Compound ginkgo nut extract, preparation method and application thereof

Abstract

The application discloses a compound ginkgo nut extract as well as a preparation method and application thereof. The compound ginkgo nut extract is prepared by mixing ginkgo nut extract, chitosan and coconut shell activated carbon according to a mass ratio of 1-5:1:1. The preparation method of the ginkgo nut extract comprises the following steps: step one, washing and detoxifying fresh ginkgo nuts, drying and crushing the ginkgo nuts in sequence to obtain ginkgo nut granular materials; step two, performing ultrasonic crushing on the ginkgo nut granular materials and performing screening treatment to obtain a ginkgo nut mixture; and step three, performing supercritical CO2 extraction on the ginkgo nut mixture, and obtaining the ginkgo nut extract by concentrating and drying the obtained extraction liquid. The compound ginkgo nut extract can effectively improve liver steatosis, liver injury, liver inflammation and fibrosis of a non-alcoholic fatty liver model mouse, and reduce the content of glutamic-pyruvic transaminase of the non-alcoholic fatty liver model mouse. In addition, the compound ginkgo nut extract can improve the expression of immune proteins in the mouse and improve the immunity of the mouse.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, in particular to a compound ginkgo nut extract and a preparation method and application thereof. BACKGROUND

[0002] Non-alcoholic fatty liver disease (NAFLD) refers to a clinical and pathological syndrome with excessive deposition of fat in hepatocytes as the main feature, which is caused by factors other than alcohol and other clear liver damage factors, and is closely related to insulin resistance and genetic susceptibility. It is an acquired metabolic stress-induced liver injury. It includes simple fatty liver (SFL), non-alcoholic steatohepatitis (NASH) and related cirrhosis. With the global epidemic trend of obesity and its related metabolic syndrome, non-alcoholic fatty liver disease has become an important cause of chronic liver disease in developed countries such as Europe and the United States and in rich areas of China. The prevalence of NAFLD in the general adult population is 10% to 30%, of which 10% to 20% are NASH, and the incidence of liver cirrhosis within 10 years is as high as 25%. Non-alcoholic fatty liver disease can directly lead to decompensated cirrhosis, hepatocellular carcinoma and recurrence of transplanted liver, and can also affect the progression of other chronic liver diseases and participate in the pathogenesis of type 2 diabetes and atherosclerosis. Therefore, non-alcoholic fatty liver disease is a new challenge in the field of modern medicine, and the harm of non-alcoholic fatty liver disease to human health will continue to increase. The body with low immunity is prone to infection or cancer, and various reasons make the immune system unable to normally play a protective role. In this case, it is easy to be infected by bacteria, viruses, fungi, etc. Therefore, low immunity aggravates the consumption of the body, so there are generally manifestations such as weak constitution, malnutrition, mental debilitation, and fatigue. Over a long period of time, it will lead to poor physical and intellectual development, and is also prone to major diseases.

[0003] Ginkgo nut (Semen Ginkgo, SG) is the mature seed of deciduous tree Ginkgo biloba L. It is harvested in autumn. Ginkgo nut contains a large amount of protein, starch, fatty oil, cyanogenic glycoside, and various amino acids, which can supplement the nutrients needed by the human body. At the same time, it has the effects of astringing lung, relieving asthma, stopping leucorrhea and shrinking urine, and can play a role in auxiliary maintenance of liver, promotion of liver metabolism, and improvement of liver detoxification capacity. It has certain benefits to the liver. Ginkgo nut can also reduce serum cholesterol, has the effect of dilating coronary artery, can improve hypertension or coronary heart disease, and has the medicinal value of lowering blood lipids. In addition, components such as ginkgolides and ginkgo phenol in ginkgo nut can enhance immunity and improve physical resistance, which is helpful for preventing diseases such as cold.

[0004] Chitosan (Chitosan, CS) has certain activity, can promote the body metabolism, also can help to enhance the body's immunity, can help to enhance the physique. And chitosan can inhibit the body to absorb and digest fat to a certain extent, reduce high-density lipoprotein, thereby helping to reduce blood lipid, it can also promote the regeneration and repair of hepatocytes, and has a certain protective effect on the liver.

