A composition for inhibiting oxidative stress damage and its application

Abstract

The present invention discloses a composition for inhibiting oxidative stress injury and its application, which relates to the technical field of biomedicine. The composition includes ginsenoside CK and biphenol compounds; the biphenol compounds are magnolol or honokiol. The present invention finds through research that the combined use of ginsenoside CK and biphenol compounds (magnolol or honokiol) has a synergistic effect, and its medicinal effect is greater than that of using them alone, and has a significant improvement effect on oxidative stress and related diseases. The composition provided by the present invention can significantly reduce the production of ROS induced by LPS, reduce the release of NO, and effectively relieve inflammation and pain caused by ROS induced by acute mechanical injury. Moreover, the composition of the present invention has a compatibility and synergistic effect, which is significantly better than using drugs alone.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a composition for inhibiting oxidative stress damage and its applications. Background Art

[0002] Oxidative stress occurs when there is an imbalance between the formation and regulation of reactive oxygen species (ROS) in cells and tissues, leading to a large accumulation of ROS. This imbalance can cause damage to important biomolecules and cells, potentially harming the entire organism. Research shows that some diseases closely related to oxidative stress include: cardiovascular diseases such as atherosclerosis, high cholesterol, stroke, heart failure, and hypertension; cancer; Parkinson's disease; Alzheimer's disease; diabetes; pain; multiple sclerosis; kidney diseases; rheumatoid arthritis; sepsis; respiratory distress syndrome; and metabolic disorders such as mitochondrial diseases, lipid metabolism disorders, and DNA repair deficiency disorders. Other chronic diseases include chronic obstructive pulmonary disease, chronic bronchitis, chronic asthma, and chronic kidney disease induced by inflammation.

[0003] Ginsenoside Compound K is the main in vivo metabolic active ingredient of the traditional Chinese medicine ginseng, and has a variety of biological activities, including anti-tumor, anti-aging, anti-inflammatory, liver-protecting, and anti-allergic pharmacological effects, etc. Chinese patents have also disclosed the applications of ginsenoside Compound K in the fields of anti-tumor (CN113527399B), pain relief (CN111558019A), anti-inflammatory (CN104622928A), liver protection (CN115645423A), skin whitening (CN114948782A), wound repair (CN114404512A), etc.

[0004] Magnolol and Honokiol are the representative active substances in the traditional Chinese medicine Magnolia officinalis. Magnolol and Honokiol have obvious pharmacological activities on the digestive, nervous, cardiovascular, and respiratory systems, etc. Secondly, they also have relatively obvious effects on glucose and lipid metabolism. In addition, they also have the effects of anti-inflammatory, analgesic, anti-pathogenic microorganism, antibacterial, anti-tumor, antioxidant, etc. Chinese patents have also disclosed the applications of Honokiol in antioxidant (CN110314116B), pain relief (CN113813325B), anti-cancer (CN115215771A), antibacterial (CN114869885A), digestion promotion (CN109521122B), and the applications of Magnolol in antioxidant (CN109589409A), pain relief (CN110075261A), bactericidal (CN111296439B), anti-cancer (CN113952351B), anti-viral (CN114907228A), etc. in related fields.

[0005] The use of drug combination can exert the synergistic effect of drugs to improve the curative effect, reduce the occurrence probability of drug resistance, and reduce the dosage of individual drugs in the composition, thereby reducing the toxic and side reactions. It has great advantages especially in the treatment of chronic diseases with complex pathogenesis regulated by multiple genes. At present, there is no report on the use of the drug composition of ginsenoside CK and magnolol / honokiol for treating inflammation or pain caused by oxidative stress injury, nor has there been a drug preparation using the above drug composition for treating inflammatory diseases or acute soft tissue injuries. Summary of the Invention

[0006] The object of the present invention is to provide a composition for inhibiting oxidative stress injury and its application to solve the problems existing in the above prior art. The composition provided by the present invention has a significant improvement effect on oxidative stress and related diseases.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a composition for inhibiting oxidative stress injury, comprising ginsenoside CK and biphenol compounds;

[0009] The biphenol compound is magnolol or honokiol.

[0010] Further, the molar ratio of ginsenoside CK to the biphenol compound is 1:(0.1 - 100).

[0011] Preferably, the molar ratio of ginsenoside CK to the biphenol compound is 1:1.

[0012] The present invention also provides the application of the above composition in the preparation of a drug for treating diseases caused by oxidative stress injury.

