CN111265558A - Application of euscaphis konishii hayata extract in preparation of diabetes drugs - Google Patents
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Abstract
The invention relates to an application of euscaphis konishii hayata extract in preparing a medicament for treating diabetes. The euscaphis konishii hayata extract is obtained by a water extraction method, a type 1 diabetes mouse model is constructed by adopting alloxan induction, a type 2 diabetes mouse model is constructed by adopting high-sugar high-fat feed for feeding and injecting small-dose streptozotocin induction, and the euscaphis konishii hayata extract can effectively reduce the blood sugar concentration of the diabetes mouse model, adjust the serum insulin content, improve the hepatic glycogen content and reduce the serum glycated protein content after being administrated to two types of diabetes mice, and can obviously improve the lipid metabolism disorder of the type 2 diabetes mouse model, so that the euscaphis konishii hayata extract has a good prevention and treatment effect on type 1 and type 2 diabetes, and can be applied to the preparation of medicaments for preventing and treating diabetes.
Description
Technical Field
The invention belongs to the field of extraction and application of plant active substances, and particularly relates to application of a euscaphis konishii hayata extract in preparation of a diabetes drug.
Background
At present, diabetes mellitus is one of the third chronic diseases after cardiovascular diseases and malignant tumors, hyperglycemia caused by insulin secretion deficiency or insulin receptor function impairment easily causes serious pathological changes and dysfunction of multiple organ tissues (such as cardiovascular and cerebrovascular diseases, kidney, nerve, retina and the like) of a human body. Type 1 diabetes is mostly caused by autoimmune system defects, genetic factors, virus infection and the like, and insulin in vivo is seriously insufficient, so that the diabetes is usually treated by directly supplementing insulin clinically; the most remarkable characteristic of type 2 diabetics is that insulin resistance is generated, and peripheral target organs (liver, muscle, adipose tissue and the like) of the diabetics cannot normally take and utilize glucose, so that the functional abnormality and metabolic disorder of the organs of the human body are caused, and even death is caused. At present, the total number of diabetes patients in China is high in the world, the incidence of diseases is in the trend of youth, and a heavy burden is brought to the development of national economy and society.
In recent years, some traditional medicinal plant-derived medicinal components show good blood sugar reducing effects, have the characteristic of small toxic and side effects, and also have good effects on improving the immunity of the organism.
Euscaphis Konishii Hayata (also known as Euscaphis Konishii Hayata) is a plant of genus Euscaphis japonica of family Staphyleaceae, named as chicken gizzard, chicken eye, and green pepper. According to the record of Chinese plant record, there are 2 staphyleaceae Euscaphis japonica plants in China, Euscaphis japonica (Thunb.) Dippel) and Fujian Euscaphis japonica (Euscaphis japonica). The euscaphis konishii hayata is a traditional Chinese herbal medicine and is mainly used for treating stomachache, traumatic injury, rheumatic pain, dizziness, diarrhea, dysentery and the like, while the euscaphis konishii hayata is mainly used for ornamental plants, the research on the medicinal value of the euscaphis konishii hayata is less at present, and the euscaphis konishii hayata are two plants belonging to. Inducing pancreatic injury by adopting alloxan, and establishing a type 1 diabetes mouse model; a mouse model of experimental type 2 diabetes is induced by feeding high-sugar high-fat feed and injecting a small dose of streptozotocin. The euscaphis konishii hayata extract is used for administration to a diabetes model mouse, and is found to be capable of remarkably inhibiting the blood sugar rise of type 1 and type 2 diabetes mice, regulating the serum insulin concentration, improving the liver glycogen content of the mice, reducing the glycated serum protein concentration, regulating the sugar metabolism of the type 1 and type 2 diabetes mice, and providing a new choice for medicines for preventing and treating hyperglycemia.
Disclosure of Invention
The invention aims to provide an application of euscaphis konishii hayata extract in preparing a medicament for treating diabetes. The euscaphis konishii hayata extract with obvious blood sugar reducing effect is obtained from euscaphis konishii hayata plant bodies through a water extraction method and is applied to the preparation of blood sugar reducing medicines.
Application of Euscaphis konishii hayata extract in preparing hypoglycemic medicine is provided.
The preparation method of the euscaphis konishii hayata extract comprises the following steps:
(1) taking euscaphis konishii hayata plant bodies, cutting up, drying at 50 ℃, and crushing to obtain euscaphis konishii hayata plant body powder;
(2) adding 100g of euscaphis konishii hayata plant body powder into 1L of water, boiling, refluxing and extracting for three times, extracting for 2 hours each time, combining extracting solutions, concentrating under reduced pressure to obtain an extract, and freeze-drying the extract to obtain the euscaphis konishii hayata extract.
