Anoectochilus roxburghii extract and application thereof in preparation of medicine for preventing and / or treating alcoholic liver disease

Abstract

The invention discloses an anoectochilus formosanus extract and application thereof in preparation of a medicine for preventing and / or treating alcoholic liver diseases, and belongs to the technical field of traditional Chinese medicine extracts. The anoectochilus formosanus extract is prepared through a water extraction process, and is obtained by taking whole anoectochilus formosanus plants as raw materials through cleaning, drying, crushing, water extraction, filtration, concentration and low-temperature storage. The invention has the core advantages of simple and convenient process, low cost, safety, no toxicity and suitability for large-scale production, and can significantly up-regulate the liver NAD + level in the alcoholic liver state, improve the mitochondrial function, inhibit inflammatory response, relieve fatty degeneration and fibrosis and effectively reverse alcohol-induced liver injury through a multi-target synergistic action mechanism. The treatment effect of the anoectochilus formosanus extract is remarkably superior to that of a single kinsenoside preparation, a more efficient, safer and cheaper medicine source for preventing and treating the alcoholic liver disease is provided, and the anoectochilus formosanus extract has important clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine extracts, and particularly relates to an Anoectochilus roxburghii extract and application thereof in preparation of a medicine for preventing and / or treating alcoholic liver disease. BACKGROUND

[0002] With the continuous rise in alcohol consumption, the incidence of alcoholic liver disease (ALD) is increasing year by year, and has become a major public health problem. Alcoholic liver disease gradually develops from fatty liver to alcoholic hepatitis, cirrhosis and even liver cancer, seriously affecting the quality of life of patients and bringing heavy economic burden to society and family.

[0003] The pathogenesis of alcoholic liver disease is complex, involving direct toxicity of acetaldehyde, a metabolite of alcohol, oxidative stress, inflammatory response, mitochondrial dysfunction and other links. As the core of cellular energy metabolism, long-term intake of alcohol will cause damage to mitochondria, leading to blocked fatty acid oxidation, excessive accumulation of fat, and consumption of a large amount of NAD + , resulting in imbalance of NAD + / NADH in hepatocytes, further exacerbating lipid metabolism disorder and energy metabolism abnormality.

[0004] At present, there are limited treatment methods for alcoholic liver disease, and there is a lack of effective therapeutic drugs. Clinical management also faces challenges such as insufficient monitoring, poor patient compliance, and low sensitivity of screening tools, often leading to delayed disease and poor prognosis.

[0005] Anoectochilus roxburghii, a rare medicinal plant of Orchidaceae and Anoectochilus, is known as "Medicine King". The Chinese Herbal Medicine records that it has the effects of clearing heat and blood, detoxifying and protecting liver. Modern research shows that Anoectochilus roxburghii is rich in anoectochilus roxburghii glycoside, flavonoids, polysaccharides and amino acids, etc. However, its application in the field of alcoholic liver disease still has technical bottlenecks: first, the effective component dissolution rate of traditional decoction is insufficient; second, single Anoectochilus roxburghii glycoside preparation cannot cover the multi-target pathological network of ALD. In addition, the extraction method of the past Anoectochilus roxburghii related extract is complex and high in cost, which limits its industrial application. Therefore, it is of great significance to develop an Anoectochilus roxburghii related extract with simple process, low cost and significant effect for the prevention and treatment of alcoholic liver disease. SUMMARY

[0006] In view of the problems of lack of existing alcoholic liver disease treatment drugs, poor effect of traditional application of Anoectochilus roxburghii and complex extraction process, the present application provides an Anoectochilus roxburghii extract and application thereof in preparation of a medicine for preventing and / or treating alcoholic liver disease, and realizes efficient, safe and low-cost liver protection effect.

[0007] To achieve the above-mentioned purpose, on the one hand, the present application provides an Anoectochilus roxburghii extract, which is prepared by water extraction process from Anoectochilus roxburghii whole plant.