[0005] Coconut shell activated carbon (Activated Carbon, AC) can help the human body to detoxify, adsorb harmful substances in the body, thereby purifying blood and digestive system, removing waste and toxins in the body, and promoting health. In addition, coconut shell activated carbon can also be used for treating diarrhea, liver and kidney diseases, food poisoning and the like. SUMMARY

[0006] In order to overcome the deficiencies of the prior art, the purpose of the present application is to provide a compound ginkgo nut extract and a preparation method and application thereof.

[0007] The present application realizes the above-mentioned purpose through the following technical scheme:

[0008] The present application provides a compound ginkgo nut extract, which comprises ginkgo nut extract, chitosan and coconut shell activated carbon mixed in a mass ratio of 1-5:1:1, and the preparation method of the ginkgo nut extract comprises the following steps:

[0009] Step one: washing and detoxifying fresh ginkgo nuts, drying and crushing to obtain ginkgo nut granules;

[0010] Step two: ultrasonic crushing the ginkgo nut granules and obtaining a ginkgo nut mixture after screening;

[0011] Step three: supercritical CO2 extraction of the ginkgo nut mixture, and the obtained extract is concentrated and dried to obtain the ginkgo nut extract;

[0012] The ginkgo nut mixture is extracted by supercritical CO2 for 0.5-2.5h under the following extraction conditions: extraction temperature of 30-50℃, extraction pressure of 20-40Mpa, entraining agent of ethanol, mass concentration of entraining agent of 87%, and flow rate of entraining agent of 0.5-2.5ml / min.

[0013] As a further optimization scheme of the present application, the compound ginkgo nut extract comprises ginkgo nut extract, chitosan and coconut shell activated carbon mixed in a mass ratio of 3-5:1:1.

[0014] As a further optimization scheme of the present application, in step two, the ultrasonic crushing is specifically that the ginkgo nut kernel granular material is placed in a 60% ethanol solution, the solid-liquid ratio is 1:30, the mixed solution is placed in an ultrasonic instrument, ultrasonic crushing is carried out twice under the temperature condition of 30-40 DEG C, the ultrasonic power is 400-600 W, and the ultrasonic crushing time is 40-50 min each time.

[0015] A preparation method of a compound ginkgo nut kernel extract is used to prepare the compound ginkgo nut kernel extract.

[0016] The compound ginkgo nut kernel extract is used for preparing a medicine for treating or preventing non-alcoholic fatty liver and immune disorder.

[0017] As a further optimization scheme of the present application, the medicinal dose of the compound ginkgo nut kernel extract is 50-150 mg / kg.

[0018] As a further optimization scheme of the present application, the medicinal dose of the compound ginkgo nut kernel extract is 100 mg / kg.

[0019] As a further optimization scheme of the present application, the immune disorder includes the immune disorder caused by excessive use of cyclophosphamide.

[0020] The present application has the following beneficial effects:

[0021] 1) The compound ginkgo nut kernel extract of the present application can effectively improve the liver fatty degeneration, liver damage, liver inflammation and fibrosis of non-alcoholic fatty liver model mice (NAFLD), and reduce the content of glutamic-pyruvic transaminase in the serum and liver of the non-alcoholic fatty liver model mice; the immune protein expression in the mice can be improved by using the compound ginkgo nut kernel extract, and the immune disorder problem of the mice can be effectively improved, and the immunity can be improved.

[0022] 2) The ginkgo nut kernel extract (SGE) in the compound ginkgo nut kernel extract of the present application has the effects of anti-inflammation, reducing blood fat, serum cholesterol and immune regulation, the chitosan (CS) has the effects of reducing blood fat, reducing blood pressure and immune regulation, and the activated carbon of coconut shell (AC) has the effects of wrapping sugar and fat, which helps to expel fat out of the body, and the scientific proportion of the three active substances is successfully explored, and the potential toxicity of the monomer is reduced.

[0023] 3) The supercritical CO2 extraction technology adopted in the present application can better remove harmful biological substances in the ginkgo nut kernel raw material and retain the activity of beneficial biological substances, and provides a more effective means for the active substances of traditional Chinese medicine to exert sufficient efficacy.

