Composition for relieving oxidative stress, nano-particles and application of composition and nano-particles

Abstract

The invention belongs to the technical field of disease treatment medicine preparation, and particularly relates to a composition for relieving oxidative stress, nano-particles and application of the composition and the nano-particles. The protopanoxadiol type saponin component (protopanoxadiol PPD or ginsenoside CK) and astragaloside are combined for use, so that a synergistic interaction effect is achieved, and the drug effect of the composition is greater than that of single use of the protopanoxadiol type saponin component (protopanoxadiol PPD or ginsenoside CK) and astragaloside; in addition, the supramolecular nanoparticles prepared by taking the chitosan as a raw material in a self-assembly mode are uniform in particle size and good in stability and dispersity, and oxidative stress of a body can be relieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease treatment drug preparation, and specifically relates to a composition for alleviating oxidative stress, nanoparticles and applications thereof. Background Art

[0002] Oxidative stress refers to the damage of cells or organisms by excessive oxides, which leads to imbalance of oxidation reaction process, resulting in damage of lipids, proteins and DNA, and then cell damage and tissue damage. Reactive oxygen species (ROS) at physiological levels are regarded as "beneficial stress" in organisms. They are characterized by participating in a variety of biochemical reactions at low to moderate levels, such as carboxylation, hydroxylation, peroxidation or regulation of signal transduction pathways, involving multiple biological processes such as nuclear transcription factors, mitogen-activated protein kinases, phosphatidylinositol-3-kinase, and nuclear factor erythroid-related factor 2. However, the increase in ROS levels, whether from endogenous (mitochondria, NADPH oxidase) or exogenous (radiation, certain drugs, food, smoking, chemicals, pollutants), can lead to a harmful oxidative stress state. This state can cause damage to cells and tissues and is closely related to the occurrence and progression of many diseases, including cardiovascular disease, neurodegenerative diseases, cancer and inflammatory diseases. In the occurrence of acute infection, inflammation and drug-induced injury diseases, oxidative stress can serve as an important pathophysiological mechanism, aggravating cell damage, affecting cell function, and thus promoting the progression of the disease. Therefore, regulating oxidative stress may become an important strategy for the prevention and treatment of various diseases.

[0003] Current antioxidant therapeutic strategies include: 1) 2 Remove superoxide radicals before they are generated, or in H 2 O 2 1) Remove hydroxyl radicals or perhydroxyl radicals before they are generated; 2) Supplement dietary antioxidants, such as GSH and Vc; 3) Control the synthesis of antioxidant enzymes, such as activating NRF2, inhibiting NOX enzymes, enhancing mitochondrial antioxidant defense capacity or reducing oxidative stress products to alleviate oxidative stress damage. However, treatment for oxidative damage usually requires comprehensive consideration of the patient's overall condition and disease characteristics, and combining the above strategies to develop a personalized treatment plan.

[0004] Traditional tonic Chinese medicine has the characteristics of multiple pathways, multiple targets, few side effects, and is easy to use for a long time. However, there are currently few systematic research reports on the role of its active pharmaceutical ingredients in regulating oxidative stress damage. Summary of the invention

[0005] The present invention proposes a synergistic enhancement method using key medicinal ingredients of tonic Chinese medicine, combining ginsenoside CK or PPD with astragaloside IV, to reduce the generation of ROS and promote the elimination of ROS, and preparing them into self-assembled nanoparticles for alleviating oxidative stress in the body.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a composition for alleviating oxidative stress, comprising protopanaxadiol saponins and astragaloside IV.

[0008] Preferably, the above-mentioned protopanaxadiol saponins are selected from protopanaxadiol PPD and / or ginsenoside CK.

[0009] More preferably, in the above composition, when the protopanaxadiol saponin is selected from protopanaxadiol PPD, the mass ratio of protopanaxadiol PPD to astragaloside IV is (1-4):(1-4); when the protopanaxadiol saponin is selected from ginsenoside CK, the mass ratio of ginsenoside CK to astragaloside IV is 1:(1-16).

[0010] More preferably, in the above composition, the mass ratio of protopanaxadiol saponins to astragaloside IV is 1:1.

[0011] Another aspect of the present invention provides a composite nanoparticle, which is prepared by subjecting the composition of the present invention to a reverse phase solvent method or a grinding method.

