Preparation method and application of plant composition silymarin and spirulina for relieving alcoholic liver injury

Abstract

The invention discloses a preparation method and application of a plant composition silymarin and spirulina for relieving alcoholic liver injury, and belongs to the technical field of food processing. The plant composition is composed of spirulina platensis powder and silymarin, can effectively improve the bioavailability of silymarin, realizes slow release of silymarin, has a synergistic effect of relieving liver function and lipid metabolism disorder, oxidative stress and inflammation caused by alcohol, and can be used for treating liver and lipid metabolism disorder. The method can also be applied to preparation of functional food for intervening and preventing alcoholic liver injury.

Description

Technical Field

[0001] The invention belongs to the technical field of health food, and specifically relates to a preparation method and application of a plant composition silymarin@spirulina for alleviating alcoholic liver damage. Background Art

[0002] Alcohol is mainly metabolized in the liver. More than 90% of alcohol consumed is oxidatively metabolized in the liver, and 5% to 10% of the alcohol is directly excreted through urine, respiratory tract and sweat. Therefore, the liver is the main target organ of alcohol damage. Excessive drinking can lead to alcoholic liver damage (ALD). Alcohol consumption is also the main cause of chronic liver disease, as people's alcohol consumption has increased in recent years. Alcoholic liver disease has become the second largest liver disease after viral liver disease, and it has become the main cause of morbidity and mortality worldwide.

[0003] Milk thistle has always been the most commonly taken dietary supplement for patients with chronic liver disease. It can activate the regeneration of liver proteins and restore the activity of liver cells damaged by alcohol and viruses. Milk thistle can also scavenge free radicals, maintain the permeability of liver cell membranes, reduce the damage of toxins to the liver, and protect people who have long-term alcohol damage to the liver and suffer from acute and chronic hepatitis, fatty liver, cirrhosis and toxic liver damage. The liver health care effect of milk thistle can be analyzed mainly from two aspects. (1) Silymarin is a lipophilic extract of milk thistle seeds. It contains a variety of flavonoid lignans, which can stabilize liver cell membranes, maintain the activity of antioxidant substances such as glutathione and superoxide dismutase, scavenge free radicals, inhibit lipid peroxidation, and reduce liver cell damage and liver tissue inflammation. (2) Silymarin can bind to and activate the estradiol receptors of hepatocytes. The activated receptors enhance the activity of RNA polymerase I in the nucleus of hepatocytes, increase ribosomal RNA transcription, increase the number of ribosomes in the cytoplasm, promote the synthesis of functional and structural proteins, and then promote the synthesis of hepatocyte DNA in a biofeedback manner, which is beneficial to the repair and regeneration of hepatocytes. These physiological effects of silymarin make it the most clinically effective natural herb for improving liver function.

[0004] Although silymarin has great potential in alleviating liver damage, its applicability has not been taken seriously due to its poor oral bioavailability. The main limiting factors of silymarin's bioavailability are its rapid metabolism and excretion rate, low permeability to intestinal epithelial cells, and poor water solubility. One of the ways to improve the oral bioavailability of silymarin is to use carriers as drug delivery systems. Traditional carriers have the potential to improve and enhance the effectiveness of hydrophobic drugs by changing the biodistribution and pharmacokinetic characteristics of silymarin, but traditional carriers cannot achieve long-term residence in the intestine, thereby increasing the effective distribution time of silymarin in the intestine. Therefore, simply encapsulating the drug in a traditional carrier is not enough to achieve long-term residence in the intestine, thereby achieving efficient delivery of active drugs to the blood and target sites.

[0005] As one of the abundant natural biomaterials in nature, microalgae has a wide variety and is easy to obtain. Photosynthetic pigments such as chlorophyll that are also rich in microalgae can be used as natural photosensitizers, and also have fluorescence imaging and photoacoustic imaging capabilities. Among microalgae, Spirulina platensis has good biocompatibility, low cost, large surface area and active surface, and high propulsion, becoming one of the most promising delivery systems in recent years. The water channels and pores on the surface of pure Spirulina platensis allow silymarin to enter, thereby further increasing the loading rate. In addition, the rich chlorophyll of pure Spirulina platensis gives it fluorescence imaging capabilities, which can be used for non-invasive tracking and real-time monitoring in vivo. The unique spiral structure of Spirulina platensis makes it easier to be captured by intestinal villi and adhere to the intestinal wall, thereby prolonging the retention time of the drug in the intestine. Therefore, the preparation of a plant composition loaded with silymarin based on microalgae is of great significance for improving the oral bioavailability of silymarin and enhancing the improvement of liver function, but there is no report on the combined use of the two in the current prior art. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a preparation method and application of silymarin@spirulina, a plant composition for alleviating alcoholic liver damage. The plant composition silymarin@spirulina can not only improve the bioavailability of silymarin, but also has a good effect of alleviating the pathogenesis of alcoholic liver damage, providing a basis for improving the utilization rate of spirulina and silymarin resources and the development of related deep-processing products.

