcumin. DETAILED DESCRIPTION

[0019] In a first aspect, the present invention proposes a new use of curcumin in regulating the oxygen supply efficiency of hemoglobin.

[0020] In the present invention, the oxygen supply efficiency of hemoglobin refers to a comprehensive evaluation of the ability of hemoglobin to bind oxygen and release oxygen to tissue cells, and is evaluated through at least two aspects, namely, the oxygen affinity of hemoglobin and the Bohr effect of hemoglobin.

[0021] The oxygen supply in the human body mainly depends on the binding and release of hemoglobin with oxygen in red blood cells. The ability of hemoglobin to bind and release oxygen is the hemoglobin oxygen affinity, also known as the oxygen carrying / release capacity, which is one of the key parameters of hemoglobin oxygen supply efficiency. The size of oxygen affinity is measured by the oxygen partial pressure (P) when the hemoglobin oxygen saturation is 50% in the oxygen dissociation curve. 50 P 50 The increase in P value means that the affinity of hemoglobin to oxygen decreases, and hemoglobin releases oxygen more easily. 50 A decrease in the value means that the affinity of hemoglobin for oxygen increases, and hemoglobin can carry oxygen more easily.

[0022] At the same time, factors that reflect the oxygen supply efficiency of hemoglobin also include the Bohr effect of hemoglobin. The Bohr effect of hemoglobin refers to the change of pH (CO2 concentration, H + concentration changes, etc.), oxygen affinity (P 50 For example, when CO2, H + The increase in concentration will cause the pH to decrease, which will cause P 50 When the pH value increases, the oxygen affinity of hemoglobin decreases. Similarly, when the pH value increases, it will cause P 50 The value decreases and the oxygen affinity of hemoglobin increases. Hemoglobin binds oxygen in the lungs and releases oxygen in peripheral tissues. However, the pH in the lungs is slightly higher, and the oxygen affinity of hemoglobin is higher here, which is conducive to binding oxygen. The pH in peripheral tissues is slightly lower, and the oxygen affinity of hemoglobin is reduced here, which is conducive to releasing oxygen. The stronger the Bohr effect of hemoglobin, the greater the gap in the oxygen affinity of hemoglobin between the lungs and peripheral tissues, which means that hemoglobin can bind more oxygen in the lungs and release more oxygen in peripheral tissues at the same time. The Bohr effect of hemoglobin is characterized by the acid-base sensitivity index (SI), that is, the larger the acid-base sensitivity index, the stronger the Bohr effect, and the acid-base (H+ The shift amplitude of the oxygen dissociation curve of hemoglobin (P 50 The larger the acid-base sensitivity index is, the smaller the Bohr effect is, and the acid-base (H + The shift amplitude of the oxygen dissociation curve of hemoglobin (P 50 The smaller the value change).

[0023] Based on this, the present invention proposes that when the ambient oxygen partial pressure is low (such as in plateau areas), if more oxygen can be absorbed from the external environment and then more of the absorbed oxygen can be supplied to peripheral tissues, it can be used as a feasible method to prevent plateau hypoxia, that is, hypoxia can be prevented by adjusting the oxygen supply efficiency of hemoglobin.

[0024] Although some drugs are not directly used to treat hypoxia, they have the effect of increasing the oxygen-carrying or oxygen-releasing capacity of hemoglobin, such as GBT440 (trade name Oxbryta) and RSR-13 (trade name Efaproxiral). GBT440 has been marketed in the United States as a drug for the treatment of sickle cell anemia. It can combine with sickle hemoglobin (HbS) to enhance the oxygen-carrying capacity of HbS, but has little effect on the oxygen-carrying capacity of normal human hemoglobin (Hemoglobin adult, HbA). RSR-13 has a regulatory effect on enhancing the oxygen-releasing capacity of hemoglobin. It was once used as an adjuvant drug for tumor chemotherapy in clinical phase III trials, but was ultimately not approved for marketing.