[0012] Preferably, the preparation method of the above-mentioned composite nanoparticles meets one or more of the following conditions: (i) the solvent used in the reverse phase solvent method or the grinding method is selected from one or more combinations of methanol, ethanol, DMF or DMSO; (ii) the pH value of the nanoparticle suspension prepared by the reverse phase solvent method or the grinding method is stable in the range of 6-9.

[0013] In another aspect, the present invention provides a medicine for treating oxidative stress damage diseases or a cosmetic for alleviating oxidative stress, which comprises the composition of the present invention and / or the composite nanoparticles of the present invention.

[0014] Preferably, the dosage forms of the above-mentioned medicines or cosmetics include liposomes, granules, tablets, capsules, injections, pills, oral solutions, tinctures, patches, sprays or creams.

[0015] In another aspect, the present invention provides use of the composition of the present invention and / or the composite nanoparticles of the present invention in the preparation of a drug for treating oxidative stress damage or a cosmetic for alleviating oxidative stress.

[0016] Preferably, the above-mentioned oxidative stress injury diseases include acute kidney injury, acute respiratory distress syndrome, myocarditis or nephritis.

[0017] The beneficial effects of the present invention include:

[0018] (1) In the present invention, the combined use of protopanaxadiol-type saponin components (protopanaxadiol PPD or ginsenoside CK) and astragaloside IV has a synergistic effect, and its efficacy is greater than that of using them alone; in addition, the supramolecular nanoparticles prepared by using them as raw materials through a self-assembly method have uniform particle size and good stability and dispersibility, which can alleviate the body's oxidative stress.

[0019] (2) The composition based on protopanaxadiol saponins and astragaloside IV provided by the present invention and its preparation can effectively alleviate acute respiratory distress syndrome (ARDS) caused by infection, acute kidney injury (AKI) caused by inflammation, and relieve myocarditis and nephritis caused by platinum drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A The results of screening of 26 tonic Chinese herbal extracts for inhibiting ROS activity; C represents blank control, M represents LPS model group; compared with group C, ###P<0.001; compared with group M, ***P<0.001;

[0021] Figure 1B The synergistic inhibitory effect of 5 active Chinese herbal medicine extracts was investigated; C represents blank control, M represents LPS model group; compared with group C, ###P<0.001; compared with group M, ***P<0.001; compared with the two groups, △Δ△P<0.001; △△P<0.01; △P<0.05; n=3; three represents Panax notoginseng, yellow represents Astragalus, human represents Ginseng, north represents Adenophora adenophora, and red represents Rhodiola rosea;

[0022] Figure 2A To investigate the ROS inhibition effect of saponin monomer components;

[0023] Figure 2B To investigate the synergistic index of ginsenoside CK and astragaloside IV in inhibiting ROS;

[0024] Figure 2C To investigate the synergistic index of ginsenoside PPD and astragaloside IV in inhibiting ROS;

[0025] FIG. 2A to FIG. 2C In the table, C represents blank control, M represents LPS group; NAC is the positive control group of N-acetylcysteine; compared with group C, ###P<0.001; compared with group M, ***P<0.001, *P<0.05; n=6;

[0026] Figure 3This is an investigation of the efficacy of ginsenoside CK combined with astragaloside IV AST in relieving AKI in mice; A is the detection of ROS in mouse serum; B is the H&E staining score of mouse kidney tissue; C represents the blank control group, M represents the LPS model group; NAC is the positive control group of N-acetylcysteine; compared with group C, ###P<0.001; compared with group M, **P<0.01;

[0027] ***P<0.001; comparison between the two groups, △△P<0.01; n=6;

[0028] Figure 4 It is a comprehensive investigation of the stability and antioxidant capacity of the composite nanoparticles of saponins in ginseng and Panax notoginseng and astragaloside IV; A is a comprehensive investigation of the average particle size of the composite nanoparticles and the ability to inhibit ROS; B is a comprehensive investigation of the Zeta potential of the composite nanoparticles and the ability to inhibit ROS;

[0029] Figure 5A The effects of different concentration ratios on particle size, distribution uniformity and Zeta potential;

[0030] Figure 5B The effects of different solvents on the particle size, distribution uniformity and Zeta potential of nanoparticles;

[0031] Figure 5C The particle size, distribution uniformity and Zeta potential of nanoparticles under different pH conditions;

[0032] Fig. 6A The effects of different concentration ratios on particle size, distribution uniformity and Zeta potential;

[0033] Figure 6B The effects of different solvents on the particle size, distribution uniformity and Zeta potential of nanoparticles;

