CN112716937B - Active ingredient composition with synergistic blood sugar reducing function and preparation method thereof - Google Patents
Active ingredient composition with synergistic blood sugar reducing function and preparation method thereof Download PDFInfo
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- CN112716937B CN112716937B CN202110189076.3A CN202110189076A CN112716937B CN 112716937 B CN112716937 B CN 112716937B CN 202110189076 A CN202110189076 A CN 202110189076A CN 112716937 B CN112716937 B CN 112716937B
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Abstract
The invention relates to an active ingredient composition with a synergistic blood sugar reducing function and a preparation method thereof. The procyanidine B1 and p-coumaric acid extracted from semen Avenae Nudae have synergistic blood sugar lowering activity, and can improve liver glucose metabolism of mice with impaired glucose tolerance, reduce insulin resistance, and improve insulin sensitivity. Meanwhile, the active ingredient combination of the invention does not influence the immune function of mice with impaired glucose tolerance, does not produce hepatotoxicity and does not influence liver metabolism under certain concentration. Therefore, the active ingredient of the invention combined with procyanidine B1 and p-coumaric acid can be used for the development of health food, special medical food and medicaments (or medicament lead compounds) for preventing and treating diseases such as impaired glucose tolerance, type II diabetes and the like, and has good application prospect.
Description
Technical Field
The invention belongs to the field of special medical food and biological medicine, and particularly relates to a composition taking procyanidine B1 and p-coumaric acid as novel active ingredients with a blood sugar reducing function and a preparation method thereof.
Background
Diabetes Mellitus (DM) is a group of metabolic diseases characterized by chronic elevated blood glucose levels, accompanied by disturbances in the metabolism of sugars, fats and proteins due to defective insulin secretion or/and insulin action. WHO-related data indicate that DM is the third place of non-infectious diseases and becomes a major health problem in the 21 st century. Of the current diabetic patients, Type 2 Diabetes mellitis, T2DM, accounts for approximately 90-95% of diabetic patients. T2DM is an abnormal state of carbohydrate metabolism following sugar loading, the pathogenesis of which is mainly related to insulin resistance and islet cell dysfunction. When blood sugar rises, glucose metabolism of insulin-sensitive cells (liver cells, skeletal muscle cells, islet cells and fat cells) is disturbed, insulin secretion of islet cells is promoted, insulin resistance is caused by gradual imbalance of insulin production and consumption, and continuous hyperglycemia and insulin resistance reduce insulin sensitivity of islet cells, so that islet beta cell function is damaged, and T2DM is further initiated. The liver is one of the most important target cells of human glucose metabolism, and glucose metabolism disorder under insulin resistance is mainly manifested by reduction of glucose uptake by liver cells, reduction of liver glycogen synthesis and increase of gluconeogenesis. Therefore, improving hepatic glucose metabolism and islet cell dysfunction are of great significance for preventing and treating occurrence and development of T2 DM.
The drugs currently used for intervention in T2DM are based primarily on three principles: inhibiting glucose absorption by the body (alpha-glucosidase inhibitors), promoting glucose utilization by target cells (metformin) and increasing insulin sensitivity of target cells (thiazolidinedione derivatives). However, the long-term taking of these drugs can cause gastrointestinal discomfort such as abdominal pain, nausea and the like, and may also cause hypoglycemia, the patient compliance is poor, and the safety and effectiveness thereof cause certain limitations on the clinical application thereof. Therefore, the search and research for developing new high-efficiency and low-toxicity hypoglycemic active ingredients have great significance.
More and more studies have shown that bioactive ingredients from natural foods or animals and plants have a positive effect on human health. In recent years, research on functional ingredients with hypoglycemic activity in the diet is receiving more and more attention, and compared with traditional hypoglycemic drugs, the functional active ingredients have the advantages of small toxic and side effects, easy absorption, long-term taking and the like.
The highland barley is an important staple food in Qinghai-Tibet plateau areas in China, and grains of the highland barley have the characteristics of high protein, high fiber, high vitamin, low fat and low sugar. A large number of epidemiological studies prove that the highland barley can play a good role in preventing and treating T2DM when being taken for a long time as dietary intervention. After the study on highland barley steamed bread diet dry, such as plum, rare plum and the like, healthy adults have postprandial blood sugar level and insulin content which are obviously lower than those of the white steamed bread group, so that the effect of reducing blood sugar is achieved. The study of the highland barley diet on the basis of the bear jasmine monarch and the like shows that 227 patients with hyperlipidemia have obvious weight, blood fat, total cholesterol and the like reduction after intervention, which indicates that the highland barley diet intervention can improve lipid metabolism. Compared with oatmeal, the highland barley oatmeal can remarkably improve the glycolipid metabolism of patients with impaired fasting blood sugar, the fasting blood sugar (FBG) and the Insulin (INS) of a subject are respectively reduced by 9.26% and 13.37%, and the Total Cholesterol (TC) and the low-density lipoprotein cholesterol (LDL-C) are respectively reduced by 7.20% and 9.42%. Liu and other researches find that highland barley dietary intervention can effectively reduce the blood sugar level of patients with impaired fasting blood sugar and promote insulin secretion.
