Compound with liver protection effect as well as preparation and application thereof

Abstract

The invention belongs to the technical field of new development of traditional Chinese medicines, and particularly relates to a compound with a liver protection effect as well as preparation and application thereof. The compound is an extracting solution obtained by mixing dendrobium nobile and rhizoma polygonati and performing water extraction. The extracting solution can be used for preparing medicines or functional food compositions for protecting liver cells from being damaged. In-vitro experiments show that the extracting solution has a remarkable protection effect on a HepG2 cell injury model, can effectively inhibit cell injury and improve the cell survival rate, and has the best activity at a certain proportion and extraction temperature. The extraction method provided by the invention is simple in process and low in cost, and the obtained product has a good application prospect in the aspect of prevention or adjuvant therapy of chemical liver injury and is suitable for the fields of medicines and health foods.

Description

Technical Field

[0001] This invention belongs to the field of new development technology of traditional Chinese medicine, specifically relating to a complex with hepatoprotective effects and its preparation and application. Background Technology

[0002] Both Dendrobium and Polygonatum are traditional Chinese medicine and food homologous substances. In traditional Chinese medicine theory, they have the effects of nourishing yin and clearing heat, and replenishing qi and nourishing yin. They are often used in health preservation diet therapy and as an adjunct treatment for diseases. Modern research shows that Dendrobium is rich in polysaccharides, alkaloids and other active ingredients, and has antioxidant and immunomodulatory effects. Polygonatum mainly contains polysaccharides, steroidal saponins and polygonatine, and other active ingredients, and has multiple effects such as antioxidant, blood sugar and lipid regulation, immune enhancement and liver protection. Traditional Chinese medicine emphasizes nourishing yin and liver to protect the liver. Its traditional effects are mainly used to improve yin deficiency constitution and promote health and longevity. It is not a targeted drug for acute or chemical liver damage. Its liver protection effect is an indirect overall conditioning rather than a direct protection against liver cell damage.

[0003] At present, there have been some studies on compound preparations containing Polygonatum, such as the combination of Polygonatum with Lycium barbarum and Polygonatum with Dioscorea opposita. However, the existing mixtures containing Polygonatum have the following shortcomings: (1) When Dendrobium and Polygonatum are extracted separately by water, the dissolution efficiency of their respective active ingredients is limited, the concentration of hepatoprotective active ingredients is low, the hepatoprotective effect is unstable in the alcoholic liver injury model, and there is a lack of synergistic regulation of the Nrf2 / NF-κB dual pathway mediated by Polygonatum steroidal saponins, resulting in insufficient anti-inflammatory and antioxidant capacity; (2) Although Polygonatum alone has antioxidant and lipid metabolism regulating effects, its direct protective effect on alcohol damage in HepG2 cells is limited; (3) The preparations of Polygonatum with Lycium barbarum and Polygonatum with Dioscorea opposita are mainly for nourishing Yin and cannot specifically intervene in alcohol-induced oxidative damage, and there is still a lack of protection against oxidative stress in HepG2 cells; (4) The research on the combination of Dendrobium with Ophiopogon japonicus, Polygonatum odoratum and other Yin-nourishing herbs only focuses on gastrointestinal protection. Therefore, exploring the synergistic potential between Polygonatum and various other substances, and developing functional foods or drugs, has significant scientific and practical value. Summary of the Invention

[0004] In view of the above problems, the present invention provides a complex with hepatoprotective effect, its preparation and application.

[0005] To achieve the above objectives, the technical solution adopted in this experiment is as follows: A liver-protective complex is obtained by water extraction of a mixture of Dendrobium and Polygonatum.

[0006] The extract is a mixture of Dendrobium and Polygonatum at a mass ratio of 1:0.2-5, which is then extracted and separated by water to obtain the supernatant.

[0007] A method for preparing the aforementioned liver-protective complex, wherein the complex is an extract obtained by water extraction of a mixture of Dendrobium and Polygonatum.

[0008] The Dendrobium and Polygonatum are mixed at a mass ratio of 1:0.2-5, and then extracted and separated by water to obtain the supernatant.