[0034] Figure 6C The particle size, distribution uniformity and Zeta potential of nanoparticles under different pH conditions;

[0035] Figure 7 The efficacy of ginsenoside CK and astragaloside IV composite nanoparticles in relieving ARDS in mice; A is the mortality rate of mice within 24 hours; B is the detection of ROS in the lung tissue of mice; compared with the blank group, ###P<0.001; compared with the model group, ***P<0.001, **P<0.01, *P<0.05; n=6;

[0036] Figure 8The efficacy of ginsenoside CK and astragaloside IV composite nanoparticles in alleviating cisplatin-induced myocardial injury in mice; A is the detection of creatine kinase CK in mouse serum; B is the detection of creatine kinase MB isoenzyme (CK-MB) in mouse serum; C is the detection of lactate dehydrogenase (LDH) in mouse serum; compared with the blank group, ###P<0.001; compared with the model group, ***P<0.001, **P<0.01, *P<0.05; compared between the two groups, △Δ△P<0.001, △P<0.05; ns means no statistical difference; n=6;

[0037] Fig. 9 This is an investigation on the efficacy of ginsenoside CK and astragaloside IV composite nanoparticles in alleviating cisplatin-induced renal injury in mice; wherein, A is the detection of creatinine (Cr) in mouse serum; B is the detection of urea nitrogen (BUN) in mouse serum; compared with the blank group, ###P<0.001; compared with the model group, ***P<0.001, **P<0.01, *P<0.05; compared between the two groups, △△P<0.01; ns means no statistical difference; n=6. DETAILED DESCRIPTION

[0038] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0039] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0040] An embodiment of the present invention provides a composition for alleviating oxidative stress, comprising protopanaxadiol saponins and astragaloside IV.

[0041] It should be noted that ginsenoside Compound K (CK) is one of the main metabolically active components of the traditional Chinese medicine ginseng or Panax notoginseng, and is a saponin of the protopanaxadiol (PPD) class, as shown in the structural formula I; ginsenoside CK has multiple biological activities, including anti-tumor, neuroprotective, anti-inflammatory, anti-allergic, anti-aging and other pharmacological effects.

[0042]

[0043] In addition, Astragaloside IV (AST), shown in structural formula II, is a representative active substance in the traditional Chinese medicine Astragalus, which has multiple pharmacological effects such as immunomodulation, ischemic protection, cardioprotection, anti-inflammatory and antiviral, anti-diabetic, anti-tumor and neuroprotection.

[0044]

[0045] It should also be noted that the compatibility combination of drugs can play a synergistic role to improve the therapeutic effect, reduce the probability of drug resistance, and reduce the dosage of a single drug in the combination, thereby reducing toxic and side effects, and has significant advantages in the treatment of complex diseases and chronic diseases. In the present invention, the protopanaxadiol type saponin component (protopanaxadiol PPD or ginsenoside CK) is used in combination with astragaloside IV to have a synergistic effect, and its efficacy is greater than that of using it alone; in addition, the supramolecular nanoparticles prepared by self-assembly using it as a raw material have uniform particle size and good stability and dispersibility, which can relieve the body's oxidative stress.

[0046] In some specific examples, in the above composition, the protopanaxadiol saponin is selected from protopanaxadiol PPD and / or ginsenoside CK.

[0047] In some specific examples, in the above-mentioned composition, when the protopanaxadiol saponin is selected from protopanaxadiol PPD, the mass ratio of protopanaxadiol PPD to astragaloside IV is (1-4): (1-4); for example, 1:2, 2:1, 3:1 or 1:3, etc.; when the protopanaxadiol saponin is selected from ginsenoside CK, the mass ratio of ginsenoside CK to astragaloside IV is 1: (1-16), for example, 1:3, 1:5, 1:7, 1:10, 1:13 or 1:15, etc.

[0048] In some specific examples, in the above composition, the mass ratio of protopanaxadiol saponin to astragaloside IV is 1:1.

[0049] The embodiment of the present invention further provides a composite nanoparticle, which is prepared by subjecting the composition of the present invention to a reverse phase solvent method or a grinding method.

[0050] It should be noted that natural small molecule self-assembled supramolecular nanoparticles have shown great potential in improving drug efficacy due to their unique advantages. Small molecule self-assembled nanoparticles have developed into a promising drug delivery system and have shown good clinical application potential due to their characteristics as pure natural products, simple preparation process, no need for carriers, few adverse reactions, good drug loading capacity, and good pharmacokinetics.