[0025] Currently, there is no drug on the market that can regulate the oxygen supply efficiency (oxygen affinity and Bohr effect) of normal hemoglobin, and there is no report on drugs that regulate the oxygen supply efficiency of hemoglobin as a treatment for high-altitude hypoxia.

[0026] The present invention identifies a highly effective regulator of hemoglobin oxygen supply efficiency, namely curcumin, which is one of the components of the orange-yellow pigment extracted from the turmeric root (Curcuma longa). The turmeric root is mainly produced in Japan, the United States, Africa, China and other places. The orange-yellow pigment extracted therefrom is a slightly acidic phenolic substance, which is composed of curcumin, demethoxycurcumin and bisdemethoxycurcumin, also known as curcumin. The World Health Organization has approved curcumin as a natural food additive. Curcumin is the main component of turmeric root that exerts pharmacological effects, among which curcumin is the most important active ingredient in curcumin. It is known to have anti-inflammatory, antioxidant, oxygen free radical scavenging, liver and kidney function protection and other effects. The molecular formula of curcumin is C 21 H 20O6, whose main chain is unsaturated aliphatic and aromatic groups, is soluble in organic solvents such as ethanol, acetic acid, acetone and chloroform, but insoluble in water. It is commercially available with a purity of 97.5% and a CAS number of 458-37-7.

[0027] The present invention verifies through experiments that curcumin can regulate the P of hemoglobin by interacting with hemoglobin. 50 value and acid-base sensitivity index SI, thereby regulating the oxygen supply efficiency of hemoglobin, and showed a significant effect in alleviating hypoxic damage in an experiment on rats raised in a simulated plateau environment, indicating that curcumin can be used as a regulator of hemoglobin oxygen supply efficiency and a drug for preventing and / or treating hypoxic damage.

[0028] The following is a more detailed description of the present invention with reference to specific embodiments, and further elaboration of the present invention, but these embodiments are by no means intended to limit the present invention.

[0029] Example 1: Experiment on the interaction between curcumin and human hemoglobin

[0030] This experiment was carried out using an SPR instrument. The specific experimental process is as follows:

[0031] (1) According to OpenSPR TM (Benchtop Surface Plasmon Resonance Instrument) Instrument Standard Operating Procedure Install the COOH chip.

[0032] (2) Run at the maximum flow rate (150 μL / min) of the assay buffer, which is HBS-ET (pH = 7.4, containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005 wt% Tween-20, 1% (v / v) DMSO).

[0033] (3) After the signal reaches the baseline, load 200 μL of an aqueous solution containing 80% (v / v) isopropanol and run for 10 s to remove bubbles. After the signal reaches the baseline, rinse the sample loop with detection buffer (HBS-ET) and evacuate it with air.

[0034] (4) After the signal reaches the baseline, adjust the flow rate of the detection buffer to 20 μL / min (the "signal reaches the baseline" mentioned in steps (3) and (4) means that after each operation, the signal returns to the baseline before proceeding to the next operation).

[0035] (5) Load 200 μL of a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and hydroxysuccinimide (NHS) (prepared by mixing 400 mM EDC solution and 100 mM NHS solution in a volume ratio of 1:1, flow rate 20 μL / min, run for 4 min).

[0036] (6) Load 200 μL of human hemoglobin solution (hemoglobin concentration is 6 mg / ml) diluted with activation buffer (sodium acetate buffer, pH=3.5, 10 mM), run for 4 min (flow rate 20 μL / min), rinse the sample loop with activation buffer, and evacuate with air.

[0037] (7) Load 200 μL of Blocking solution (1 M ethanolamine aqueous solution, flow rate 20 μL / min, run for 4 min), rinse the sample loop with detection buffer (HBS-ET), and evacuate with air.

[0038] (8) Observe the baseline for 5 minutes to ensure stability.

