Use of Cimicifuga extract, Ganoderma lucidum polysaccharide and NADPH in combination in the preparation of drugs for treating severe sepsis

Abstract

The present invention relates to the field of biomedicine, and more particularly to the use of a Cimicifuga extract, Ganoderma lucidum polysaccharide, and NADPH in combination for the preparation of a medicament for treating severe sepsis. The present invention provides the use of a Cimicifuga extract, Ganoderma lucidum polysaccharide, and NADPH in combination for the preparation of a medicament for treating severe sepsis. The combined use of these three active substances not only has excellent anti-inflammatory effects but also protects body organs, effectively blocks the progression of sepsis, and treats severe sepsis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to use of a Cimicifuga extract, Ganoderma lucidum polysaccharide and NADPH in combination in preparing a drug for treating severe sepsis. Background Art

[0002] Sepsis can be categorized as acute inflammatory response syndrome, simple sepsis, severe sepsis, or septic shock. Severe sepsis, formerly known as sepsisemia, primarily refers to a secondary systemic inflammatory response. It is triggered by infection, inducing a systemic inflammatory response and leading to organ dysfunction. If left uncontrolled, it can lead to sepsis and, in severe cases, symptoms such as septic shock. The mortality rate from shock is as high as 50%. Statistical analysis shows that approximately 31.5 million people worldwide develop sepsis each year, resulting in over 5 million deaths. This is also a significant contributor to the high mortality rate among ICU patients.

[0003] The pathogenic mechanism of sepsis has not yet been fully elucidated. It is currently generally accepted that when the body is subjected to trauma, infection, or even shock, various humoral and cellular systems are activated, leading to the synthesis, expression, and release of multiple inflammatory mediators and cytokines. Through a series of chain reactions involving inflammatory mediators, a biological amplification effect (i.e., a "cascade effect" or "cytokine storm") is produced, which exacerbates the inflammatory response and causes uncontrolled local and systemic inflammatory responses. This leads to extensive damage to vascular endothelial cells, further promoting leukocyte adhesion and initiating the coagulation system, causing damage to organs and tissues throughout the body, and ultimately leading to multiple organ dysfunction. Sepsis is characterized by the following characteristics: it is self-inflicted damage caused by excessive activation of the body's inflammatory response due to factors such as severe infection; it often presents with high-output, low-resistance hemodynamics and a hypermetabolic state, accompanied by multiple organ dysfunction and a high mortality rate.

[0004] Therefore, how to develop relevant drugs that can effectively treat severe sepsis is an urgent problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] The present invention aims to provide a use of a Cimicifuga extract, Ganoderma lucidum polysaccharide and NADPH in combination for preparing a drug for treating severe sepsis.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of Cimicifuga heracleifolia extract, Ganoderma lucidum polysaccharide and NADPH in combination for preparing a medicine for treating severe sepsis.

[0008] Preferably, the mass ratio of the Cimicifuga extract, Ganoderma lucidum polysaccharide and NADPH is 6-10:6-10:2-4.

[0009] Preferably, the preparation method of the Cimicifuga extract is:

[0010] (1) mixing Cimicifuga heracleifolia with water and extracting the mixture, filtering the mixture to obtain an extract, and concentrating the extract to obtain a water extract;

[0011] (2) The aqueous extract is mixed with ethanol, allowed to stand and then centrifuged, and the precipitate is dried to obtain a Cimicifuga extract.

[0012] Preferably, in step (1), the mass ratio of the Cimicifuga heracleifolia to water is 1:5-9; and the temperature of the water is 75-85°C.

[0013] Preferably, the extraction time in step (1) is 6 to 10 hours; and the extract is concentrated to 1 / 8 to 1 / 4 of the original volume.

[0014] Preferably, the volume ratio of the water extract to ethanol in step (2) is 1:6-10; and the volume concentration of the ethanol is 70-80%.

[0015] Preferably, the standing time in step (2) is 6 to 10 hours; the centrifugal speed is 4500 to 5500 rpm, and the time is 35 to 45 minutes.

