Application of fructus alpiniae oxyphyllae flavone extract in preparation of medicine for treating diabetes and complications thereof, Parkinson's disease and pulmonary fibrosis disease

Abstract

The invention belongs to the field of research and development of new drugs, and particularly relates to application of a fructus alpiniae oxyphyllae flavone extract (AFOAO) in preparation of drugs for treating diabetes and complications thereof, Parkinson's disease and pulmonary fibrosis diseases. The fructus alpiniae oxyphyllae flavone extract is prepared from 17 components including kaempferol, luteolin, phloretin, catechin and the like. A series of systematic experiments show that a group of specific fructus alpiniae oxyphyllae flavone extracts (mainly comprising 16 active flavones such as kaempferol, apigenin and rutin) can simultaneously improve symptoms such as diabetes and complications thereof, Parkinson's disease and pulmonary fibrosis, and have various pharmacological activities. The invention officially embodies the treatment characteristic of'one medicine with multiple effects' of the traditional Chinese medicine, and has a great application prospect in preparation of medicines for treating diabetes and complications thereof, Parkinson's disease, pulmonary fibrosis and other diseases.

Description

Technical Field

[0001] The invention belongs to the field of new drug research and development, and specifically relates to an application of an Alpinia oxyphylla flavone extract (AFOAO) in preparing a drug for treating diabetes and its complications, Parkinson's disease and pulmonary fibrosis. Background Art

[0002] Although diabetes and its complications, Parkinson's disease, and pulmonary fibrosis involve different organ systems, these diseases have significant common features in pathological progression, which are mainly reflected in key biological processes such as chronic inflammation, oxidative stress, apoptosis, fibrosis, and metabolic disorders. In terms of chronic inflammation, the activation of NF-κB and NLRP3 inflammasomes leads to the release of proinflammatory factors, which triggers insulin resistance in diabetes, neuronal damage in Parkinson's disease, and fibroblast proliferation in pulmonary fibrosis. Oxidative stress is manifested as excessive production of ROS and weakened antioxidant defense, leading to apoptosis of pancreatic β cells, neuronal death in the substantia nigra, and ECM deposition in the lungs. The fibrotic process promotes organ sclerosis through the TGF-β / Smad signaling pathway and EMT, which manifests as diabetic nephropathy, gliosis, and pulmonary interstitial fibrosis. The imbalance between apoptosis and autophagy leads to insufficient insulin secretion, motor dysfunction, and alveolar repair disorders through abnormal Bcl-2 / Bax and mTOR pathways. Metabolic disorders are manifested as insulin resistance and mitochondrial dysfunction, leading to organ damage, neuronal energy crisis, and fibroblast activation. Targeting these common mechanisms, traditional Chinese medicine can synergistically intervene through multiple targets: flavonoids and alkaloids are anti-inflammatory, polysaccharides are antioxidant, saponins are anti-fibrotic, and polyphenols regulate metabolism, providing a theoretical basis for the development of a "one medicine, multiple diseases" treatment strategy.

[0003] As the main active ingredient of the traditional Chinese medicine Alpinia oxyphylla, Alpinia oxyphylla flavonoids contain a variety of flavonoid compounds and have multiple activities. It is expected to provide new ideas for the treatment of metabolic and degenerative diseases and has important development value. It is based on this that the technical solution of the present invention is proposed. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a use of a flavonoid extract of Alpinia oxyphylla in the preparation of a drug for treating diabetes and its complications, Parkinson's disease and pulmonary fibrosis. The flavonoid extract of Alpinia oxyphylla includes (in order to clearly illustrate various extracts, the present invention provides Chinese name, English name, molecular formula, molecular weight and retention time):

[0005] 1. Kaempferol (flavonol compound), Kaempferol, C 15 H 10 O6, 286.04824 and 15.481min.

[0006] 2. Luteolin (flavonoid compound), C 15 H 10 O6, 286.05153 and 13.481 min.

[0007] 3. Phloretin (dihydrochalcone compound), C 15 H 14 O5, 274.08772 and 13.036 min.

[0008] 4. 7-Hydroxy-3-[4-hydroxy-3-(3-methyl-2-buten-1-yl)phenyl]-8-(3-methyl-2-buten-1-yl)-4H-chromen-4-one (complex flavonoid derivative), C 25 H 26 O4, 390.18376 and 20.802 min.

[0009] 5. Catechin (flavan-3-ol compound), C 15 H 14 O6, 290.08262 and 13.098 min.

[0010] 6. 5,7-dihydroxy-2-phenyl-4H-chromen-4-one (flavonoid basic skeleton), C 15 H 10 O4, 254.05803 and 16.816 min.

[0011] 7. Rutin (quercetin-3-O-rutinoside), C 27 H 30 O 16 , 610.15386 and 12.918 min.

[0012] 8. Miquelianin (flavonoid glycoside), C 21 H 18 O 13 , 478.07528 and 13.217 min.

[0013] 9. Isokaempferide (O-methylated flavonol), C 16 H12 O6, 300.06401 and 15.888 min.

[0014] 10. Trifolin (Kaempferol-3-O-galactoside), C 21 H 20 O 11 , 448.10115 and 13.59 min.

[0015] 11. (2S,3S,4S,5R,6S)-6-{[5,7-Dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yl]oxy}-3,4,5-trihydroxyoxane-2-carboxylic acid (flavone glucuronic acid derivative), C 21 H 18 O 12 , 462.08061 and 12.682 min.

[0016] 12. Quercetin (classical flavonol), C 15 H 10 O7, 302.04308 and 14.594 min.

[0017] 13. Genistein (isoflavones), C 15 H 10 O5, 270.05318 and 16.956 min.