[0039] (9) The analyte curcumin was dissolved in DMSO and then diluted to different concentrations using the assay buffer (HBS-ET) (see Table 1 for concentrations). Figure 1 ), and then the following operations were performed on each concentration of curcumin solution: loading the sample at a flow rate of 20 μL / min for 240 s at 25°C to allow hemoglobin to bind to the curcumin solution; then continuing to load the detection buffer without curcumin at the same flow rate of 20 μL / min to elute the hemoglobin bound to curcumin for 240 s to dissociate hemoglobin and curcumin.

[0040] (10) The binding reaction signal detected by the SPR instrument was plotted using the analysis software TraceDrawer (RidgeviewInstruments ab, Sweden) and the analysis method One To One analysis model to obtain a curve of the response value (RU) corresponding to the binding reaction signal versus time, which is the curve of the binding and dissociation of hemoglobin and curcumin according to the concentration gradient, see Figure 1 .

[0041] This experiment further investigated the binding and dissociation curves of human hemoglobin and curcumin ( Figure 1 ) was analyzed and the binding rate constant Ka was calculated to be 1.92×10 3 mol -1 s -1 , dissociation rate constant Kd = 2.51 × 10 -2 s -1 , and the dissociation equilibrium constant (also called affinity constant) KD = Kd / Ka = ​​1.31×10 -5 mol. Combining the KD value with the curve of different concentrations of curcumin and human hemoglobin, the response value of the binding curve of different concentrations of curcumin and human hemoglobin is positively correlated with the concentration of curcumin, indicating that curcumin can bind to human hemoglobin in a curcumin concentration gradient ( Figure 1The higher the concentration of curcumin, the greater the response value of the binding reaction signal, and the more hemoglobin binds to curcumin).

[0042] Example 2: Oxygen Dissociation Assay (ODA) Experiment

[0043] Using a UV-visible spectrophotometer, the spectrum of each group of samples was scanned in the 350nm-700nm band under controlled gas environment conditions. The specific method is as follows:

[0044] 1. Preparation of each group of samples

[0045] Blank group: 3 μM human hemoglobin solution, which was obtained by dissolving human hemoglobin in buffer A (buffer A containing 130 mM NaCl, 5 mM KCl and 30 mM TES) at pH=7.4.

[0046] Experimental group: The buffer A solution of human hemoglobin and the DMSO solution of curcumin were mixed evenly at room temperature at a molar ratio of human hemoglobin:curcumin=1:3. After mixing, the concentration of human hemoglobin was 3 μM, and the concentration of curcumin was 9 μM.

[0047] 2. Testing process

[0048] 200 μL of each of the two groups of samples were added to a 96-well plate and placed in a 25°C, normoxic environment (i.e., an atmospheric environment with an altitude of 0 m, an ambient atmospheric pressure of 760 mmHg, and an ambient oxygen partial pressure of 159 mmHg) for 1 hour to allow the samples to reach oxygen saturation (hemoglobin becomes oxyhemoglobin). Nitrogen gas was then continuously introduced into the samples (20 L / min), and a spectral scan was performed every 6 minutes (the temperature during the scan was 37°C), which was called one cycle, and a total of 25 cycles were performed. The five-wavelength method (see formula A) was used to substitute the absorbance values ​​measured after each cycle into formula A (A576, A700, A630, and A560 in formula A refer to the absorbance values ​​at wavelengths of 576 nm, 700 nm, 630 nm, and 560 nm, respectively. The five-wavelength method also has an absorbance at 540 nm, but it is not used in this formula). The oxygenated hemoglobin content (oxyHb%) in each group of samples after each cycle was calculated. The results are shown in Tables 1 and Figure 2 .