[0016] Preferably, the medicine is a preparation prepared with Cimicifuga extract, Ganoderma lucidum polysaccharide and NADPH as active ingredients, and pharmaceutically acceptable excipients or auxiliary ingredients.

[0017] Preferably, the preparation is an oral preparation or an injectable preparation.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Sepsis-induced degradation of the endothelial polysaccharide coating can lead to increased capillary permeability, which in turn causes a decrease in effective circulating blood volume, tissue hypoperfusion, edema, and hypoxia, accelerating the development of sepsis. NADPH can reduce vascular permeability, thereby blocking the development of sepsis and has the effect of protecting vascular endothelial cells and reducing myocardial damage. Cimicifuga extract can significantly reduce the mortality rate of septic mice and alleviate the organ damage associated with septic mice through intervention with cimicifuga glucoside. Ganoderma lucidum polysaccharide downregulates the expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, while upregulating the expression of the anti-inflammatory cytokine IL-10. The combined use of the above three active substances not only has excellent anti-inflammatory effects, but also can protect the body's organs, effectively block the development of sepsis, and treat severe sepsis. DETAILED DESCRIPTION

[0020] The present invention provides the use of Cimicifuga heracleifolia extract, Ganoderma lucidum polysaccharide and NADPH in combination for preparing a medicine for treating severe sepsis.

[0021] In the present invention, the mass ratio of the Cimicifuga extract, Ganoderma lucidum polysaccharide and NADPH is 6-10:6-10:2-4; preferably 7-9:7-9:3; more preferably 8:7-9:3; more preferably 8:8:3.

[0022] In the present invention, the preparation method of the Cimicifuga extract is:

[0023] (1) mixing Cimicifuga heracleifolia with water and extracting the mixture, filtering the mixture to obtain an extract, and concentrating the extract to obtain a water extract;

[0024] (2) The aqueous extract is mixed with ethanol, allowed to stand and then centrifuged, and the precipitate is dried to obtain a Cimicifuga extract.

[0025] In the present invention, the mass ratio of the Cimicifuga heracleifolia to water in step (1) is 1:5-9; preferably 1:6-8; and more preferably 1:7.

[0026] In the present invention, the temperature of the water is 75-85°C, preferably 77-83°C, more preferably 79-81°C, and even more preferably 80°C.

[0027] In the present invention, the extraction time in step (1) is 6 to 10 hours, preferably 7 to 9 hours, and more preferably 8 hours.

[0028] In the present invention, the extract is concentrated to 1 / 8 to 1 / 4 of the original volume; preferably 1 / 7 to 1 / 5; more preferably 1 / 6.

[0029] In the present invention, the volume ratio of the water extract to ethanol in step (2) is 1:6-10; preferably 1:7-9; more preferably 1:8.

[0030] In the present invention, the volume concentration of ethanol is 70-80%; preferably 72-78%; more preferably 74-76%; and more preferably 75%.

[0031] In the present invention, the standing time in step (2) is 6 to 10 hours, preferably 7 to 9 hours, and more preferably 8 hours.

[0032] In the present invention, the centrifugal speed is 4500-5500 rpm, and the time is 35-45 min; preferably, the speed is 4700-5300 rpm, and the time is 37-43 min; further preferably, the speed is 4900-5100 rpm, and the time is 39-41 min; more preferably, the speed is 5000 rpm, and the time is 40 min.

[0033] In the present invention, the medicine is a preparation prepared by taking Cimicifuga extract, Ganoderma lucidum polysaccharide and NADPH as active ingredients, and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0034] In the present invention, the preparation is an oral preparation or an injection preparation; preferably an injection preparation.

[0035] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] In the following examples, the Ganoderma lucidum polysaccharide was purchased from Baoji Liupanyun Biotechnology Co., Ltd. NADPH was purchased from Aibixin (Shanghai) Biotechnology Co., Ltd.