[0008] In another aspect, the present application provides a preparation method of the above-mentioned Anoectochilus roxburghii extract, comprising the following steps: S1, selecting Anoectochilus roxburghii whole plant, washing thoroughly and drying; S2, adding water to the dried Anoectochilus roxburghii according to a solid-liquid ratio of 1:2, crushing and filtering, and collecting the aqueous solution; S3, concentrating the aqueous solution in S2, filtering again, collecting the refined aqueous solution, and the refined aqueous solution is the Anoectochilus roxburghii extract solution.

[0009] In another aspect, the present application provides the use of the above-mentioned Anoectochilus roxburghii extract in the preparation of a medicament for preventing or treating alcoholic liver disease.

[0010] Preferably, the alcoholic liver disease includes alcohol-induced fatty liver, liver inflammation, hepatocyte damage and liver fibrosis.

[0011] Preferably, the medicament plays a role by up-regulating the liver NAD + level under alcohol liver state, improving mitochondrial biogenesis and fusion and fission function, and inhibiting the expression of inflammatory factors.

[0012] Therefore, the Anoectochilus roxburghii extract and the use thereof in the preparation of a medicament for preventing and / or treating alcoholic liver disease have the following beneficial effects: (1) The extraction method of the present application is simple and easy to operate, uses water as the extraction medium, has no organic solvent residue, is safe and non-toxic, reduces production cost, and is suitable for industrialization and popularization.

[0013] (2) Compared with a single Anoectochilus roxburghii glycoside preparation, the Anoectochilus roxburghii extract of the present application forms a multi-target treatment network through the synergistic effect of composite active ingredients, has better effects in improving liver damage, inhibiting inflammation, regulating mitochondrial function and NAD + metabolism, and can more comprehensively play a liver protection role.

[0014] (3) The extract of the present application provides a new source of preventive and therapeutic medicaments for alcoholic liver disease, has the characteristics of high efficiency, safety and low cost, can cover multiple pathological stages from fatty liver to liver fibrosis, can significantly reduce the burden of alcoholic liver disease on the public health system, and has important clinical application value.

[0015] The technical solutions of the present application will be further described in detail below with the aid of the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Comparison of the gross morphology of the livers of mice in each group; Figure 2 Comparison of H&E stained liver sections from different groups of mice (scale bar: 50) ); Figure 3 Statistical graphs showing the results of serum LDL-C and ALT levels in mice in each group; where A represents the serum LDL-C level and B represents the ALT level in mice; Figure 4 The results show the mRNA and protein expression of TNF-α and IL-6 in the livers of mice in each group; where A represents the TNF-α mRNA level, B represents the IL-6 mRNA level, and C represents the protein expression results. Figure 5 The results show the detection of liver fibrosis in mice in each group; where A represents the level of Collagen I mRNA, B represents the level of α-SMA mRNA, and C represents the results of immunohistochemistry and liver tissue staining. Figure 6 The results of PGC-1α protein expression and immunohistochemical staining in the livers of mice in each group are shown; where A is the expression level of PGC-1α protein detected by Western blot, and B is the result of PGC-1α immunohistochemical staining. Figure 7 The results show the mRNA and protein expression of DRP1 and MFN1 in the liver of mice in each group; where A is the DRP1 mRNA level, B is the MFN1 mRNA level, C is the immunohistochemical staining result, and D is the DRP1 and MFN1 protein expression levels. Figure 8 NAD in the livers of mice in each group + Results of content and NAD+ / NADH ratio detection; where A represents mitochondrial NAD+. + Level, B is NAD + / NADH ratio. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0019] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0020] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0021] Example 1 The preparation method of Anoectochilus roxburghii extract includes the following steps: (1) Select high-quality whole plants of *Anoectochilus roxburghii* and thoroughly wash them with deionized water to remove surface mud, impurities, and residual contaminants. Place the samples at 45°C. Dry in a fume hood for 2 hours until the difference between two consecutive weighings does not exceed 0.5 mg.

[0022] (2) Take dried Anoectochilus roxburghii, add deionized water at a solid-liquid ratio of 1:2 (mass-volume ratio, g / mL), pulverize with a high-speed pulverizer, filter with gauze, and collect the first aqueous solution.