The euscaphis konishii hayata plant is any one or more of branches, leaves, roots, flowers, seeds, seed coats, fruits and fruit peels of euscaphis konishii hayata.
The invention has the beneficial effects that: the euscaphis konishii hayata extract can obviously inhibit the blood sugar rise of a diabetic mouse, regulate the concentration of serum insulin, improve the content of liver glycogen of the mouse, reduce the concentration of glycated serum protein, regulate the sugar metabolism of type 1 and type 2 diabetic mice, improve the pancreatic pathological changes of the type 1 and type 2 diabetic mice, and can be used for preventing and treating diabetes.
Drawings
FIG. 1 shows the effect of extracts from various parts of Euscaphis konishii on pancreatic tissues of type 1 diabetic mice. a: normal control group, b: model group, c: positive control group, d, pericarp extract group, e: shoot extract group, f: leaf extract group, g: a seed extract group.
FIG. 2 the effect of extract of various parts of Euscaphis konishii on pancreatic tissue of type 2 diabetic mice. a: normal control group, b: model group, c: positive control group, d, pericarp extract group, e: shoot extract group, f: leaf extract group, g: a seed extract group.
Detailed Description
For further disclosure, but not limitation, the present invention is further described in detail below with reference to examples.
The Euscaphis Konishii for the following experiments was derived from Tiantai diagenesis of shorea, fujian, province.
Example 1
Taking dried euscaphis konishii hayata pericarp, chopping, drying at 50 ℃, and crushing to obtain powder. Boiling and reflux-extracting the euscaphis konishii hayata pericarp powder for three times according to the proportion of adding 100g of water into 1L of water, extracting for 2 hours each time, combining extracting solutions, concentrating under reduced pressure to obtain an extract, and freeze-drying the extract to obtain the euscaphis konishii hayata pericarp extract.
Example 2
Taking dried euscaphis konishii hayata branches, chopping, drying at 50 ℃, and crushing to obtain powder. Boiling and reflux-extracting the euscaphis konishii hayata branch powder for three times according to the proportion of adding 100g of water into 1L of water, extracting for 2 hours each time, combining extracting solutions, concentrating under reduced pressure to obtain an extract, and freeze-drying the extract to obtain the euscaphis konishii hayata branch extract.
Example 3
Taking dried euscaphis konishii hayata leaves, cutting up, drying at 50 ℃, and crushing to obtain powder. Boiling and reflux-extracting the euscaphis konishii hayata leaf powder for three times according to the proportion that 100g of euscaphis konishii hayata leaf powder is added into 1L of water, extracting for 2 hours each time, combining extracting solutions, concentrating under reduced pressure to obtain an extract, and freeze-drying the extract to obtain the euscaphis konishii hayata leaf extract.
Example 4
Taking dried euscaphis konishii hayata seeds, cutting up, drying at 50 ℃, and crushing to obtain powder. Boiling and reflux-extracting the euscaphis konishii hayata seed powder for three times according to the proportion that 100g of euscaphis konishii hayata seed powder is added into 1L of water, extracting for 2 hours each time, combining extracting solutions, concentrating under reduced pressure to obtain an extract, and freeze-drying the extract to obtain the euscaphis konishii hayata seed extract.
Example 5 [ pharmacodynamic experiment ]
1 reagent and medicine
The euscaphis konishii hayata extract prepared in the example 1-4 is added into 0.5 wt% of sodium carboxymethyl cellulose solution to prepare 1g/Kg of euscaphis konishii hayata extract suspension for standby. The positive control was metformin hydrochloride, purchased from the great day pharmaceutical group. Model 580 blood glucose test instrument was purchased from Jiangsu Yuejong medical instruments, Inc. The mouse insulin ELISA kit and the glycated serum protein content determination kit are purchased from Shanghai enzyme-linked bioscience, Inc., and the liver glycogen, low-density lipoprotein cholesterol (LDL-C) kit, high-density lipoprotein cholesterol (HDL-C) kit, Total Cholesterol (TC) kit and Triglyceride (TG) kit are purchased from Nanjing institute of bioengineering.