In conclusion, the highland barley has good application prospect for improving impaired glucose tolerance and preventing diabetes as dietary intervention. In recent years, many scholars have conducted a series of studies on the active ingredient of highland barley for improving T2DM, and it is considered that soluble polysaccharides, polyphenols, resistant starch, fatty acids, etc. may be contained. However, the research focuses on the hypolipidemic activity, and no report is found on the specific active ingredients capable of directly regulating the carbohydrate metabolism in the highland barley. Among the active ingredients related to the invention, the procyanidine B1 (derived from other plants) has not been reported to be relevant in the aspects of reducing blood sugar and improving impaired glucose tolerance. The reports of coumaric acid (derived from other plants) on reducing blood sugar and improving impaired glucose tolerance focus on achieving the effect of preventing diabetes through reducing blood fat or regulating lipid metabolism, and the reports of reducing blood sugar, improving impaired glucose tolerance and preventing diabetes through regulating carbohydrate metabolism are not available. In addition, the combination of the procyanidine B1 and the p-coumaric acid provided by the invention has no report on the effects of reducing the blood sugar level, promoting the absorption of glucose by liver cells and regulating the glycometabolism in the aspect of liver glycogen synthesis, so that the impaired glucose tolerance is improved and the diabetes is prevented.
Disclosure of Invention
The invention aims to provide an active ingredient composition with a synergistic hypoglycemic function.
Another object is to provide a method for preparing a composition of active ingredients extracted from grains with a synergistic hypoglycemic effect.
Specifically, the active ingredient composition is procyanidin B1 and p-coumaric acid. Preferably, the mass ratio of the procyanidin B1 to the p-coumaric acid in the active ingredient composition is (0.1-5) to (0.1-5), more preferably, the mass ratio is 1: 20. 20: 1. 1:10, 10:1, 1:5, 5:1, 1:2, 2:1 or 1: 1.
Specifically, the active ingredient combination has the synergistic hypoglycemic activity, can improve the hepatic glucose metabolism of mice with impaired glucose tolerance, reduce insulin resistance and improve insulin sensitivity. Meanwhile, the combination of the active ingredients does not influence the immune function of mice with impaired glucose tolerance, does not produce hepatotoxicity and does not influence liver metabolism under certain concentration.
The invention aims to provide a preparation method of an active ingredient composition with a hypoglycemic function, which comprises the following steps:
(1) raw material treatment: weighing dried cereal grains, pulverizing into coarse powder, and putting into an extraction tank;
(2) degreasing treatment: leaching the coarse powder with petroleum ether, collecting the petroleum ether, and volatilizing the petroleum ether from the leached coarse powder for later use;
(3) and (3) ethyl acetate extraction: adding ethyl acetate into coarse powder after petroleum ether leaching and volatilizing, continuously extracting in a constant-temperature water bath, and collecting supernatant;
(4) n-butanol extraction: adding n-butanol into the coarse powder after ethyl acetate extraction, continuously extracting in a constant-temperature water bath, and collecting supernatant;
(5) extracting with 80% ethanol: adding 80% ethanol into the coarse powder after n-butanol extraction, and continuously extracting in constant temperature water bath. Collecting the supernatant;
(6) extracting with 40% ethanol: adding 40% ethanol into the coarse powder after 80% ethanol extraction, continuously extracting in a constant-temperature water bath, and collecting supernatant;
(7) extracting distilled water: adding distilled water into the coarse powder after 40% ethanol extraction, continuously extracting in a constant-temperature water bath, and collecting supernatant;
(8) evaporating solvent from the supernatant obtained by extracting the above solvents, and lyophilizing to obtain extracts, which are sequentially named as A, B, C, D, E, F.
The invention aims to provide a preparation method of an active ingredient composition with a hypoglycemic function, which comprises the following steps:
(1) raw material treatment: weighing 60 kg of dried grain seeds, crushing the weighed grain seeds into 60-mesh coarse powder, and putting the coarse powder into an extraction tank;
(2) degreasing treatment: leaching the coarse powder with petroleum ether for 3-4 times at a ratio of 1:10 (w/v), collecting the petroleum ether, and volatilizing the petroleum ether from the leached coarse powder for later use;
(3) and (3) ethyl acetate extraction: adding ethyl acetate into the coarse powder after petroleum ether leaching and volatilizing at a ratio of 1:10 (w/v), continuously extracting for 2 times in a constant-temperature water bath at 40 ℃ for 4 hours, and combining and collecting supernate;
(4) n-butanol extraction: adding n-butanol into the coarse powder extracted by ethyl acetate at a ratio of 1:10 (w/v), continuously extracting for 2 times in a constant-temperature water bath at 40 ℃ for 4 h, and combining and collecting the supernatant;
(5) extracting with 80% ethanol: adding 80% ethanol into the coarse powder at a ratio of 1:10 (w/v), continuously extracting for 2 times in a constant temperature water bath at 40 deg.C for 4 hr, and mixing to collect supernatant;
(6) extracting with 40% ethanol: adding 80% ethanol-extracted coarse powder into 40% ethanol at a ratio of 1:10 (w/v), continuously extracting for 2 times in constant temperature water bath at 40 deg.C for 4 hr, mixing, and collecting supernatant;
(7) extracting distilled water: adding distilled water into the coarse powder extracted by 40% ethanol at a ratio of 1:10 (w/v), continuously extracting for 2 times in a constant-temperature water bath at 80 ℃ for 4 h, and mixing and collecting supernatant;
(8) evaporating the solvent from the supernatant obtained by extracting the above solvents at a temperature lower than 60 deg.C, and lyophilizing to obtain extracts, which are sequentially named as A, B, C, D, E, F;
the extract B and the extract E are highland barley active ingredient compositions rich in procyanidine B1 and p-coumaric acid respectively.
The cereal grain can be semen Avenae Nudae, herba Avenae Fatuae, fructus Hordei vulgaris, semen Tritici Aestivi, semen glycines, semen Maydis, jowar, herba Avenae Fatuae, semen Fagopyri Esculenti, semen oryzae Sativae, indica Rice, rye, and semen Tribuli.
The grain in the present invention does not mean only seeds of gramineous plants, but may be:
cereal: including rice (indica rice, japonica rice, rice), wheat (wheat, barley, oat, rye), corn, sorghum, millet, yellow rice, buckwheat, etc.;
a soybean protein: including small red beans, kidney beans, and the like;
potatoes: including sweet potato (sweet potato or purple sweet potato), potato, etc.