[0018] 14. Formononetin (methylated isoflavone), C 16 H 12 O4, 268.07372 and 18.166 min.

[0019] 15. Biochanin A (isoflavone derivative), C 16 H 12 O5, 284.06886 and 18.415 min.

[0020] 16. Vanillin (benzaldehyde compound), C8H8O3, 120.02149 and 10.63 min.

[0021] 17. 5-O-Methylgenistein (methylated isoflavone), C 16 H 12 O5, 284.06892 and 17.161 min.

[0022] The preparation method of the Alpinia oxyphylla flavonoid extract is as follows:

[0023] (1) Extraction: Take 100 g of Alpinia oxyphylla medicinal materials, add them to 1000 g of a 70 vol% methanol aqueous solution for mixing, heat under reflux for 1.5 h and then filter, collecting the filtrate; the medicinal residues are added again to 1000 g of a 70 vol% methanol aqueous solution, repeat the extraction for 1.5 h and then filter, and combine the two filtrates to obtain an extract;

[0024] (2) Concentration: Concentrate the extract under reduced pressure at 60 °C until there is no alcohol smell to obtain a concentrated solution;

[0025] (3) Purification: Pass the concentrated solution through an AB-8 macroporous adsorption resin column, first wash off impurities with deionized water, and then elute the target components with 70 vol% ethanol, collecting the eluate;

[0026] (4) Drying: Concentrate the eluate under reduced pressure at 50 °C, and then dry it under vacuum to obtain a solid powder;

[0027] (5) Pulverization: Further grind the solid powder into a fine powder, and pass through an 80-mesh sieve to obtain the Alpinia oxyphylla extract.

[0028] Among them, diabetic complications include diabetic liver injury, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, diabetic cognitive impairment, and so on.

[0029] Preferably, the drug is a drug for reducing fasting blood glucose;

[0030] And / or, the drug is a drug for reducing random blood glucose;

[0031] And / or, the drug is a drug for reducing glycated hemoglobin.

[0032] Preferably, the drug is a drug for improving oral glucose tolerance;

[0033] And / or, the drug is a drug for improving insulin tolerance;

[0034] And / or, the drug is a drug for reducing the insulin resistance index.

[0035] Preferably, the drug is a drug for improving hepatic steatosis caused by diabetes;

[0036] And / or, the drug is a drug for improving liver fibrosis caused by diabetes.

[0037] Preferably, the drug is a drug for improving glomerular and tubular fibrosis caused by diabetes;

[0038] And / or, the drug is a drug for improving thickening of the glomerular basement membrane and expansion of the mesangial matrix caused by diabetes;

[0039] And / or, the drug is a drug for improving tubulointerstitial injury caused by diabetes.

[0040] Preferably, the drug is a drug for improving insufficient retinal blood perfusion caused by diabetes;

[0041] And / or, the drug is a drug for improving loss of ganglion cells, loss and disordered arrangement of cells in the inner nuclear layer and outer nuclear layer caused by diabetes.

[0042] Preferably, the drug is a drug for improving myocardial fibrosis caused by diabetes.

[0043] Preferably, the drug is a drug for improving cognitive impairment caused by diabetes;

[0044] And / or, the drug is a drug for improving learning and memory dysfunction caused by diabetes;

[0045] And / or, the drug is a drug for improving synaptic damage of hippocampal and cortical neurons caused by diabetes.

[0046] Preferably, the drug is a drug for improving motor dysfunction;

[0047] And / or, the drug is a drug for treating dopaminergic neuron damage.

[0048] Preferably, the drug is a drug for treating pulmonary fibrosis.

[0049] The beneficial effects of the present invention are as follows:

[0050] A series of systematic experiments were carried out in the present invention, and it was found that a group of specific extracts of flavonoids from Alpinia oxyphylla Miq. (mainly including 16 active flavonoids such as kaempferol, apigenin, rutin, etc.) can simultaneously improve symptoms such as diabetes and its complications, Parkinson's disease, and pulmonary fibrosis, and it has a variety of pharmacological activities. The present invention formally embodies the treatment characteristics of "one drug with multiple effects" of traditional Chinese medicine, and has great application prospects in the preparation of drugs for diseases such as diabetes and its complications, Parkinson's disease, and pulmonary fibrosis. Description of the Drawings

[0051] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0052] Figure 1 It is the result diagram of the effect of AFOAO on the body weight of db / db mice; wherein: Figure 1 A is the diagram of the dynamic change of the body weight of mice in each group during the 12-week intervention; Figure 1 B is the analysis diagram of the area under the curve of the body weight change curve.

[0053] The data are expressed as mean ± standard error (Mean ± SEM), n = 10. Con: normal control group; db / db: diabetic model group; db / db + AFOAO: AFOAO treatment group; AUC: area under the curve (Area Under Curve).

[0054] Figure 2 It is the result diagram of the effect of AFOAO on the fasting blood glucose, random blood glucose and glycosylated hemoglobin of db / db mice; wherein: Figure 2 A is the diagram of the dynamic change of the fasting blood glucose of mice in each group during the 12-week intervention; Figure 2 B is the analysis diagram of the area under the curve of the fasting blood glucose change curve; Figure 2 C is the diagram of the random blood glucose level of mice in each group after the last treatment; Figure 2 D is the diagram of the glycosylated hemoglobin level of mice in each group after the last treatment.

[0055] The data are expressed as mean ± standard error (Mean ± SEM), n = 10. ***P < 0.001, vs Con; #P < 0.05, ##P < 0.01, P < 0.001, vs db / db. Con: normal control group; db / db: diabetic model group; db / db + AFOAO: AFOAO treatment group; AUC: area under the curve (Area Under Curve).