[0049]

[0050] Table 1 Changes in oxygenated hemoglobin content

[0051]

[0052]

[0053] In this embodiment, the uninterrupted introduction of nitrogen can deoxygenate the oxyhemoglobin. Figure 2 The results in Table 1 and Table 2 are the changes in the content of oxygenated hemoglobin in each group with the deoxygenation time (nitrogen introduction time). The results show that under the same deoxygenation environment, compared with the human hemoglobin in the blank group, the content of oxygenated hemoglobin in the experimental group (human hemoglobin sample mixed with curcumin) decreases more slowly, which means that its deoxygenation rate slows down in the same oxygen partial pressure environment; it shows that curcumin can slow down the oxygen release process of oxygenated hemoglobin, so that oxygenated hemoglobin can still release more oxygen for tissue use when it reaches hypoxic tissue through blood circulation, so as to alleviate tissue hypoxia.

[0054] This experiment also shows that due to the introduction of nitrogen, the oxygen partial pressure in each group of samples at the end of the experiment was low, but at this time the oxygenated hemoglobin content of the experimental group was still higher than that of the blank group (86.83% VS. 62.08%), indicating that in an environment with low oxygen partial pressure, the hemoglobin treated with curcumin (a mixed solution of curcumin and hemoglobin) still reserves more oxygen, allowing oxygenated hemoglobin to reach hypoxic tissues smoothly through the blood circulation, and in an environment with lower oxygen partial pressure, it can still release more oxygen to hypoxic tissues for use by tissues to alleviate tissue hypoxia.

[0055] Example 3: Experiment on the effect of curcumin on the oxygen supply efficiency of human hemoglobin

[0056] This example was carried out using a BLOODOX-2018 oxygen carrying / release analyzer purchased from Beijing Ruanlong Biotechnology Co., Ltd. DMSO solutions with different curcumin concentrations were mixed with buffer A solutions containing human hemoglobin at pH values ​​of 7.2, 7.4, and 7.6, respectively, to prepare mixed solutions with different final concentrations (i.e., 9 mixed solution samples), the final concentration of human hemoglobin in the mixed solutions was 10 μM, and the final concentrations of curcumin were 0 μM, 10 μM, and 30 μM, respectively, which were defined as a control group, a low-dose group, and a high-dose group, respectively. During the test, 4 mL of the mixed solution sample was added to the sample pool of the analyzer, and air was passed through for 30 minutes. After reaching oxygen saturation, nitrogen was introduced for deoxygenation, and an oxygen dissociation curve was drawn. According to the oxygen dissociation curve, the oxygen partial pressure value corresponding to the oxygen saturation of 50% for each sample was obtained, which is P 50 Based on the pH values ​​of samples at three pH environments: pH = 7.2, 7.4, and 7.6 50 The acid-base sensitivity index (SI) was calculated by 50酸 -P 50碱 ) / P 50中 ), where P 50酸 , P 50碱 , P 50中The pH values ​​are 7.2, 7.6, and 7.4 respectively. 50 Each sample was tested 4 times, and the results are shown in Table 2 and Table 3 (the high-dose group is representative in Table 3).

[0057] Table 2 P of human hemoglobin after interaction with different concentrations of curcumin 50 Value (mmHg, pH = 7.4)

[0058]

[0059]

[0060] The data in Table 2 show that curcumin can increase the P 50 The value decreased, indicating that the oxygen affinity of hemoglobin increased, hemoglobin was able to carry more oxygen, and the effect of high-dose curcumin was more obvious.

[0061] Table 3 Acid-base sensitivity index (SI) of human hemoglobin after the action of curcumin

[0062]

[0063] The data in Table 3 show that the acid-base sensitivity index SI of human hemoglobin can be significantly increased after the interaction with curcumin, and the Bohr effect can be enhanced, indicating that curcumin can make hemoglobin bind more oxygen in the lungs and release more oxygen in peripheral tissues to relieve hypoxia of cells and tissues.