2 method of experiment
2.1 animal model establishment
After one week of adaptive feeding of male Kunming mice, 10 mice are randomly selected as a normal blank group and fed with common feed, the other mice are divided into two groups, one group is fed with common feed, the other group is fed with high-sugar and high-fat feed (lard 15%, sucrose 15%, cholesterol 1%, yolk powder 10%, bile salt 0.2%, and basal feed 58.8%), and after one month, the two groups of mice respectively carry out type 1 and type 2 diabetes mouse modeling.
2.1.11 establishment of diabetes model mouse
After fasting overnight, carrying out intraperitoneal injection by adopting alloxan solution (250mg/kg), after 72h, fasting overnight, cutting the tail and taking blood to measure the blood sugar value of the mouse, wherein the mouse with the blood sugar value being more than or equal to 20mmol/L is determined as the mouse with successful type 1 diabetes molding.
2.1.22 establishment of diabetes model mouse
After fasting overnight, carrying out intraperitoneal injection by using streptozotocin solution (60mg/kg), continuously feeding the mice fed with the high-sugar and high-fat feed for 15 days, taking blood from tail veins to measure the blood sugar value of the mice, wherein the mice with the blood sugar value being more than or equal to 16mmol/L are determined as the mice with successful type 2 diabetes modeling.
2.2 animal experiments
Model groups, positive control groups (metformin 250mg/kg), euscaphis konishii hayata peel extracts, branch extracts, leaf extracts and seed extracts (1g/kg) administration groups are respectively arranged on type 1 and type 2 diabetes mellitus mice which are successfully modeled, and 10 mice are respectively modeled. The normal blank group and type 1 diabetic mice were fed with normal diet, and type 2 diabetic mice were fed with high-sugar and high-fat diet. The administration group of the euscaphis konishii hayata peel extract, the branch extract, the leaf extract and the seed extract and the positive control group are subjected to intragastric administration according to 0.1mL/10g, the normal control group and the model group are subjected to intragastric administration according to 0.1mL/10g, 0.5 wt% sodium carboxymethylcellulose solution is administered once a day, and the blood sugar is measured once a week.
2.3 blood index detection
Fasting is carried out overnight before blood sugar detection, and the fasting blood sugar of the mice is detected by cutting the tail and taking blood in the morning the next day. On the last day of administration, after mice were fasted overnight without water deprivation, the eyes were removed and blood was collected, and the animals were sacrificed by dislocation of cervical vertebrae. The whole blood of the mouse is collected by an EP tube coated by 1 wt% of heparin sodium, after standing for 45min, the blood serum is taken after centrifugation for 10min at 3000 r/min, and the measurement of the blood serum insulin level and the content of the glycated protein is carried out according to the instruction of a kit.
2.4 liver homogenate index detection
After a mouse is dislocated and dies, the liver is taken and washed by ice normal saline, filter paper is used for wiping, 0.1g of the liver is weighed and added with 900mL of ice normal saline to prepare 10% liver homogenate, the liver homogenate is centrifuged at 5000 r/min for 10min, and then clear liquid is taken, and the contents of hepatic glycogen, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and Malondialdehyde (MDA) are measured according to a kit method.
2.5 data processing
Data result in
One-way ANOVA statistical analysis was performed on the experimental data using SPSS 21.0.
2.6 histopathological Studies
Mouse pancreatic tissue was fixed in 10 wt% formalin, washed overnight with running water, dehydrated embedded in a tissue dehydrator (LeicaTP1020), and sectioned and HE stained. The method comprises the following specific steps: (1) and (3) dehydrating: the dehydration was carried out in accordance with 60 vol% ethanol 1h, 70 vol% ethanol 1h, 85 vol% ethanol 2h, 95 vol% ethanol 2h, 100 vol% ethanol I1h and 100 vol% ethanol II 1 h. (2) And (3) transparency: and carrying out transparency on xylene I for 45min and xylene II for 45 min. (3) Embedding: paraffin I1h and Paraffin II were embedded for 1h (56-62 ℃) and then solidified on a cold stage. (4) Slicing and baking: the embedded wax block was sliced with a microtome (Leica RM2235) to a thickness of 5 μm, gently developed with a brush pen in warm water at 50 ℃ and laid on a glass slide, and dried in an incubator at 60 ℃. (5) Dewaxing: the dried slices are placed in dimethylbenzene I and dimethylbenzene II for 10min respectively, absolute ethyl alcohol and 95 vol% ethyl alcohol for 5min respectively, 80 vol% ethyl alcohol for 3min and 70 vol% ethyl alcohol for 3min, and dewaxing is carried out. (6) Dyeing: placing the slices into hematoxylin staining solution for staining for 5min, placing the slices into flowing water for rinsing, placing the slices into 1 vol% hydrochloric acid for differentiation for a few seconds, and performing flowing water for rinsing for 15min to stain cell nuclei; dyeing with eosin dye solution for 1-2min, and washing in running water. (7) Dehydration, transparency and mounting: soaking 80 vol% ethanol, 95 vol% ethanol and absolute ethanol for 3min in sequence for dehydration, then soaking xylene I and xylene II for 5min respectively for transparence, and dropping a drop of resin for mounting. After being dried, the slices can be observed under a microscope.