[0007] The invention provides a plant composition for alleviating alcoholic liver damage. The composition consists of pure Spirulina alba powder and silymarin.

[0008] Furthermore, the mass ratio of pure Spirulina apicula to silymarin in the composition is 1:1 to 1.2.

[0009] Furthermore, the preparation process of the pure Spirulina alba powder is as follows:

[0010] The pure Spirulina platensis cultured in Zarrouk medium is centrifuged, the supernatant is removed, the precipitate is washed with water, and then the washed precipitate is freeze-dried to obtain the pure Spirulina platensis powder.

[0011] Further, the Zarrouk medium includes the following substances per 1L: 0.8-1.0g NaCl, 0.02-0.04g CaCl2, 2-2.5g NaNO3, 0.01-0.02g FeSO4·7H2O, 0.06-0.08g EDTA-2Na, 0.8-1.0g K2SO4, 0.1-0.2g MgSO4·7H2O, 16-16.8g NaHCO3, 0.3-0.5g K2HPO4, 0.01-0.02g NH4VO3, 0.08-0.1g K2Cr(SO4)4·24H2O, 0.03-0.05g NiSO4·7H2O, 0.01-0.02g Na2WO4·2H2O, 0.02-0.04g Co(NO3)2·6H2O, 0.02~0.04g Ti2(SO4)3, 0.2~0.22g ZnSO4·7H2O, 0.06~0.08g CuSO4·5H2O, 0.01~0.02g MoO3, 2.5~2.86g H3BO3, 1.5~1.81g MnCl2·4H2O.

[0012] The invention provides application of the plant composition in the field of health care products.

[0013] Furthermore, the health care product refers to a health care product that has an auxiliary protective effect on chemical liver damage.

[0014] The invention provides an application of the plant composition in the field of medicine preparation.

[0015] Furthermore, the drug is used to alleviate liver damage caused by alcohol and / or treat lipid metabolism disorders, oxidative stress or inflammation caused by alcohol.

[0016] The present invention also provides a method for preparing the above-mentioned plant composition, comprising the following steps:

[0017] S1, mixing pure Spirulina alba powder with water to obtain a pure Spirulina alba solution;

[0018] S2, dissolving silymarin in ethanol to obtain a silymarin solution;

[0019] S3. Mix the spirulina solution and silymarin solution, stir for a certain period of time, centrifuge, remove the supernatant, and freeze-dry the precipitate to obtain a plant composition.

[0020] Furthermore, the concentration of pure Spirulina in the pure Spirulina solution in step S1 is 40-500 μg / mL.

[0021] Furthermore, the concentration of silymarin in the silymarin solution in step S2 is 500-2000 μg / mL.

[0022] Furthermore, in step S3, the mixing volume ratio of the pure Spirulina solution and the silymarin solution is 10:1-2.

[0023] Furthermore, the mass ratio of pure Spirulina apicula to silymarin in the plant composition of step S3 is 1:1 to 1.2.

[0024] Furthermore, the centrifugation condition in step S3 is 3000-8000 r / min for 5-10 min.

[0025] Furthermore, the stirring temperature in step S3 is 20-30° C., and the stirring time is 12-24 h.

[0026] Beneficial effects of the present invention:

[0027] The present invention prepares a plant composition by natural pure Spirulina and silymarin, realizes synergistic effect, and can more effectively alleviate liver cell damage caused by alcohol. The plant composition has no side effects on the body, and can also prolong the concentration of silymarin in the blood, further alleviate liver function and lipid metabolism disorders, oxidative stress, and inflammation caused by alcohol, and achieve the effect of sobering up and protecting the liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the silymarin concentration-absorbance standard curve;

[0029] Figure 2 It is the encapsulation rate result diagram of silymarin encapsulated by freeze-dried spirulina and non-freeze-dried spirulina, wherein SP is the precipitate of spirulina after washing, and SP freeze-dried powder is freeze-dried spirulina powder;

[0030] Figure 3 The figure shows the encapsulation efficiency of silymarin encapsulated by Spirulina at different reaction times;

[0031] Figure 4 This is the result diagram of the encapsulation rate of Spirulina freeze-dried powder for different concentrations of silymarin;

[0032] Figure 5 are the UV spectra and fluorescence spectra of SP, silymarin and silymarin@SP, where Figure 5 A is the UV spectrum and Figure 5 B is the fluorescence spectrum;