[0023] (3) Place the first aqueous solution in a rotary evaporator and heat it at 40°C. Concentrate to 1 / 3 of the original volume under 0.08 MPa conditions, then use 0.22 The purified aqueous solution is collected by membrane filtration.

[0024] (4) Dispense the purified aqueous solution into cryovials and store at -80°C. Long-term storage in an ultra-low temperature freezer yields the desired extract of *Anoectochilus roxburghii* (KPE).

[0025] Test Example 1 Select qualified SPF-grade male C57 mice, 8 weeks old, weighing approximately 20g. The participants were randomly divided into four groups: ① Normal group (control group, equal-calorie liquid diet, Ctrl); ② Alcohol group (alcoholic liquid diet, EtOH); ③ Alcoholic diet + Anoectochilus roxburghii glycoside treatment group (EtOH + Kin); ④ Alcoholic diet + Anoectochilus roxburghii extract treatment group (EtOH + KPE).

[0026] The alcoholic liver disease mouse model was established according to Liber-DeCarli liquid diet for five weeks. From the second week, the mice in the treatment group of alcohol diet + anoectochilus roxburghii glycosides and the treatment group of alcohol diet + anoectochilus roxburghii extract were respectively given intragastrically administered with anoectochilus roxburghii glycosides (16 mg / kg) or anoectochilus roxburghii extract (1.25 g / kg) medicine every morning. On the day of administration, the animals in each group were closely observed and recorded within 0-4 h after administration. After the end of the test, the body weight of all mice was weighed using an electronic balance, and the mice were sacrificed by intraperitoneal injection of sodium pentobarbital anesthesia, and gross dissection was performed. The location, size, color, adhesion, etc. of the organs were observed by naked eye, and the texture of the surface and section of the organs was checked for abnormal changes.

[0027] Test test: (1) Systematic observation of the general activity of mice in each group during the experiment found that: The normal group (Ctrl) mice showed active behavior, smooth fur, obvious circadian rhythm, frequent autonomous activity at night, and normal food and water intake. The alcohol group (EtOH) mice showed obvious behavioral abnormalities, including decreased activity, fluffy fur, daytime lethargy, and decreased food intake, which are typical symptoms of alcohol poisoning. Although the mice in the anoectochilus roxburghii glycoside treatment group (EtOH+Kin) showed some improvement, some individuals still showed intermittent decreased activity. The activity status of mice in the anoectochilus roxburghii extract treatment group (EtOH+KPE) was significantly improved compared to the alcohol group, showing increased autonomous activity, restored fur condition, and increased food intake.

[0028] (2) The results of gross dissection examination are shown in Table 1. Figure 1 Among them, the liver of the alcohol group (EtOH) mice was obviously swollen, with a blunt edge, a yellow-white surface, a tight capsule, and a significantly increased weight after weighing, with a soft texture. The liver of the mice in the anoectochilus roxburghii extract treatment group (EtOH+KPE) was less swollen, and the surface color was significantly improved compared to the alcohol group, with an improvement effect better than that of the mice in the anoectochilus roxburghii glycoside treatment group (EtOH+Kin) and close to the normal group.

[0029] (3) H&E staining to detect steatosis: Fresh mouse liver was taken, fixed, paraffin-embedded, and sectioned 4 μm thick. Dewaxed twice in xylene for 10 min each time. Rehydrated in a series of ethanol (100%, 95%, 85%, 75%) for 3 min per gradient. Soaked in distilled water for 2 min. Stained with hematoxylin dye for 5 min, and washed with distilled water to remove the floating color. Differentiated for 10 s with differentiation solution, and immersed in tap water for 2 min each time. Added eosin dye for 30 s, and quickly dehydrated after pouring off the excess dye. Gradient ethanol (75%, 85%, 95%, and 100% ethanol ) was immersed for 2-3 s each time, and 100% ethanol ) Soak 1 min, xylene transparent twice, 1 min each time, neutral gum fixation, and observe under a microscope.