[0056] Figure 3 It is the result diagram of the effect of AFOAO on the insulin resistance of db / db mice; wherein: Figure 3 A is the diagram of the glucose tolerance result of mice in each group at the end of the treatment; Figure 3 B is the insulin tolerance of mice in each group; Figure 3 C is Figure 3 A and Figure 3 The area under the curve diagram of B; Figure 3 D is the insulin resistance index of mice in each group.

[0057] Data are expressed as Mean±SEM, n = 10. Con: normal control group; db / db: diabetic model group; db / db+AFOAO: AFOAO treatment group; AUC: Area Under Curve; HOMA-IR: insulin resistance index.

[0058] Figure 4 It is the result graph of the effect of AFOAO on liver injury in db / db mice; among them: Figure 4 A is the gross liver map of mice in each group; Figure 4 B is the HE staining and Masson staining of liver tissues of mice in each group.

[0059] Con: normal control group; db / db: diabetic model; db / db+AFOAO: AFOAO treatment group.

[0060] Figure 5 It is the result graph of the effect of AFOAO on kidney injury in db / db mice; among them: Figure 5 A is the gross kidney map of mice in each group; Figure 5 B is the HE staining and Masson staining of kidney tissues of mice in each group.

[0061] Con: normal control group; db / db: diabetic model; db / db+AFOAO: AFOAO treatment group.

[0062] Figure 6 It is the result graph of the effect of AFOAO on retinopathy in db / db mice; among them: Figure 6 A is the representative OCTA picture of mice in each group; Figure 6 B is the HE staining of retina tissues of mice in each group.

[0063] Con: normal control group; db / db: diabetic model; db / db+AFOAO: AFOAO treatment group.

[0064] Figure 7 It is the result graph of the effect of AFOAO on myocardial lesions in db / db mice, that is, the HE staining map of heart tissues of mice in each group.

[0065] Con: normal control group; db / db: diabetic model; db / db+AFOAO: AFOAO treatment group.

[0066] Figure 8 It is the result graph of the cognitive ability of AFOAO on db / db mice detected by novel object recognition; among them: Figure 8 A is the schematic diagram of the novel object recognition experiment; Figure 8 B is the result graph of the recognition index of mice after a 0.5 h interval;Figure 8 C is the graph of the recognition index results of mice after a 24-hour interval.

[0067] Con: normal control group; db / db: diabetic model; db / db+AFOAO: AFOAO treatment group.

[0068] Figure 9 It is the graph of the results of the Morris water maze experiment to detect the cognitive ability of AFOAO on db / db mice; among them: Figure 9 A is the schematic diagram of the Morris water maze experiment; Figure 9 B is the spatial learning curve during the training of Con, DACD, and DACD+AFOAO mice in the place navigation experiment (MWM Training), measured by the latency to find the hidden platform; Figure 9 C is the latency of Con, DACD, and DACD+AFOAO mice to find the platform in the (MWMprobe) test; Figure 9 D is the percentage of time spent by Con, DACD, and DACD+AFOAO mice in different quadrants in the spatial exploration experiment (MWMprobe).

[0069] N = 10, the data are expressed as mean ± SEM, **P<0.01; ***P<0.001 vs Con; #P<0.05; P<0.001 vs DACD. Con: normal control group; DACD: diabetic cognitive dysfunction group; DACD+AFOAO: AFOAO treatment group.

[0070] Figure 10 It is the graph of observing the morphology of hippocampal and cortical neurons of mice in each group by HE staining.

[0071] Con: normal control group; DACD: diabetic cognitive dysfunction group; DACD+AFOAO: AFOAO treatment group.

[0072] Figure 11 It is the graph of the effect of AFOAO on the PD-like motor dysfunction of 6-OHDA mice; among them: Figure 11 A is the turning latency of mice in the pole climbing experiment; Figure 11 B is the total time of mice climbing the pole; Figure 11 C is the number of times the two front feet of each group of mice touch the cylinder wall within 5 minutes in the cylinder experiment; Figure 11 D is the falling latency of each group of mice in the rotarod experiment; Figure 11 E is the number of rotations per minute towards the damaged side of each group of mice within 30 minutes in the amphetamine rotation experiment.

[0073] Con: Normal control group; 6-OHDA: 6-OHDA group; 6-OHDA+AFOAO: AFOAO treatment group.

[0074] Figure 12 It is the result diagram of the effect of AFOAO on the damage of dopaminergic neurons in the substantia nigra-striatal pathway of mice induced by 6-OHDA.

[0075] Con: Normal control group; 6-OHDA: 6-OHDA group; 6-OHDA+AFOAO: AFOAO treatment group; TH: Tyrosine hydroxylase, a specific marker of dopaminergic neurons.

[0076] Figure 13 It is the result diagram of the effect of AFOAO on bleomycin-induced pulmonary fibrosis in mice; among them: Figure 13 A is the gross picture of the lung tissue of mice in each group; Figure 13 B is the HE and Masson staining of the lung tissue of mice in each group; Figure 13 C is the immunohistochemical staining of fibrosis markers (α-SMA, Collagen I and Fibronectin) in the lung tissue of mice in each group.

[0077] Con: Normal control group; BLM: Bleomycin group; BLM+AFOAO: AFOAO treatment group. Detailed implementation manners

[0078] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other implementation manners obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope protected by the present invention.

[0079] Example 1: Diabetes-related experiments

[0080] (1) Influence on body weight

[0081] Experimental method: 3-month-old C57BL / 6J mice were selected as the normal control group (Con), and age-matched db / db mice were used as the diabetes model group. The model group was randomly divided into two groups: diabetes control group (db / db) and AFOAO treatment group (db / db+AFOAO). The treatment group was orally administered 50 mg / kg of Alpinia oxyphylla flavonoid extract (AFOAO) every day, and the control group was given the same volume of pure water, and the intervention continued for 12 weeks.