The grain particles in the invention can also be replaced by other plant source materials, including various fruits, plant source medicinal materials and the like. Can be grape seed, sorghum bran, Photinia serrulata flower, blackcurrant leaf, etc.; or herba Hedyotidis Diffusae and folium Eucommiae.
The invention also aims to provide the application of the composition or the composition prepared by the preparation method in health-care food and special medical food for reducing blood sugar, improving insulin resistance and insulin sensitivity or medicines for reducing blood sugar or improving impaired glucose tolerance taking the composition as a lead.
The invention also aims to provide a health food, a special medical food or a medicament for reducing blood sugar or improving impaired glucose tolerance, which contains the active ingredient composition with the blood sugar reducing function or the composition prepared by the method.
The invention also aims to provide the application of the composition with the active ingredients with the hypoglycemic function in preventing and treating diseases such as impaired glucose tolerance or type II diabetes.
Another object of the present invention is to provide a health food, a special medical food or a medicament for preventing and treating impaired glucose tolerance or type ii diabetes, which comprises the aforementioned composition as an active ingredient, or procyanidin B1 or p-coumaric acid alone as an active ingredient.
It is another object of the present invention to use the aforementioned composition as a health food or medicine in an aqueous suspension, solution or solid state.
The health product or medicine also comprises pharmaceutically acceptable adjuvants.
The auxiliary materials are any one or the combination of at least two of excipient, diluent, carrier, flavoring agent, adhesive and filler.
The invention has the following beneficial effects:
1. provides highland barley active ingredient composition with effects of improving insulin resistance and improving carbohydrate metabolism, namely procyanidin B1 and p-coumaric acid. Compared with the traditional hypoglycemic drugs, the active ingredients of the hypoglycemic drugs have the advantages of small toxic and side effects and higher safety. Therefore, the invention has important significance for treating impaired glucose tolerance and preventing T2DM from forming new medicaments and health-care foods.
2. Provides a preparation process for extracting active ingredients with the blood sugar reducing function from grains. The method has important significance for developing and producing the medicine or health-care food with the function of reducing blood sugar, which contains the procyanidine B1 and the p-coumaric acid as active ingredients.
3. Provides an application of a synergistic hypoglycemic active ingredient composition in preparing medicaments or health-care foods for improving impaired glucose tolerance or preventing T2 DM. Can be used for medicines or health foods developed based on highland barley and having blood sugar lowering effect, and contains the above procyanidin B1 and p-coumaric acid as active ingredients.
4. Provides the application of the active ingredients with the synergistic hypoglycemic activity in preparing medicaments or health-care foods for preventing and treating diseases such as T2DM and the like.
Drawings
FIG. 1 is a flow chart of the preparation process of highland barley active ingredient extract with different polarities
FIG. 2 shows the effect of successive extracts of highland barley of different concentrations on the glucose consumption (A) and glycogen content (B) of insulin resistant HepG2 cells; n: a normal cell group; m: a group of model cells; met. metformin positive control group.
FIG. 3 shows that the continuous extract B of highland barley and E synergistically regulate the sugar metabolism of HepG 2. (A) Index of synergy of extracts B to F on glucose consumption by insulin resistant HepG2 cells; (B) the combination of extract B and E had an effect on insulin resistance against HepG2 cell sugar consumption; (C) the combination of extract B and E has the effect on the glycogen content of insulin resistant HepG2 cells; (D) the effect of extract B in combination with E on the rate-limiting enzyme activity of insulin resistance HepG2 cell carbohydrate metabolism.
FIG. 4 identification of compound B from continuous extract of highland barley. (A) A TIC total ion flow diagram in a negative ion mode; (B) compound 1 MS chromatogram; (C) MS/MS chromatogram of compound 1; (D) MS chromatogram of compound 2; (E) MS of Compound 2/MS chromatogram.
FIG. 5 Proanthocyanidins B1, effect of linoleic acid and extract E isolated fractions on glucose consumption (A) and glycogen content (B) in insulin resistant HepG2 cells.
FIG. 6 isolation and identification of active ingredient E-1 compound. (A) A Sephadex LH-20 chromatographic column separation diagram; (B) an E-1 component TIC total ion flow diagram in a negative ion mode; (C) an HPLC profile of the E-1 component; (D) a mass spectrum of the E-1 component (MS (-))); (E) secondary mass spectrum of E-1 component (MS/MS (-)).
FIG. 7 the effect of procyanidin B1 on insulin resistance HepG2 cell sugar consumption (A) and glycogen content (B) before and after combination with p-coumaric acid.
FIG. 8 Effect of procyanidin B1 in combination with Paracoumaric acid on sugar metabolism rate-limiting enzyme Activity
FIG. 9 Effect of procyanidin B1 and p-coumaric acid on expression of proteins of carbohydrate metabolism pathway PI3K/Akt/GSK-3 beta, AMPK/GLUT2 and AMPK/PCK1 in insulin resistant HepG2 cells.
Figure 10 effects of procyanidin B1 on coumaric acid on KM mice with impaired glucose tolerance. (A) Oral glucose tolerance (OGTT) and insulin tolerance (IPITT) tests; (B) measuring the content of glycogen in liver, the content of insulin in pancreas and the activity of rate-limiting enzyme of carbohydrate metabolism in liver of the mouse; NC: normal group mice; MC: model group mice; met mice from the metformin gavage group.
FIG. 11H & E staining results of procyanidin B1 and KM mice with impaired glucose tolerance under the action of coumaric acid on liver (A) and pancreas (B).
Detailed Description
The invention discloses an active ingredient composition with a synergistic hypoglycemic function and a preparation method thereof, and a person skilled in the art can use the contents to appropriately improve process parameters for realization.