[0030] The results are shown in Figure 2 . The alcohol group (EtOH) mice showed disorder of liver lobule structure, obvious ballooning degeneration of hepatocytes, a large number of vacuoles in the cytoplasm, and inflammatory cell infiltration around the central vein. The kinin treatment group (EtOH+Kin) mice showed mild fatty degeneration and local point necrosis. The KPE treatment group (EtOH+KPE) mice showed orderly arrangement of liver cords, almost no fatty degeneration, and no inflammatory cell infiltration.

[0031] (4) Serological detection: The prepared clear and non-hemolytic mouse serum was taken out from the refrigerator and restored to room temperature, and then gently shaken. The working solution was configured, and the working solution and serum were added in order. The parameters were set on the enzyme marker, and then the measurement was performed. After the standard curve was drawn, the specific values of LDL-C and ALT in each serum sample were calculated according to the change of absorbance.

[0032] The results of serological detection are shown in Figure 3 , wherein A is the LDL-C level of mouse serum, and B is the ALT level of mouse serum. .

[0033] The LDL-C level of the alcohol group (EtOH) mice was significantly increased, indicating obvious lipid metabolism disorder. The LDL-C level of the kinin treatment group (EtOH+Kin) mice was decreased, but still higher than that of the normal group (Ctrl). The LDL-C level of the KPE treatment group (EtOH+KPE) mice was significantly decreased and close to the normal level. The LDL-C level of the alcohol group (EtOH) mice was significantly increased, indicating obvious liver cell damage.

[0034] The ALT level of the kinin treatment group (EtOH+Kin) mice was lower than that of the alcohol model group, but still slightly higher than that of the normal group. The ALT level of the KPE treatment group (EtOH+KPE) mice was decreased and close to the normal level. The LDL-C and ALT levels of the KPE treatment group were significantly better than those of the kinin treatment group.

[0035] (5) mRNA detection: Fluorescent real-time quantitative PCR. The required cDNA, SYBR Mixture, primers and DEPC water were placed on ice in advance and quickly transferred to the eight-tube array according to the sample order. The reaction system was prepared, and the total volume was 10 The template DNA is 1-20 ng cDNA or 10-100 ng DNA. After the system is prepared, mix thoroughly. Add the sample to the reaction wells, centrifuge, and remove air bubbles. Place the eight-cell array with the sample on a real-time PCR instrument and set the corresponding reaction parameters. Set the melting curve. Analyze the experimental results: Observe the melting curve, paying attention to non-specific amplification or primer dimers. Use 2 -ΔΔCT The Ct values ​​of the target genes in the samples were compared to analyze their expression differences.

[0036] Western Blot analysis: The denatured protein sample was transferred from -20°C to... Remove from the refrigerator and quickly place on ice. Once the sample has thawed, add it to the lanes of the stacking gel, loading 40 μL of sample into each well. The voltage was set at 80V, and after 30 minutes of electrophoresis, the voltage was increased to 120V to continue protein separation. The final electrophoresis time was adjusted according to the molecular weight of the proteins. After electrophoresis, the membrane was transferred.

[0037] Before transfer, hydrate the PVDF membrane by soaking it in methanol for about 30-50 seconds. Afterward, rinse it with deionized water and place it in transfer buffer. After electrophoresis, remove the PAGE gel and arrange it in a "sandwich" configuration in the transfer holder, with the gel side connected to the negative electrode and the PVDF membrane side connected to the positive electrode, ensuring no air bubbles or wrinkles remain between the sandwich components. Place the transfer holder in the transfer tank, pour in the transfer buffer, and perform wet transfer using the transfer system at 100V for 1 hour. After transfer, remove the PVDF membrane and soak it in TBS for 10 minutes on a shaker at room temperature. Discard the TBS and add 10 mL of blocking buffer (5% BSA dissolved in TBST), incubating slowly at room temperature for 1 hour. Place the PVDF membrane in a solution prepared with 5% TBST. In the antibody, placed at 4 Incubate overnight on a shaker. Wash three times with 0.1% TBST to remove residual nonspecifically bound or unbound membranes. Antibody washing: Wash for 15 minutes each time, 3 times. Secondary antibody incubation: Place the PVDF membrane in goat anti-rabbit secondary antibody prepared with 0.5% TBST and incubate on a shaker at room temperature for 1 hour. Wash three times with 0.1% TBST to remove residual non-specifically bound or unbound goat anti-rabbit secondary antibody from the membrane, 15 minutes each time, 3 times.