[0037] Example 1

[0038] A method for preparing a drug for treating severe sepsis, comprising the following steps:

[0039] (1) Mix Cimicifuga heracleifolia with 75°C water in a mass ratio of 1:5 and extract for 6 h, filter to obtain an extract, and concentrate the extract to 1 / 4 of its original volume to obtain a water extract;

[0040] (2) mixing the aqueous extract with 70% ethanol in a volume ratio of 1:6, allowing to stand for 6 hours, and then centrifuging at 4500 rpm for 35 minutes. The precipitate was dried at 65° C. to a constant weight to obtain a Cimicifuga extract;

[0041] (3) The Cimicifuga extract, Ganoderma lucidum polysaccharide and NADPH were mixed in a mass ratio of 6:6:2 to obtain a drug for treating severe sepsis.

[0042] Example 2

[0043] A method for preparing a drug for treating severe sepsis, comprising the following steps:

[0044] (1) Mix Cimicifuga heracleifolia and 85°C water in a mass ratio of 1:9 and extract for 10 h, filter to obtain an extract, and concentrate the extract to 1 / 8 of its original volume to obtain a water extract;

[0045] (2) mixing the aqueous extract with 80% ethanol at a volume ratio of 1:10, allowing to stand for 10 hours, and then centrifuging at 5500 rpm for 35 minutes. The precipitate was dried at 65° C. to a constant weight to obtain a Cimicifuga extract;

[0046] (3) The Cimicifuga extract, Ganoderma lucidum polysaccharide and NADPH were mixed in a mass ratio of 6:6:2 to obtain a drug for treating severe sepsis.

[0047] Example 3

[0048] A method for preparing a drug for treating severe sepsis, comprising the following steps:

[0049] (1) Mix Cimicifuga heracleifolia with 80°C water in a mass ratio of 1:7 and extract for 8 h, filter to obtain an extract, and concentrate the extract to 1 / 6 of its original volume to obtain a water extract;

[0050] (2) mixing the aqueous extract with 75% ethanol at a volume ratio of 1:8, allowing to stand for 8 hours, and then centrifuging at 5000 rpm for 40 minutes. The precipitate was dried at 65° C. to a constant weight to obtain a Cimicifuga extract;

[0051] (3) The Cimicifuga extract, Ganoderma lucidum polysaccharide and NADPH were mixed in a mass ratio of 8:8:3 to obtain a drug for treating severe sepsis.

[0052] Experimental Example 1

[0053] Comparative test:

[0054] (1) Experimental groups:

[0055] The medicine obtained in Example 3 was used as the experimental group.

[0056] In control group 1, other methods were the same as those in Example 3, except that the Cimicifuga extract purchased in the prior art (Baoji Liupanyun Biotechnology Co., Ltd.) was used.

[0057] In control group 2, other methods were the same as those in Example 3, except that the extract obtained in step (1) was concentrated and then directly dried at 65° C. to constant weight to obtain the Cimicifuga extract.

[0058] Control group 3: other methods were the same as those in Example 3, except that no Cimicifuga extract was added.

[0059] Control group 4: other methods were the same as those in Example 3, except that NADPH was not added.

[0060] The blank control group 1 did not receive any treatment;

[0061] The LPS group was treated with LPS only without any other drugs.

[0062] (2) Experimental methods

[0063] The density is 30×10 4 RAW264.7 cells at a concentration of 10 cells / mL were seeded into 96-well plates and cultured for 24 hours. The drugs obtained from each experimental group were added after the final concentration was 12 μg / ml. The treatment time was 1 hour, and then the cells were exposed to LPS at a final concentration of 100 ng / ml for 18 hours.

[0064] The 96-well plate was then washed twice with PBS, and MTT (5 mg / ml) was added to the cells, followed by an additional 4-hour incubation. The cells were dissolved in DMSO, and absorbance was measured at 570 nm. Cell viability was determined based on the absorbance. Two parallel sets of experiments were performed for each experimental group, and the results were averaged. The results are shown in Table 1.