[0009] The powders of Dendrobium and Polygonatum are mixed at a mass ratio of 1:0.2-5. The mixture is then added to water and extracted at room temperature (25℃) to 100℃ for 20 min. The resulting upper extract is centrifuged at high speed and the supernatant is collected. The supernatant is then filtered using a filter membrane, and the filtrate is the extract, which is ready for use. The mass ratio of water to the mixture is 50-20:1.

[0010] The upper extract was centrifuged at 10,000 r / min for 10 min, and the supernatant was collected and then centrifuged at 10,000 r / min for 5 min. The collected supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was the extract.

[0011] Application of the aforementioned hepatoprotective complex, specifically its application in liver protection and immune regulation.

[0012] The application of the complex in the preparation of drugs or health products for alcoholic liver injury.

[0013] The alcohol concentration in the alcoholic liver injury was 200-1000 nM; the lipopolysaccharide (LPS) concentration was 1-10 μg / mL.

[0014] The extract showed good cell survival rate when applied to HepG2 cells damaged by a certain concentration of alcohol / LPS; it could alleviate cell damage caused by the addition of alcohol / LPS.

[0015] Furthermore, the extract was applied to HepG2 cells at a concentration of 0.675-10 mg / mL (a composite extract); the alcohol concentration used for damage was 200-1000 nM; and the LPS concentration used for damage was 1-10 μg / mL. The results showed that it could alleviate cell damage caused by the addition of alcohol / LPS, and also alleviate oxidative stress caused by alcohol stimulation, thus reducing NO secretion. This indicates that the composite extract has a protective effect on the liver and can alleviate damage from harmful substances such as alcohol, providing a new approach for the development of liver-protective functional foods.

[0016] Advantages of this invention: 1. This invention differs from the traditional Chinese medicine (TCM) methods of extracting single or simple mixtures. It obtains an extract through extraction at a specific temperature and in a specific ratio, combining multiple active ingredients such as Dendrobium polysaccharides, Dendrobium alkaloids, Polygonatum polysaccharides, and steroidal saponins. Compared to simply mixing the powders of both herbs in deionized water for direct extraction, and compared to separately extracting Dendrobium and Polygonatum separately in hot water and then mixing the extracts, the extract prepared by hot water co-extraction of the two powders in a specific ratio promotes the synergistic dissolution of Dendrobium polysaccharides and Polygonatum steroidal saponins, achieving synergistic extraction and enhancement of active ingredients. Simultaneously, it avoids component loss and ratio deviations caused by step-by-step extraction. The resulting composite extract exhibits superior hepatoprotective and immunomodulatory activities compared to the control group obtained by simply mixing single extracts or directly extracting the powders at room temperature (see Example 6 for details). Furthermore, it has low operating costs, is easy to scale up for production, and has good industrialization prospects, laying a solid technical foundation for future development.

[0017] 2. This invention provides scientific evidence for the traditional theory of "medicine and food sharing the same origin". Through the activity determination of the HepG2 cell model, it provides scientific basis for the liver-protective effect of Dendrobium and Polygonatum, provides ideas for the development of functional foods and health foods, and also broadens the path for high-value utilization of these two traditional resources. Attached Figure Description

[0018] Figure 1 The graph shows the effect of different ratios of Dendrobium-Polygonatum compound extract obtained by mixing and extracting at different temperatures and in different proportions on the survival rate of HepG2 cells in Example 2 of the present invention. Among them, A is the effect of different ratios of extract on cell survival rate at room temperature (25℃), B is the effect of different ratios of extract on cell survival rate at 60℃, and C is the effect of different ratios of extract on cell survival rate at 100℃.

[0019] Figure 2 This is a graph showing the protection rate of high-concentration and medium-concentration Dendrobium-Polygonatum compound extracts on HepG2 cells after alcohol damage in Example 3 of the present invention.

[0020] Figure 3 This is a graph showing the protection rate of high-concentration and medium-concentration Dendrobium-Polygonatum compound extracts on HepG2 cells after LPS damage in Example 4 of this invention.