[0024] 4) The raw material of the compound ginkgo nut extract of the present application is easy to obtain and widely used. In view of its activity in the animal body, it is expected to be developed into a biological and medical preparation for treating or preventing non-alcoholic fatty liver and immune disorders. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Statistical graphs of single-factor experiments of supercritical carbon dioxide method for extracting ginkgo nut extract (SGE) on the extraction rate of ginkgo nut extract (SGE) (A: time; B: flow rate of entraining agent; C: pressure; D: temperature);

[0026] Figure 2 Morphology of compound ginkgo nut extract (morphology of ginkgo nut extract (SGE), chitosan (CS) and activated carbon (AC) mixed in a mass ratio of 1:1:1; 3:1:1; 5:1:1);

[0027] Figure 3 Effect of compound ginkgo nut extract on non-alcoholic fatty liver mice (NAFLD), effect of compound ginkgo nut extract (100 mg / kg) on body weight (A), liver morphology (B), liver weight (C) and ALT (D) of non-alcoholic fatty liver mice;

[0028] Figure 4 Effect of compound ginkgo nut extract on non-alcoholic fatty liver treatment groups (SGE / CS / AC (1:1:1), SGE / CS / AC (3:1:1) and SGE / CS / AC (5:1:1)), (A) H&E staining, Oil Red O staining and Masson staining results of liver of mice in each treatment group; (B) statistical results of Oil Red O staining of liver of mice in each treatment group; (C) statistical results of Masson staining of liver of mice in each treatment group;

[0029] Figure 5 Immune regulation of compound ginkgo nut extract on immunocompromised mice (INC), (A) immune fluorescence staining of CD8 and CD3 of spleen of mice in each treatment group (SGE / CS / AC (1:1:1), SGE / CS / AC (3:1:1) and SGE / CS / AC (5:1:1), EE-SGE / CS / AC (3:1:1)); statistical results of CD3 (B) and CD8 (C) of spleen of mice in each treatment group; detection results of immunoglobulin LgG (D) and LgM (E) in serum of mice in each treatment group.

[0030] Figure 6For the evaluation of the safety of the compound ginkgo nut extract in vivo, (A) H&E staining of the heart, liver, spleen, lung and kidney of mice after treatment with the compound ginkgo nut extract (100 mg / kg); (B) WBC, (C) RBC and (D) LYM blood biochemical index detection of mice after treatment with the compound ginkgo nut extract (100 mg / kg). DETAILED DESCRIPTION

[0031] The application will be described in further detail below with reference to the drawings. It is necessary to point out here that the following detailed description is only used to further illustrate the application and cannot be understood as limiting the scope of protection of the application. Those skilled in the art can make some non-essential improvements and adjustments to the application according to the above application content.

[0032] 1. Materials

[0033] The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used are commercially available products unless otherwise specified.

[0034] 2. Methods

[0035] 2.1 Preparation of ginkgo nut extract by supercritical CO2 extraction technology

[0036] 1) Raw material cleaning: wash the ginkgo nut with water to clean it and remove the poison for standby use;

[0037] 2) Raw material drying: dry the ginkgo nut;

[0038] 3) Raw material grinding: grind the ginkgo nut to obtain ginkgo nut granules;

[0039] 4) Ultrasonic crushing: place the ginkgo nut granules in a 60% ethanol solution with a solid-liquid ratio of 1:30, and place the mixed solution in an ultrasonic instrument under the condition of a temperature of 30-40°C for ultrasonic crushing twice, with an ultrasonic power of 400-600W and an ultrasonic crushing time of 40-50min each time. After ultrasonic crushing, ginkgo nut coarse powder is obtained;

[0040] 5) Raw material sieving: sieve the ginkgo nut coarse powder (20 mesh sieve) to obtain a ginkgo nut mixture;

[0041] 6) Supercritical CO2 extraction: send the ginkgo nut mixture sieved in step 5) into a supercritical CO2 extraction device for extraction. After the obtained extract is concentrated and dried, ginkgo nut extract powder is obtained;

[0042] The filtrate was subjected to supercritical CO2 extraction for 0.5–2.5 h under the following conditions: extraction temperature of 30–50 °C, extraction pressure of 20–40 MPa, entrainer of ethanol with a mass concentration of 87%, and entrainer flow rate of 0.5–2.5 ml / min.