[0033] Figure 6Fourier transform infrared spectra of SP, silymarin and silymarin@SP;

[0034] Figure 7 is the cumulative release curve of silymarin@SP in the simulated gastrointestinal digestion process;

[0035] Figure 8 This is a graph showing the effect of oral administration of silymarin@spirulina of the present invention on the content of silymarin in the blood of rats;

[0036] Fig. 9 This is a graph showing the results of the biochemical analysis test of serum of mice after oral administration of silymarin@spirulina of the present invention, wherein graphs A to F correspond to the indicators of ALT, AST, TG, T-CHO, LDL-C, and HDL-C, respectively;

[0037] Fig.10 This is a graph showing the results of analyzing the oxidative stress level in the liver tissue of mice after oral administration of silymarin@spirulina of the present invention, wherein graphs A to F correspond to the indicators of GSH, MDA, SOD, CAT, ADH, and ALDH, respectively;

[0038] Fig.11 This is a graph showing the results of the determination of pro-inflammatory factors in mouse serum after oral administration of silymarin@Spirulina of the present invention, wherein graphs A to C correspond to IL-1β, IL-6, and TNF-α indicators, respectively;

[0039] Fig.12 This is a graph showing the effect of silymarin@Spirulina of the present invention on pathological changes in the liver of mice after oral administration;

[0040] Fig.13 This is a diagram showing the effect of silymarin@Spirulina of the present invention on mouse liver immunohistochemistry after oral administration;

[0041] Fig.14 Definition of contours for different types of interactions between silymarin and SP: ideal additive action curves and contour calculation method;

[0042] Fig.15 Figure 2 shows the validation test results of the interaction between silymarin and SP. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to these embodiments.

[0044] Source of raw materials

[0045] The lyophilized Spirulina powder is prepared as follows: pure Spirulina cultured in Zarrouk medium is centrifuged, the precipitate is washed three times with distilled water, and the washed precipitate is freeze-dried to obtain the lyophilized Spirulina powder;

[0046] Zarrouk medium formula (1L): 1.0g NaCl, 0.04g CaCl2, 2.5g NaNO3, 0.01g FeSO4·7H2O, 0.08g EDTA-2Na, 1.0g K2SO4, 0.2g MgSO4·7H2O, 16.8g NaHCO3, 0.5g K2HPO4 and 2mL trace element nutrient solution (0.02g NH4VO3, 0.1g K2Cr(SO4)4·24H2O, 0.05g NiSO4·7H2O, 0.02g Na2WO4·2H2O, 0.04g Co(NO3)2·6H2O, 0.04g Ti2(SO4)3, 0.22g ZnSO4·7H2O, 0.08g CuSO4·5H2O, 0.02g MoO3, 2.86g H3BO3, 1.81g MnCl2·4H2O).

[0047] Spirulina was purchased from Jiangsu Blue Ocean Technology Co., Ltd.; silymarin was a common commercial product with CAS registration number: 65666-07-1.

[0048] Detection Methods

[0049] Drawing of Silymarin Standard Curve: Dissolve Silymarin powder in ethanol to obtain (1 g / mL) Silymarin mother solution, then dilute the Silymarin mother solution with deionized water to concentrations of 0.1, 0.4, 0.8, 1.6 and 2 mg / mL. Record the absorbance of the diluted solution under ultraviolet 285 nm, and draw a Silymarin concentration-absorbance standard curve. See the Silymarin concentration-absorbance standard curve for details. Figure 1 , the silymarin concentration-absorbance standard curve is: y=2.4715x-0.0354. Based on the standard curve, silymarin can be quantified according to the absorbance value of silymarin.

[0050] Example 1

[0051] Weigh 500 μg of spirulina freeze-dried powder and washed spirulina precipitate respectively, add 9 mL of ultrapure water to prepare spirulina suspension. Then add 1 mL of silymarin solution (concentration of 1000 μg / mL, solvent is ethanol), stir for 12 hours, centrifuge at 5000 r / min for 10 minutes, measure the silymarin concentration of the supernatant, and calculate the concentration of silymarin in the supernatant according to the formula. Figure 1The entrapped silymarin content was calculated using a standard curve; the washed spirulina precipitate was obtained by filtering pure Spirulina cultured in Zarrouk medium and then washing with ultrapure water.

[0052] Test results see Figure 2 The encapsulation rate of freeze-dried Spirulina powder is 71.83%, which is 30.29% higher than that of non-freeze-dried Spirulina. Therefore, freeze-dried Spirulina has more advantages in encapsulating silymarin.