[0021] Figure 4 The graph shows the effect of high and medium concentrations of Dendrobium-Polygonatum compound extract on the NO secretion of HepG2 cells in Example 5 of this invention. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings, and the scope of protection of the present invention is not limited to the following embodiments.

[0023] This invention uses Dendrobium and Polygonatum as raw materials, extracts them with deionized water, and obtains a compound extract using a specific ratio (Dendrobium:Polygonatum mass ratio 1:0.2-1:5) and temperature (room temperature, 60℃ or 100℃). The obtained extract is non-toxic to HepG2 cells in the concentration range of 0.675-10 mg / mL. At the same time, using HepG2 cells as a model, the cytotoxicity, alcohol / LPS damage protection rate, NO secretion and other indicators were measured. Finally, it was proved that the Dendrobium-Polygonatum compound extract has high experimental safety and liver protection and immune enhancement effects.

[0024] The specific method is as follows: Dendrobium and Polygonatum are prepared into powder using a pulverizer, and deionized water is added to extract for 20 min under different temperature stirring conditions. The resulting upper extract is centrifuged twice at high speed (10000 r / min) and the supernatant is collected. Then, it is filtered using a 0.22 μm filter membrane and finally stored at 4 ℃ for later use.

[0025] Extracts of different concentrations (2.5 and 10 mg / mL) were applied to HepG2 cells damaged by 800 nM, showing good repair activity. At concentrations of 2.5 mg / mL and above, the extracts effectively protected hepatocytes from LPS-induced death, and at these concentrations, they also stimulated HepG2 cells to secrete NO, regulating the immune response. These experimental results provide methods and guidance for developing novel functional foods and health products based on traditional Chinese medicine for liver protection and immune enhancement.

[0026] Example 1: Preparation of Dendrobium-Polygonatum composite extract under different conditions Temperatures were set (room temperature, hot water 60 ℃, boiling water 100 ℃), and the mass ratio of Dendrobium to Polygonatum was set at each temperature (Dendrobium:Polygonatum 5:1, 2:1, 1:1, 1:2, 1:5), resulting in 15 groups. Each group contained 6 g of the drug powder, which was added to 100 mL of deionized water. After thorough mixing, the mixture was extracted for 20 min under different treatment conditions. Following extraction, the mixture was centrifuged at 10000 r / min for 10 min. The supernatant was then transferred to another centrifuge tube, balanced again, and centrifuged at 10000 r / min for 5 min. The supernatant obtained after two centrifugations was filtered through a 0.22 μm filter into an EP tube, yielding at least 5 mL of filtrate. The filtrates were labeled 1-15 and stored at 4 ℃ for later use.

[0027] Example 2: Determination of the viability of HepG2 cells by Dendrobium-Polygonatum compound extracts obtained at different temperatures and ratios. After HepG2 cells were cultured to a confluence of over 90%, they were digested, counted, and diluted to 1×10⁶ cells / cells. 5Cells were repeatedly pipetted to ensure thorough mixing, and 200 μL of each well was seeded into a 96-well plate and cultured for 24 h. After cell attachment, except for the normal group, different concentrations of Dendrobium-Polygonatum composite extract obtained in Example 1 at different temperatures and ratios were added to each well of the experimental group. (The extracts were diluted with DMEM complete medium, with a stock solution concentration of 60 mg / mL, and prepared into final concentrations of (10, 5, 2.5, 1.25, 0.675 mg / mL) after isochronous dilution. After culturing for another 24 h, the medium was removed, MTT solution was added, and incubation was continued for 4 h. The medium was then slowly removed, DMSO was added, and the plates were shaken thoroughly. The absorbance of each well was measured at 570 nm. With the cell viability of the normal medium group as 100%, the relative cell viability (%) was calculated according to the formula: (A measurement well - A blank well) / (A control well - A blank well).

[0028] Experimental results are as follows Figure 1 The figures show that the cell survival rate was higher than 95% when the concentration of different extracts was below 10 mg / mL, indicating that the compound Chinese medicine extracts prepared within this concentration range were non-toxic to HepG2 cells and had high safety.