It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention.
While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Example 1 preparation of hypoglycemic active ingredient in highland barley
(1) Raw material treatment: weighing 60 kg of dried highland barley seeds, crushing the highland barley seeds into coarse powder of 60 meshes, and putting the coarse powder into an extraction tank;
(2) extraction: firstly, leaching coarse powder for 3-4 times by using petroleum ether at a ratio of 1:10 (w/v), and volatilizing the petroleum ether from the leached highland barley coarse powder for degreasing. Removing petroleum ether from the petroleum ether extract and drying to obtain an extract A.
And secondly, continuously extracting the degreased and volatilized highland barley powder by respectively adopting ethyl acetate, n-butyl alcohol, 80% ethanol, 40% ethanol and distilled water.
The specific extraction process comprises the following steps:
a) adding ethyl acetate into the highland barley coarse powder after being rinsed and volatilized by petroleum ether at a ratio of 1:10 (w/v), continuously extracting for 2 times in a constant-temperature water bath at 40 ℃, combining and collecting supernate, evaporating and volatilizing a solvent in a rotary manner, and freeze-drying to obtain an extract B;
b) adding n-butanol into the coarse powder of highland barley extracted by ethyl acetate at a ratio of 1:10 (w/v), continuously extracting for 4 h in a constant-temperature water bath at 40 ℃, extracting for 2 times, combining and collecting supernate, evaporating to remove solvent in a rotary manner, and freeze-drying to obtain an extract C;
c) adding 80% ethanol into the highland barley coarse powder extracted by n-butanol at a ratio of 1:10 (w/v), continuously extracting for 2 times in a constant-temperature water bath at 40 ℃ for 4 h, combining and collecting supernate, evaporating to remove solvent, and freeze-drying to obtain an extract D;
d) adding 80% ethanol-extracted highland barley coarse powder into 40% ethanol at a ratio of 1:10 (w/v), continuously extracting for 4 h in a constant-temperature water bath at 40 deg.C for 2 times, mixing and collecting supernatant, evaporating solvent, and freeze-drying to obtain extract E;
e) adding distilled water into the highland barley coarse powder extracted by 40% ethanol at a ratio of 1:10 (w/v), continuously extracting for 2 times in a constant-temperature water bath at 80 ℃ for 4 h, combining and collecting supernate, evaporating the solvent in a rotary manner, and freeze-drying to obtain an extract F.
Wherein the ethyl acetate extract and the 40% ethanol extract are highland barley crude extracts rich in procyanidine B1 and p-coumaric acid, and the extraction rates are 0.66% and 1.01%, respectively. The extraction process is shown in FIG. 1.
Example 2 Single component assay for modulating glucose metabolism in insulin resistant HepG2 cells
Selecting human liver cancer cell line HepG2 to culture in DMEM medium (DMEM complete medium for short) containing 10% fetal calf serum, 100U/mL penicillin and 100 mug/mL penicillin, and 5% CO at 37 ℃2And culturing in an incubator with relative humidity of 90%.
HepG2 cells in logarithmic growth phase were taken at a density of 3.5X 104and/mL, inoculating the cells into a 6-well plate, removing the old culture medium after the cells are attached to the wall, and adding the cells into a newly prepared serum-free DMEM culture medium for starvation culture for 12 hours. Cells were randomly divided into a blank control group and a model group.
Model group cells were induced with 1.0. mu.M insulin for 24 h to construct a HepG2 insulin resistance model. The model group cells were then divided into sample treatment groups: continuously extracting components (25 mug/mL, 50 mug/mL and 100 mug/mL) from highland barley with various concentrations; the control group (serum-free DMEM medium) and the positive control group (1.0 mM metformin) were incubated for 24 h, and the glucose consumption and glycogen content of the cells were measured using the kit.
The effect of component compatibility on cell sugar consumption is evaluated by a Chou-Talalay combined index method. The Chou-Talalay combined index method is a simple, accurate and comprehensive method for quantitatively analyzing drug interaction. According to the equation of middle efficiency fa/fu = (D/D)m)mAfter taking the logarithm of both sides, the logarithm of both sides is given by y = log (fa/fu), x = log D, a = -mlogDmB = m, yielding the regression line equation y = bx + a. The formula can calculate the effective dose of the two medicines used singly and in combination, and the required dose when different effects are generated, namely the effective mapping method.
Calculating the Combination Index (CI) of two drugs1/Dx1+ D2/Dx2+α·(D1·D2)/(Dx1·Dx2) Corresponding table 1 is as follows:
TABLE 1 Combined index CI vs. interaction
As shown in fig. 2, extract a showed no significant change in cellular sugar consumption with increasing concentration, and glycogen content was only significantly increased at 100 μ g/ml with an increase of less than 20% with treatment with different concentrations of successive extracts a-F. Extract C showed a significant increase in cellular sugar consumption and glycogen content (P) only at a concentration of 100. mu.g/ml<0.01) showed no dose-dependent effect and had less than 20% amplification of sugar consumption. Sugar consumption and glycogen content in insulin resistant HepG2 cells with increasing concentration in the successive extracts B, D, E, F treatment: (P <0.01), and exhibits a dose-dependent effect. In conclusion, the continuous highland barley extracts B, D, E and F can obviously promote the sugar consumption and glycogen content of the insulin resistant HepG2 cells.
Example 3 synergistic component screening for modulation of glucose metabolism in insulin resistant HepG2 cells
Pairwise matching the components B, D, E and F of the highland barley continuous extracts screened in the experiment (B: D: E: F = 1: 2: 1: 4), and evaluating the interaction among various components by using a Chou-Talalay combined index method based on the glucose consumption index of the highland barley continuous extracts to insulin resistance HepG2 cells.
