CN111419852A

CN111419852A - Application of 3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid in preparation of hypoglycemic drugs

Info

Publication number: CN111419852A
Application number: CN202010433041.5A
Authority: CN
Inventors: 鲁曼霞; 刘向前; 杨阳
Original assignee: Hunan University of Chinese Medicine
Current assignee: Hunan University of Chinese Medicine
Priority date: 2020-05-21
Filing date: 2020-05-21
Publication date: 2020-07-17

Abstract

The invention discloses application of 3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid in preparation of a hypoglycemic drug, and innovatively discovers that 3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid has remarkable hypoglycemic activity, so that the invention provides a brand-new application of 3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid in preparation of the hypoglycemic drug, and has a wide application prospect in preparation of the drug for preventing and treating diabetes.

Description

Application of 3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid in preparation of hypoglycemic drugs

Technical Field

The invention belongs to the field of medicine application, and particularly relates to application of 3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid in preparation of a hypoglycemic medicine.

Background

At present, diabetes is an endocrine metabolic disease seriously threatening human health, the incidence rate is on the rise year by year, the diabetes is the first place in China, about 95 percent of the diabetes is type II diabetes, the type II diabetes is non-insulin-dependent diabetes, the etiology and pathogenesis of the diabetes are complex, islet β cell apoptosis and Insulin Resistance (IR) are the research core of the type II diabetes, islet β cell apoptosis is not only the direct cause of the type II diabetes, but also plays an important role in the generation and development of the type II diabetes, nuclear factor-kappa B (nuclear factor-kappa B, NF-kappa B) participates in the gene transcription of immune-mediated inflammation and various physiological and pathological processes, researches show that NF-kappa B is closely related to diabetes and obesity, NF-kappa B also participates in insulin resistance, insulin β cell function damage, further causes generation of transmembrane caspase, inflammatory cell I-460-serine-beta-transferase, NO-beta.

3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid, white crystal, colorless needle crystal in methanol solvent, m.p. of 232-234 ℃, structural formula shown in formula 1;¹H-NMR and¹³the C-NMR data are shown in Table 1.

TABLE 1 preparation of compounds of formula 1¹H-NMR and¹³C-NMR data (Pyridine-d)₅,ppm)

Note (s: singlet; brs: broad singlet; m: multiplet;. signal crossover)

The research shows that the impressicin (3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid) is a compound with an anti-inflammatory effect, can obviously reduce the secretion of a RAW264.7 macrophage strain TNF- α, I L-1 β and HMGB1 under L PS stimulation, has obvious effect and presents a dose-dependent relationship, and the preliminary study on the mechanism of inhibiting the HMGB1 shows that the release of the HMGB1 can be reduced, but the total amount of the HMGB1 is not influenced, and the steady-state level of the HMGB1 in the RAW264.7 nucleus is not changed.

Disclosure of Invention

The invention discloses a novel discovery that 3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid has remarkable hypoglycemic activity, and therefore, the invention aims to provide a brand-new application of 3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid in preparing hypoglycemic drugs.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid in the preparation of hypoglycemic drugs.

Preferably, the 3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid is applied to preparation of a medicament for preventing and treating diabetes.

The medicament for preventing and treating diabetes is a medicament capable of inhibiting the activities of α -glucosidase, α -amylase and PTP 1B.

The medicament for preventing and treating diabetes is a medicament capable of promoting insulin secretion under the stimulation of glucose.

The medicine for preventing and treating diabetes is a medicine capable of partially recovering cell viability in RIN-m5F cells subjected to cell factor I L-1 β and IFN-gamma induced RIN-m5F apoptosis treatment in a concentration-dependent manner.

The medicine for preventing and treating diabetes is a medicine capable of reducing Caspase-3 activity in RIN-m5F cells subjected to cell apoptosis treatment by cytokine I L-1 β and IFN-gamma induction RIN-m5F in a concentration-dependent manner.

The medicine for preventing and treating diabetes mellitus is a medicine capable of regulating the NO level in RIN-m5F cells subjected to cell apoptosis treatment by cytokine I L-1 β and IFN-gamma induction RIN-m5F in a concentration-dependent manner.

The medicine for preventing and treating diabetes mellitus is a medicine capable of regulating the ROS level in RIN-m5F cells subjected to cell factor I L-1 β and IFN-gamma induction RIN-m5F cell apoptosis treatment in a concentration-dependent mode.

Preferably, in the medicament for preventing and treating diabetes, the mass percentage of 3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid is 0.1-100%.

Preferably, the medicament for preventing and treating diabetes further comprises a pharmaceutically acceptable adjuvant or carrier.

The application of the invention can be combined with the prior pharmaceutically acceptable auxiliary materials or carriers to prepare any pharmaceutically acceptable dosage form.