[0051] In some specific examples, the preparation method of the composite nanoparticles satisfies one or more of the following conditions:

[0052] (i) The solvent used in the reverse phase solvent method or the grinding method is selected from one or more combinations of methanol, ethanol, DMF or DMSO; specifically, DMSO or ethanol may be preferred;

[0053] (ii) The pH value of the nanoparticle suspension obtained by the reverse phase solvent method or the grinding method is stable in the range of 6-9, such as 7 or 8.

[0054] The embodiments of the present invention also provide a drug for treating oxidative stress damage diseases or a cosmetic (such as lotion, cream or ointment, etc.) for alleviating oxidative stress, which includes the pharmaceutical composition of the present invention and / or the composite nanoparticles of the present invention.

[0055] In some specific examples, the dosage forms of the above-mentioned medicines or cosmetics include liposomes, granules, tablets, capsules, injections, pills, oral solutions, tinctures, patches, sprays or creams.

[0056] The embodiments of the present invention also provide a use of the pharmaceutical composition of the present invention and / or the composite nanoparticles of the present invention in the preparation of a drug for treating oxidative stress damage diseases or a cosmetic for alleviating oxidative stress.

[0057] In some specific examples, the oxidative stress injury diseases include acute kidney injury (AKI), acute respiratory distress syndrome (ARDS), myocarditis or nephritis.

[0058] It should be noted that acute kidney injury (AKI) includes acute kidney injury caused by inflammation, acute respiratory distress syndrome (ARDS) includes acute respiratory distress syndrome caused by infection, and myocarditis or nephritis includes myocarditis or nephritis caused by platinum chemotherapy.

[0059] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0060] In the following examples, ginsenoside CK (Cat. No. S29921), protopanaxadiol (Cat. No. B21619), astragaloside IV (Cat. No. B20564), and other ginsenoside standards used were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; DCFH-DA reactive oxygen species detection kit (CA1410) was purchased from Beijing Solebold Biotechnology Co., Ltd.; mouse creatine kinase (CK) ELISA kit (JL18284-96T), mouse creatine kinase MB isoenzyme (CK-MB) ELISA kit (JL12422-96T) were purchased from Shanghai Jianglai Biotechnology Co., Ltd.; lactate dehydrogenase (LDH) activity kit (BC0685-100T / 48S) was purchased from Beijing Solebold Technology Co., Ltd. Creatinine kit (C011-2-1) and urea nitrogen (C013-2-1) kit were purchased from Nanjing Jiancheng Bioengineering Institute.

[0061] In the following examples, RAW 264.7 cells are mouse mononuclear macrophage leukemia cells (Cat. No.: CL-0190), purchased from Wuhan Pronocell Life Science Co., Ltd.; Kunming mice and ICR mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Diet management was carried out according to the feeding standards under SPF conditions, and the experimental operations were in accordance with the requirements of the "Guide to the Use and Care of Laboratory Animals and Animal Experiment Management Regulations".

[0062] 1. Traditional Chinese Medicine Synergistic Trial

[0063] In the following example, the preparation method of the ethanol extract of traditional Chinese medicine is as follows: the medicinal material is crushed by a powder grinder, passed through a 100-mesh sieve, and a 70% ethanol solution is added at a ratio of 10:1 (V / W). Ultrasonic extraction is performed at 200w for 1h, and centrifugation is performed at 4000rpm for 10min. The supernatant of the extract is placed in a 40°C oven to evaporate the solvent to obtain a dry powder of the medicinal material extract, which is redissolved in DMSO to prepare a 1g / mL stock solution, which is sealed and stored at -20°C.

[0064] Example 1 Investigation of the synergistic effect of tonic Chinese herbal extracts in inhibiting ROS

[0065] The protective effects of Chinese herbal ethanol extracts and their combinations on improving oxidative stress damage were evaluated by LPS-induced acute cell damage, as follows:

[0066] When the RAW 264.7 cells were grown to 80% density, 5 × 10 4The cell suspension was inoculated into a 96-well culture plate containing DMEM high-glucose medium (Punosai, PM150210) and cultured overnight; LPS (1 μg / ml) was used to induce acute oxidative stress damage in the cells, and the corresponding drugs 1 mg / mL (different Chinese medicine ethanol extracts or combinations) were given for intervention; 12 hours after administration, the cell culture supernatant was discarded, and the ROS content in the cells was determined by the DCFH-DA method, specifically including: adding 5 μM DCFH-DA probe (Beijing Solebow Biotechnology Co., Ltd., CA1410) to the well plate and incubating at 37°C in the dark for 30 minutes; after sufficient washing with PBS, the fluorescence value was determined using a multifunctional microplate reader under the conditions of excitation wavelength 488 nm and emission wavelength 525 nm, and statistical analysis was performed.