[0082] Results: As Figure 1As shown in Figure A, the body weight of mice in the db / db group before intervention was significantly higher than that in the normal control group, but there was no statistical difference compared with the db / db+AFOAO group. After AFOAO intervention, the body weight of the treatment group showed an obvious downward trend since the second week. Further analysis by the area under the curve (AUC), as Figure 1 shown in Figure B: Although the cumulative increase in body weight of mice in the db / db+AFOAO group was lower than that in the db / db group, the difference between groups did not reach statistical significance. The above results indicate that AFOAO has no significant improvement effect on diabetes-related weight gain.

[0083] (2) Effects on fasting blood glucose, random blood glucose and glycated hemoglobin

[0084] Experimental methods

[0085] Measurement of fasting blood glucose: Mice were fasted for 14 h before measurement, 24 h after the last administration. The tail vein blood of mice was measured before treatment, and at 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks of treatment using a blood glucose detector.

[0086] Measurement of random blood glucose: After the last administration, the blood glucose of mice in each group was measured at a randomly selected time using a blood glucose detector.

[0087] Measurement of glycated hemoglobin: After the end of the entire treatment cycle, mouse blood was collected into an anticoagulation tube, and anticoagulated whole blood was collected. 1 ml of blood was taken, centrifuged at 1000×g for 10 minutes, the bottom erythrocytes were collected, and the supernatant plasma was discarded. The erythrocytes were washed twice with 1 ml of precooled PBS, centrifuged at 1000×g for 10 minutes, and the supernatant was discarded. The erythrocytes were then resuspended with 1 mL of precooled PBS and ultrasonically disrupted. Centrifuged at 10000 rpm for 10 minutes, and the supernatant was collected. The glycated hemoglobin level of mice was measured using a glycated hemoglobin test kit. The specific method is as follows,

[0088] 1. Set the standard wells, sample wells, and blank wells and record their positions. To reduce experimental errors, it is recommended to set duplicate wells for the standards and samples.

[0089] 2. Sample addition: Add 100 μL of each gradient standard to the standard wells, add 100 μl of appropriately diluted test samples to the sample wells, and add 100 μL of sample diluent to the blank wells. Cover with a plastic film and incubate at 37 °C for 90 minutes.

[0090] 3. Wash the plate twice: Remove the plastic film, aspirate or shake off the liquid in the enzyme-linked immunosorbent assay (ELISA) plate, and pat it 2-3 times on a clean absorbent paper. Add 350 μL of washing buffer to each well without soaking, discard the liquid in the wells, and pat it 2-3 times on the absorbent paper. Repeat this washing step 2 times.

[0091] 4. Add biotin-antibody working solution: Add 100 μL of biotin-antibody working solution to each well. Cover with a film and incubate statically at 37 °C for 60 minutes.

[0092] 5. Wash the plate 3 times: Remove the film, aspirate or shake off the liquid in the ELISA plate, and pat it 2 - 3 times on a clean absorbent paper. Add 350 μL of washing buffer to each well, soak for 1 minute, discard the liquid in the well, and pat it 2 - 3 times on the absorbent paper. Repeat the washing step 3 times.

[0093] 6. Add HRP-streptavidin (SABC): Add 100 μL of SABC working solution to each well. Cover with a film and incubate statically at 37 °C for 30 minutes.

[0094] 7. Wash the plate 5 times: Remove the film and wash the plate 5 times with washing buffer, referring to step 5 for the method.

[0095] 8. Add TMB chromogenic substrate: Add 90 μL of TMB chromogenic substrate to each well, cover with a film, and incubate statically at 37 °C in the dark for 10 - 20 minutes. Turn on the ELISA reader and preheat it for 15 min.

[0096] 9. Add reaction termination solution: After color development, do not discard the liquid in the well. Add 50 μL of reaction termination solution to each well. The color will immediately change from blue to yellow. The order of adding the termination solution is the same as the order of adding the TMB substrate.

[0097] 10. Measurement of OD value: Immediately read the OD450 value at 450 nm with an ELISA reader.

[0098] Results: As Figure 2 shown in A, the fasting blood glucose of db / db group mice before intervention was significantly higher than that of the normal control group, but there was no statistical difference from the db / db + AFOAO group. After AFOAO intervention, the fasting blood glucose of the treatment group mice showed an obvious downward trend since the 2nd week. Further analysis by the area under the curve (AUC), as Figure 2 shown in B, the cumulative growth of fasting blood glucose in db / db + AFOAO group mice was significantly lower than that in the db / db group, and the result was statistically significant. The results suggest that AFOAO can significantly reduce the fasting blood glucose of diabetic mice. In this example, the random blood glucose and glycated hemoglobin of each group of mice were also detected and found (as Figure 2 shown in C, Figure 2 D), the random blood glucose and glycated hemoglobin of diabetic mice were significantly higher than those of the normal group, and after AFOAO treatment, the random blood glucose and glycated hemoglobin of diabetic mice were significantly reduced. The above results comprehensively show that AFOAO can significantly improve the blood glucose level of diabetic mice.

[0099] (III) Effects on glucose tolerance, insulin tolerance, and insulin resistance index

[0100] Experimental methods

[0101] Glucose tolerance test: Before the experiment, the mice were fasted for 14 h and 24 h after the last administration. After measuring the fasting blood glucose of the mice, a glucose solution prepared with double-distilled water at a dose of 1 g / kg was intragastrically administered (stored at room temperature for 12 h). Subsequently, the tail vein blood of the mice was collected at 15, 30, 60, and 120 min using a blood glucose detector to measure the blood glucose.