3, experimental results:
3.1 Effect of Toonae sinensis extract on type 1 diabetes mouse model
Compared with a normal group, blood sugar concentrations of a model group and mice of each administration group are very high at 0d (P is less than 0.01), which indicates that a type 1 diabetes model is successfully constructed, the mice after molding have the symptoms of polydipsia, polyphagia, diuresis and weight reduction, but the symptoms of the mice of the positive metformin group and the mice of the administration group of the euscaphis konishii hayata extract are gradually relieved in the experimental process, and the mice of each group do not die, after 14d, the blood sugar concentration of the mice of each administration group of the euscaphis konishii hayata has obvious reduction (P is less than 0.05), and after 21d, the blood sugar concentration of the mice of the administration group of the euscaphis konishii hayata extract has the same effect as that of the metformin group (P is more than 0.05), thus prompting that the euscaphis konishii hayata extract has better blood sugar reduction effect.
TABLE 1 Effect of Toonae japonica extract on blood glucose in type 1 diabetes model mice: (
n＝10)
Note: comparison with model groups:*P＜0.05，**p is less than 0.01; compared with the normal control group:#P＜0.05，##P＜0.01。
the serum insulin and the liver glycogen content of the model group mice are obviously lower than those of the normal group, while the serum glycated protein concentration is higher than that of the normal group, which reflects that the islet structure of the type 1 diabetic mice is damaged by alloxan to cause sugar metabolism disorder. After the euscaphis konishii hayata extract is administrated, the serum insulin concentration and the liver glycogen content of a diabetic mouse can be obviously improved, and the glycosylated serum protein level is reduced, so that the euscaphis konishii hayata extract can relieve the glycometabolism disorder of a type 1 diabetic mouse.
TABLE 2 Effect of Toona sinensis extract on carbohydrate metabolism in type 1 diabetes model mice: (
n＝10)
Note: comparison with model groups:*P＜0.05，**p is less than 0.01; compared with the normal control group:#P＜0.05，##P＜0.01。
as can be seen from FIG. 1, the pancreatic islets of the mice in the normal control group have normal shapes, regular boundaries, circular or quasi-circular shapes, abundant cytoplasm and clear cell boundaries; the number of pancreatic islets of mice in a alloxan-induced type 1 diabetes model group is reduced, the shape is irregular, the edges are irregular, the boundaries are not clear, and a severe inflammatory infiltration phenomenon occurs; the pancreatic islet forms in the pancreatic tissues of mice of each extract administration group of euscaphis konishii hayata are relatively regular, compared with a model group, the boundaries of exocrine glands and pancreatic islets are relatively clear, the number of cells in the pancreatic islets is increased, the cell arrangement is also improved, and the extract of euscaphis konishii hayata has a protection effect on the pancreatic islet cells of type 1 diabetes mice.
3.2 Effect of Toonae sinensis extract on type 2 diabetes mouse model
Feeding the mice with high-sugar and high-fat feed, and injecting small dose of streptozotocin to cause pancreatic injury to construct type 2 diabetes mellitus mice. Mice were tested for blood glucose and blood lipid levels using euscaphis konishii hayata extract for 4 weeks (see tables 3 and 4).
When the drug is administered for 0d, compared with the normal group, the blood sugar concentration of the mice in the model group and each administration group is obviously increased (P is less than 0.01), and the blood sugar concentration of the model group is maintained at a higher level during the experiment, which indicates that the model of the type 2 diabetes is successfully modeled. After the administration for 21 days, the blood sugar of mice of the administration group of the euscaphis konishii hayata extract has significant difference (P is less than 0.05) compared with that of a model group, and after the administration for 28 days, the blood sugar concentration of the mice of each administration group of the euscaphis konishii hayata has very significant difference (P is less than 0.01) compared with that of the model group, and the result indicates that the euscaphis konishii hayata extract has good blood sugar reducing effect and has a certain aging relation.