As shown in the results of FIG. 3A, the CI values of the B + E combination at all concentrations are less than 1 (CI: 0.66-0.70), and the B + E combination shows synergistic effect at all concentrations by comparing the synergy index tables. Meanwhile, the effective value is calculated to be 50 mug/ml. The glucose consumption values in the insulin resistance HepG2 cells before and after the B + E combination are determined under the medium-effective value concentration and the maximum concentration, and the results show that the glucose consumption values in the B + E combination insulin resistance HepG2 cells are larger than a single component under the two concentrations (figure 3B) and show very significant difference (theP <0.01), which shows that the B + E combination can remarkably improve the glucose absorption and utilization of insulin resistant HepG2 cells, and shows the synergistic effect of improving the glucose absorption. In addition, intracellular glycogen content values results show (FIG. 3C) that glycogen content in insulin resistant HepG2 cells at both concentrations after B + E combination was higher than the average of the effects of the two individual components before combination (glycogen content)B+ E>1/2 glycogen contentB+1/2 glycogen contentE) And show significant differences: (P <0.05), which shows that the increase of glycogen content of the B + E combination in insulin resistant HepG2 cells is obviously stronger than the average value of the combination under the same concentration, and the B + E combination shows synergistic effect on promoting the increase of glycogen content of the cells. Taken together, the B + E combination showed a synergistic effect in promoting both insulin resistance HepG2 cell sugar consumption and intracellular glycogen synthesis with a median effect value of 50. mu.g/ml. .
In addition, a rate-limiting enzyme for sugar metabolism, a rate-limiting enzyme for glycogen synthesis: glycogen synthase (GCS), glycogen degradation rate-limiting enzyme: glycogen phosphorylase a (gpa), gluconeogenesis rate-limiting enzyme (PEPCK) phosphoenolpyruvate carboxylase: the results show (fig. 3D) that GCS activity in cells after combination is significantly higher than any single component: (P <0.01), the activity of GPa is obviously lower than that of the single component (a)P <0.05) without significant change in PEPCK activity. The composition is shown to synergistically regulate the glucose metabolism of insulin resistant HepG2 cells by promoting the sugar consumption and the glycogen synthesis.
EXAMPLE 4 identification of synergistic Components active Compounds
Identifying the types of the active component compounds by using a UPLC-MS/MS method, wherein the chromatographic conditions are as follows: a Nexera UHPLC-30A system adopts a Waters HSS T3 chromatographic column (150 mm multiplied by 3 mm, 1.8 mu m) and the mobile phase is acetonitrile (A) -0.1% acetic acid water solution (B); gradient elution: 0-15 min, 50% A, 10-13 min, 95% A, 13-15 min, 0% A. The flow rate was 0.3 mL/min. Mass spectrum conditions: tripleTOF5600 + AB SCIEX ™ mass spectrometer, run in negative ion mode (ESI, mz 100-.
The electrospray capillary voltage, the declustering voltage, the collision energy and the six polarity voltages were 4500V (negative), 60V, 35V and 15V, respectively. The ion source and capillary temperatures were set at 110 ℃ and 500 ℃, respectively. The flow rates of the atomizer and the desolventizing gas were set to 60L/h and 50L/h, respectively, for nitrogen and hydrogen.
And (3) comparing the UPLC-MS mass spectrogram obtained by artificial spectrogram analysis in combination with the mass spectrogram of a Respect GNPS library, and selecting a compound with the similarity higher than 80% in the mass spectrogram library to qualitatively determine the type of the compound. The relative percentages of compounds were determined from data in the total ion flow graph.
As shown in fig. 4, the B component has a better response to the negative ion detection of the mass spectrum, and the corresponding mass spectrogram results are extracted from the peak with the highest response value in the total ion flow diagram, which shows that 2 m/z molecules are obtained and identified as 577 and 289 two molecular ions (fig. 4A), respectively, and the compounds are identified as procyanidin B1 and linoleic acid based on the matching of molecular mass and a database. And further performing mass spectrum fragmentation secondary mass spectrum on the two molecular ion peaks, wherein characteristic fragment ions obtained by fragmentation of ion fragments of the compound 1 at m/z 577 are MS/MS 451, 425, 407 and 289, wherein the fragment ions MS/MS 451 are obtained by fission of a heterocycle to lose 1,3, 5-trihydroxybenzene structures, the relative molecular mass difference is 126 Da, and the fragment ions MS/MS 425 and 407 are respectively obtained by further fragmentation of the heterocycle to lose H2Characteristic fragment ions of O. In addition, fragment ions of MS/MS 289 are catechin structure characteristic units, and compared with related documents, the fragment ions are characteristic fragments obtained by cracking procyanidine B1 under a negative ion mode, and the compound can be identified as procyanidine B1 based on the results. In addition, Compound 2 is at m/z 279The ion fragments are cleaved to give characteristic fragment ions of MS/MS 148, 190 and 238, and the cleavage characteristics can be identified as linoleic acid, again as can be seen in comparison to the relevant references. The content of procyanidin B1 was determined to be 14.80% by peak area comparison.
The E fraction was separated by Sephadex LH-20 column chromatography and 3 fractions were collected in total (FIG. 6A; i.e., E-1, E-2, E-3). The B fraction was taken to identify the effect of the isolated fractions of compounds Procyanidin B1 (PB), Linoleic Acid (LA) and E on glucose consumption and glycogen content in insulin resistant HepG2 cells (FIG. 5). Each component can obviously promote the consumption of cell sugar and the content of glycogen (P<0.01) wherein procyanidin B1 and E-1 fractions were significantly increased in insulin resistance HepG2 cell sugar consumption and glycogen content compared to fractions B and E, respectively (P <0.05), indicating that both are active ingredients in the corresponding components B and E-1, respectively.