[0102] Insulin tolerance test: Before the experiment, the mice were fasted for 14 h and 24 h after the last administration. After measuring the fasting blood glucose, the mice were intraperitoneally injected with an insulin solution prepared with normal saline at a dose of 0.75 U / kg. Subsequently, the tail vein blood of the mice was collected at 15, 30, 60, and 120 min using a blood glucose detector to measure the blood glucose.

[0103] Insulin resistance index (Homeostatic model assessment of insulin resistance, HOMA-IR): After the end of the entire treatment cycle, the mice were fasted for 14 h and 24 h after the last administration. The tail vein blood of the mice was collected using a blood glucose detector to measure the blood glucose. Subsequently, the blood of the mice was collected into an anticoagulant tube and centrifuged at 6,000 rpm for 15 min at 4°C to collect the plasma. Subsequently, the insulin level in the plasma of the mice was measured using an ELISA kit. The specific method is as follows: Add 50 μL of diluted sample or standard product to the sample well; add 50 μL of sample diluent to the blank well; add 100 μL of the corresponding detection antibody labeled with horseradish peroxidase to each sample well, seal the well with a sealing film, and incubate in a 37°C biochemical incubator for 60 min; discard the liquid, pat dry on the absorbent paper, add 250 μL of washing solution to each well, let stand for 1 min, discard the washing solution, and pat dry on the absorbent paper. Repeat the washing of the plate 5 times; add 50 μL of substrate reaction solutions A and B to each well, incubate at 37°C in the dark for 15 min; add 50 μL of termination solution to each well, and measure the absorbance value of each well at a wavelength of 450 nm using a multifunctional microplate reader within 15 min; according to the standard curve simulation equation drawn from the standard product, calculate the insulin concentration in each sample. Insulin resistance coefficient (HOMA-IR) = fasting blood glucose × fasting insulin ÷ 22.5.

[0104] Results: As Figure 3 shown in A, compared with the mice in the normal control group, the blood glucose level of the db / db group mice increased sharply after intragastric administration of 1 g / kg glucose solution and then slowly returned to a level close to that before intragastric administration; while AFOAO treatment could significantly reduce the increase in blood glucose level after glucose intragastric administration in mice and improve the oral glucose tolerance of diabetic mice; Insulin tolerance is another important index for detecting insulin resistance in mice. As Figure 3As shown in Figure B, compared with the normal control group of mice, after the db / db group of mice received an intraperitoneal injection of 0.75 U / kg insulin, the blood glucose level decreased slowly. By 15 min, the blood glucose level reached the lowest point, but it was still higher than the normal blood glucose level. Subsequently, the blood glucose level increased slowly. Compared with the db / db group of mice, the blood glucose levels at each time point in the AFOAO treatment group of mice decreased to a lower level, suggesting that AFOAO can improve insulin tolerance in diabetic mice. The area under the blood glucose curve more intuitively reflected the above results( Figure 3 C). Subsequently, determination of the insulin resistance index in each group of mice found that, compared with the normal control group of mice, the insulin resistance index in the db / db group of mice was significantly increased; while after AFOAO treatment, the insulin resistance index could be significantly decreased( Figure 3 D).

[0105] Example 2: Experiments related to diabetic liver injury

[0106] Experimental method: 3-month-old C57BL / 6J mice were selected as the normal control group (Con), and age-matched db / db mice were used as the diabetic model group. The model group was randomly divided into two groups: the diabetic control group (db / db) and the AFOAO treatment group (db / db + AFOAO). The treatment group was given 50 mg / kg of Alpinia oxyphylla flavonoid extract (AFOAO) by oral gavage every day, and the control group was given an equal volume of pure water for continuous intervention for 12 weeks. The liver was taken for gross photography, and then paraffin sections, HE and Masson staining were performed for analysis.

[0107] Results: As Figure 4 shown in Figure A, compared with the normal group of mice, the liver volume of the db / db group of mice was significantly increased, the color became lighter, showing obvious granularity; while after AFOAO treatment, the above pathological changes could be restored to a certain extent, approaching the normal group. Through HE staining to analyze the liver tissue structure of each group of mice, it was found that compared with the Con group, the hepatocytes of the db / db group of mice showed obvious fatty degeneration (the liver cell nucleus was squeezed to one side, and the cells were vacuolated), and after AFOAO treatment, the fatty degeneration of the liver in db / db mice could be significantly improved (as Figure 4 shown in Figure B). Subsequently, through Masson staining to analyze the liver fibrosis situation, it was found that compared with the Con group of mice, obvious fibrotic lesions occurred near the hepatic lobules in the db / db group of mice, while there were no obvious fibrotic lesions in the AFOAO treatment group. The above results comprehensively showed that AFOAO could significantly improve diabetic-related hepatic fatty degeneration and fibrosis.

[0108] Example 3: Experiments related to diabetic nephropathy

[0109] Experimental method: 3-month-old C57BL / 6J mice were selected as the normal control group (Con), and age-matched db / db mice were used as the diabetic model group. The model group was randomly divided into two groups: the diabetic control group (db / db) and the AFOAO treatment group (db / db+AFOAO). The treatment group was orally administered 50 mg / kg of Alpinia oxyphylla Miq. flavonoid extract (AFOAO) daily, and the control group was given an equal volume of pure water. The intervention lasted for 12 weeks. The kidneys were photographed macroscopically, and then paraffin sections, HE and Masson staining were performed for analysis.