[0029] Example 3: Determination of the protective effect of high and medium concentrations of Dendrobium-Polygonatum compound extract on HepG2 cells after alcohol damage. In this experiment, HepG2 cells in the logarithmic growth phase were damaged by 800 nM ethanol, and after 4 hours, samples of different concentrations were added. The study included a control group using pure DMEM complete culture medium, groups 1-15 (with groups 1-5 containing extracts of Dendrobium:Polygonatum mass ratios of 5:1, 2:1, 1:1, 1:2, and 1:5 at room temperature; groups 6-10 containing corresponding proportions at 60℃; and groups 11-15 containing corresponding proportions at 100℃), groups 1-15 (with groups 1-5 containing extracts of Dendrobium:Polygonatum mass ratios of 5:1, 2:1, 1:1, 1:2, and 1:5 at room temperature; groups 6-10 containing corresponding proportions at 60℃; and groups 11-15 containing corresponding proportions at 100℃), groups 1-15 (with groups 1-5 containing extracts of Dendrobium:Polygonatum mass ratios of 5:1, 2:1, 1:1, 1:2, and 1:5 at room temperature; groups 6-10 containing corresponding proportions at 60℃; and groups 11-15 containing corresponding proportions at 100℃), and a damage group receiving only pure culture medium after injury. The aim was to investigate the cell-protective function of the Dendrobium-Polygonatum composite extract after alcohol injury. The specific experimental procedure involved dividing the 96-well plates into four groups: ① blank control group (DMEM complete medium), ② high-concentration extraction solution groups (10 mg / mL, 1-15), ③ medium-concentration extraction solution groups (2.5 mg / mL, 1-15), and ④ alcohol-damaged group (800 nM alcohol, i.e., ethanol group). After the cells in each group adhered and reached the logarithmic growth phase, they were subjected to a damage treatment for 4 h. The medium was then replaced (for the control group, extraction solution group, and damage group), and the cells were cultured for another 24 h. The medium in the wells was then removed. The MTT assay was performed according to the method described in Example 2.

[0030] In addition, samples of extracts with different concentrations, both high and medium, were prepared, and the results were as follows: Figure 2 As shown in the figure, each group includes: a blank control group (control), high-concentration groups (10 mg / mL) and medium-concentration groups (2.5 mg / mL) of different extracts (1-15), and an alcohol-damaged group (ethanol group). The figure shows that under high-concentration (10 mg / mL) conditions, all 15 extracts maintained cell survival rates above 90% after alcohol damage, indicating good hepatoprotective activity. Especially under medium-concentration (2.5 mg / mL) conditions, all extracts effectively protected alcohol-damaged hepatocytes, with samples 7 and 12 (Dendrobium:Polygonatum mass ratio 2:1 extracted at 60℃ and 100℃) showing the most significant effects. In contrast, the protection rate of samples extracted at room temperature (1-5) under medium concentration was slightly lower than that extracted with hot water, indicating that appropriately increasing the extraction temperature helps enhance the hepatoprotective activity of the extracts.

[0031] Example 4: Determination of the protective effect of high and medium concentrations of Dendrobium-Polygonatum compound extract on HepG2 cells after LPS damage. In this experiment, HepG2 cells were pretreated with two different concentrations (10 mg / mL and 2.5 mg / mL) of extract for 24 h, followed by induction with 10 μg / mL LPS for 12 h. The aim was to investigate the protective efficacy of Dendrobium-Polygonatum compound extract against LPS-induced hepatocyte inflammatory damage. The specific experimental procedures were as follows: 96-well plates were divided into ① a blank control group (DMEM complete medium), ② high-concentration extract groups (10 mg / mL, divided into two 96-well plates) ③ medium-concentration extract groups (2.5 mg / mL, divided into two 96-well plates) ④ an LPS-damaged group (10 μg / mL LPS added alone). After cell adhesion, the extract groups and the blank control group were added to DMEM complete medium containing the corresponding samples and cultured for 24 h. Subsequently, the medium was removed, and all groups except the control group were added to 10 μg / mL LPS and cultured for another 12 h. Cell viability was determined according to the MTT assay described in Example 3. (For specific instructions, please refer to the experimental setup described above.)