[0067] The test results of different Chinese herbal medicine ethanol extracts are as follows Figure 1A As shown, the results showed that compared with the blank group (C), the RAW 264.7 cells in the model group (M) produced a large amount of ROS under the induction of LPS, while the positive control 10μM N-acetylcysteine ​​group (NAC) could effectively clear some ROS; among the 26 Chinese herbal medicine ethanol extracts (1 mg / mL), only Astragalus, Ginseng, Adenophora, Panax notoginseng and Rhodiola rosea had significant scavenging effects on ROS (p<0.05, p<0.01).

[0068] In addition, the above five Chinese herbal medicine ethanol extracts were combined in a mass ratio of 1:1 to investigate whether they have a synergistic effect. Figure 1B As shown, the results showed that compared with the inhibitory effect of 1 mg / mL of the extract alone, the inhibitory effect of most combinations was improved after the two extracts were used in combination at 0.5 mg / mL (p<0.05, p<0.01); among them, the synergistic effect of the combination of Astragalus and ginseng extract was the most significant (p<0.001).

[0069] 2. Synergistic Test of Chinese Herbal Medicine Extracts

[0070] Example 2 Screening of the active ingredients of Astragalus membranaceus for inhibiting ROS and investigation of the synergistic index

[0071] The main active ingredients in ginseng, Panax notoginseng and Astragalus are all saponins. In order to clarify the antagonistic ROS effect of the key active ingredients in the extracts, the same method as in Example 1 was adopted to select representative saponin components (5 μM) for evaluation of ROS inhibitory activity, including protopanaxadiol (PPD type) saponins: ginsenosides Rb1, Rd, Rg3, F2, Rh2, CK and PPD; protopanaxatriol (PPT type) saponins: Re, Rg2, Rg1, Rh1, F1, PPT; Ro type ginsenoside: Ro; and astragaloside IV (AST) (all purchased from Shanghai Yuanye Biotechnology Co., Ltd.).

[0072] Test results such as Figure 2A As shown in the results, ginsenoside CK and PPD in the protopanaxadiol type saponins have significant scavenging efficiency for ROS (p<0.001), while ginsenoside Rg1 and PPT in the protopanaxadiol type have weak scavenging activity (p<0.05). In addition, astragaloside IV (AST) also has strong inhibitory activity (p<0.001). Based on the above results, ginsenoside CK, the metabolic active product of protopanaxadiol type saponins, and its aglycone PPD were finally selected as the key active ingredients for antagonizing ROS.

[0073] Then, astragaloside IV (AST) was further combined to conduct a subsequent synergistic index (CI value) investigation, specifically including: 5μM AST was combined with 0.156μM to 5μM ginsenoside CK to investigate the ROS scavenging effect using the same method as above. The synergistic index (CI value) of the two-drug combination against ROS was analyzed by CompuSyn software. (Among them, CI<1 represents synergistic effect, CI=1 represents additive effect, and CI>1 represents antagonistic effect).

[0074] Test results such as Figure 2B As shown in Figure 2, under certain concentration compatibility conditions, the combination of ginsenoside CK and AST has a synergistic effect, and the compatibility ratio is CK:AST = 1:1 to 1:16; among them, the activity is best under the ratio of CK:AST = 1:1. Figure 2C As shown, the combination of 0.312-5 μM ginsenoside PPD and 5 μM AST also has a synergistic effect, and the compatibility ratio of PPD:AST = 1:1, 1:2, 1:4, 2:1, 4:1 has a good synergistic effect, among which 1:1 has the best effect.

[0075] Example 3 Effect of Ginsenoside CK Combined with Astragaloside IV on Alleviating LPS-Induced AKI in Mice

[0076] Male Kunming mice were injected intraperitoneally with LPS (10 mg / kg) to induce acute kidney injury (AKI). Specifically, 36 mice were randomly divided into 6 groups (6 mice in each group): blank control group (C), LPS model group (M), positive control group (N-acetylcysteine, NAC, 100 mg / kg), ginsenoside CK group (CK, 20 mg / kg), astragaloside IV group (AST, 20 mg / kg) and ginsenoside CK and astragaloside IV combined administration group (CK 10 mg / kg + AST 10 mg / kg); except for the control group, all groups were given intraperitoneal drug intervention treatment immediately after modeling; 24 hours after infection, the ROS level in mouse serum was determined by the reactive oxygen species kit (Beijing Solebow Biotechnology Co., Ltd., CA1410); at the same time, the kidney tissues were fixed with formalin for subsequent H&E staining analysis.