[0013] Further, the diseases include inflammation or pain caused by oxidative stress injury.

[0014] The present invention also provides a drug for treating diseases caused by oxidative stress injury, comprising the above composition.

[0015] Further, the drug further comprises a pharmaceutically acceptable excipient.

[0016] Further, the dosage form of the drug includes granules, tablets, capsules, injections, pills, oral liquids, tinctures, patches, sprays or creams.

[0017] The present invention discloses the following technical effects:

[0018] The present invention has found through research that the combined use of ginsenoside CK and biphenol compounds (magnolol or honokiol) has a synergistic effect, and its pharmacodynamic effect is greater than that of using them alone, and it has a significant improvement effect on oxidative stress and related diseases. The composition provided by the present invention can significantly reduce the production of ROS induced by LPS, reduce the release of NO, and effectively relieve inflammation and pain caused by acute mechanical injury. Moreover, the composition of the present invention has a synergistic effect in compatibility, which is significantly better than using the drugs alone. Brief Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0020] Figure 1 Effect of ginsenoside CK on inhibiting the production of ROS in LPS-induced RAW264.7 cells;

[0021] Figure 2 Effect of magnolol and honokiol on inhibiting the production of ROS in LPS-induced RAW264.7 cells;

[0022] Figure 3 Investigation results of the combination index (CI) of the combination of ginsenoside CK and magnolol or honokiol in scavenging ROS; among them, A is the drug combination of ginsenoside and magnolol, and the data on each scatter data label in A are the concentrations of magnolol, and the data unit is μM; B is the drug combination of ginsenoside and honokiol, and the data on each scatter data label in B are the concentrations of honokiol, and the data unit is μM;

[0023] Figure 4 Result of the combination of ginsenoside CK and magnolol in synergistically inhibiting the production of NO in LPS-induced RAW264.7 cells;

[0024] Figure 5 Effect of the drug combination of ginsenoside CK and honokiol on improving pain in rats with soft tissue injury;

[0025] Figure 6 Effect of the drug combination of ginsenoside CK and honokiol on substance P in the plasma of rats with soft tissue injury;

[0026] Figure 7 Effect of the drug combination of ginsenoside CK and honokiol on bradykinin in the plasma of rats with soft tissue injury;

[0027] Figure 8 Effect of the combination of ginsenoside CK and honokiol on prostaglandin E2 in the plasma of rats with soft tissue injury Detailed implementation mode

[0028] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0029] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0031] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation modes of the present invention specification, which are obvious to those skilled in the art. Other implementation modes obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0032] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0033] Term description:

[0034] The ginsenoside CK referred to in the present invention has the following structural formula:

[0035]

[0036] The magnolol referred to in the present invention has the following structural formula:

[0037]

[0038] The honokiol referred to in the present invention has the following structural formula:

[0039]

[0040] In the following examples, ginsenoside CK, honokiol, and magnolol used in the experiments were purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with the product numbers S29921-1g, S31396-1g, and S31381-1g, respectively; RAW 264.7 cells, which are mouse monocyte macrophage leukemia cells, were purchased from Wuhan Punosai Life Science Co., Ltd., with the product number CL-0190; SD rats (180 - 220 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The SD rats were under standard pathogen-free conditions and their diet was managed according to the feeding standards. All experimental operations complied with the requirements of the "Guidelines for the Use and Maintenance of Laboratory Animals and the Regulations for Animal Experiment Management".

[0041] Example 1

[0042] To investigate the protective effects of ginsenoside CK, honokiol, and magnolol against oxidative stress injury at the cellular level, RAW 264.7 cells were used to induce the production of ROS by lipopolysaccharide (LPS). The release of ROS in the cells was detected using a ROS detection kit, and the inhibitory effect of combined drug administration on LPS-induced cellular oxidative stress was investigated. Specifically, RAW 264.7 cells were seeded in 96-well plates and cultured overnight. In the experiment, LPS (1 μg / mL) was used to establish a model, and at the same time, serial concentrations of ginsenoside CK, honokiol, and magnolol, as well as combinations of the two types of drugs, were administered. After 8 hours of treatment, 5 μmol of DCFH-DA probe (Reactive Oxygen Species Assay Kit, Beijing Solarbio Science & Technology Co., Ltd., CA1410) was loaded into the cells, and incubation was continued in the dark for 30 minutes. After washing three times with pre-cooled PBS, a multifunctional microplate reader was used to measure the change in fluorescence value (λEx: 488 nm, λEm: 525 nm). The combination index (CI value) after the combined intervention of the two types of drugs was analyzed using CompuSyn software. Among them, <1 represents a synergistic effect, =1 represents an additive effect, and >1 represents an antagonistic effect.