[0043] 2.2 Optimization of Supercritical CO2 Extraction of Ginkgo Nut Extract Using Response Surface Methodology

[0044] 2.2.1 Single-factor influence experiment

[0045] Temperature: Temperature is an important factor affecting the extraction of ginkgo kernels. High temperature helps to improve the extraction efficiency of active substances, but excessively high temperature can also damage the structure of the extract. Therefore, the single-factor experimental temperature was set at five gradients of 30, 35, 40, 45 and 50℃ (extraction pressure of 30 MPa, entrainer flow rate of 1.5 mL / min and extraction time of 1.5 h) to evaluate the effect of extraction temperature.

[0046] Pressure: The single-factor pressure was set to five gradients of 20, 25, 30, 35, and 40 MPa (temperature 40℃, entrainer flow rate 1.5 mL / min, extraction time 1.5 h) to observe the effect of extraction pressure.

[0047] Entrainer flow rate: The single-factor flow rate was set to five gradients of 0.5, 1, 1.5, 2, and 2.5 mL / min (temperature 40℃, pressure 30 MPa, extraction time 1.5 h) to evaluate the effect of entrainer flow rate.

[0048] Extraction time: Time also affects extraction efficiency. Generally speaking, the longer the extraction time, the better the extraction effect of the compound. Therefore, the effects of extraction time of 30, 60, 90, 120 and 150 min (temperature of 40℃, pressure of 30 MPa and entrainer flow rate of 1.5 mL / min) on the content of ginkgo kernel extract were studied.

[0049] Experimental conclusions: such as Figure 1 As shown, for the single-factor experiments, the extraction effect of ginkgo kernels was best at a temperature of 45℃, followed by a pressure of 40 MPa, an entrainer flow rate of 1 mL / min, and an extraction time of 2 h. Among the four factors, temperature had a relatively small effect on the extraction rate. Therefore, the temperature was fixed at 45℃, and the following three-factor, three-level response surface optimization experiment was conducted.

[0050] It should be noted that in each embodiment of the single-factor influence experiment and the subsequent three-factor influence experiment, the ultrasonic crushing in step 4) of preparing the ginkgo kernel extract is as follows: the ginkgo kernel granular material is placed in a 60% ethanol solution with a solid-liquid ratio of 1:30, the mixed solution is placed in an ultrasonic instrument, ultrasonic crushing is performed twice at 37°C, the ultrasonic power is 550W, and the ultrasonic crushing time is 45min each time.

[0051] 2.2.2. Temperature is a quantitative variable, and the influence of pressure, entrainer flow rate, and extraction time is studied.

[0052] Based on the results of the single-factor experiment, the extraction temperature is set to 45°C, and the pressure (X1), entrainer flow rate (X2), and extraction time (X3) are selected as variables. A three-factor three-level experimental design and response surface determination are used.

[0053] Table 1: Experimental design and response value determination of supercritical CO2 extraction of SGE

[0054]

[0055]

[0056] Experimental conclusion: The optimal extraction conditions of ginkgo kernel extract (SGE) are as follows: time 2h, entrainer flow rate 1mL / min, pressure 35Mpa, temperature 45°C, and the extraction rate of ginkgo kernel extract (SGE) is 8.07%-8.16%.

[0057] 2.3 Comparative analysis of ginkgo kernel extract prepared by alcohol extraction and supercritical CO2 extraction technology

[0058] 2.3.1 Preparation of ginkgo kernel extract (SGE) by supercritical CO2 extraction technology

[0059] The supercritical CO2 extraction technology scheme in Example 14 in step 2.2.2 above is used to prepare ginkgo kernel extract, i.e. ginkgo kernel mixture 10g is extracted by supercritical CO2 under the following conditions: extraction temperature 45°C, extraction pressure 38Mpa, entrainer ethanol, entrainer mass concentration 87%, entrainer flow rate 1.3mL / min, and extraction time 2.1h. The ethanol dosage is 200mL, and after drying, 0.805g of extract powder is obtained, and the extraction rate of ginkgo kernel extract (SGE) is 8.05%.