[0053] Example 2

[0054] Weigh 500 μg of spirulina freeze-dried powder and add 9 mL of ultrapure water to prepare spirulina suspension. Then add 1 mL of silymarin solution (concentration of 1000 μg / mL), stir for 2, 4, 8, 12 and 24 hours respectively, centrifuge at 5000 r / min for 10 minutes, measure the silymarin concentration of the supernatant, and calculate the concentration of silymarin in the supernatant according to the formula. Figure 1 The content of silymarin encapsulated was calculated based on the standard curve.

[0055] Test results see Figure 3 The drug loading efficiency of Spirulina increases with the increase of reaction time. The drug loading efficiency after 12h reaction is 71.83%, and the drug loading efficiency after 24h reaction is 74.96%, and the difference between the two is not obvious. Based on this, the encapsulation of Spirulina freeze-dried powder is selected to be stirred for 12h.

[0056] Example 3

[0057] Weigh 500 μg of spirulina freeze-dried powder and add 9 mL of ultrapure water to prepare spirulina suspension. Then add 1 mL of silymarin solution of different concentrations (concentrations are 200, 400, 800, 1600 and 2000 μg / mL, solvent is ethanol), stir for 12 hours, centrifuge at 5000 r / min for 10 minutes, measure the silymarin concentration of the supernatant, and calculate the concentration of silymarin in the supernatant according to the concentration of silymarin solution. Figure 1 The content of silymarin encapsulated was calculated based on the standard curve.

[0058] Test results see Figure 4 , with the increase of the initial concentration of silymarin, the encapsulation efficiency gradually increased. When the concentration of silymarin was greater than 800μg / mL, the drug loading rate could reach more than 70%, but when the concentration continued to increase, the drug loading rate did not increase significantly. Therefore, the silymarin concentration of 800μg / mL was selected as the initial concentration, at which the drug loading rate was 70.83%.

[0059] Example 4

[0060] 500 μg of spirulina freeze-dried powder was weighed and added to 9 mL of ultrapure water to prepare a spirulina suspension. Then 1 mL of silymarin solution (concentration of 800 μg / mL, solvent: ethanol) was added, stirred for 12 h, centrifuged at 5000 r / min for 10 min, and the precipitate was dried to obtain a plant composition, which was named silymarin@spirulina.

[0061] Example 5

[0062] This example is a verification of the drug loading performance of Spirulina, wherein SP is freeze-dried Spirulina powder, silymarin is silymarin, and silymarin@SP is silymarin@Spirulina prepared in Example 4.

[0063] The UV spectra of SP, silymari and silymarin@SP were measured by UV spectrophotometer and fluorescence spectrophotometer ( Figure 5 A) and fluorescence spectra ( Figure 5 B) Among them, Figure 5 A shows that silymarin@SP has the main characteristic absorption peaks of SP (chlorophyll: 420-450nm; phycocyanin: 620nm; allophycocyanin: 675nm) and the main characteristic absorption peak of silymarin (285nm). Figure 5 B shows that silymarin@SP retains the fluorescence of SP, indicating that SP successfully encapsulated silymarin.

[0064] Fourier transform infrared spectrometer at 400-4000 cm -1 Infrared spectra of SP, silymari and silymarin@SP were detected within the range ( Figure 6 ), showing that silymarin@SP has the main characteristic absorption peaks of SP and silymarin, indicating that SP successfully encapsulated silymarin.

[0065] Example 6

[0066] This example is a test of the in vitro simulated gastrointestinal digestion release performance of silymarin@Spirulina prepared in Example 4.

[0067] The determination process is as follows: weigh 1 mg of silymarin@Spirulina and disperse it in 5 mL of simulated gastric fluid, stir at 37°C for 2 hours. Then add 10 mL of simulated intestinal fluid and react again for 8 hours. Centrifuge at 5000 r / min for 10 minutes to collect the supernatant at different time points, and measure the silymarin concentration in the supernatant. Figure 1 The release rate of silymarin in Spirulina during simulated gastrointestinal digestion was calculated using a standard curve; simulated gastric fluid (R30386) and simulated intestinal fluid (R30384) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0068] The release curve of silymarin is shown in Figure 7 After 2 hours of digestion in gastric juice, a total of about 17.22% of silymarin was released in the simulated gastric environment. As the digestion time of silymarin@spirulina in the simulated intestinal fluid increased, the release of silymarin continued to accumulate. After 8 hours of digestion in the intestinal fluid, the cumulative release of silymarin reached 65.57%. It is proved that spirulina can effectively prevent silymarin from being degraded by gastric acid, achieve long-term retention of the carrier in the intestinal tract and slowly release silymarin.

[0069] Example 7

[0070] Pharmacokinetic experiments were conducted on silymarin and silymarin@Spirulina (Example 4).