[0038] Chemiluminescence (ECL): Using the HRP-ECL luminescence method, solution A and solution B are mixed in a 1:1 ratio, uniformly dropped onto a PVDF membrane, placed in a gel imaging system for analysis and scanning, and marker scanning is performed.

[0039] The results of mRNA and protein expression of TNF-α and IL-6 in the liver of mice in each group are shown in Table 1 and Table 2, respectively. Figure 4 .

[0040] The KPE treatment group (EtOH+KPE) can reduce the mRNA up-regulation of TNF-α and IL-6 induced by alcohol, and the effect is significantly better than that of the kin treatment group (EtOH+Kin). The protein expression detection results are consistent with the above findings. The alcohol group (EtOH) can see the increase of TNF-α and IL-6 in liver tissue, while the KPE treatment group (EtOH+KPE) only shows weak protein expression, close to the normal level. These results jointly confirm that KPE can effectively inhibit the inflammatory response of the liver induced by alcohol from multiple aspects.

[0041] (6) The mRNA detection method is the same as that in test (5).

[0042] Immunohistochemical staining: Put the glass slide with tissue into a 60 C oven and bake for 15 minutes. Use xylene solution to deparaffinize the tissue, and immerse the glass slide with tissue in xylene solution , for 12 min respectively, and then use gradient alcohol into water, with alcohol gradient of 100% , 7 min, 95%, 90%, 80% and 70% alcohol solution, each time for 5 min, and then immerse in distilled water for hydration, with hydration time of 3 min. Pour appropriate amount of antigen repair solution into the staining tank. Take the glass slide out of the distilled water, and put the glass slide with tissue into the repair solution, and heat it in the microwave oven at high fire for 7 min. After the liquid boils, keep the repair solution boiling for 3 min to complete the repair.

[0043] After the repair is completed, cool the slice in water bath, and let the protein renature. PBS clean. Use an immunohistochemical pen to draw a closed circle around the tissue to prevent the loss of incubation liquid. Drop about 50 of peroxidase blocking solution (reagent 1) on the tissue section, and place the section in a wet box to keep it moist, and let it stand at room temperature for 30 min. PBS clean, drop about 50 of normal immune animal serum (reagent 2) on the section, and place the section in a wet box in parallel, and let it stand at room temperature for 30 min. 4 Incubate the first antibody overnight. Incubate the second antibody for 1 h, and drop about 50 of streptavidin-peroxidase solution (reagent 4) on the section.​​Place in a humidified chamber and let stand at room temperature for 10 minutes. Develop with DAB. Counterstain the nucleus with hematoxylin stain for 30 seconds. Mount with neutral resin, taking care to avoid air bubbles. After mounting, observe the tissue staining under a microscope. Acquire and photograph the stained slides.

[0044] Masson staining: section thickness 4 Paraffin sections were routinely dewaxed and dissolved in distilled water. Immediately before use, Weigert iron-hematoxylin staining solution was prepared by mixing reagents A1 and A2 in a 1:1 ratio and applied to the sections for staining for 5-10 minutes. Excess staining solution was washed away with distilled water, followed by differentiation with acidic differentiation solution for 5-15 seconds, and then washed with distilled water for 30 seconds. Masson's blue solution was added for blueing again for 3-5 minutes, followed by washing with distilled water for 30 seconds. Finally, Ponceau S and fuchsin staining solution was applied for staining for 5-10 minutes.

[0045] In the above procedure, prepare the weak acid working solution according to a distilled water:weak acid solution ratio of 2:1. Wash with the weak acid working solution for 30 seconds. Discard excess liquid, then treat with phosphomolybdic acid solution for 1-2 minutes. Wash with the weak acid working solution for 30 seconds. Discard excess liquid, then stain with aniline blue staining solution for 1-2 minutes. Wash with the weak acid working solution for 30 seconds. Perform rapid dehydration with 95% ethanol for 2-3 seconds, followed by dehydration twice with anhydrous ethanol, 5-10 seconds each time. Clear with xylene twice, 1-2 minutes each time, then mount with neutral resin. Observe and photograph under a microscope.