[0032] Experimental results are as follows Figure 3 As shown, under both high and medium concentration conditions, the composite extracts of different samples could effectively protect liver cells from LPS-induced damage, resulting in a cell survival rate of over 90%, indicating that it has a good protective effect.

[0033] Example 5: Determination of NO secretion in HepG2 cells stimulated by high and medium concentrations of Dendrobium-Polygonatum compound extract. This experiment aimed to determine whether the Dendrobium-Polygonatum compound extract possessed immunomodulatory activity, analyzed by measuring the NO content secreted by cells. The experiment included: ① a negative control group (DMEM complete medium), ② high-concentration extract groups (1-15, 10 mg / mL), ③ medium-concentration extract groups (1-15, 2.5 mg / mL), and ④ a positive control group (LPS, 10 μg / mL). Cells were cultured at a rate of 1×10⁶ cells / mL. 5 Cells were seeded at 200 μL / well in 96-well plates and cultured for 24 h until the logarithmic growth phase. Each group was then replaced with DMEM complete medium containing the corresponding sample or LPS. After another 24 h of culture, the cell supernatant from each well was collected for NO determination. Because NO is highly reactive and easily oxidized, its detection is usually achieved indirectly by measuring nitrite (NO2-) levels. We used the Griess method to detect NO2- content, which indirectly reflects the amount of NO produced. During the assay, cell supernatant from each well was sequentially transferred to a new 96-well plate. After transfer, an equal volume of nitric oxide detection solution was added to each well of the new 96-well plate. The entire procedure was performed under dark conditions, and the absorbance at 570 nm was measured. Finally, the NO secretion amount for each sample was calculated based on the standard curve.

[0034] Experimental results are as follows Figure 4 The results showed that different sample extracts had different effects on stimulating NO secretion in HepG2 cells. Among them, 60 ℃ and 100 ℃ conditions could better stimulate NO secretion, indicating that the immune enhancement effect was better. The best efficacy was achieved when the ratio of Dendrobium to Polygonatum was 2:1 by mass.

[0035] Example 6: Comparative Experiment of Single Extract and Different Mixing Methods To verify the superiority of the combined extraction process of Dendrobium and Polygonatum, the following five control groups were set up, with the optimal conditions (60℃, Dendrobium:Polygonatum mass ratio 2:1, i.e., sample No. 7) as the representative: (A) Dendrobium extract alone (60℃, 6 g Dendrobium powder added to 100 mL deionized water, preparation method as in Example 1); (B) Polygonatum extract alone (60℃, 6 g Polygonatum powder added to 100 mL deionized water); (C) Equal volume mixture after separate extraction (Denimium and Polygonatum were extracted separately at 60℃, and mixed in corresponding volumes at a mass ratio of 2:1); (D) Direct water extraction of mixed powders at room temperature (25℃, Dendrobium:Polygonatum = 2:1); (E) The combined extraction solution of the present invention (sample No. 7, 60℃, Dendrobium:Polygonatum = 2:1). Each group was tested for cytotoxicity, alcohol damage protection rate, LPS damage protection rate, and NO secretion at two concentrations of 10 mg / mL and 2.5 mg / mL, following the methods described in Examples 2, 3, 4, and 5.

[0036] Cytotoxicity results showed that at a concentration of 10 mg / mL, the cell survival rate of each group was higher than 90%, and there was no obvious cytotoxicity in any group. Specific data are shown in Table 1.

[0037] Table 1. Survival rate of HepG2 cells in each group (cytotoxicity assay, 10 mg / mL)

[0038] Results of alcohol damage protection experiment (800 nM alcohol damage, 10 mg / mL sample treatment): The composite co-extraction solution (E) of this invention showed the highest cell survival rate, which was significantly better than single extraction and other control methods, indicating that the composite co-extraction process has a significant synergistic effect. Specific data are shown in Table 2.