[0110] Results: As Figure 5 shown in Figure A, compared with the normal group of mice, the kidney volume of db / db group mice was significantly increased; compared with the db / db group, the kidney volume of AFOAO treatment group mice showed no significant change. Through HE staining analysis of the renal tissue structure of each group of mice, it was found that compared with the Con group, the glomeruli of db / db group mice showed obvious thickening of the basement membrane and expansion of the mesangial matrix; obvious interstitial damage occurred in the renal tubular area. Compared with db / db group mice, AFOAO treatment could improve the above-mentioned pathology to a certain extent. By evaluating the renal tissue fibrosis pathology of each group of mice through Masson staining, it was found that compared with the Con group, obvious fibrosis lesions appeared in both the glomeruli and renal tubules of db / db group mice; while AFOAO treatment could significantly improve the fibrosis of the glomeruli and renal tubules. The above results comprehensively showed that AFOAO could improve diabetic nephropathy.

[0111] Example 4: Experiments related to diabetic retinopathy

[0112] Experimental method: 3-month-old C57BL / 6J mice were selected as the normal control group (Con), and age-matched db / db mice were used as the diabetic model group. The model group was randomly divided into two groups: the diabetic control group (db / db) and the AFOAO treatment group (db / db+AFOAO). The treatment group was orally administered 50 mg / kg of Alpinia oxyphylla Miq. flavonoid extract (AFOAO) daily, and the control group was given an equal volume of pure water. The intervention lasted for 12 weeks. At the end of the treatment, OCTA examination was performed on the fundus of each group of mice, and paraffin sections and HE staining were performed on the retinal tissue.

[0113] Results: As Figure 6As shown in Figure A, the OCTA results showed that compared with the Con group, there was obvious perfusion deficiency in the superficial blood flow and retinal blood flow of db / db group mice; while after AFOAO treatment, the above-mentioned pathology could be improved. These results indicate that AFOAO can improve the retinal blood flow in diabetic mice, and it can be predicted that it has the effect of reducing retinal ischemic injury caused by diabetes. Subsequently, through HE analysis of the retinal tissue structure of mice in each group, it was found that compared with the Con group, there was obvious loss of ganglion cells in the optic nerve of db / db group mice, the inner nuclear layer and the outer nuclear layer were both thinned, the number of cells in the layer decreased, and the arrangement was disordered; while AFOAO could significantly improve the above-mentioned pathology (such as Figure 6 shown in Figure B). The above results comprehensively indicate that AFOAO can improve diabetic retinopathy.

[0114] Example 5: Experiments related to diabetic cardiomyopathy

[0115] Experimental method: Three-month-old C57BL / 6J mice were selected as the normal control group (Con), and age-matched db / db mice were used as the diabetic model group. The model group was randomly divided into two groups: the diabetic control group (db / db) and the AFOAO treatment group (db / db + AFOAO). The treatment group was given 50 mg / kg of Alpinia oxyphylla flavonoid extract (AFOAO) by oral gavage every day, and the control group was given the same volume of pure water, and the intervention lasted for 12 weeks. At the end of the treatment, paraffin sections and HE staining were performed on the heart tissues of mice in each group.

[0116] Results: As Figure 7 shown, through Masson staining for pathological analysis of cardiac fibrosis in mice in each group, it was found that compared with the Con group, obvious fibrotic lesions appeared in the hearts of db / db group mice; while AFOAO could significantly improve the above-mentioned pathology. The above results comprehensively indicate that AFOAO can improve diabetic myocardial lesions.

[0117] Example 6: Experiments related to diabetic cognitive impairment

[0118] (I) Experimental method: Three-month-old C57BL / 6J mice were selected as the normal control group (Con), and age-matched db / db mice were used as the diabetic cognitive impairment model group (DACD group). The model group was randomly divided into two groups: the diabetic cognitive impairment control group (DACD) and the AFOAO treatment group (DACD + AFOAO). The treatment group was given 50 mg / kg of Alpinia oxyphylla flavonoid extract (AFOAO) by oral gavage every day, and the control group was given the same volume of pure water, and the intervention lasted for 12 weeks. At the end of the treatment, the mice in each group were subjected to a novel object recognition test, and the specific operation was as Figure 8A. Specifically, on the first day of the experiment, the mice were placed in a 30x30x30 cm cubic box for habituation. On the second day, two identical objects were placed in two different corners of the box. The mice were allowed to explore for 10 minutes and then returned to their cages for a retention interval of 0.5 h or 24 h. One of the two identical objects was replaced with a new object for the novel object recognition test. After the retention interval, the animals were returned to the test site for 5 minutes of exploration. Effective exploration was defined as the mouse touching the object with its nose or focusing its attention within less than 1 cm of the object (turning around, climbing, and sitting on the object were all considered ineffective). The exploration time of the old and new objects was scored using the discrimination index DI = (TN - Tf) / (Tn + Tf) x 100%.

[0119] Results: As Figure 8 shown in Figure 8 B and

[0120] (2) Experimental method: Three-month-old C57BL / 6J mice were selected as the normal control group (Con), and age-matched db / db mice were used as the diabetic cognitive impairment model group (DACD group). The model group was randomly divided into two groups: the diabetic cognitive impairment control group (DACD) and the AFOAO treatment group (DACD + AFOAO). The treatment group was orally administered 50 mg / kg of Alpinia oxyphylla flavonoid extract (AFOAO) daily, and the control group was given an equal volume of pure water for continuous intervention for 12 weeks. At the end of the treatment, the Morris water maze test was performed on each group of mice. The specific operation is shown in Figure 9 Figure A. Specifically, the Morris water maze experiment was carried out in a circular pool with a diameter of 120 cm. The water height in the pool was 40 cm, and the water temperature was 23 ± 1°C. White milk powder was used to make the water opaque. The swimming pool was in a laboratory with several visual cues, divided into four equal quadrants. A circular platform with a diameter of 8 cm was placed in the middle of a fixed quadrant of the pool, only 1 cm above the water surface. During the hidden platform training, the mice were trained 5 times a day for 5 consecutive days. Each mouse had to swim for 60 seconds to find the platform. If the mouse did not find the platform within the specified time, it was picked up and placed on the platform for 5 seconds. The interval between each trial was 20 min. On the 6th day, the platform was removed, and all the mice were given 1 exploration trial for 60 s each. If the mouse failed to pass through the platform area, the attention to the platform was defined as 60 seconds. The latency to reach the platform area, the number of times passing through the platform area, and the percentile time spent in different quadrants were recorded and analyzed.