[0046] Sirius Red staining: Sections are 4 μm thick. Paraffin sections are routinely dewaxed to distilled water. Prepare iron-hematoxylin staining solution immediately before use. Add the solution and stain for 5-10 minutes, then wash with distilled water for 10-20 seconds to remove excess staining solution. Wash with tap water for 5-10 minutes to regain blue color, then wash three times with distilled water for 5-10 seconds each time. Stain with Sirius Red solution for 10-15 minutes; for tissues that stain easily, the staining time can be controlled within 5-10 minutes. Quickly rinse the sections with distilled water to remove excess staining solution. Dehydrate rapidly with a series of 75% ethanol solutions, clear with xylene, and mount with neutral resin. Observe and photograph under a microscope.

[0047] Fibrotic lesion detection results as follows Figure 5 As shown, A represents Collagen I mRNA levels, and B represents... mRNA levels, C represents immunohistochemistry and liver tissue staining results. The liver of the alcohol group (EtOH) mice showed typical fibrosis pathological features: the hepatic lobule structure was disordered, and a large amount of collagen fibers was deposited around the portal area and central vein. Immunohistochemical detection showed that the positive rates of Collagen I and a-SMA were significantly increased. The positive rate of the kinin treatment group (EtOH+Kin) mice was lower than that of the alcohol model group, but was still slightly higher than that of the normal group. The liver fibrosis of the KPE treatment group (EtOH+KPE) mice disappeared, which was close to that of the normal mice.

[0048] (7) Regulating mitochondrial biogenesis.

[0049] Protein expression detection and immunohistochemical staining were consistent with the foregoing test methods.

[0050] The results of Western blot and immunohistochemical staining are shown in Figure 6 , wherein A is the Western blot detection of PGC-1a protein expression, and B is the PGC-1a immunohistochemical staining result. It is shown that the PGC-1a protein expression in the nucleus of the liver cells of the alcohol group (EtOH) is significantly reduced; the PGC-1a nuclear expression intensity of the KPE treatment group (EtOH+KPE) is restored to the normal level. It is proved that the KPE has a significant improvement effect on the mitochondrial biogenesis disorder induced by alcohol, and its mechanism is closely related to the regulation of the PGC-1a-mediated mitochondrial biogenesis pathway. It is suggested that the KPE can promote mitochondrial biogenesis by activating PGC-1a.

[0051] (8) Regulating mitochondrial fusion and fission. mRNA detection, protein expression detection and immunohistochemical staining are consistent with the foregoing test methods.

[0052] The results are shown in Figure 7 , wherein A is the DRP1 mRNA level, B is the MFN1 mRNA level, C is the immunohistochemical staining result, and D is the DRP1 and MFN1 protein expression. .

[0053] The qPCR detection showed that the KPE treatment group (EtOH+KPE) can significantly inhibit the up-regulation of DRP1 mRNA expression and the down-regulation of MFN1 mRNA expression induced by alcohol, and its effect is better than that of the kinin treatment group (EtOH+Kin). The immunohistochemical results further confirmed that the DRP1 protein expression in the liver tissue of the alcohol group (EtOH) was significantly enhanced; while the DRP1 expression of the KPE treatment group (EtOH+KPE) was significantly weakened, which was close to the normal level (Ctrl group). It is shown that the KPE treatment group (EtOH+KPE) has a significant regulatory effect on the mitochondrial fission abnormality induced by alcohol, and the KPE can effectively correct the mitochondrial fission abnormality induced by alcohol.

[0054] (9) NAD + Content detection: after pre-cooling the PBS on ice, weigh about 10-30 mg of tissue sample, cut it with scissors, and put it in a homogenizer. Add 200 of NAD extracting solution per 10 mg of tissue. Homogenize at room temperature or on ice. + / NADH extracting solution at room temperature or on ice. Then, centrifuge at 12,000 g for 5-10 minutes, and take the supernatant as the sample to be detected. Refer to the NAD + / NADH detection kit (product number: S0175) for subsequent detection.