[0039] Table 2. HepG2 cell survival rate in each group (alcohol injury protection experiment, 800 nM alcohol + 10 mg / mL sample).

[0040] LPS damage protection experiment results (10 μg / mL LPS, 10 mg / mL sample treatment): The composite extract (E) of this invention showed the highest cell survival rate, which was significantly better than single extraction and other control methods, further demonstrating the advantages of composite extraction in anti-inflammatory and liver protection. Specific data are shown in Table 3.

[0041] Table 3. HepG2 cell survival rate in each group (LPS damage protection experiment, 10 μg / mL LPS + 10 mg / mL sample).

[0042] NO secretion measurement results (10 mg / mL sample, without damage treatment): The compound extract (E) of this invention stimulated HepG2 cells to secrete the highest amount of NO, which was significantly better than other control groups. This indicates that the synergistic dissolution of active ingredients after compound extraction significantly enhanced the immunomodulatory activity. Specific data are shown in Table 4.

[0043] Table 4. NO secretion levels in HepG2 cells of each group (10 mg / mL sample).

[0044] In summary, the compound extract prepared from Dendrobium and Polygonatum showed good cellular safety at a concentration of 10 mg / mL under different conditions and in various proportions during the activity assay. It was able to repair alcohol-damaged HepG2 cells and alleviate LPS-induced cell death. Furthermore, the extract stimulated HepG2 cells to secrete a certain amount of NO, indicating a good immunostimulatory effect and the ability to enhance the body's immune activity. Moreover, the compound extraction according to the proportions of this invention exhibited stronger hepatoprotective and immune-enhancing activities than extractions of Dendrobium and Polygonatum alone, or extractions of both separately followed by a mixture.

[0045] Therefore, this invention demonstrates the potential of Dendrobium-Polygonatum as a product with liver-protecting and immune-enhancing functions, and provides ideas for the new development of traditional Chinese medicine.

Claims

1. A complex with hepatoprotective effects, characterized in that: The complex is an extract obtained by water extraction of a mixture of Dendrobium and Polygonatum.

2. The hepatoprotective complex according to claim 1, characterized in that: The extract is a mixture of Dendrobium and Polygonatum at a mass ratio of 1:0.2-5, which is then extracted and separated by water to obtain the supernatant.

3. A method for preparing the hepatoprotective complex according to claim 1, characterized in that: The complex is an extract obtained by water extraction of a mixture of Dendrobium and Polygonatum.

4. The method for preparing the hepatoprotective complex according to claim 3, characterized in that: The Dendrobium and Polygonatum are mixed at a mass ratio of 1:0.2-5, and then extracted and separated by water to obtain the supernatant.

5. The method for preparing the hepatoprotective complex according to claim 4, characterized in that: The powders of Dendrobium and Polygonatum are mixed at a mass ratio of 1:0.2-5. The mixture is then added to water and extracted at room temperature to 100℃ for 20-60 min. The resulting upper extract is centrifuged at high speed and the supernatant is collected. The supernatant is then filtered using a filter membrane, and the filtrate is the extract, which is ready for use. The mass ratio of water to the mixture is 50-20:

1.

6. The method for preparing the hepatoprotective complex according to claim 5, characterized in that: The upper extract is centrifuged at 8000-12000 r / min for 10-30 min, and the supernatant is collected and then centrifuged at 8000-12000 r / min for 5-20 min. The collected supernatant is filtered through a 0.22 μm filter membrane, and the filtrate is the extract.

7. The application of the hepatoprotective complex according to claim 1, characterized in that: Application of the complex in liver protection and immune regulation.

8. The application of the hepatoprotective complex according to claim 7, characterized in that: The application of the complex in the preparation of drugs or health products for alcoholic liver injury.

9. The application of the hepatoprotective complex according to claim 8, characterized in that: The alcohol concentration in the alcoholic liver injury was 200-1000 nM; the concentration of lipopolysaccharide (LPS, an inflammation-inducing substance) was 1-10 μg / mL.

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