[0121] Results: In the MWM Training (place navigation experiment) test, it was found that DACD+AFOAO mice spent less time navigating to the platform than DACD mice (as shown in Figure 9 Figure B), indicating enhanced spatial learning ability. In the MWM probe (spatial exploration experiment) test, compared with DACD mice, DACD+AFOAO mice had a shorter latency to find the platform (as shown in Figure 9 Figure C) and spent more time in the target quadrant (as shown in Figure 9 Figure D), indicating enhanced spatial memory ability. The above results comprehensively suggest that AFOAO can improve the learning and memory dysfunction of DACD mice to a certain extent.

[0122] (III) Experimental method: Three-month-old C57BL / 6J mice were selected as the normal control group (Con), and age-matched db / db mice were used as the diabetic cognitive impairment model group (DACD group). The model group was randomly divided into two groups: the diabetic cognitive impairment control group (DACD) and the AFOAO treatment group (DACD+AFOAO). The treatment group was orally gavaged with 50 mg / kg of Alpinia oxyphylla Miq. flavonoid extract (AFOAO) daily, and the control group was given an equal volume of pure water. The intervention continued for 12 weeks. At the end of the treatment, the brain tissues of mice in each group were taken for paraffin section, and HE was used to analyze the hippocampal and cortical structures of mice in each group.

[0123] Results: As shown in Figure 10 Figure, the neurons in the hippocampus and cortex of Con group mice were in good morphological condition and synapses were visible; the neurons in the hippocampus and cortex of DACD group mice were shrunken and no synapses were visible; while a small number of synapses were visible in the hippocampal and cortical neurons of the AFOAO treatment group. The above results suggest that AFOAO can protect the synaptic function of neurons.

[0124] Example 7: Experiments related to Parkinson's disease

[0125] (I) Experimental method: Twenty-four 3-month-old C57BL / 6J mice were randomly divided into a Con group (normal control group), a 6-OHDA group (PD model group), and a 6-OHDA+AFOAO group (AFOAO treatment group). Construction of the 6-OHDA model: Using stereotaxic injection technology, unilateral damage to the dorsal striatum of mice was performed at the following coordinates relative to the forebrain and dura mater surface (AP: +0.65 mm; ML: +2.0 mm; DV1: -4 mm; DV2: -3.5 mm). Under pentobarbital sodium anesthesia, a subthreshold dose of 6-OHDA (10 μg of 6-OHDA dissolved in 4 μL of physiological saline containing 0.01% ascorbic acid, 2 μL each time) was injected into the dorsal striatum at a constant speed within 2 minutes, and left for 3 minutes to allow the toxin to diffuse from the injection site before slowly withdrawing the needle. Drug treatment was given on the second day after 6-OHDA injection. Specifically: the AFOAO treatment group (administered 50 mg / kg / day of AFOAO by gavage), and both the Con group and the 6-OHDA group were gavaged with an equal volume of pure water. After 4 weeks of drug treatment, the pole climbing test, cylinder test, rotarod test, and amphetamine rotation test were used to comprehensively evaluate the 6-OHDA model and the therapeutic effect of AFOAO.

[0126] Results: As Figure 11 shown, compared with the Con group, the mice in the 6-OHDA group showed obvious motor dysfunction. The pole climbing test showed that the pole turn latency and total pole climbing time of the mice in the 6-OHDA group were significantly longer than those of the mice in the Con group; the cylinder test showed that the number of times the two front feet of the mice in the 6-OHDA group climbed on the cylinder wall was significantly lower than that of the Con group; the rotarod test showed that the latency to fall of the mice in the 6-OHDA group was significantly shorter than that of the mice in the Con group; the amphetamine rotation test showed that the number of rotations of the mice in the 6-OHDA group towards the damaged side was significantly higher than that of the mice in the Con group. After treatment with AFOAO, the above-mentioned motor dysfunctions of the mice in the 6-OHDA group were significantly improved. The above results suggest that AFOAO can improve the PD-like motor dysfunction induced by 6-OHDA in mice.

[0127] (II) Experimental method: The model construction and treatment were the same as Figure 11 , and at the end of treatment, the substantia nigra and striatum of mice in each group were taken for paraffin section and TH immunohistochemical staining analysis.

[0128] Results: TH is a specific marker for dopaminergic neurons and can reflect the number and status of such neurons. As Figure 12As shown, compared with the Con group, a large amount of TH signal was lost in the substantia nigra and striatum of mice in the 6-OHDA group; while the TH signal in the substantia nigra and striatum of mice in the AFOAO treatment group was significantly enhanced. The results indicate that AFOAO has a protective effect on the damage of dopaminergic neurons in the substantia nigra-striatum pathway induced by 6-OHDA in mice.

[0129] Example 8: Experiments related to pulmonary fibrosis

[0130] Experimental method: Male C57BL / 6J mice (8 weeks old, body weight about 18 g) were randomly divided into 3 groups, with 8 mice in each group, including: normal group (Con), bleomycin group (BLM, 5 mg / kg), and AFOAO administration group (50 mg / kg).

