Anti-hypoxia morin and nanoparticles thereof

Abstract

The invention belongs to the technical field of functional factor development, and particularly relates to novel anti-hypoxia application of MO. According to the invention, the MO-DEX NPs nanoparticles are also prepared, and the dispersity of MO in an aqueous solution is improved by the nanoparticles. The in-vitro and in-vivo anti-hypoxia activity of MO and the nanoparticles thereof is verified by establishing an in-vitro CoCl2 induced PC12 cell chemical hypoxia model and an in-vivo low-pressure hypoxia mouse model, and it is found that the nanoparticles can significantly improve PC12 cell activity decline caused by hypoxia; the compound can relieve damage of mouse hematopoietic system, brain, lung, heart and other tissues and abnormal energy metabolism caused by hypobaric hypoxia, and is expected to be used for preventing and treating mountain sickness.

Description

Technical Field

[0001] The invention belongs to the technical field of functional factor development, and specifically relates to mulberry pigment, its nanoparticles and new application thereof in preventing or alleviating hypoxia symptoms. Background Art

[0002] Hypoxia refers to a pathological process of abnormal tissue metabolism and damage caused by insufficient oxygen supply or oxygen utilization disorder. Acute or long-term exposure to the low oxygen environment of the plateau can easily cause damage to the central nervous system, respiratory circulatory system, and cardiovascular system. Mountain sickness is a general term for various clinical manifestations caused by the failure of acute hypoxia acclimatization in people who rush to the plateau. The pathology is complex and there is no specific medicine. At present, the main diagnostic and therapeutic drugs on the market for preventing or alleviating hypoxic damage are mostly chemical control agents based on acetazolamide and dexamethasone. Most of them have side effects, such as causing gastrointestinal discomfort and indigestion, which limits their application in routine prevention. Traditional Chinese medicine compound preparations based on plateau plants such as Rhodiola rosea and saffron face problems such as resource shortages, high prices, and over-development that will affect the plateau environment. Therefore, finding natural anti-hypoxic drugs with abundant resources, low toxicity and side effects, and reliable efficacy is of great significance for improving the health of people exposed to acute plateau hypoxia.

[0003] Morin (MO) is a light yellow pigment extracted from the bark of medicinal plants in the Moraceae family. It has pharmacological functions such as anti-inflammatory, is inexpensive, widely available, and has few toxic and side effects. However, its hydrophobicity and poor stability limit its application in food and medicine.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In the first aspect, the inventors have demonstrated for the first time that MO can resist hypoxia, especially hypobaric hypoxia, which can prevent or alleviate at least one of the following symptoms:

[0006] 1) Damage to the heart, lungs, brain and / or hematopoietic system caused by hypoxia

[0007] 2) Abnormal energy metabolism caused by hypoxia, such as abnormal increase in lactate dehydrogenase activity in serum and brain, heart, and lung tissues

[0008] 3) Altitude sickness.

[0009] On the other hand, the present invention aims to provide a flavonoid polymer nanoparticle encapsulating MO that improves the water solubility and bioavailability of MO, and at the same time broadens the new application of MO in the field of anti-hypoxia. The nanoparticle can be composed of dextran (wall material) and morin (core material), and the dextran can be dextran (abbreviated as DEX), and the formula weight of DEX is 4-6KDa, preferably 5KDa (i.e., 5000Da), for example, MO is encapsulated with DEX to obtain morin dextran nanoparticles (abbreviated as MO-DEX NPs).

[0010] The nanoparticles can be prepared by anti-solvent precipitation method, for example, DEX aqueous solution is gradually added to morin ethanol solution, the added DEX can be 10-15 times the mass of MO, preferably 12 times; the DEX ethanol solution can be 15-25 times the volume of the aqueous solution, preferably 20 times.

[0011] As an example, the specific preparation method of MO-DEX NPs having anti-hypoxia activity of the present invention is as follows:

[0012] Step 1: Weigh 5 mg of MO and dissolve it in 100 mL of ethanol. Stir thoroughly to dissolve it.

[0013] Step 2: Weigh 60 mg of DEX and dissolve it in 5 mL of water. Stir thoroughly to dissolve.

[0014] Step 3: Add the DEX aqueous solution dropwise into the MO solution under magnetic stirring and continue stirring at room temperature;

[0015] Step 4: Centrifuge the mixed solution obtained in step 3 and collect the precipitate to obtain MO-DEX NPs.