[0071] Six male SD rats were purchased from Liaoning Changsheng Experimental Animal Co., Ltd. After three days of adaptive feeding, they were randomly divided into two groups (n=3), a silymarin group and a silymarin@Spirulina group.

[0072] The experiment started after fasting for 24 hours but not drinking water. The silymarin group was given a dose of 0.028g / kg; the silymarin@Spirulina group was given a dose of 0.046g / kg, both by intragastric gavage, with a dose of 2mL. Blood samples were collected at 0, 2, 4, and 8 hours after administration.

[0073] Liquid chromatography was used to detect the content of silymarin in blood, wherein the liquid chromatography conditions were as follows: chromatographic column: C18 column (4 μm, 4.6×150 mm); mobile phase: (A): dichloromethane: methanol: acetonitrile: water (5:85:45.5:4.5, v / v), (B): dichloromethane: methanol: acetonitrile: water (22:28:45.5:4.5, v / v); gradient elution: 0-8 min, 0% A; 8-20 min, 100% B.

[0074] The blood silymarin content was used to evaluate the bioavailability of silymarin. Figure 8 As shown, the retention time of silymarin in plasma in the silymarin@SP group was significantly longer than that in the silymarin group, indicating that silymarin@SP is beneficial for delaying the release of silymarin and can significantly improve the bioavailability of silymarin in plasma.

[0075] To further verify the effect of the composition of the present invention, the specific experiments are as follows:

[0076] C57BL / 6 male mice (60 mice), 8 weeks old. First, all mice were subjected to a 5-day liquid feed adaptation period, during which they were fed with Lieber-DeCarli liquid control feed. After the liquid feed adaptation, all mice were randomly divided into a blank group (control), a model group (ethanol), a positive drug group (positive), a Spirulina group (SP), a silymarin group (silymarin) and a silymarin@Spirulina group (silymarin@SP), with 10 mice in each group.

[0077] The blank group (control) was fed with Lieber-DeCarli liquid control feed (TP4030C, purchased from Nantong Trophy Company); and the solvent (normal saline) was given by gavage at the same time, once a day, with a dose of 0.2 mL.

[0078] The model group (ethanol) was fed with Lieber-DeCarli alcohol liquid model feed (TP4030B, purchased from Nantong Trophy Company); and the solvent (normal saline) was given by gavage once a day, with a dose of 0.2 mL.

[0079] The positive group (positive) was given Lieber-DeCarli alcohol liquid model feed, and a commercially available silymarin product was used as a positive control (purchased from Puritan's Pride) at a dosage of 0.2 g / kg. The mice were given intragastric administration once a day.

[0080] The Spirulina group (SP) was given the Lieber-DeCarli alcohol liquid model feed and 0.13 g / kg Spirulina powder by intragastric gavage once a day.

[0081] The silymarin group was given the Lieber-DeCarli alcohol liquid model feed and 0.2 g / kg of Euglena powder by intragastric administration once a day.

[0082] The silymarin@Spirulina group (silymarin@SP) was given the Lieber-DeCarli alcohol liquid model feed and 0.33 g / kg of Euglena powder by oral gavage once a day.

[0083] The experiment lasted for 16 days, and the mice were weighed every 2 days. During the feeding period, the mice were gavaged before 9:00 am every day. On the 16th day, except for the blank group, 0.2 mL of 31.5% ethanol was gavaged at 7:00 am, and the blank group was gavaged with 0.2 mL of 45% maltose solution. The mice in each group were killed after fasting for 9 hours, and the serum and liver were collected for biochemical index analysis and pathological analysis.

[0084] Example 8

[0085] This example is a serum biochemical analysis. The blood collected in Example 7 was stored in an anticoagulant tube, left to stand at 4°C for 6 hours, and then centrifuged at 5000r / min for 20 minutes. The supernatant was aspirated into a sterile centrifuge tube and stored in a -80°C refrigerator for later use. The kit was purchased from Nanjing Jiancheng Biological Co., Ltd., and the ALT, AST, TG, T-CHO, LDL-C, and HDL-C indicators were measured according to the manufacturer's instructions. The results are shown in Fig. 9 and Table 1 below, where * indicates compared with “control”, * indicates p<0.05, and ** indicates p<0.01; “#” indicates compared with ethanol, # indicates p<0.05, and ## indicates p<0.01.