Further identifying the type of the compound of the E-1 component by using UPLC-MS/MS, wherein the E-1 component has better response to the negative ion detection of mass spectrum, selecting the peak with the highest response value in the total ion flow diagram, and the corresponding mass spectrum result shows that 1 molecular ion with m/z of 164 is obtained (figures 6B and D), and characteristic fragment ions of the molecular ion are obtained by cracking at m/z 164 as MS/MS147 and 119 (figure 6E), and the compound is identified as p-coumaric acid by the comparison literature result. The relative purity of p-coumaric acid in the E-1 component was 90.64% based on peak area comparison (FIG. 6C). In conclusion, the active compounds in highland barley which have the function of synergistically improving insulin resistance HepG2 cell carbohydrate metabolism are procyanidine B1 and p-coumaric acid.
Example 5 the Effect of procyanidin B1 in combination with p-coumaric acid in synergistically promoting insulin resistance HepG2 cell carbohydrate consumption and intracellular glycogen content
Based on the separated and identified active ingredients, the invention further measures the sugar consumption and the glycogen content of the insulin resistant HepG2 cells before and after the combination of the active ingredients, thereby verifying the synergistic effect of the active ingredients. As shown in fig. 7 (a), at two concentration doses, Procyanidin B1 (PB) was combined with p-Coumaric Acid (CA) with significantly higher sugar consumption in the cells than the single component: (a)P <0.01) indicating that the combined use of the two can synergistically promote insulin resistance HepG2 cellsSugar consumption of (2). The experimental results further verify that procyanidin B1 and p-coumaric acid are the active ingredients of components B and E, respectively. In addition, as shown in fig. 7 (B), cellular glycogen content was significantly higher at the same concentration after PB and CA combination at both concentration doses than the single component: (B)P <0.05), exhibit a synergistic effect in promoting intracellular glycogen synthesis. In conclusion, the results further verify that the procyanidine B1 and the p-coumaric acid composition have the effect of synergistically improving insulin resistance HepG2 cell sugar consumption.
Example 6 Proanthocyanidins B1 acting in synergistic modulation of insulin resistance HepG2 cellular carbohydrate metabolism with p-coumaric acid
To further verify the effect of the composition on glucose metabolism pathway of insulin resistant HepG2 cells, the glucose metabolism rate-limiting enzyme activity of insulin resistant HepG2 cells under the intervention effect of the composition is determined, and the result is shown in FIG. 8, the GCS activity in the cells after the composition stem is remarkably increased compared with the single component intervention (the single component intervention)P <0.01), the activity of GPa is obviously reduced by (P <0.01), the PEPCK enzyme activity has no significant difference, which indicates that the combination of the procyanidin B1 and the p-coumaric acid can synergistically promote the glucose consumption and the cellular glycogen synthesis.
In addition, Western blot was used to determine the expression of proteins in the signaling pathways corresponding to glucose consumption, glycogen synthesis and gluconeogenesis pathways, AMPK/GLUT2, PI3K/Akt/GSK-3 β and AMPK/PCK1, in insulin resistant HepG2 cells treated with the above combinations (FIG. 9). The result shows that the expression quantity of glycogen synthesis signal pathway node protein PI3K and Akt in cells under the combined intervention of procyanidine B1 and p-coumaric acid is obviously increased compared with the average expression quantity of single components (the expression quantity of each component is the average value of the expression quantity of each component)P <0.01), the GSK-3 β expression was significantly reduced compared to the mean of the expression of the individual components (fig. 9A;P < 0.05）。
in addition, the expression level of the gluconeogenesis signal pathway node protein PCK1 after combination does not exceed the expression level of the protein when the protein acts alone, and the synergistic effect of the combination is shown to mainly influence the pathway not to be the gluconeogenesis effect. The PB and CA combination showed a greater protein expression level in cells than AMPK and GLUT2 alone (2 × relative grayscale value PB + CA/(relative grayscale value PB + relative grayscale value CA) > 1) (fig. 9B). Therefore, the composition realizes synergistic improvement of HepG2 insulin resistance against cellular sugar metabolism by remarkably up-regulating protein expression of an AMPK/GLUT2 node of a sugar consumption signal pathway and protein expression of a PI3K/Akt/GSK-3 beta node of a glycogen synthesis signal pathway.
Example 7 Effect of procyanidin B1 and p-coumaric acid and composition thereof on improving impaired glucose tolerance in KM mice
Males, 115 KM mice at 5 weeks of age, were taken and after 1 week of adaptive feeding, were randomly divided into normal and model groups according to body weight. Normal group is fed with common feed, model group is fed with high-sugar and high-fat feed, and free feeding is adopted. Model group mice were induced with high-glucose and high-lipid diet for 4 weeks and then further induced to develop impaired glucose tolerance by a single injection of 30 mg/kg bw of low dose STZ, fasted (without water deprivation) for 12 hours after one week, and fasting blood glucose and random oral glucose tolerance (OGTT) of the mice were measured.
Selecting fasting blood glucose of mice<7.0 mmol/L, 7.8-11.1 mmol/L of blood sugar 2 h after meal, and the area AUC under the OGTT curve is obviously different from that of the normal group (1:)P <0.05) are impaired glucose tolerance mice. Animals were then randomly assigned to 4 groups based on blood glucose values: the test method comprises the following steps of (1) a negative control group (a model group), active ingredients (procyanidine B1, p-coumaric acid and procyanidine B1+ p-coumaric acid; the low dose of 100 mg/kg b.w. and the high dose of 300 mg/kg b.w. are respectively set), a positive control group (200 mg/kg b.w. metformin) and 8 animals in each group, wherein the normal mice randomly selected before modeling are used as a blank control group, the stomach is continuously perfused for 4 weeks, and after the stomach perfusion is finished, oral glucose tolerance (OGTT) and insulin tolerance (IPITT) experiments are respectively carried out.