Preferably, the dosage form of the medicament for preventing and treating diabetes is tablets, granules, capsules, dripping pills, oral liquid or injections.

Has the advantages that:

(1) the compound of the formula 1 has obvious effect of inhibiting α -glucosidase, α -amylase and PTP 1B.

(2) The compound of formula 1 of the present invention is effective in promoting insulin secretion under glucose stimulation.

(3) The compound of formula 1 of the present invention can partially restore cell viability in cytokine I L-1 β and IFN- γ -induced RIN-m5F apoptosis-treated RIN-m5F cells in a concentration-dependent manner, and can restore viability to the level of the group not treated with the cytokine.

(4) The compound of formula 1 of the present invention is capable of regulating the level of NO in RIN-m5F cells treated with cytokine I L-1 β and IFN-. gamma.inducing apoptosis of RIN-m5F cells in a concentration-dependent manner.

(5) The compound of formula 1 of the present invention can reduce Caspase-3 activity in cytokine I L-1 β and IFN-gamma induced RIN-m5F apoptosis treated RIN-m5F cells in a concentration-dependent manner.

(6) The compound of formula 1 of the present invention is capable of regulating ROS levels in concentration-dependent manner in RIN-m5F cells treated with cytokine I L-1 β and IFN- γ induced apoptosis of RIN-m5F cells.

In conclusion, the 3 α, 11 α -dihydroxy-lupin-20 (29) -en-28-oic acid of the present invention has significant hypoglycemic activity.

Drawings

FIG. 1 is a graph showing the effect of different concentrations of the compound of formula 1 on the activity of RIN-m5F cells in example 2.

FIG. 2 is a graph showing the results of varying concentrations of the compound of formula 1 on glucose-stimulated insulin secretion in example 3; culturing RIN-m5F cells at basal (4mM) or stimulated (20mM) glucose concentration in the presence of a compound of formula 1; p <0.05, relative to vehicle-treated control; bars represent SEM (n ═ 3).

FIG. 3 is a graph showing the effect of different concentrations of the compound of formula 1 on cytokine-induced apoptosis in example 4; wherein-represents untreated, + represents treated, P <0.05 compared to control of blank treatment; # P <0.01 compared to cytokine treated group; bars represent SEM (n ═ 3).

FIG. 4 is a graph showing the effect of various concentrations of the compound of formula 1 in example 5 on cytokine-induced NO production in RIN-m5F cells; wherein-represents untreated, + represents treated, P <0.05 compared to control of blank treatment; # P <0.01 compared to cytokine treated group; bars represent SEM (n ═ 3).

FIG. 5 is a graph showing the effect of different concentrations of the compound of formula 1 in example 6 on cytokine-induced Caspase-3 activity in RIN-m5F cells; wherein-represents untreated, + represents treated, P <0.05 compared to control of blank treatment; # P <0.01 compared to cytokine treated group; bars represent SEM (n ═ 3).

FIG. 6 is a graph of the results of the effect of varying concentrations of the compound of formula 1 in example 7 on cytokine-induced ROS levels in RIN-m5F cells; wherein-represents untreated, + represents treated, P <0.05 compared to control of blank treatment; # P <0.01 compared to cytokine treated group; bars represent SEM (n ═ 3).

Detailed Description

The method for extracting the compound shown in the formula 1 specifically comprises the following steps:

(1) flash extraction: extracting acanthopanax gracilistylus leaves by using a flash extractor, taking methanol and/or ethanol as a solvent, extracting for 1-3 times at 30-80 ℃, combining, decompressing and recovering the solvent to obtain an extract;

(2) degreasing: degreasing the extract by using petroleum ether;

(3) extraction: extracting the degreased extract for 1-3 times at 30-80 ℃ by using methanol, and recovering the methanol from the extracting solution to obtain an extract;

(4) separation: subjecting the obtained extract to macroporous resin, gradient eluting with 30-100% ethanol, collecting 60-100% ethanol fraction, mixing, recovering ethanol, and drying to obtain dry extract; mixing the dry extract with silica gel, subjecting to silica gel column chromatography, gradient eluting with chloroform and methanol at volume ratio of 25: 1-10: 1, wherein 10-18: 1 part is subjected to gradient elution with chloroform, methanol and water at volume ratio of 15-20: 1: 0.1, 8-10: 1: 0.3, collecting 18-22: 1: 0 part, removing solvent, concentrating, and crystallizing to obtain compound of formula 1.

Example 1

α -glucosidase inhibition assay acarbose was used as a positive control (except that acarbose was used instead of the compound of formula 1).