[0077] Test results such as Figure 3 The results of the determination of ROS content in the peripheral blood of mice are shown in Figure 3 As shown in A, the results showed that 100 mg / kg NAC or different dosing treatments significantly reversed the level of ROS in peripheral blood, and the effect of the combined administration group was significantly better than that of the single administration group (p<0.01). H&E staining showed that LPS stimulation led to enlarged glomeruli and vacuolar degeneration of tubular epithelial cells, swelling, deformation, shedding, inflammatory cell infiltration and large aggregation in damaged tissues, and glomeruli showed swelling, atrophy and other phenomena. Tissue damage was scored according to the blind evaluation method (0, no damage; 1, <25%; 2, 25-50%; 3, 51-75%; 4, >75%). The results are shown in Figure 3 As shown in B, both CK and AST alone can significantly alleviate the above pathological characteristics (p<0.01), but the improvement effect of the combined administration group (CK+AST) is more obvious (p<0.001), and there is a significant difference compared with the single use group (p<0.01).

[0078] Example 4 Investigation of the stability and antioxidant capacity of ginsenoside and astragaloside IV composite nanoparticles

[0079] Based on the fact that the combination of some saponin components and astragaloside IV in Example 2 can produce a synergistic effect, ginsenosides and astragaloside IV were self-assembled into composite nanoparticles by a reverse solvent precipitation method, and their stability and antioxidant capacity were comprehensively evaluated; specifically, astragaloside IV (10 mg / mL) and saponin components (10 mg / mL) were dissolved in DMSO, and then dropped into water under 200w ultrasonic conditions to prepare a supramolecular true solution without adjusting the pH value. After removing the solvent by dialysis (8 hours), the particle size and Zeta potential value were determined by the DLS method; at the same time, based on the method of Example 1, the anti-ROS ability at the cellular level was detected.

[0080] Test results such as Figure 4 As shown in the data, among the 14 ginsenoside components, only ginsenoside CK and ginsenoside diol PPD self-assembled with astragaloside AST to form stable nanoparticles (Zeta potential value <-20) and had good antioxidant capacity.

[0081] Example 5 Preparation of Ginsenoside and Astragaloside I Composite Nanoparticles

[0082] Using the anti-solvent precipitation method of Example 4, ginsenoside CK or PPD was mixed with astragaloside IV in different ratios, or dissolved in different organic solvents, or dropped into aqueous solutions of different pH values ​​under ultrasonic conditions to investigate its effect on the formation of nano-supramolecular.

[0083] The ratio of astragaloside AST and ginsenoside CK is as follows:

[0084] First, the particle size and Zeta position of the composite nanoparticles formed by different ratios of astragaloside AST and ginsenoside CK are shown in Figure 2. Figure 5A As shown, the results showed that when the optimal ratio of astragaloside IV to ginsenoside CK was 10:10 (i.e. 1:1), the composite nanoparticles formed had the smallest particle size (174.5nm) and the most stable Zeta point (-27.6mV).

[0085] Secondly, the particle size and Zeta potential of the composite nanoparticles prepared by different organic solvents such as dimethyl sulfoxide (DMSO), methanol (MeOH), ethanol (EtOH), and dimethylformamide (DMF) are shown in Figure 2. Figure 5B As shown, the results show that different organic solvents dimethyl sulfoxide (DMSO), methanol (MeOH), ethanol (EtOH), dimethylformamide (DMF) can be used to prepare composite nanoparticles. Based on the particle size and Zeta potential value, DMSO solvent is preferred.

[0086] Third, the particle size and Zeta potential of the composite nanoparticles prepared at different solution pH values ​​are shown in Figure 5C As shown, the results showed that the Zeta potential of the nanoparticles was relatively stable in the neutral to alkaline pH range (pH 7-11), with pH 7 being preferred.

[0087] The ratio of astragaloside IV and protoginsenoside PPD is as follows:

[0088] First, the particle size and Zeta position of the composite nanoparticles formed by different ratios of astragaloside AST and ginsenoside PPD are shown in Figure 2. Fig. 6AAs shown, the results showed that when the optimal ratio of astragaloside IV to protoginsenoside PPD was 10:10 (i.e. 1:1), the nanoparticles formed had the smallest particle size (189.3nm) and the most stable Zeta point (-25.8mV).