[0060] 2.3.2 Preparation of ginkgo kernel extract (EE-SGE) by alcohol extraction

[0061] The white ginkgo kernel mixture 10 g obtained in step 2.1 above was subjected to alcohol extraction, 200 mL of 87% ethanol was used, heated reflux ultrasonic extraction was carried out for 2.1 h, the ultrasonic power was 420 W, the temperature was 45°C, the filtrate was combined and filtered, the filtrate was concentrated under reduced pressure, and the concentrated solution was dried to obtain an extract powder 0.643 g, and the extraction rate of white ginkgo kernel extract (EE-SGE) was 6.43%.

[0062] Experimental conclusion: the extraction rate of white ginkgo kernel extract (SGE) prepared by supercritical CO2 extraction technology (Example 14) was 8.05%; while the extraction rate of white ginkgo kernel extract (EE-SGE) prepared by alcohol extraction was 6.43%, which showed that the white ginkgo kernel extract prepared by supercritical CO2 extraction technology had higher efficiency, and it could be seen that supercritical CO2 extraction method had obvious advantages compared with alcohol extraction method.

[0063] 2.3 Preparation of compound white ginkgo kernel extract

[0064] The white ginkgo kernel extract (SGE) prepared according to the above step 2.2 (Example 14) was mixed with chitosan (CS) and activated carbon (AC) according to the mass ratio of 1-5:1:1 to obtain a compound white ginkgo kernel extract (for example, SGE / CS / AC (1:1:1), SGE / CS / AC (3:1:1), SGE / CS / AC (5:1:1)) as shown in Table 1. Figure 2

[0065] 3. Effect verification experiment of compound white ginkgo kernel extract

[0066] 3.1 Preparation of mouse model (NAFLD) fed with high-fat feed and detection of related indexes after treatment

[0067] Control group (Control): 8-week-old C57BL / 6 male mice were fed with ordinary feed; distilled water was administered by gavage at the same time every day, and the body weight was recorded every week for 16 weeks.

[0068] Model group (NAFLD): 8-week-old C57BL / 6 male mice were fed with 60% high-fat feed every day; distilled water was administered by gavage at the same time every day, and the body weight was recorded every week for 16 weeks.

[0069] Treatment group: 8-week-old C57BL / 6 male mice were fed with 60% high-fat feed every day; 100 mg / kg of compound white ginkgo kernel extract (the mass ratio of SGE, CS and AC was 1:1:1; 3:1:1; 5:1:1) was administered by gavage at the same time every day for the prevention / treatment of non-alcoholic fatty liver, and the body weight was recorded every week for 16 weeks.

[0070] The experimental results are shown in Table 2. Figure 3 ​As shown: Changes in mouse body weight are as follows Figure 3 As shown in Figure A, the body weight of mice in the model group (NAFLD) was significantly higher than that of mice in the control group (control group) (model group = 176.4% of control group). The body weight of mice in the SGE / CS / AC (1:1:1) compound ginkgo seed extract treatment group was 120.9% of that of the control group, while the body weight of mice in the SGE / CS / AC (3:1:1) and SGE / CS / AC (5:1:1) compound ginkgo seed extract treatment groups was similar to that of the control group.

[0071] like Figure 3 B and 3C showed that the liver size and weight of mice in the NAFLD model group (2.32g±0.11) were significantly larger than those in the control group (1.27g±0.05), while the liver weight of mice in the SGE / CS / AC (3:1:1) and SGE / CS / AC (5:1:1) compound ginkgo seed extract treatment groups were similar to those in the control group, and there was no significant difference between the two groups.

[0072] In addition, alanine aminotransferase (ALT) is one of the important indicators for assessing liver function, such as... Figure 3 The D-scan showed that ALT levels were significantly increased in NAFLD model mice, indicating that the mice's livers had suffered some damage. In contrast, ALT levels decreased in mice treated with compound ginkgo seed extract (all three treatment groups). Among them, the ALT levels in mice treated with SGE / CS / AC (3:1:1) and SGE / CS / AC (5:1:1) compound ginkgo seed extract were not significantly different from those in the control group.