[0043] For the drug combination described in this example, the administration concentrations of single-dose ginsenoside CK were 0.05 μM, 0.5 μM, and 5 μM; the administration concentrations of single-dose honokiol or magnolol were 0.05 μM, 0.5 μM, and 5 μM. The drug concentrations of ginsenoside CK and magnolol in the compatibility combination group were 0.025 μM, 0.25 μM, and 2.5 μM, respectively.

[0044] The results were as Figure 1-3 shown. In RAW 264.7 cells, different concentrations of ginsenoside CK ( Figure 1 ) honokiol, and magnolol ( Figure 2)Both can significantly reduce the generation of ROS induced by LPS, indicating that ginsenoside CK, magnolol or honokiol can improve oxidative stress injury. After the combined use of ginsenoside CK and magnolol or honokiol, as Figure 3 shown ( Figure 3 in which A is the CI index of ginsenoside and honokiol, Figure 3 and B is the CI index of ginsenoside and magnolol in ### ), under some concentration compatibility conditions, the combined use of the two groups of drugs has a synergistic inhibitory effect on LPS-induced ROS, and the synergistic ratio basically remains inhibitory. Specifically, the compatibility ratio of 0.25 μM ginsenoside CK combined with magnolol or honokiol with synergistic effect is 1:0.1 - 10; the compatibility ratio of 0.025 μM ginsenoside CK combined with magnolol or honokiol with synergistic effect is 1:1 - 100. The results show that medium-concentration and low-concentration ginsenoside CK and magnolol or honokiol have certain synergistic effects in a relatively wide concentration ratio range. Among them,

[0045] Example 2

[0046] To investigate the inhibitory effect of the synergistic compatibility of ginsenoside CK and honokiol on the related inflammation caused by oxidative stress, the method of Example 1 was also referred to. An inflammation model was constructed by inducing RAW 264.7 cells with LPS (10 μg / mL). At the same time, different concentrations of ginsenoside CK, magnolol and their combined formulations were given respectively. After 8 hours of treatment, a nitric oxide content detection kit (Beyotime Biotechnology Co., Ltd., S0021S) was used to detect the generated nitric oxide (NO).

[0047] The results are as Figure 4 shown. A large amount of inflammatory substance nitric oxide is released in cells under LPS stimulation. When 5 μM ginsenoside CK and 5 μM magnolol are used alone, they can reduce the production of nitric oxide in cells, while at a low dose (0.5 μM), neither of them has an obvious inhibitory effect. In comparison, in the combined use group of ginsenoside CK and magnolol, whether it is 2.5 μM ginsenoside CK combined with 0.25 μM or 2.5 μM magnolol, or the combination of 0.25 μM ginsenoside and 2.5 μM magnolol, it can significantly reduce the release of the inflammatory substance nitric oxide compared with the single use. It can be seen that the pharmaceutical composition has a synergistic effect in improving the inflammation caused by oxidative stress injury. Among them, ### compared with the blank group, P < 0.001; * compared with the lipopolysaccharide group, P < 0.05; ** compared with the lipopolysaccharide group, P < 0.01; *** compared with lipopolysaccharide, P < 0.001.

[0048] Example 3

[0049] To investigate the protective effect of the combination of ginsenoside CK and honokiol on oxidative stress injury at the animal level, a rat soft tissue injury model was constructed, and a comprehensive evaluation was carried out on the improvement of pain and inflammatory indexes. Specifically, 54 male SD rats weighing 200 - 220 g were randomly divided into 9 groups: blank control group, model group, ibuprofen positive control group (30 mg / kg), low-dose ginsenoside CK group (12.5 mg / kg), high-dose ginsenoside CK group (25 mg / kg), low-dose honokiol group (12.5 mg / kg), high-dose honokiol group (25 mg / kg), low-dose combination of ginsenoside CK and honokiol group (6.25 + 6.25 mg / kg), and high-dose combination of ginsenoside CK and honokiol group (12.5 + 12.5 mg / kg), with 6 rats in each group. Intraperitoneal injection was used for drug administration. The blank control group and the model group were given the same volume of physiological saline in the same way.