[0055] Take 655 of NADH preparation solution. After dissolving 5 mg of NADH provided by the kit, 10 mM NADH standard is obtained. Store the 10 mM NADH standard in -80 darkness. Dilute the 10 mM NADH standard with NAD + / NADH extracting solution to 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 mM concentration gradients. When detecting, add 20 of the standard to each well of the 96-well plate, which is equivalent to 0, 5, 10, 20, 40, 80, 120, 160, and 200 pmol of NADH per well.

[0056] The point with a concentration of 0 mM is a blank control point containing only NAD + / NADH extracting solution. Dilute the alcohol dehydrogenase with the reaction buffer by 45 times, for example, 2 of alcohol dehydrogenase is added to 88 of reaction buffer, and 90 of alcohol dehydrogenase working solution is obtained. Take 20 of the sample to be detected after dilution with NAD + / NADH extracting solution into the 96-well plate. Take 50-100 of the sample to be detected into a centrifuge tube, and heat it at 60 in a water bath or PCR instrument for 30 minutes to decompose NAD + . If insoluble substances are generated after heating, centrifuge at 10,000 x g at room temperature or 4 for 5 minutes, and take 20 of the supernatant after dilution with NAD + / NADH extracting solution as the sample to be detected into the 96-well plate.

[0057] Calculate the average absorbance at each point in the standard group, subtract the absorbance of the blank control group, and you will get the absorbance of each standard. Plot a standard curve with NADH concentration on the x-axis and absorbance on the y-axis. .

[0058] Calculate NAD in tissue samples based on standard curve. + And the total NADH concentration or the NADH concentration. Calculate the NAD content in the sample according to the formula. + The amount and NAD + The ratio of / NADH.

[0059] ; ; in, Oxidized form The total amount of reduced NADH.

[0060] The results are as follows Figure 8 As shown, A represents mitochondrial NAD+. + Level, B is NAD + The ratio of liver NAD+ to NADH. This indicates the liver NAD+ levels in the alcohol (EtOH) group. + The level was significantly lower than that of the normal control group, and NAD was also significantly lower. + The significantly decreased NAD / NADH ratio indicates that alcohol consumption significantly affects the liver's redox balance. Drug treatment can effectively restore NAD / NADH levels. + The levels were measured, with the group treated with *Anoectochilus roxburghii* extract (EtOH+KPE) showing the most significant effect, while also lowering NAD5 levels. + The NADH ratio increased. While the treatment group treated with *Anoectochilus roxburghii* glycosides (EtOH+Kin) showed some improvement, the effect was weaker than that of the *Anoectochilus roxburghii* extract treatment group. These results confirm that *Anoectochilus roxburghii* extract can effectively reverse alcohol-induced NAD+ deficiency. + Exhaustion, NAD + Returned to normal levels.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A Chrysoplenium extract, characterized in that: The gold potted plant extract is prepared by water extraction process from the whole plant of gold potted plant.

2. The method of claim 1, wherein the Anoectochilus roxburghii extract is prepared by the steps of: The method comprises the following steps: S1, selecting the whole plant of gold potted plant, washing thoroughly and drying; S2, adding water to the dried gold potted plant according to the solid-liquid ratio of 1:2, crushing and filtering, and collecting the aqueous solution; S3, concentrating the aqueous solution collected in S2, filtering again, collecting the refined aqueous solution, and the refined aqueous solution is the gold potted plant extract solution.

3. The use of the gold potted plant extract of claim 1 in the preparation of a medicine for preventing or treating alcoholic liver disease.

4. Use according to claim 3, characterized in that: The alcoholic liver disease includes alcohol-induced fatty liver, liver inflammation, liver cell damage and liver fibrosis.

5. Use according to claim 3, characterized in that: The drug exerts its effect by up-regulating liver NAD + levels, improving mitochondrial biogenesis and fusion-fission function, and suppressing inflammatory factor expression under alcoholic liver conditions.

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