[0086] Table 1

[0087] Control Ethanol Positive SP Silymarin Silymarin@SP ALT 5.91±0.68 36.74±6.34 19.02±8.62 16.09±2.13 15.27±4.86 14.01±1.87 AST 9.08±0.51 16.91±1.07 7.98±0.63 4.49±0.29 6.59±0.41 6.52±0.09 TG 0.59±0.01 2.33±0.02 1.68±0.06 1.46±0.01 1.20±0.02 0.97±0.01 T-CHO 0.48±0.01 1.03±0.02 0.86±0.03 0.74±0.02 0.71±0.01 0.58±0.02 LDL-C 0.08±0.004 0.28±0.01 0.22±0.01 0.18±0.01 0.18±0.01 0.15±0.003 HDL-C 3.42±0.09 1.49±0.01 1.88±0.11 2.09±0.03 2.12±0.02 2.45±0.002

[0088] Serum ALT and AST levels are often used as indicators to evaluate liver function. Fig. 9 As shown in AB, compared with the control group (5.91 and 9.08), the levels of ALT and AST in the serum of mice in the ethanol group increased significantly to 36.74 and 16.91, indicating liver damage in mice. The levels of the above indicators in the serum of mice in the silymarin@SP group decreased significantly to 14.01 and 6.52, and the effect was the most obvious. This shows that silymarin@SP has the ability to alleviate alcoholic liver damage. Fig. 9 As shown in CD, the levels of TG and T-CHO in the serum of mice in the ethanol group increased from 0.59 and 0.48 to 2.33 and 1.03, indicating fatty degeneration of the liver, while the levels of the above indicators in the serum of mice in the silymarin@SP group were significantly reduced to 0.97 and 0.58, and the effect was the most obvious. This shows that silymarin@SP has the effect of alleviating lipid accumulation in the liver. Fig. 9 As shown in EF, LDL-C is "bad" cholesterol, HDL-C is "good" cholesterol, and the level of LDL-C in the serum of mice in the ethanol group increased from 0.08 to 0.28, and the level of HDL-C decreased from 3.42 to 1.49, while the levels of the above indicators in the serum of mice in the silymarin@SP group were significantly improved to 0.15 and 2.45, and the effect was the most obvious. This shows that silymarin@SP has the ability to alleviate alcoholic liver damage.

[0089] Example 9

[0090] This example is an analysis of oxidative stress levels in liver tissue. Part of the liver tissue in Example 7 was taken to prepare a 10% liver tissue homogenate and the protein was quantified and then frozen at -80°C. The kit was purchased from Nanjing Jiancheng Biological Co., Ltd., and the GSH, MDA, SOD, CAT, ADH, and ALDH indicators were measured according to the manufacturer's instructions.

[0091] Alcohol is mainly metabolized in the liver, which can easily cause oxidative stress in the liver. The changes in GSH, MDA, SOD and CAT in the liver can fully reflect the level of oxidative stress in the liver. ADH can convert ethanol into acetaldehyde, which can be further oxidized into acetate by ALDH. The levels of ADH and ALDH can fully reflect the ability to regulate alcohol. Fig.10 and Table 2, where * indicates compared with “control”, * indicates p<0.05, ** indicates p<0.01; “#” indicates compared with ethanol, # indicates p<0.05, ## indicates p<0.01.

[0092] Table 2

[0093] Control Ethanol Positive SP Silymarin Silymarin@SP GSH 160.36±2.46 67.73±0.1 100.99±12.45 78.15±1.59 72.33±9.43 110.01±0.97 MDA 0.62±0.06 1.91±0.003 0.97±0.01 1.57±0.04 1.02±0.03 0.82±0.001 SOD 209.38±18.89 90.46±19.68 138.55±9.39 112.74±16.97 128.57±28.89 197.83±27.78 CAT 46.38±3.31 20.24±0.30 41.24±4.19 41.86±1.98 39.54±0.63 42.32±4.06 ADH 2.95±0.20 1.75±0.16 3.95±0.15 2.49±0.11 3.10±0.39 4.57±0.12 ALDH 2.97±0.17 3.86±0.15 8.22±0.16 6.27±0.44 7.81±0.62 9.17±0.07

[0094] The activities or contents of GSH, MDA, SOD, CAT, ADH, and ALDH in mouse liver are as follows: Fig.10 As shown in AD, compared with the control group, the levels of GSH, SOD and CAT in the liver of mice in the ethanol group were significantly decreased from 160.36, 209.38 and 46.38 to 67.73, 90.46 and 20.24, and the MDA level was significantly increased from 0.62 to 1.91. SOD and CAT are considered to be the main enzymes in the antioxidant defense system. The reduction of their activity can lead to the accumulation of free radicals and aggravate the damage of liver cells. MDA is a sign of peroxidative damage to liver cells. In the liver of mice in the silymarin@SP group, the levels of GSH, SOD and CAT were significantly increased to 110.01, 197.83 and 42.32, and the level of MDA was significantly decreased to 0.97, and the effect was the most obvious. It shows that silymarin@SP can effectively alleviate liver oxidative stress damage. Fig.10 As shown in EF, compared with the ethanol group, the ADH and ALDH levels in the silymarin@SP group were significantly increased from 1.75 and 3.86 to 4.57 and 9.17, indicating that silymarin@SP may help restore liver health by normalizing the alcohol metabolism system.