[0064] In the present invention, the theoretical oxygen release capacity value is defined as the amount of hemoglobin bound to oxygen and released to tissues and cells calculated based on the oxygen dissociation curve. In the experiment of this embodiment, the oxygen dissociation curve under the conditions of pH = 7.6 and alveolar oxygen partial pressure of 100 mmHg and 60 mmHg was used to simulate the oxygen carrying state of hemoglobin in the lungs of the plain and plateau, and the oxygen dissociation curve under the conditions of pH = 7.2 and peripheral tissue oxygen partial pressure of 40 mmHg and 30 mmHg was used to simulate the oxygen release state of hemoglobin in the peripheral tissues of the plain and plateau. The oxygen saturation difference corresponding to the alveolar oxygen partial pressure (100 mmHg) and the peripheral tissue oxygen partial pressure (40 mmHg) in the oxygen dissociation curve simulating the plain was used as the theoretical oxygen release capacity value under the plain environment; the oxygen saturation difference corresponding to the alveolar oxygen partial pressure (60 mmHg) and the peripheral tissue oxygen partial pressure (30 mmHg) in the oxygen dissociation curve simulating the plateau was used as the theoretical oxygen release capacity value under the plateau environment. The results are shown in Table 4.

[0065] Theoretical oxygen release capacity of plain: △SO2=SO 2(pH＝7.6-100mmHg) -SO 2(pH＝7.2-40mmHg)

[0066] Theoretical oxygen release capacity of plateau: △SO2=SO2(pH＝7.6-60mmHg) -SO 2(pH＝7.2-30mmHg)

[0067] Table 4 Theoretical oxygen release capacity (△SO2) data after human hemoglobin and curcumin react

[0068]

[0069]

[0070] The data in Table 4 show that the theoretical oxygen release capacity of hemoglobin increased after the action of curcumin, among which the theoretical oxygen release capacity value △SO2 in the plain environment increased by 38.6%, and the theoretical oxygen release capacity value △SO2 in the plateau environment increased by 45.4%.

[0071] The above results show that curcumin can reduce the P 50 value, increasing its oxygen-carrying capacity in the lungs; at the same time, curcumin can increase the acid-base sensitivity index SI and enhance the ability of hemoglobin to release oxygen in peripheral tissues; whether in plain or plateau environments, curcumin increases the theoretical oxygen release capacity of hemoglobin, and the increase is greater in plateau environments. This result shows that curcumin can be used as an effective regulator of hemoglobin oxygen supply efficiency and is more suitable for plateau environments.

[0072] Example 4: Animal experiment on curcumin preventing high altitude hypoxia

[0073] 1. Experimental process

[0074] Using a multi-factor composite simulated medical experimental chamber (purchased from Guizhou Fenglei Aviation Ordnance Co., Ltd.), at 25°C, the low-pressure and low-oxygen environment at an altitude of 6000m was simulated, and the experimental animals (Wistar rats, SPF grade, male, 200-220g) were placed in the experimental chamber for feeding. The animals were divided into four groups: a normoxic control group, a hypoxic control group, a hypoxic high-dose medication group (curcumin dosage 100mg / kg), and a hypoxic low-dose medication group (curcumin dosage 50mg / kg). The normoxic control group had an atmospheric environment at an altitude of 0m, an ambient atmospheric pressure of 760mmHg, and an ambient oxygen partial pressure of 159mmHg; the hypoxic group simulated an atmospheric environment at an altitude of 6000m, with an ambient atmospheric pressure of 349mmHg and an ambient oxygen partial pressure of about 70mmHg. Curcumin was dissolved in physiological saline containing 1wt% sodium carboxymethyl cellulose as a solvent. The drug administration operation is as follows: first, the simulated altitude of the experimental chamber is raised from 0m to 3500m, and the rats are gavaged once; then the simulated altitude of the experimental chamber is further raised to 6000m, and the rats are fed. After that, the simulated altitude of the experimental chamber is lowered to 3500m every day, and the rats are gavaged once. The time for the rats to be in the atmospheric environment of the simulated altitude of 3500m is generally no more than 1 hour per day. After the drug administration, the simulated altitude is raised to 6000m. After 7 days of feeding, various indicators are tested, and the results are shown in Tables 5-12.