After thoroughly mixing 20. mu. L α -glucosidase (0.25U) solution and 60. mu. L compound of formula 1, incubation was carried out at 37 ℃ for 18 minutes, then 50. mu. L p-nitrophenyl- α -d-glucopyranoside (pNPG) solution (5mM) was added, the mixture was further incubated at 37 ℃ for 25 minutes, and 120. mu. L0.1.1M Na was added₂CO₃The reaction was terminated and absorbance was measured at 405nm in a microplate reader, the results of which are shown in Table 2.

α -Amylase inhibition assay acarbose was used as a positive control (except that acarbose was used instead of the compound of formula 1).

125 μ L α -amylase (3U/m L) and 125 μ L formula 1 compounds at 37 ℃ temperature in 10 minutes, then 125 μ L2% starch solution is added to the test tube, and further temperature in 30 minutes, adding 250 μ L48 mM dinitrosalicylic acid reagent to stop the reaction, and immediately its boiling water bath for 15 minutes, cooling to room temperature, distilled water dilution, and at 540nm to measure the absorbance, the results are shown in Table 2.

PTP1B inhibition assay: ursolic acid was used as a positive control (except that the compound of formula 1 was replaced with ursolic acid).

To each of 96 wells in a microtiter plate (final volume: 100. mu. L) was added 10. mu. L of the compound of formula 1 and PTP1B (2.5 ng/. mu. L), a solution containing 50mM citrate (pH 6.0), 0.1M NaCl, 1M buffer methylenediamine tetraacetic acid and 2. mu. L100 mM dithiothreitol the samples were then incubated at room temperature for 1 hour and the absorbance measured at 540nm, the results of which are shown in Table 2.

Half maximal Inhibitory Concentration (IC) according to enzyme inhibitory activity₅₀) Comparison of the results As shown in Table 2, the compound of formula 1 exhibited α -glucosidase inhibitory activity, IC₅₀The value is 19.21 +/-0.55 mu g/m L, and the inhibition activity of the compound is stronger than that of positive control acarbose (IC)₅₀661.73 + -0.48 μ g/m L.) inhibitory Activity of the Compound of formula 1 against α -Amylase (IC)₅₀44.39 + -0.71 μ g/m L) higher than the positive control acarbose (IC)₅₀854.43 + -0.81 μ g/m L.) inhibitory Activity of a Compound of formula 1 on PTP1B (IC)₅₀10.62 +/-0.82 mu g/m L) is higher than that of positive control ursolic acid (IC)₅₀31.11±0.47μg/mL)。

TABLE 2 inhibitory Activity of compounds of formula 1 on α -glucosidase, α -amylase and PTP1B

Note that data for 50% inhibitory concentration (μ g/m L) were calculated from the inhibition curve and expressed as mean ± SEM (n-3). 1,2 was used as a positive control in each assay P <0.05 compared to the positive control in the assay.

Example 2

Culturing rat islet β cytoma cell (RIN-m5F) in RPMI1640 containing 10% bovine serum albumin and 1% antifungal antibiotic, and determining cytotoxicity by MTT method, regulating cell density of RIN-m5F cell in logarithmic growth phase to 2 × 10⁵One/well, seeded in 48-well plates, RIN-m5F cellsTreatment with varying concentrations of the compound of formula 1 (5, 10 and 20 μ M) for 24 hours, 300 μ L MTT solution (0.5mg/M L) was added to each well, and after incubating the cells for 4 hours, the supernatant was filtered and formazan crystals were solubilized in 200 μ L DMSO. the plates were incubated gently with shaking for 10 minutes, and then absorbance was measured at 570nm using a microplate reader (Thermo Scientific TMMultiskan TMFC, USA.) IC was calculated using GraphPad prism version 7.01(GraphPad Software, San Diego, CA, USA)₅₀The value is obtained. The effect of different concentrations of the compound of formula 1 on the activity of RIN-m5F cells is shown in fig. 1, while Vehicle is a blank control, and all samples treated with different concentrations of the compound of formula 1 showed cell viability higher than 90% and showed no cytotoxicity.

Example 3

Insulin secretion test (GSIS) under glucose stimulation with glibenclamide (Gly) as positive control, and collecting 2 × 10 log-phase RIN-m5F cells⁵The concentration per well was seeded in 48-well plates. The wells were then washed with Krebs-Ringer bicarbonate buffer (KRB; 5mM KCl, 115mM NaCl; 24mM NaHCO) at 37 deg.C₃，2.5mM CaCl₂25mM HEPES, 1 g/L BSA; pH 7.4) for 3 washes and preincubation for 1h glucose medium (RPMI-1640 glucose concentration 4mM or glucose concentration 20mM) was added and placed at 37 ℃ with 5% CO₂After 24h incubation in the incubator, after the addition of different concentrations of the compound of formula 1 or positive control in groups, the cells were incubated for 12h then the supernatant was removed from each well and centrifuged (5 min at 2000rpm at 4 ℃) then the insulin concentration was determined with rat insulin E L ISA kit (a L PCO Co, selemm, new hampshire, usa).