[0089] Secondly, the particle size and Zeta potential of the composite nanoparticles prepared by different organic solvents such as dimethyl sulfoxide (DMSO), methanol (MeOH), ethanol (EtOH), and dimethylformamide (DMF) are shown in Figure 2. Figure 6B As shown, the results show that organic solvents dimethyl sulfoxide (DMSO), methanol (MeOH), ethanol (EtOH), and dimethylformamide (DMF) can be used to prepare composite nanoparticles. Based on the particle size and Zeta potential value, dimethyl sulfoxide or ethanol is preferred.

[0090] Third, the particle size and Zeta potential of the composite nanoparticles prepared at different solution pH values ​​are shown in Figure 6C As shown, the results show that the Zeta potential of nanoparticles is relatively stable in the neutral to alkaline pH range (pH 7-11), and pH 7 is also preferred.

[0091] 3. Animal Testing

[0092] In order to investigate the effect of the composite nanoparticles at the animal level, the composite nanoparticles were prepared according to Example 4, and the above-mentioned optimal conditions were selected: astragaloside IV and ginsenoside CK were preferably used for preparation, with a ratio of 10:10, DMSO was used as the solvent, and pH = 7; specifically, astragaloside IV (10 mg / mL) and ginsenoside CK (10 mg / mL) were dissolved in DMSO, and then dropped into water under ultrasonic conditions to prepare a supramolecular true solution; after the solvent was removed by dialysis (8 hours), the suspension was concentrated to obtain a composite nanoparticle suspension of astragaloside IV (62.5 μg / mL) and saponin component (62.5 μg / mL).

[0093] Example 6: Efficacy of Ginsenoside CK and Astragaloside I Composite Nanoparticles in Alleviating ARDS in Mice

[0094] Thirty SPF male Kunming mice (20-25 g / mouse) were randomly divided into five groups, namely, blank group, model group, levofloxacin positive drug group (Lev, 60 mg / kg), high-dose group (0.5 mg / kgAST+0.5 mg / kg CK) and low-dose group (0.1 mg / kgAST+0.1 mg / kg CK) of AST+CK composite nanoparticle suspension. The mice were infected with Pseudomonas aeruginosa PA14 (Pseudomonas aeruginosa PA-14, provided by Professor Bai Fang's laboratory of Nankai University) to construct an acute lung injury model. Except for the blank group, the mice in the other groups were anesthetized with 4% chloral hydrate, and 10 μL of the above bacterial suspension (5×10 7 / 20μL) induced acute lung infection and ARDS in mice; drug intervention was performed by tail vein injection at the same time as modeling, and the death of mice within 24 hours was recorded. After 24 hours, lung tissues of mice in each group were embedded to prepare frozen sections, and ROS staining analysis was performed on tissue sections using DCFH-DA probe.

[0095] The results are as follows Figure 7 As shown in the results, the mortality rate of the model group (M) was significantly increased compared with the blank group (C), and different doses of AST+CK composite nanoparticles could improve the survival rate of mice ( Figure 7 A); In addition, the DCFH-DA staining results of lung tissue ROS showed that compared with the blank group (C), the ROS in the model group (M) increased significantly, and the positive drug group (Lev) and the high and low AST+CK composite nanoparticle groups could significantly reduce the ROS in the lung tissue of mice with acute lung injury ( Figure 7 B)(p<0.05,p<0.001).

[0096] The above data show that the composite nanoparticles have a good ROS scavenging effect and can reduce the mortality rate of ARDS mice.

[0097] Example 7 Ginsenoside CK and Astragaloside I Composite Nanoparticles Alleviate Cisplatin-Induced Oxidative Stress Damage

[0098] In order to investigate the protective effect of ginsenoside CK and astragaloside IV composite nanoparticles on drug-induced oxidative stress injury, a cisplatin-induced myocardial injury model was constructed; specifically, 6-8 week old ICR mice were randomly divided into 6 groups, blank control group (C), cisplatin-induced model group (M), N-acetylcysteine ​​control group (NAC, 100 mg / kg), CK (10 mg / kg), AST (10 mg / kg), and CK (4 mg / kg) + AST (4 mg / kg) composite nanoparticle administration group, with 6 mice in each group; 5 mg / kg of cisplatin was intraperitoneally injected into mice, once every other day, for a total of 7 times, to induce subacute myocardial injury; the composite nanoparticle administration group was injected with the drug via tail vein starting from the 7th day, the blank group and the model group were given the same volume of normal saline in the same way, and the positive drug group was given 100 mg / kg NAC; 28 days later, blood samples were collected to detect the important biochemical indicators of myocardial injury, serum creatine kinase (CK), mouse creatine kinase (CK-MB) and lactate dehydrogenase (LDH) activity. At the same time, serum creatinine (CRE) and urea nitrogen (BUN), two kidney injury indicators, were detected.