[0075] 2.1 Changes in rat body weight

[0076] Table 5 Body weight changes of rats

[0077]

[0078]

[0079] The results in Table 5 show that after 7 days of feeding, the average weight of the normoxic control group increased by 5.00±1.83g, and the average weight of the hypoxic control group decreased by 4.00±1.83g, indicating that in the 6000m plateau environment, the weight of the experimental animals will decrease due to plateau hypoxia. After intervention with different concentrations of curcumin, the weight of the animals in the low-dose group and the high-dose group increased compared with the hypoxic control group, and the difference was significant. The above results show that curcumin intervention can effectively alleviate the weight loss caused by plateau hypoxia.

[0080] 2.2 Changes of blood routine parameters in rats

[0081] After 7 days of feeding, the rats were cannulated in the carotid artery and blood was collected for routine blood tests, including white blood cell count (WBC), red blood cell count (RBC), hemoglobin content (HGB), hematocrit (HCT), and platelet count (PLT). The results are shown in Table 6.

[0082] Table 6 Blood routine test results

[0083]

[0084] After acute high-altitude hypoxia (normoxic control group VS. hypoxic control group), the body compensates for changes in blood routine parameters, which are manifested as increased red and white blood cells, increased hematocrit, increased hemoglobin and thrombocytopenia due to complex mechanisms such as dehydration and compensatory regulation. In the long run, it will lead to more serious pathological changes, causing chronic inflammation, abnormal coagulation function and irreversible lesions of cardiopulmonary function. The data in Table 6 show that compared with rats in the normoxic control group, rats in the hypoxic control group under low atmospheric pressure showed obvious compensatory changes in blood routine parameters, and after the administration of curcumin, the amplitude of compensatory changes was significantly alleviated, indicating that curcumin can alleviate the degree of hypoxia in rats by regulating the oxygen supply efficiency of hemoglobin, thereby preventing and alleviating the occurrence of acute hypoxic injury, and significantly reducing the degree of compensatory changes in blood routine parameters of the body. Therefore, curcumin can be used as a regulator of hemoglobin oxygen supply efficiency for the prevention and / or treatment of acute hypoxic injury at high altitude.

[0085] 2.3 Analysis of whole blood gas in rat carotid artery

[0086] After 7 days of feeding, the carotid artery was cannulated and blood was collected for blood gas parameter testing. The results are shown in Tables 7-8.

[0087] Table 7 Arterial oxygen partial pressure (PO2 / mmHg)

[0088]

[0089] Table 8 Arterial oxygen saturation (SO2 / %)

[0090]

[0091] The general situation is that after acute high-altitude hypoxia occurs, the arterial oxygen partial pressure of rats decreases significantly, causing a decrease in arterial oxygen saturation, and the body suffers from acute high-altitude hypoxia; this experiment's normoxic control group and hypoxic control group also verified this. However, this experiment shows that after curcumin was given to rats raised in a hypoxic environment, the arterial oxygen partial pressure increased to a certain extent compared with the hypoxic control group, but there was no significant difference, indicating that curcumin does not supply oxygen to the body by promoting respiration. After curcumin was given, arterial oxygen saturation increased significantly, that is, the oxygen carrying capacity of arterial blood was increased by curcumin's regulation of hemoglobin oxygen supply efficiency. The results show that curcumin, as a hemoglobin oxygen supply efficiency regulator, can improve the arterial oxygen saturation of rats and prevent and / or treat the occurrence of acute high-altitude hypoxia.

[0092] 2.4 Rat carotid artery whole blood biochemical analysis

[0093] After 7 days of feeding, carotid artery cannulation was performed, and whole blood was collected. After plasma was separated, whole blood biochemical analysis was performed, including blood glucose (GLU), creatinine (CRE), alanine aminotransferase (AST), and aspartate aminotransferase (ALT).