The fasting blood glucose and postprandial blood glucose values of the mice were measured. The mouse is anesthetized by using excessive pentobarbital sodium, then sacrifice is carried out, serum is taken, an ELISA kit is used for measuring the content of insulin in the serum, the content of TG is measured by a GPO-PAP method, the content of TC is measured by a CHOD-PAP method, the content of LDL-C and HDL-C is measured by a clearing method, and the activities of glutamic-pyruvic transaminase and glutamic oxalacetic transaminase are measured by a rate method. The liver, thymus and spleen of the mice are taken to measure the liver index, thymus index and spleen index. The kit method is used for measuring the content of glycogen in the liver, the content of insulin in pancreas and the activity of rate-limiting enzyme of carbohydrate metabolism in the liver. HE staining was selected for histological examination of liver and pancreas.
As shown in the following Table 2, the weight average of the groups with different dosages of each active ingredient after the completion of the gavage is smaller than that of the mice in the model group, and the differences are significant. Wherein the mice with Procyanidin B1 (PB) high dose group showed the greatest weight loss. In addition, the weight of mice in the composition-treated group at a low dose was lower than that in the single-component-treated group, and a synergistic effect of reducing the weight of mice was exhibited. In addition, the thymus index and spleen index of the mice in the MC group are obviously lower than those of the mice in the NC group, which shows that the immunologic function of the mice with impaired glucose tolerance is reduced, and the thymus index of the active ingredients is increased to different degrees after gastric lavage, which shows that the immunologic function of the mice with impaired glucose tolerance can be enhanced to a certain degree.
As shown in fig. 10A below, the area under the AUC curve corresponding to oral glucose tolerance (OGTT) of the mice after gastric lavage with each active ingredient is significantly reduced compared with the MC group of mice, wherein the blood glucose value of the PB group of mice is reduced by the largest extent, and there is no significant difference compared with the NC group of mice, which indicates that the proanthocyanidin B1 can reverse the impaired oral glucose tolerance of the mice. Furthermore, there was a significant reduction in the area under the AUC curve after PB and CA combination compared to the pre-combination mean (P <0.01) indicating that the combination can synergistically improve the impaired oral glucose tolerance of mice, wherein the area of the OGTT AUC of the mice under the intragastric administration of the PB + CA combination under high dose has no significant difference compared with the mice of an NC group, indicating that the combination can synergistically improve the impaired oral glucose tolerance of the mice under the high dose and shows a reversal effect.
The area under the AUC curve corresponding to oral insulin tolerance (IPITT) of mice after gastric lavage of each active component is obviously reduced (P <0.01), indicating that each intervention group can improve the impaired insulin tolerance of the mice with impaired glucose tolerance. The AUC area corresponding to mouse IPITT after the combination of PB and CA is obviously lower than that of the CA group, but compared with the PB group, the AUC area is not obviously different, and no synergistic effect is shown.
The fasting blood sugar (FBG) and the postprandial blood sugar (PBG) of the mice with each active component under gavage are obviously reducedP <0.01), indicating that each intervention group was able to significantly reduce the blood glucose in IGT mice (fig. 10B). FBG index results show that the PB group fasting blood glucose under high doseThe values were not significantly different compared to the NC group mice, indicating that PB reverses fasting glucose to normal levels in IGT mice. The FBG values of mice in the high dose group intervention showed no synergy as indicated, but also reached normal levels.
The PBG index result shows that the postprandial blood glucose values of the PB and CA groups have no obvious difference compared with the NC group mice under high dose, which indicates that the PBG index result can reverse the postprandial blood glucose of the IGT mice to a normal level. Significant reduction of PB + CA composition compared to single component PBG (a)P <0.05) and no significant difference in the high dose group compared to the NC group mice, indicating that the combination shows a synergistic effect in lowering postprandial blood glucose and can reverse postprandial blood glucose to normal levels at high doses.
From the serum insulin content, each intervention group was able to significantly reduce the insulin content. Especially, compared with a single component, the PB + CA composition can obviously improve the insulin content under two doses, and shows obvious synergistic effect, so that the normal insulin level is achieved.
In conclusion, the procyanidine B1 and the p-coumaric acid can improve the impaired oral glucose tolerance and the impaired oral insulin tolerance of IGT mice, and have a remarkable effect on reducing the blood glucose level of the mice. Wherein procyanidin B1 has strong effect in reversing fasting blood glucose level, and has strong effect in lowering insulin level on coumaric acid. Compared with single component, the combined high-dose group comprehensively shows synergistic effect in the aspects of improving oral glucose tolerance and regulating postprandial blood glucose level, and can reverse insulin resistance to reach normal blood glucose and insulin level.
Glycogen content in mouse liver was significantly increased after gavage of each active ingredient (fig. 10C;P <0.01) and the content of hepatic glycogen is obviously higher than that of each single component after the composition intervenes, and the synergistic effect is shown. Meanwhile, after the intervention of the composition group, the GCS activity of the liver of a mouse is obviously increased, the GPa activity is obviously reduced, and the PEPCK activity is obviously reduced (P <0.01). The GCS enzyme activity in the liver of the mouse in the composition group is obviously higher than that of the single component intervention group, the GPa enzyme activity is obviously lower than that of the single component intervention group, and the result is consistent with the result of the early cell experiment. Indicating procyanidinsB1 and p-coumaric acid achieve the effect of improving the glucose metabolism of mice with impaired glucose tolerance by synergistically promoting blood glucose reduction and liver glycogen synthesis.