[0016] Preferably, the MO used in step 1 has a purity of 99.8%, and the ethanol specification is analytical grade;

[0017] Preferably, in step 3, the magnetic stirring speed is 1200 r / min, the dropping speed of the DEX solution is less than 1 mL / min, and the stirring time is 2 h;

[0018] Preferably, in step 4, the centrifuge speed is 12000 r / min and the centrifugation time is 20 min.

[0019] The present invention has the following advantages:

[0020] 1: The morin and glucan used in the present invention are both natural biomolecules, the raw materials are safe and degradable, and are easily available and inexpensive;

[0021] 2: The present invention develops a stable mulberry pigment delivery system, improves the water solubility and bioavailability of mulberry pigment, and provides the possibility for the application of mulberry pigment in the fields of food and medicine;

[0022] 3: The present invention evaluated the anti-hypoxic function of mulberry pigment and mulberry pigment glucan nanoparticles by establishing a CoCl2-induced PC12 cell chemical hypoxia model and a low-pressure hypoxia mouse model. The experiments showed that pretreatment with mulberry pigment and mulberry pigment glucan nanoparticles could significantly inhibit the decrease in PC12 cell activity caused by CoCl2 chemical induction, and could significantly alleviate the damage caused by low-pressure hypoxia to the mouse hematopoietic system and organ tissues, and regulate the abnormal energy metabolism of mice caused by low-pressure hypoxia. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 SEM images of MO and MO-DEX NPs: (A) SEM image of MO; (B) SEM image of MO-DEX NPs;

[0024] Figure 2 Contact angles of MO and MO-DEX NPs: (A) contact angle of MO; (B) contact angle of MO-DEX NPs;

[0025] Figure 3 Effects of MO-DEX NPs on organ indexes of mice treated with hypobaric hypoxia: (A) brain index; (B) lung index; (C) heart index;

[0026] Figure 4 Effects of MO-DEX NPs on peripheral blood counts of mice treated with hypobaric hypoxia: (A) red blood cells; (B) hemoglobin; (C) average hemoglobin content;

[0027] Figure 5 Effects of MO-DEX NPs on MDA in serum, brain, lung and heart of mice treated with hypobaric hypoxia: (A) MDA content in serum; (B) MDA content in brain; (C) MDA content in lung; (D) MDA content in heart;

[0028] Figure 6 Effects of MO-DEX NPs on LDH activity in serum, brain, lung and heart of mice treated with hypobaric hypoxia: (A) LDH activity in serum; (B) LDH activity in brain; (C) LDH activity in lung; (D) LDH activity in heart;

[0029] The significance mark # in each figure indicates the comparison between the NC group and the MC group, and * indicates the comparison between other experimental groups and the MC group. The more # and * there are, the stronger the significance is. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with the drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention belong to the protection scope of the present invention.

[0031] Example 1: Preparation of MO-DEX NPs

[0032] Take 5 mg of morin and dissolve it in 100 mL of anhydrous ethanol, and 60 mg of dextran (DEX, formula weight is 5 KDa) and dissolve it in 5 mL of water. After stirring them evenly, the dextran solution was added dropwise to the morin anhydrous ethanol solution at a speed lower than 1 mL / min, and then stirred at a constant speed on a magnetic stirrer for 2 h. The collected mixed solution was centrifuged at a speed of 12000 r / min for 20 min, and the obtained precipitate was MO-DEX NPs.

[0033] Test Example 1: Determination of the physicochemical properties of MO-DEX NPs of the present invention

[0034] The encapsulation efficiency and loading rate of MO-DEX NPs were indirectly calculated by measuring the content of MO in the supernatant obtained by centrifugation using a UV-visible spectrophotometer, and the particle size, PDI and Zeta potential of MO-DEX NPs were measured by DLS. The results are shown in Table 1:

[0035] Table 1. Physicochemical parameters of MO-DEX NPs

[0036]

[0037] Experimental Example 2: Characterization of MO-DEX NPs of the present invention

[0038] The MO-DEX NPs prepared in Example 1 were characterized by observing their microscopic morphology using SEM, measuring their hydrophilic angle using a surface tension meter, and performing structural analysis using an XRD diffractometer and a Fourier transform infrared spectrometer.

[0039] The microscopic morphology of MO-DEX NPs is shown in Figure 1 As shown, the surface of MO presents a rod-like structure of irregular size, while MO-DEX NPs present a regular spherical structure with uniform distribution and no obvious aggregation phenomenon. The change in their microscopic morphology indicates that the nanoparticles prepared by this method can wrap MO.