[0095] Example 10

[0096] This example is the detection of pro-inflammatory factors in serum. The kit was purchased from Shanghai Ke Ai Bo Biotechnology Co., Ltd., and the content of pro-inflammatory factors IL-1β, IL-6, and TNF-α in serum was measured according to the manufacturer's instructions.

[0097] The abnormal increase of IL-1β, IL-6, and TNF-α indicates the occurrence of tissue inflammation. Fig.11 and Table 3, where * indicates compared with “control”, * indicates p<0.05, ** indicates p<0.01; “#” indicates compared with ethanol, # indicates p<0.05, ## indicates p<0.01.

[0098] Table 3

[0099] Control Ethanol Positive SP Silymarin Silymarin@SP IL-1β 132.47±11.42 212.71±11.17 175.49±2.21 189.84±14.34 172.81±9.25 155.56±9.51 IL-6 120.41±29.55 227.04±19.96 173.21±16.96 199.79±20.33 182.64±4.32 152.22±2.82 TNF-α 106.37±18.64 251.06±20.46 168.06±16.16 171.31±12.31 159.86±21.52 145.48±31.88

[0100] Changes in serum proinflammatory factors such as Fig.11 As shown in AC, compared with the control group, the levels of pro-inflammatory cytokines in the serum of the ethanol group increased from 132.47, 120.41 and 106.37 to 212.71, 227.04 and 251.06, but the silymarin@SP group reversed this result, and the effect was the most obvious, which significantly reduced the contents of IL-1β, IL-6 and TNF-α to 155.56, 152.22 and 145.48. This shows that silymarin@SP can alleviate alcoholic liver injury through anti-inflammatory effects.

[0101] Embodiment 11

[0102] This example is the pathological changes of mouse liver. Hematoxylin and eosin (H&E) staining was used to observe the pathological changes of liver tissue, and the staining samples were prepared by Wuhan Saiweier Biotechnology Co., Ltd.

[0103] Pathological changes in mouse liver Fig.12 As shown in the figure, compared with the control group, the liver of the ethanol group showed a variety of abnormal symptoms, including irregular arrangement of hepatocytes, cytoplasmic lipid vesicles, and inflammatory infiltration. However, these symptoms in the silymarin@SP group were significantly alleviated, and the effect was the most obvious. This shows that silymarin@SP can alleviate the changes in liver cases caused by ethanol.

[0104] Example 12

[0105] This example is the immunohistochemistry of mouse liver. F4 / 80, KI-67, and CD45 antibodies were used to perform immunohistochemical staining on the liver, and the staining samples were prepared by Wuhan Saiweier Biotechnology Co., Ltd.

[0106] Immunohistochemical analysis of mouse liver Fig.13As shown in the figure, compared with the control group, the infiltration of F4 / 80, KI-67, and CD45 cells in the liver of the ethanol group increased, but the expression of these markers in the silymarin@SP group decreased most significantly, indicating that the infiltration of inflammatory cells decreased. This indicates that silymarin@SP can alleviate alcoholic liver injury through anti-inflammatory effects.

[0107] Example 13

[0108] This example is a calculation of drug synergy, which is based on the literature "Several Calculation Methods for Drug Synergy". Ⅰ ) and Spirulina (D Ⅱ ) are composed of two active substances that can alleviate alcoholic liver damage, and their effective doses are Z1 * =560μg and Z2 * =500μg, the activity ratio of silymarin and spirulina is R=Z1 * / Z2 * =1.12. If silymarin and spirulina are used together, the dosages Z1 and Z2 are additive and must satisfy Z1+R×Z2=Z1 * (Equation 1), variable behavior Z1 / Z1 * +Z2 / Z2 * =1(Equation 2).