[0073] like Figure 4 As shown in Figure A, the results of H&E staining of mouse liver sections showed that the hepatocytes of mice in the Control group were neatly arranged, with clear nuclei and no obvious necrosis; the liver lobule structure of mice in the NAFLD model group was abnormal, the hepatocytes were significantly enlarged and disordered, and the nuclei were smaller; compared with the model group (NAFLD), the compound ginkgo seed extract treatment groups (three treatment groups) improved the liver structure, especially the SGE / CS / AC (3:1:1) and SGE / CS / AC (5:1:1) treatment groups, whose liver structure was relatively normal and the hepatocytes were neatly arranged.

[0074] like Figure 4 As shown in A and 4B, the Oil Red O staining results showed that obvious vacuoles appeared in the cells of the NAFLD model group, with a large number of red lipid droplets inside the cells, and obvious ballooning changes in the hepatocytes. After intervention with compound ginkgo seed extract, the white bubbles in the hepatocytes became smaller, and the volume and number of red lipid droplets were also significantly reduced.

[0075] like Figure 4 As shown in A and 4CFigure 4 Masson staining results of C showed that blue collagen deposition was observed in the liver of the model group (NAFLD), and the collagen fiber tissue proliferation of the treatment groups of the compound ginkgo nut extract (three treatment groups) was significantly improved, especially the SGE / CS / AC (3:1:1) and SGE / CS / AC (5:1:1) treatment groups had the most significant comprehensive improvement effect.

[0076] 3.2 Mouse model (INC) constructed by injection of cyclophosphamide and detection of related indexes after treatment

[0077] Control group (Control): 8-week-old C57BL / 6 male mice were fed with ordinary feed, and normal saline was injected intraperitoneally for 3 consecutive days; distilled water was administered by gavage at the same time every day.

[0078] Model group (INC): 8-week-old C57BL / 6 male mice were injected intraperitoneally with cyclophosphamide at a dose of 60 mg / kg.d for 3 consecutive days to construct a mouse model with low immunity; distilled water was administered by gavage at the same time every day.

[0079] Treatment group: 8-week-old C57BL / 6 male mice were injected intraperitoneally with cyclophosphamide at a dose of 60 mg / kg.d for 3 consecutive days to construct a mouse model with low immunity; 24 hours after modeling, 100 mg / kg of the compound ginkgo nut extract (the mass ratio of SGE, CS and AC is 1:1:1; 3:1:1; 5:1:1) was administered by gavage at the same time every day for 30 days for immune regulation; in order to compare the effect of the ginkgo nut extract prepared by the supercritical CO2 technology, the ginkgo nut extract prepared by the alcohol extraction method (step 2.3.2 alcohol extraction method) was also used, and the treatment group (EE-3:1:1 or EE-SGE / CS / AC (3:1:1)) was obtained by mixing chitosan (CS) and activated carbon (AC) from coconut shell at a mass ratio of 3:1:1.

[0080] The experimental results are shown in Figure 5 Figure 5 A shows that, compared with the control group (Control), the expression amount of CD3 and CD8 cells in the immune low model group (INC) is significantly reduced, which is manifested as a significant decrease in green fluorescence (CD3) and red fluorescence (CD8), indicating that the immunity of the mouse is reduced; the use of the compound ginkgo nut extract can significantly improve the expression amount of CD3 Figure 5 A and 5B) and CD8 Figure 5 A and Figure 5 ​The expression of C) has different degrees of improvement, wherein the SGE / CS / AC (3:1:1) and SGE / CS / AC (5:1:1) compound ginkgo nut extract treatment groups have the most obvious improvement effect, and the SGE / CS / AC (1:1:1) and EE-SGE / CS / AC (3:1:1) have less effect. It can be known from the comparison of SGE / CS / AC (3:1:1) and EE-SGE / CS / AC (3:1:1) that the treatment effect of the ginkgo nut extract prepared by supercritical CO2 technology is obviously better than that of the ginkgo nut extract prepared by alcohol extraction method (step 2.3.2 alcohol extraction method). Therefore, it can be shown that the supercritical CO2 technology can better remove harmful biological substances and retain beneficial biological substance activity in the ginkgo nut raw material, and provide a more effective means for the active substances of traditional Chinese medicine to exert sufficient efficacy.