As shown in FIG. 10B, the serum lipid metabolism indices (TC, TG, LDL-C, HDL-C) of the mice were improved to different degrees after the gavage of each active ingredient. The serum TC content of the PB, CA and combined mice under high dose is not obviously different compared with that of NC mice, which indicates that the PB, CA and combined mice can reverse the normal level. Meanwhile, the serum TG content of the mice is remarkably reduced after the gavage of each active component, wherein the serum TG content of the mice of the PB group and the composition group is not remarkably different from that of the mice of the NC group, which indicates that the PB group and the composition group can reverse TG to normal level. In addition, the serum LDL-C content of the mice under the gastric lavage of each active component is also obviously reduced (P <0.01), HDL-C content is significantly increased: (P <0.01). In addition, the liver index of the mice decreased after gavage of each active ingredient, showing various degrees of relief of liver damage (table 2). In conclusion, the procyanidine B1 and the p-coumaric acid can not only remarkably regulate the carbohydrate metabolism of IGT-damaged mice, but also have remarkable regulation effect on lipid metabolism. Wherein procyanidin B1 has stronger action than p-coumaric acid, and the composition shows synergistic effect in down-regulating TC, TG and LDL-C indexes.
TABLE 2 procyanidin B1 and Effect of p-coumaric acid on body weight, organ index and activity of aspartate Aminotransferase (AST) and glutamate-pyruvate transaminase (ALT) in mice with impaired glucose tolerance
HE staining results showed (fig. 11A) that the MC group of mouse hepatocytes exhibited diffuse steatosis and were cytosolic filled with round lipid droplets of varying sizes and ring shapes. The active ingredients in the intervention group of mice have normal hepatocyte structures, lipid drops are occasionally seen in cytoplasm, and the intervention group of mice has a dose-dependent effect, wherein the procyanidine B1 high-dose group has the most obvious effect, and the liver lipid accumulation degree of the mice in the composition intervention group is less than that of the mice in the single-component intervention group.
The insulin content in the pancreas of mice in each intervention group was significantly higher than that of mice in the MC group (fig. 10C;P <0.01) the effect of the composition intervention group is stronger than that of a PB group and weaker than that of a CA group, and after statistical analysis of data, the content of pancreatic insulin of mice in the composition intervention group has no significant difference compared with the average value of 2 single-component intervention groups, and the composition intervention group can play a role in addition.
HE staining results showed (fig. 11B) that the distribution of islets in the MC group was indistinct from the pancreatic perimeter, some of the islet cells were visually impaired, and the cell morphology was spindle or irregular. The islet cell structure of the mice in each active ingredient intervention group is improved compared with that in the model group, and the composition intervention group has a dose-dependent effect. In conclusion, the procyanidine B1 and p-coumaric acid show a synergistic improvement effect on hyperinsulinemia of mice with impaired glucose tolerance besides the hypoglycemic activity.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. The preparation method of the active ingredient composition with the blood sugar reducing function is characterized by comprising the following steps:
(1) raw material treatment: weighing 60 kg of dried grain particles, crushing into 60-mesh coarse powder, and putting into an extraction tank;
(2) degreasing treatment: leaching the coarse powder for 3-4 times by using petroleum ether at a ratio of 1:10 (w/v), and volatilizing the petroleum ether from the leached highland barley coarse powder for later use; the petroleum ether extract is A;
(3) and (3) ethyl acetate extraction: adding ethyl acetate into the coarse powder after petroleum ether leaching and volatilizing at a ratio of 1:10 (w/v), continuously extracting for 2 times in a constant-temperature water bath at 40 ℃ for 4 hours, and merging and collecting supernatant B;
(4) n-butanol extraction: adding n-butanol into the coarse powder extracted by ethyl acetate at a ratio of 1:10 (w/v), continuously extracting for 2 times in a constant-temperature water bath at 40 ℃ for 4 h, and combining and collecting supernatant C;
(5) extracting with 80% ethanol: adding 80% ethanol into the coarse powder with a ratio of 1:10 (w/v), continuously extracting for 2 times in a constant temperature water bath at 40 deg.C for 4 h, and mixing to collect supernatant D;
(6) extracting with 40% ethanol: adding 80% ethanol-extracted coarse powder into 40% ethanol at a ratio of 1:10 (w/v), continuously extracting for 2 times in constant temperature water bath at 40 deg.C for 4 hr, mixing, and collecting supernatant E;
(7) extracting distilled water: adding distilled water into the coarse powder extracted by 40% ethanol at a ratio of 1:10 (w/v), continuously extracting for 2 times in a constant-temperature water bath at 40 ℃ for 4 h, and combining and collecting supernatant F;
(8) respectively rotary evaporating the supernatant B, C, D, E, F at a temperature below 60 deg.C, and freeze drying to obtain extract B, C, D, E, F;
the grain particles are highland barley;
the active ingredient composition with the hypoglycemic function is a composition prepared by an extract B and an extract E in a ratio of 1: 1;
the extract B and the extract E are respectively active ingredient compositions rich in procyanidine B1 and p-coumaric acid.
2. An active ingredient composition having a hypoglycemic function, characterized in that the extract B and the extract E prepared by the preparation method of claim 1 are formulated in a ratio of 1: 1.
3. Use of a composition according to claim 2 for the preparation of a medicament for lowering blood glucose or improving impaired glucose tolerance.
4. A medicament having an active ingredient with a hypoglycemic function, characterized in that the composition of claim 2 is used as an active ingredient.
5. Use of a composition according to claim 2 for the preparation of a medicament for the prevention and treatment of impaired glucose tolerance or type ii diabetes.
6. The use of claim 5, wherein the medicament further comprises a pharmaceutically acceptable excipient.
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| CN110787197A (en) * | 2019-11-11 | 2020-02-14 | 中国科学院西北高原生物研究所 | Quinoa bran extract and extraction method and application thereof |
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