[0131] Establishment of pulmonary fibrosis model and administration protocol: After C57BL / 6J mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (50 mg / kg), the neck skin of the mice was disinfected with 75% alcohol. The skin was incised about 1 cm with sterile surgical instruments, and the muscle was bluntly separated to expose the trachea. 50 μL of bleomycin solution (5 mg / kg) was injected into the trachea to induce pulmonary fibrosis, and mice with pulmonary fibrosis were obtained. In the normal group and the bleomycin group, the mice with pulmonary fibrosis were given intragastric administration of an equal volume of pure water, and the mice in the administration group were given intragastric administration of 50 mg / kg AFOAO. After continuous administration for 21 days, the lung tissues of mice in each group were taken for gross photography, HE, Masson, and staining analysis of fibrosis markers (α-SMA, Collagen I, and Fibronectin).

[0132] Results: The gross results of the lung tissues of mice in each group showed that the lung tissues of mice in the normal group were pink, without edema or lesions. The lung tissues of mice induced by bleomycin showed edema, tissue consolidation, and hemorrhagic transformation. In the AFOAO administration group, the degree of lung injury caused by bleomycin was alleviated (as Figure 13 shown in Figure 13 A); Through the analysis of tissue pathology, the results showed ( Figure 13 B and

[0133] C), after administration of AFOAO, the lung tissue structure was more complete compared with the bleomycin model group, the collagen deposition decreased, the levels of α-SMA and Fibronectin decreased, and the degree of fibrosis was significantly alleviated, indicating that AFOAO can effectively improve bleomycin-induced pulmonary fibrosis. The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, and all should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. Use of a flavonoid extract of Alpinia oxyphylla Miq. in the preparation of a medicament for treating diabetes and its complications, Parkinson's disease and pulmonary fibrosis diseases, characterized in that, The fructus alpiniae oxyphyllae flavone extract includes: Kaempferol, luteolin, phloretin, 7-hydroxy-3-[4-hydroxy-3-(3-methylbut-2-en-1-yl)phenyl]-8-(3-methylbut-2-en-1-yl)-4H-chromen-4-one, catechin, 5,7-dihydroxy-2-phenyl-4H-chromen-4-one, rutin, quercetin-3-O-glucuronide, isokaempferide, trifolin, (2S,3S,4S,5R,6S)-6-{[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yl]oxy}-3,4,5-trihydroxyoxane-2-carboxylic acid, quercetin, genistein, formononetin, biochanin A, vanillin, 5-O-methylgenistein.

2. Use of the flavonoid extract of Alpinia oxyphylla Miq. according to claim 1 in the preparation of a medicament for treating diabetes and its complications, Parkinson's disease and pulmonary fibrosis diseases, characterized in that, The drug is a drug for reducing fasting blood glucose; and / or, the drug is a drug for reducing random blood glucose; and / or, the drug is a drug for reducing glycated hemoglobin.

3. Use of the fructus alpiniae oxyphyllae flavone extract according to claim 1 in the preparation of a medicament for treating diabetes and its complications, Parkinson's disease and pulmonary fibrosis diseases, characterized in that, The drug is a drug for improving oral glucose tolerance; and / or, the drug is a drug for improving insulin tolerance; and / or, the drug is a drug for reducing insulin resistance index.

4. Use of the fructus alpiniae oxyphyllae flavonoid extract according to claim 1 in the preparation of a medicament for treating diabetes and its complications, Parkinson's disease and pulmonary fibrosis diseases, characterized in that, The drug is a drug for improving hepatic steatosis caused by diabetes; and / or, the drug is a drug for improving liver fibrosis caused by diabetes.

5. Use of the flavonoid extract of Alpinia oxyphylla Miq. according to claim 1 in the preparation of a medicament for treating diabetes and its complications, Parkinson's disease and pulmonary fibrosis diseases, characterized in that, The drug is a drug for improving glomerular and tubular fibrosis caused by diabetes; and / or, the drug is a drug for improving glomerular basement membrane thickening and mesangial matrix expansion caused by diabetes; and / or, the drug is a drug for improving tubulointerstitial injury caused by diabetes.

6. Use of the flavonoid extract of Alpinia oxyphylla Miq. according to claim 1 in the preparation of a medicament for treating diabetes and its complications, Parkinson's disease and pulmonary fibrosis diseases, characterized in that, The drug is a drug for improving retinal blood perfusion insufficiency caused by diabetes; and / or, the drug is a drug for improving optic ganglion cell loss, inner nuclear layer and outer nuclear layer cell loss and disordered arrangement caused by diabetes.

7. Use of the semen myristicae flavone extract according to claim 1 in the preparation of a medicament for treating diabetes and its complications, Parkinson's disease and pulmonary fibrosis diseases, characterized in that, The drug is a drug for improving myocardial fibrosis caused by diabetes; 8. Use of the flavone extract of Alpinia oxyphylla Miq. according to claim 1 in the preparation of a medicament for treating diabetes and its complications, Parkinson's disease and pulmonary fibrosis diseases, characterized in that, The drug is a drug for improving cognitive impairment caused by diabetes; and / or, the drug is a drug for improving learning and memory dysfunction caused by diabetes; and / or, the drug is a drug for improving hippocampal and cortical neuron synaptic injury caused by diabetes.

9. Use of the fructus alpiniae oxyphyllae flavone extract according to claim 1 in the preparation of a medicament for treating diabetes and its complications, Parkinson's disease and pulmonary fibrosis diseases, characterized in that, The drug is a drug for improving motor dysfunction; and / or, the drug is a drug for treating dopaminergic neuron injury.

10. Use of the fructus alpiniae oxyphyllae flavone extract according to claim 1 in the preparation of a medicament for treating diabetes and its complications, Parkinson's disease and pulmonary fibrosis diseases, characterized in that, The drug is a drug for treating pulmonary fibrosis.

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