[0040] In order to confirm that MO has been encapsulated in MO-DEX NPs, XRD and infrared spectra of MO, DEX and MO-DEX NPs were measured. The results showed that compared with MO and DEX, no new absorption peaks were found in the infrared spectrum of MH-DEX NPs, indicating that the formation of covalent bonds was not involved in the preparation of nanoparticles, and the spectrum of MO-DEX NPs was basically similar to that of DEX, indicating that DEX successfully encapsulated the MO core material as a wall material. The XRD spectrum also confirmed the result. In the diffraction pattern of MO, there are many sharp diffraction peaks, while in the diffraction pattern of DEX, no sharp characteristic peaks are shown. After the formation of MO-DEX NPs, the unique crystallization peak of MO disappears, and only a broad and weak flat peak appears at 2θ of 17.64°. Combined with the morphological changes of MO-DEX NPs and the changes in the characteristic peaks of FT-IR, it can be shown that MO is successfully loaded as a core material.

[0041] In order to confirm that MO-DEX NPs improve the poor hydrophilicity of MO, the contact angles of MO and MO-DEX NPs were measured. Figure 2 .

[0042] Experimental Example 3: Protective effect of MO-DEX NPs of the present invention on hypoxic injury of PC12 cells in vitro

[0043] PC12 cells were cultured at 6×10 3 The cells were inoculated at a density of 100 μmol / well in a 96-well culture dish, and 1640 culture medium (containing 10% fetal bovine serum) was added for 12 hours. Then, different concentrations of MO and MO-DEX NPs solutions (MO-DEX NPs concentration was calculated based on MO equivalent concentration) were added to the culture medium, and PBS was used as a blank control. The culture was continued for 12 hours. After 12 hours, CoCl2 solution was added and continued to be cultured for 24 hours. The final concentration of CoCl2 solution was 300 μmol / L. After 24 hours, CCK-8 solution was prepared to replace the original culture medium. After incubation for 1.5 hours, the absorbance value was measured at a wavelength of 450 nm using an enzyme reader to calculate the cell activity.

[0044] The results show:

[0045] In the MO equivalent concentration range of 1.25-40μg / mL, MO and MO-DEX NPs showed no obvious cytotoxicity, and as the concentration of MO increased to 80μg / mL, the activity of PC12 cells in the MO group decreased significantly, and after being prepared as MO-DEX NPs, its biocompatibility was significantly improved and cell viability was significantly restored. After CoCl2 chemically induced hypoxia treatment, the activity of PC12 cells in the model group decreased significantly (P<0.001), while MO and MO-DEX NPs pretreatment groups both had a certain damage alleviation effect (P<0.05), and at the same MO equivalent concentration, the anti-hypoxia effect of MO-DEX NPs was more significant.

[0046] Experimental Example 4: Anti-hypoxia effect of MO-DEX NPs of the present invention in vivo

[0047] The present invention adopts a low-pressure hypoxia-induced mouse injury model to evaluate the in vivo anti-hypoxic effect of MO-DEX NPs, which is mainly evaluated by measuring the damage to the mouse organs and blood circulation system after hypoxia treatment.

[0048] 1. Drugs and Reagents

[0049] Test drug: MO-DEX NPs of Example 1;

[0050] Positive drug: Salidroside.

[0051] 2. Experimental animals: SPF male Kunming mice aged 6 to 8 weeks and weighing 18-20 g.

[0052] 3. Preparation of serum and sample tissue

[0053] (1) Preparation of serum samples

[0054] The whole blood of the mice was placed in a 4°C refrigerator for 6 hours, and then centrifuged at 4000 r / min for 10 minutes. The supernatant was the serum sample.

[0055] (2) Preparation of tissue homogenate

[0056] Take out the tissues and organs stored at -80℃ and use pre-cooled physiological saline according to the weight of the tissues:

[0057] The cells were homogenized with normal saline at a ratio of 1:9, and then centrifuged at 4000 r / min for 10 min. The supernatant was the tissue sample.

[0058] (3) Determination of biochemical indicators

[0059] The levels of malondialdehyde (MDA) and the energy metabolism index lactate dehydrogenase (LDH) in serum, brain, lung and heart tissue homogenates were determined according to the corresponding kit instructions.