[0094] Table 9 Plasma biochemical test results

[0095]

[0096] After acute high-altitude hypoxia, rats' liver and kidney functions and metabolism were disturbed, as reflected by decreased serum glucose, increased creatinine, and increased aminotransferase (AST & ALT) levels. After administration of curcumin, changes in glucose, creatinine, and aminotransferase levels in rats raised in a high-altitude environment were significantly alleviated, and even recovered to levels comparable to those in the normoxic control group, indicating that curcumin, as a hemoglobin oxygen supply efficiency regulator, can prevent and / or treat liver and kidney dysfunction caused by high-altitude hypoxia in rats.

[0097] 2.5 Analysis of blood oxygen supply efficiency in rats

[0098] After 7 days of feeding, the carotid artery was cannulated and blood was collected. The blood was tested using the BLOODOX-2018 oxygen carrying / release analyzer, and the oxygen dissociation curves simulating different acid-base environments in the body were drawn to obtain the changes in parameters related to oxygen supply efficiency (P 50 Value, SI, △SO2), the results are shown in Table 10-Table 12.

[0099] Table 10 Rat arterial blood P 50 Value (mmHg) data

[0100]

[0101]

[0102] Table 11 Data of rat arterial acid-base sensitivity index (SI)

[0103]

[0104] Table 12 Data on the theoretical oxygen release capacity of rat arterial blood at plateau

[0105]

[0106] After acute high altitude hypoxia occurs, the body needs more oxygen to maintain basic life activities. In addition to the compensatory increase in the number of red blood cells, compensatory P 50The value of the oxygen release capacity of hemoglobin increased (the oxygen affinity of hemoglobin decreased), and the theoretical oxygen release capacity value also decreased significantly, indicating that the theoretical amount of oxygen released by hemoglobin to the body decreased under the high altitude environment. In this case, there was no difference in the acid-base sensitivity index (Bohr effect) between the hypoxic control group and the normoxic control group, indicating that after acute high altitude hypoxia occurs, the body itself cannot adjust its Bohr effect to enhance oxygen supply efficiency. After administration of curcumin, P 50 The increase in the value was significantly reduced, especially when the curcumin concentration was 100 mg / kg, no obvious P 50 The value increased compensatorily, indicating that P 50 The value can even be maintained at a level comparable to that of the normoxic control group. At the same time, after administration of curcumin, SI and theoretical oxygen release capacity values ​​were significantly increased compared with the hypoxic control group, indicating that curcumin theoretically significantly increases the amount of oxygen released by hemoglobin to the body under high altitude conditions, and hemoglobin can provide the body with more oxygen and better relieve hypoxia. That is, curcumin is an effective regulator of hemoglobin oxygen supply efficiency, which can prevent and / or treat acute high altitude hypoxia by regulating the oxygen supply efficiency of hemoglobin.

[0107] After the two concentration gradients of curcumin were administered to rats for intervention, the rats survived well, and various indicators were between the normoxic blank group and the hypoxic control group. At the same time, the concentrations involved in the present invention were lower than the safe concentrations (IC 50 =1500mg / kg).

[0108] In summary, in vitro and in vivo experiments have shown that curcumin can bind to human hemoglobin in a concentration gradient and reduce the P 50 value (increase oxygen affinity), increase acid-base sensitivity index (enhance Bohr effect), increase theoretical oxygen release capacity value, and enhance oxygen supply efficiency. In the acute hypobaric hypoxic rat model, curcumin can prevent and / or treat the compensatory changes of the body caused by acute hypoxia, and this effect is inseparable from its regulatory effect on the oxygen supply efficiency of hemoglobin. Therefore, curcumin is an effective regulator of hemoglobin oxygen supply efficiency, and curcumin can be used as an effective drug for preventing and / or treating the adverse reactions of acute high altitude hypoxia.

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the contents of the present invention.

Claims

1. Application of curcumin in the preparation of drugs for the treatment of acute high altitude hypoxia.

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