[0065] Table 1 Cell survival rate questionnaire

[0066]

[0067]

[0068] As shown in Table 1, the experimental group had the highest cell survival rate, reaching 126.3%, indicating that the drug prepared by the present invention has a protective and growth-promoting effect on cells. From the control group, it can be seen that the addition of NADPH has a greater impact on cell survival, while the addition of Cimicifuga extract has a smaller impact on cell survival.

[0069] Experimental Example 2

[0070] 42 mice were randomly divided into 7 groups, with 6 mice in each group. Specifically, they included: experimental group, control group 1 to control group 4 (same as experimental example 1); model group: treated with LPS only; blank control group 2: no treatment.

[0071] Mice in the experimental and control groups 1 to 4 were injected with the drugs prepared by the corresponding methods (10 mg / kg, dissolved in 5% DMSO) for 1 hour. The experimental, control groups 1 to 4, and the model group were challenged with LPS (injection; 10 mg / kg). The blank control group 2 did not receive any treatment.

[0072] The serum of mice in each group was collected, and the levels of TNF-α, IL-6, and IL-10 in the serum were detected according to the instructions of the ELISA detection kit. Two parallel experiments were set up for each experimental group, and the results were averaged, as shown in Table 2.

[0073] Table 2 TNF-α, IL-6, and IL-10 level detection results

[0074] Group IL-1α (pg / ml) IL-6 (pg / ml) IL-10 (pg / ml) Blank control group 2 8.34 11.95 112.37 Model Group 90.42 58.5 45.17 Experimental group 18.64 18.67 99.51 Control group 1 32.65 36.84 79.37 Control group 2 35.81 35.34 80.43 Control group 3 49.43 38.52 72.22 Control group 4 23.35 21.52 95.51

[0075] As shown in Table 2, TNF-α and IL-6 levels were reduced, while IL-10 levels were increased in the experimental group, indicating that the combined use of Cimicifuga extract, Ganoderma lucidum polysaccharide, and NADPH has an inhibitory effect on systemic inflammation in LPS-infected mice with severe sepsis. The addition of NADPH had a smaller effect than the addition of Cimicifuga extract. A comparison of the data from Control Groups 1 and 3 shows that the preparation method of Cimicifuga extract significantly affects its effectiveness. Only the extraction method of the present invention can achieve the optimal effectiveness and enable a synergistic effect between Cimicifuga extract, Ganoderma lucidum polysaccharide, and NADPH.

[0076] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a combination of Cimicifuga extract, Ganoderma lucidum polysaccharide, and NADPH in the preparation of a drug for treating severe sepsis, wherein the mass ratio of the Cimicifuga extract, Ganoderma lucidum polysaccharide, and NADPH is 6-10:6-10:2-4; The preparation method of the Cimicifuga extract is as follows: (1) Mix Cimicifuga heracleifolia with water and extract, filter to obtain an extract, and concentrate the extract to obtain a water extract; (2) The aqueous extract is mixed with ethanol, allowed to stand and then centrifuged, and the precipitate is dried to obtain a Cimicifuga extract.

2. The use according to claim 1, characterized in that In step (1), the mass ratio of the Cimicifuga heracleifolia to water is 1:5-9; and the temperature of the water is 75-85°C.

3. The use according to claim 1, characterized in that The extraction time in step (1) is 6 to 10 hours; the extract is concentrated to 1 / 8 to 1 / 4 of the original volume.

4. The use according to claim 1, characterized in that In step (2), the volume ratio of the water extract to ethanol is 1:6-10; and the volume concentration of the ethanol is 70-80%.

5. The use according to claim 1, characterized in that The standing time in step (2) is 6 to 10 hours; the centrifugal speed is 4500 to 5500 rpm, and the time is 35 to 45 minutes.

6. The use according to claim 1, characterized in that The medicine is a preparation prepared by taking Cimicifuga extract, Ganoderma lucidum polysaccharide and NADPH as active ingredients, and adding pharmaceutically acceptable excipients or auxiliary ingredients.

7. The use according to claim 6, characterized in that The preparation is an oral preparation or an injection preparation.

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