[0060] 4. Experimental methods: The animals were randomly divided into groups, 14 in each group, and kept in two cages. The groups were as follows: normal control group (NC), hypobaric hypoxia injury model group (MC), salidroside positive control group (PC), MO-DEX NPs low-dose group (NPs-L), MO-DEX NPs medium-dose group (NPs-M), MO-DEX NPs high-dose group (NPs-H), and the concentration of MO was calculated by the loading rate of the dose of the NPs-M group and named as the MO control group (MO). The oral dose and treatment method of each group of mice are shown in the following table:

[0061] Table 2. Oral administration of mice in each group

[0062]

[0063] 5. Using the MO-DEX NPs of Example 1, animal experiments were conducted in accordance with the method of Section 4 of Test Example 4. The mice were gavaged continuously for 14 days according to the treatment method of Table 2, and the drugs were gavaged at 8-9 am every day. The weight changes of the mice were recorded every day during the gavage period, and the weight change curve of the mice was drawn. Then, the mice were treated with hypobaric hypoxia according to the method of this Test Example 4.5, that is, after being placed at room temperature for one hour, the hypobaric hypoxia-treated mice were placed in a hypobaric oxygen chamber, and the nitrogen and oxygen airflows were quickly adjusted so that the altitude in the oxygen chamber was uniformly increased to 5000m at a speed of 10m / s and maintained for 48h. The mice in the NC group were placed in a normoxic environment. After the hypoxia treatment, the mice were taken out, blood was taken from the eyeballs, and then the mice were killed by dislocating the neck, and various physiological and biochemical indicators in the mice were measured.

[0064] 6. Effects of MO-DEX NPs on organ indices of hypobaric hypoxia mice

[0065] Animal experiments were conducted using the MO-DEX NPs of Example 1 in the manner of Experimental Example 4.5. After the mice were killed by cervical dislocation, the heart, lung, and brain tissues were immediately removed, the fat was removed, and the remaining blood was washed with pre-cooled sterile saline. The surface moisture of the organs was absorbed with filter paper, and the organs were weighed to calculate the organ index of the mice.

[0066] Organ index = organ weight / mouse body weight × 100%,

[0067] The experimental results are as follows Figure 3 As shown in AC, after low-pressure hypoxia treatment, the brain and lung indexes of mice in the MC group were significantly increased (P<0.001), and the heart index was significantly increased (P<0.01), while the MO and various doses of MO-DEX NPs pretreatment groups were able to significantly reduce the brain, lung, and heart indexes, showing a good anti-hypoxia protective effect.

[0068] 7. Effects of MO-DEX NPs on peripheral blood counts of hypobaric hypoxic mice

[0069] The MO-DEX NPs of Example 1 were used to conduct animal experiments in the manner of Experimental Example 4.5, that is, after collecting whole blood from mice by eye sampling, 20 μL of whole blood was immediately taken out and diluted in a buffer solution containing an anticoagulant, and then a blood routine test was performed using a blood automatic biochemical analyzer to analyze the contents of red blood cells (RBC), hemoglobin (HGB) and mean hemoglobin (MCH) in the whole blood.

[0070] The experimental results are as follows Figure 4 As shown in AC, compared with the NC group, the hemoglobin level of mice in the MC group increased significantly (P<0.05), the average hemoglobin content increased significantly (P<0.01), and although the red blood cell content did not change significantly, it showed a significant upward trend compared with the NC group. The MO and MO-DEXNPs pretreatment groups can significantly improve the damage caused by hypoxia to the hematopoietic system, significantly alleviate the sudden increase in the average hemoglobin level, downregulate hemoglobin expression, and reduce the red blood cell content.

[0071] 8. Effects of MO-DEX NPs on MDA content in serum, brain, lung and heart of hypobaric hypoxic mice

[0072] The MO-DEX NPs of Example 1 were used to conduct animal experiments in the manner of Experimental Example 4.5, and the levels of the antioxidant index MDA in serum and brain, lung, and heart tissue homogenates were measured according to the malondialdehyde (MDA) kit test manual.

[0073] The experimental results are as follows Figure 5 As shown in the data, after hypobaric hypoxia treatment, the MDA content in the serum of mice increased significantly from 7.31±1.36nmol / mL in the NC group to 10.32±0.67nmol / mL in the MC group (P<0.001). Compared with MC, the PC group (8.52±1.41nmol / mL, P<0.01), the MO group (8.14±1.23nmol / mL, P<0.001) and the MO-DEX NPs groups (P<0.001) significantly reduced the lipid peroxidation damage caused by hypoxia. Figure 5 (B) is the result of determination of MDA content in brain tissue. Compared with the NC group (4.03±0.36nmol / mgprot), the MDA content in brain tissue of the MC group increased significantly to 5.43±0.75nmol / mgprot (P<0.001), while the NPs-H group (4.40±0.69nmol / mgprot, P<0.05) showed good anti-hypoxic injury effect on brain tissue. Figure 5(C) shows the MDA content in lung tissue. Compared with the NC group (2.77±0.36nmol / mgprot), the MDA content in the MC group was also significantly increased (3.93±0.73nmol / mgprot, P<0.01), while the PC group (3.09±0.30nmol / mgprot, P<0.05), MO group (3.01±0.64nmol / mgprot, P<0.05), NPs-L group (2.92±0.80nmol / mgprot, P<0.01) and NPs-M group (2.96±0.61nmol / mgprot, P<0.01) had a significant inhibitory effect on the increase of its content. Figure 5 (D) is the MDA content in the heart. The MDA content in the MC group (5.63±0.82nmol / mgprot, P<0.01) was significantly higher than that in the NC group (4.44±0.44nmol / mgprot, P<0.05). The PC, NPs-M, and NPs-H groups were able to effectively alleviate the cardiac oxidative damage caused by hypoxia. In this experiment, it was found that MO-DEX NPs showed better anti-hypoxia efficacy than the MO group, and could exert higher biological activity at the same dose.