[0050] The rats were anesthetized with 10% chloral hydrate at a dose of 3 mg / kg body weight. All the hair on the right buttock and the posterior side of the thigh of the anesthetized rats was shaved off with a razor, and then the fine hair was removed with 8% sodium sulfide. After hair removal, except for the blank control group, the right lower limbs of all rats were placed flat and fixed on the flat plate of the acute soft tissue injury modeling device. The free fall method was selected for modeling. The hitting site was the outer side of the middle of the right calf. A 100 g weight was freely dropped from a height of 100 cm and continuously hit 5 times. After hitting, swelling, cyanosis, and ecchymosis were observed with the naked eye, but there was no skin breakage, unilateral hindlimb lameness, but no fracture or dislocation signs, and no other abnormal conditions occurred, indicating that the modeling was successful. The successfully modeled rats were immediately given drugs once according to the above grouping after modeling, and then given drugs again 24 hours later. One hour after the drug administration was completed, the pain values of the animals in each group were measured using a pressure application measurement system (Ugo Basile, Italy, 38500PAM). Finally, the animal plasma was collected from the abdominal aorta to measure the related indexes of substance P (SP), bradykinin (BK), and prostaglandin E2 (PGE2). The rat substance P (SP) ELISA kit, product number: MM-0444R1, was purchased from Jiangsu Enzyme Immunoassay Industry Co., Ltd.; the rat bradykinin (BK) ELISA kit, product number: JL21045, was purchased from Shanghai Jianglai Biotechnology Co., Ltd.; the rat prostaglandin E2 (PGE2) ELISA kit, product number: MM-0068R1, was purchased from Jiangsu Enzyme Immunoassay Industry Co., Ltd. The measurement methods were all carried out according to the kit instructions.

[0051] The results are as Figure 5As shown, compared with the blank group, the pain pressure perception value of the model group was significantly reduced, indicating successful model construction. Compared with the model group, the positive drug ibuprofen could significantly relieve the pain of rats. Similarly, ginsenoside CK and honokiol could also improve the pain symptoms in a concentration gradient manner. Moreover, the combined use of ginsenoside CK and honokiol had better effects than the single administration group. In addition, the determination of pain markers, substance P ( Figure 6 ) and bradykinin ( Figure 7 ) in the plasma of rats also showed that the combined drug group had significantly better pain relief effect than the single use group.

[0052] The detection results of related prostaglandin indexes for the inflammation caused by soft tissue injury are as Figure 8 shown. In the model group, the prostaglandin level was significantly increased. After drug intervention, high-dose ginsenoside CK (25 mg / kg) and high- and low-dose honokiol (25 mg / kg, 12.5 mg / kg), as well as the combined use group of ginsenoside CK and honokiol (12.5 mg / kg + 12.5 mg / kg; 6.25 mg / kg + 6.25 mg / kg) could effectively inhibit the production of prostaglandins; and the combined use group of ginsenoside CK and honokiol (12.5 mg / kg + 12.5 mg / kg) had significantly better effects than the single use group of honokiol (25 mg / kg). The above results indicate that the drug combination also has a synergistic effect in improving the diseases caused by oxidative stress injury at the animal level. Among them, ### Compared with the blank group, P < 0.001; * compared with the model group, P < 0.05; ** compared with the model group, P < 0.01; *** compared with the model group, P < 0.001.

[0053] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A composition for inhibiting oxidative stress damage, characterized in that, It includes ginsenoside CK and biphenol compounds; The biphenol compounds are magnolol or honokiol; The concentration of ginsenoside CK is 0.025 - 0.25 μM, and the concentration of the biphenol compounds is 0.025 - 2.5 μM.

2. The composition according to claim 1, wherein The molar ratio of ginsenoside CK to the biphenol compounds is 1:

1.

3. Use of a composition according to any one of claims 1-2 in the preparation of a medicament, characterized in that, The drug is used for treating soft tissue injuries.

4. A drug for treating soft tissue injuries, characterized in that, It includes the composition according to any one of claims 1 - 2.

5. The drug according to claim 4, characterized in that, The drug further includes pharmaceutically acceptable excipients.

6. The drug according to claim 4, wherein, The dosage forms of the drug include granules, tablets, capsules, injections, pills, oral liquids, tinctures, patches, sprays or creams.

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