[0099] Serum creatine kinase (CK), mouse creatine kinase (CK-MB) and lactate dehydrogenase (LDH) activity test results are as follows Figure 8 As shown in the figure, the results showed that compared with the blank group, the CK, CK-MB and LDH activity indicators of the model group were significantly increased, while the NAC positive group only improved the CK indicator (p<0.05); in contrast, in the separate nanoparticle groups of ginsenoside CK and astragaloside IV, only 10 mg / kg ginsenoside CK improved the CK-MB indicator (p<0.01), while other indicators did not show significant changes; and the ginsenoside CK and astragaloside IV composite nanoparticle group could effectively prevent the increase of the above indicators (p<0.001), and its effect was significantly better than that of the single use group (p<0.05, p<0.001); indicating that the combination of ginsenoside CK and astragaloside IV can better improve cisplatin-induced myocardial oxidative stress injury.

[0100] Similarly, the test results of serum creatinine (CRE) and urea nitrogen (BUN), two indicators of kidney damage, are as follows Fig. 9As shown in the results, the CRE and BUN indexes in the cisplatin model group increased significantly, and the NAC positive group had a correction after intervention (p<0.01); in the single nanoparticle groups of ginsenoside CK and astragaloside IV, only the BUN index changed significantly (p<0.05, p<0.01), while the CRE index did not change significantly; on the contrary, the composite nanoparticle administration group had a significant correction of both CRE and BUN (p<0.001); and the effect of the composite nanoparticle administration group was significantly better than that of the single use group (p<0.01). The above results indicate that ginsenoside CK and astragaloside IV composite nanoparticles can improve cisplatin-induced renal injury and have a synergistic effect.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A composition for alleviating oxidative stress, characterized in that: Including protopanaxadiol saponins and astragaloside IV.

2. The composition according to claim 1, characterized in that The protopanaxadiol saponin is selected from protopanaxadiol PPD and / or ginsenoside CK.

3. The composition according to claim 2, characterized in that When the protopanaxadiol saponin is selected from protopanaxadiol PPD, the mass ratio of protopanaxadiol PPD to astragaloside IV is (1-4): (1-4)； When the protopanaxadiol saponins are selected from ginsenoside CK, the mass ratio of ginsenoside CK to astragaloside IV is 1:(1-16).

4. The composition according to claim 1 or 2, characterized in that The mass ratio of protopanaxadiol saponins to astragaloside IV is 1:

1.

5. Composite nanoparticles, characterized in that: The invention is prepared by subjecting the composition according to any one of claims 1 to 4 to a reverse phase solvent method or a grinding method.

6. The composite nanoparticle according to claim 5, characterized in that The preparation method of the composite nanoparticles meets one or more of the following conditions: (i) the solvent used in the reverse phase solvent method or the grinding method is selected from one or more combinations of methanol, ethanol, DMF or DMSO; (ii) The pH value of the nanoparticle suspension obtained by the reverse phase solvent method or the grinding method is stable in the range of 6-9.

7. A drug for treating oxidative stress damage or a cosmetic for alleviating oxidative stress, characterized in that: The method comprises the composition according to any one of claims 1 to 4 and / or the composite nanoparticles according to any one of claims 5 to 7.

8. The medicine or cosmetic according to claim 7, characterized in that: The dosage forms of drugs or cosmetics include liposomes, granules, tablets, capsules, injections, pills, oral solutions, tinctures, patches, sprays or creams.

9. Use of the composition according to any one of claims 1 to 4 and / or the composite nanoparticles according to any one of claims 5 to 6 in the preparation of a medicament for treating oxidative stress damage diseases or a cosmetic for alleviating oxidative stress.

10. The use according to claim 9, characterized in that: Diseases resulting from oxidative stress damage include acute kidney injury, acute respiratory distress syndrome, myocarditis, or nephritis.

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