[0074] 9. Effects of MO-DEX NPs on LDH activity in serum, brain, lung and heart of hypobaric hypoxic mice

[0075] The MO-DEX NPs of Example 1 were used to conduct animal experiments in the manner of Experimental Example 4.5, and the activity of LDH, an energy metabolism index, in serum and brain and lung tissue homogenates was measured according to the test kit of lactate dehydrogenase (LDH).

[0076] like Figure 6 As shown in (A), after hypobaric hypoxia treatment, compared with the NC group (344.9±44.42U / L),

[0077] The LDH activity in the MC group increased significantly to (429.3±18.75U / L, P<0.001). Compared with the MC group, both the MO and MO-DEXNPs groups showed good efficacy in regulating energy metabolism (P<0.001) and were able to inhibit the increase in LDH activity caused by hypoxia. Figure 6 (B) is the activity of LDH in brain tissue. As can be seen from the figure, low-pressure hypoxia caused the LDH activity in the mouse brain tissue to increase significantly to 44.94±6.56U / gprot (P<0.001), while the NPs-M group and NPs-H group were able to alleviate the abnormal energy metabolism and tissue damage caused by hypoxia. Figure 6(C) and (D) measured the LDH activity in the lung and heart, respectively. The figure shows that the LDH activity in the heart and lung tissues of the MC group mice increased significantly (P<0.05), and MO-DEX NPs were able to alleviate the damage to varying degrees. In addition, at the same dose in the MO and MO-DEX NPs groups, MO-DEX NPs showed stronger anti-hypoxia activity.

[0078] In summary, the present invention has developed a stable mulberry pigment delivery system for preventing or alleviating hypoxic damage. The mulberry pigment glucan nanoparticles prepared by the present invention are uniform in size, have good encapsulation efficiency for mulberry pigment, and can significantly improve the hydrophobicity of mulberry pigment to improve its bioavailability. In vitro experiments have shown that MO-DEX NPs exhibit better biocompatibility and can significantly improve the decreased activity of PC12 cells induced by CoCl2. The in vivo hypobaric hypoxia mouse model has demonstrated the in vivo anti-hypoxic activity of MO-DEX NPs, and MO-DEX NPs exhibit a superior anti-hypoxic effect relative to MO. Therefore, the present invention is different from previous studies on nanoparticle systems, and provides a new strategy for improving the bioavailability of mulberry pigment, and provides a more promising oral drug delivery system for preventing or alleviating hypoxic damage.

Claims

1. Application of mulberry pigment MO in the preparation of anti-hypoxia products.

2. The use according to claim 1, characterized in that: The anti-hypoxia product can prevent or alleviate at least one of the following symptoms: 1) Damage to the heart, lungs, brain and / or hematopoietic system caused by hypoxia 2) Abnormal energy metabolism caused by hypoxia 3) Altitude sickness.

3. The use according to the preceding claim, characterized in that The abnormal energy metabolism is manifested as abnormal increase in lactate dehydrogenase activity in serum, brain, heart and / or lung tissue.

4. A method for preparing nanoparticles, comprising encapsulating MO with dextran DEX to obtain nanoparticles MO-DEX NPs.

5. The method according to the preceding claim, characterized in that The MO-DEX NPs were prepared by an anti-solvent precipitation method.

6. The method according to the preceding claim, characterized in that The method comprises gradually adding a dextran DEX aqueous solution into an ethanol solution of morin.

7. The method according to claim 6, characterized in that: The added DEX is 10-15 times, preferably 12 times, the mass of MO; the formula weight of DEX is 4-6KDa, preferably 5KDa.

8. The method according to claim 6, characterized in that: The volume of the ethanol solution is 15-25 times, preferably 20 times, of the volume of the aqueous solution.

9. Nanoparticles MO-DEX NPs obtained by the method of any preceding claim.

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