[0081] Figure 5 The results of D and 5E show that the immunoglobulin LgG and LgM of the INC model group mice are lower than those of the control group (Control). After the intervention of the compound ginkgo nut extract, the serum immunoglobulin LgG and LgM of the mice are higher than those of the INC model group, and the effect of the SGE / CS / AC (3:1:1) and SGE / CS / AC (5:1:1) compound ginkgo nut extract intervention is the best. Therefore, it can be shown that the compound ginkgo nut extract can effectively improve the immune function of the INC mice, and by comparing SGE / CS / AC (3:1:1) and EE-SGE / CS / AC (3:1:1), it is proved again that the supercritical CO2 technology can better remove harmful biological substances and retain beneficial biological substance activity in the ginkgo nut raw material, and provide a more effective means for the active substances of traditional Chinese medicine to exert sufficient efficacy.

[0082] As shown in Figure 6 A, the H&E staining results show that the compound ginkgo nut extract (100 mg / kg.d) of SGE / CS / AC (1:1:1), SGE / CS / AC (3:1:1) and SGE / CS / AC (5:1:1) has no obvious damage to the heart, liver, spleen, lung and kidney of mice, and no inflammatory infiltration is observed. The blood biochemical indicators show that the different proportions of compound ginkgo nut extract have no significant effect on the white blood cells (WBC, Figure 6 B), red blood cells (RBC, Figure 6 C), and lymphocytes (LYM, Figure 6 D) in the serum, which shows that the compound ginkgo nut extract has low toxicity and high biological safety.

[0083] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the patent scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the protection scope of the present application.

Claims

1. A compound ginkgo kernel extract, characterized in that, The compound ginkgo kernel extract is a mixture of ginkgo kernel extract, chitosan, and coconut shell activated carbon in a mass ratio of 1-5:1:

1. The preparation method of the ginkgo kernel extract includes the following steps: Step 1: Wash, detoxify, dry, and crush fresh ginkgo kernels to obtain ginkgo kernel granules. Step 2: Ginkgo kernel particles are ultrasonically crushed and then sieved to obtain a ginkgo kernel mixture; Step 3: The mixture of ginkgo kernels is subjected to supercritical CO2 extraction. The resulting extract is concentrated and dried to obtain ginkgo kernel extract, which is used to prepare drugs for the treatment or prevention of non-alcoholic fatty liver and immune disorders. The mixture of ginkgo kernels was subjected to supercritical CO2 extraction for 2.1 h under the following conditions: extraction temperature of 45℃, extraction pressure of 38 MPa, entrainer of ethanol, entrainer mass concentration of 87%, and entrainer flow rate of 1.3 ml / min.

2. The compound ginkgo kernel extract according to claim 1 ， The compound ginkgo kernel extract is characterized by being a mixture of ginkgo kernel extract, chitosan, and coconut shell activated carbon in a mass ratio of 3-5:1:

1.

3. The compound ginkgo kernel extract according to claim 1 ， The feature is that, in step two, the ultrasonic crushing specifically involves placing ginkgo kernel particles in a 60% ethanol solution with a material-to-liquid ratio of 1:30, placing the mixed solution in an ultrasonic instrument, and performing ultrasonic crushing twice at a temperature of 30-40°C, with an ultrasonic power of 400-600W, and each ultrasonic crushing time being 40-50 minutes.

4. The use of the compound ginkgo seed extract as described in any one of claims 1-3 in the preparation of drugs for treating or preventing non-alcoholic fatty liver disease and immune dysregulation, characterized in that, Drugs for the treatment or prevention of non-alcoholic fatty liver and immune disorders were prepared using compound ginkgo kernel extract as the main component.

5. The application according to claim 4 ， The compound ginkgo kernel extract is characterized in that the medicinal dosage is 50-150 mg / kg.

6. The application according to claim 5 ， Its features are, The medicinal dosage of the compound ginkgo kernel extract is 100 mg / kg.

7. The application according to claim 4 ， Its features are, The immune dysregulation includes immune dysregulation caused by excessive use of cyclophosphamide.

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