[0109] Cell experiments were used to test whether silymarin and spirulina had synergistic effects. LO2 cells were cultured at 1×10 5 The cells were seeded at a density of 100 μg / mL / well in a 96-well plate and incubated at 37°C for 24 hours to allow the cells to adhere to the wall. The cells were divided into a blank group (control), a model group (ethanol), a spirulina group (SP) and a silymarin group (silymarin). Except for the control group, 100 μL of 0.8 mol / L ethanol solution was added in sequence to damage the group for 12 hours. Then the silymarin group was added with a silymarin solution prepared with a concentration of 80 μg / mL (Z1) dissolved in DMEM culture medium. Then to achieve the same effect, according to Equation 2, it can be calculated that the amount of SP added (Z2) = 430 μg / mL. In the rectangular coordinate system with Z1 and Z2 as axes, Equation 2 is obviously a straight line, and its intercepts on the two coordinate axes are Z1 and Z2 respectively. * and Z2 * ( Fig.14 ). All the dose combinations represented by the points on this line satisfy the additivity. If the dose combination of Z1 and Z2 is administered to achieve the specified effect level, it proves that the two drugs are additivity. The points of Z1 and Z2 can be Fig.14If they fall below the straight line, they are mutually cooperative, and if they fall above the straight line, they are mutually antagonistic.

[0110] Drug synergy calculations are shown in Fig.15 After 12 hours of injury with 0.8 mol / L ethanol solution, the survival rate of LO2 cells was 51.05%, indicating that the alcohol injury model was successfully established. After incubation with 80 μg / mL (Z1) silymarin solution for 12 hours, the cell survival rate increased to 67.69%, proving that silymarin solution can alleviate alcohol-induced liver cell damage. According to equation 2, when the addition amount of SP (Z2) is 430 μg / mL, it can play an additive role. The experimental results show that with the increase of SP concentration, the ability to alleviate alcohol-induced liver cell damage also increases. When the concentration of SP is 300 μg / mL, the cell survival rate increases to 71.01%. When the SP concentration is further increased to 430 μg / mL, the cell survival rate increases to 77.46%. The inhibition ability of these two concentrations is greater than 67.69%, and Z1 / Z1 can be obtained. * +Z2 / Z2 * <1, so SP and silymarin play a synergistic role in alleviating alcohol-induced liver cell damage.

[0111] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A plant composition for alleviating alcoholic liver damage, characterized in that: The composition consists of pure Spirulina alba powder and silymarin; The mass ratio of pure Spirulina apicula to silymarin in the composition is 1:1-1.

2.

2. The plant composition according to claim 1, characterized in that The preparation process of the pure Spirulina alba powder is as follows: The pure Spirulina platensis cultured in Zarrouk medium is centrifuged, the supernatant is removed, the precipitate is washed with water, and then the washed precipitate is freeze-dried to obtain the pure Spirulina platensis powder.

3. The plant composition according to claim 2, characterized in that The Zarrouk medium contains the following per 1L: 0.8~1.0g NaCl, 0.02~0.04g CaCl2, 2~2.5g NaNO3, 0.01~0.02g FeSO4·7H2O, 0.06~0.08g EDTA-2Na, 0.8~1.0g K2SO4, 0.1~0.2g MgSO4·7H2O, 16~16.8g NaHCO3, 0.3~0.5g K2HPO4, 0.01~0.02g NH4VO3, 0.08~0.1gK2Cr(SO4)4·24H2O, 0.03~0.05g NiSO4·7H2O, 0.01~0.02gNa2WO4·2H2O, 0.02~0.04g Co(NO3)2·6H2O, 0.02~0.04g Ti2(SO4)3, 0.2~0.22g ZnSO4·7H2O, 0.06~0.08g CuSO4·5H2O, 0.01~0.02g MoO3, 2.5~2.86gH3BO3, 1.5~1.81g MnCl2·4H2O.

4. The use of the plant composition of claim 1 in the field of health care products, characterized in that: The health care products refer to health care products that have auxiliary protective effects on chemical liver damage.

5. The use of the plant composition according to claim 1 in the field of drug preparation, characterized in that: The drug is used to alleviate liver function damage caused by alcohol and / or treat lipid metabolism disorders, oxidative stress or inflammation caused by alcohol.

6. The method for preparing the plant composition according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, mixing pure Spirulina alba powder with water to obtain a pure Spirulina alba solution; S2, dissolving silymarin in ethanol to obtain a silymarin solution; S3. Mix the spirulina solution and silymarin solution, stir for a certain period of time, centrifuge, remove the supernatant, and freeze-dry the precipitate to obtain a plant composition.

7. The preparation method according to claim 6, characterized in that The concentration of pure Spirulina in the pure Spirulina solution in step S1 is 40-500 μg / mL.

8. The preparation method according to claim 6, characterized in that The concentration of silymarin in the silymarin solution of step S2 is 500-2000 μg / mL.

9. The preparation method according to claim 6, characterized in that: The centrifugal condition in step S3 is 3000-8000 r / min for 5-10 min.

10. The preparation method according to claim 6, characterized in that: The stirring temperature in step S3 is 20-30° C., and the stirring time is 12-24 hours.

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