TABLE 3 Effect of Toonae japonica extract on blood glucose in type 2 diabetes model mice: (
n＝10)
Note: comparison with model groups:*P＜0.05，**p is less than 0.01; compared with the normal control group:#P＜0.05，##P＜0.01。
serum insulin, glycosylated serum protein and glycogen content of each group of mice are measured, and it is found that the mice in the model group have hyperinsulinemia, the concentration of the glycosylated serum protein is higher than that in the normal group, but the glycogen content is lower than that in the normal group, and the glucose metabolism disorder caused by the pancreatic islet function damage of the type 2 diabetic mice is reflected. After the euscaphis konishii hayata extract is administrated, the serum insulin concentration of a diabetic mouse can be obviously reduced, the hepatic glycogen content is increased, and the glycosylated serum protein level is reduced, so that the euscaphis konishii hayata extract also has a regulating effect on glucose metabolism disorder of a type 2 diabetic mouse.
TABLE 4 Effect of Toona sinensis extract on carbohydrate metabolism in type 2 diabetes model mice: (
n＝10)
Note: comparison with model groups:*P＜0.05，**p is less than 0.01; compared with the normal control group:#P＜0.05，##P＜0.01。
in addition, the content measurement of Total Cholesterol (TC), Triglyceride (TG), high density lipoprotein (HLD-C) and low density lipoprotein (LDL-C) in serum is carried out according to the kit instructions, and the TG, TC and LDL-C of the model group mice are obviously increased (P is less than 0.05 or P is less than 0.01) and HDL-C is obviously reduced compared with the normal group, which indicates that the hyperlipidemia symptom of the type 2 diabetes mice can be caused by the high-sugar high-fat feed. After the euscaphis konishii hayata extracts are administrated, the increase of TG, TC and LDC-C can be obviously inhibited, the HDL-C content (P is less than 0.05 or P is less than 0.01) in serum is increased, and the euscaphis konishii hayata extracts can also effectively improve the lipid metabolism disorder of diabetic mice.
TABLE 5 Effect of Toonae japonica extract on blood lipid of type 2 diabetes model mice: (
n＝10)
Note: comparison with model groups:*P＜0.05，**p is less than 0.01; compared with the normal control group:#P＜0.05，##P＜0.01。
as can be seen from FIG. 2, the pancreatic islets of the mice in the normal control group have normal shapes, regular boundaries, circular or quasi-circular shapes, abundant cytoplasm and clear cell boundaries; the pancreas tissue islet morphology of the STZ combined high-sugar and high-fat induced type 2 diabetes model group mice is irregular, and vacuole and inflammatory infiltration phenomena occur; compared with a model group, the islet morphology of the mice of each administration group of the euscaphis konishii hayata extract is more regular, the phenomena of vacuole and inflammatory infiltration are reduced, and the euscaphis konishii hayata extract has a protection effect on islet cells of the mice with type 2 diabetes.
4 conclusion
The euscaphis konishii hayata extract can obviously inhibit the blood sugar rise of type 1 and type 2 diabetes mellitus mice, improve the liver glycogen content and the serum insulin concentration of the mice, reduce the concentration of glycated serum protein, regulate the carbohydrate metabolism of the type 1 and type 2 diabetes mellitus mice, improve the pancreatic pathological changes of the type 1 and type 2 diabetes mellitus mice, and can be used for preventing and treating diabetes mellitus.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (3)
1. The application of the euscaphis konishii hayata extract is characterized by comprising the application of the euscaphis konishii hayata extract in preparing a medicine for treating diabetes.
2. Use according to claim 1, characterized in that: the preparation method of the euscaphis konishii hayata extract comprises the following steps:
(1) taking euscaphis konishii hayata plant bodies, cutting up, drying at 50 ℃, and crushing to obtain euscaphis konishii hayata plant body powder;
(2) boiling and reflux-extracting the euscaphis konishii hayata plant body powder for three times according to the proportion that 100g of euscaphis konishii hayata plant body powder is added into 1L of water, extracting for 2 hours each time, combining extracting solutions, concentrating under reduced pressure to obtain an extract, and freeze-drying the extract to obtain the euscaphis konishii hayata extract.
3. Use according to claim 2, characterized in that: the euscaphis konishii hayata plant is one or more of branches, leaves, roots, flowers, seeds, seed coats, fruits and fruit peels of euscaphis konishii hayata.
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Application publication date: 20200612 |