As shown in fig. 2, the compound of formula 1 (Imp) at various concentrations released insulin significantly higher than the blank control (Veh) under glucose challenge and, at a concentration of 20 μ M, significantly higher than the positive control glibenclamide (Gly).

Example 4

The combination of Cytokines (Cytokines) I L-1 β and IFN-gamma induces apoptosis of RIN-m5F cells, and logarithmic growth phase of RIN-m5F cells as 2 × 10⁵Concentration per well was inoculated into 48-well plates combined cytokines (recombinant human I L-1 β 10ng/m L and rat IFN-. gamma.100 ng/m L, R) were added&D Systents, maijin li, MN, USA) to induce RIN-M5F apoptosis, followed by treatment with different concentrations of the compound of formula 1 (5, 10 and 20 μ M) for 24h, cytokine-induced cell death was measured by MTT assay addition of 300 μ L MTT solution (0.5mg/M L), incubation of cells for 4h, filtration of supernatant, and solubilization of formazan crystals in 200 μ L DMSO, incubation with uniform shaking for 10min, followed by measurement of absorbance at 570nm using a microplate reader (Thermo scientific multiman TMFC, USA), calculation of IC using GraphPad Prism version 7.01(GraphPad software, San Diego, CA, USA)₅₀The value is obtained.

As shown in fig. 3, cytokine-treated RIN-M5F cells resulted in a significant decrease in cell viability to 57.61 ± 2.73% compared to the blank group whereas cell viability returned to 106.34 ± 2.28% higher than that of the positive control recombinant human insulin N-nitro-L-arginine methyl ester L-NAME (cell viability returned to 62.33 ± 1.85% at a concentration of 20 μ M) at a concentration of 20 μ M compound treatment of the sample, these results indicate that the compound of formula 1 can partially restore cell viability in cytokine-treated RIN-M5F cells in a concentration-dependent manner and can restore viability to a level of the non-cytokine-treated group.

Example 5

The NO levels were determined by measuring the concentration of nitrite in whole cell extracts and cell culture media RIN-m5F cells in the logarithmic growth phase were made into cell suspensions, 100 μ L cell suspensions were added to the well plates at 2 × 10⁵The concentration per well was seeded in 48-well plates at 37 ℃ in 5% CO₂After incubation for 24h in a saturated humidity incubator, the well plates were removed, the compound sample mother liquor of formula 1 was mixed with serum-free medium to a final concentration of 5 μ M, 10 μ M and 20 μ M, placed in the incubator for incubation, then I L-1 β (10ng/M L) and IFN- γ (100ng/M L) were added and incubation continued for 24h, the 96 well plates were removed, 100 μ L of the cell supernatant per well was aspirated and mixed with 100 μ L Griess reagent in the dark for 10min, sodium nitrite was used to generate a standard curve, the optical density of the sample at 520nm was measured, the corresponding NO content was calculated according to the standard curve regression equation and experimental group.

As shown in fig. 4, NO production was significantly increased to 63.35 ± 1.26% (P <0.01) in the cytokine-induced group compared to the blank group, but the concentration of the compound of formula 1 was 20 μ M, the level of NO production was reduced to 47.65 ± 1.37%, and the NO production in cytokine-treated RIN-M5F cells was reduced in a concentration-dependent manner, which showed better inhibitory NO production effect than the positive control, recombinant human insulin N-nitro-L-arginine methyl ester (59.12 ± 1.45%) at the same concentration.

Example 6

RIN-m5F cells in logarithmic growth phase were cultured at 2 × 10⁵The concentration per well was seeded in 48-well plates. 5% CO at 37 ℃₂Culturing in a saturated humidity incubator for 24h, discarding old culture solution, performing group treatment, adding culture solution to a control group, adding culture solution with the concentration of a compound of formula 1 of 5 μ M, 10 μ M and 20 μ M to the experimental group, continuously incubating for 1h, then adding I L-1 β (10ng/M L) and IFN- γ (100ng/M L) to continuously incubate for 24h, digesting cells with trypsin and collecting cells, centrifuging (centrifuging at 4 ℃ and 2000rpm for 5min), discarding supernatant, washing with Phosphate Buffer Solution (PBS), adding 50 μ L lysate to 100 ten thousand cells, performing ice-bath lysis for 15 min.4 ℃ and 16000g for 15min, transferring supernatant to a centrifuge tube with precooled ice bath, and applying Caspase-3 colorimetric assay kit (Abcam, Cambridge, MA, USA) to RIN-M5-5F cell lysate (2 × 10 per well in 6)⁵). Spectrophotometric detection based on the chromophore p-nitroaniline (p-NA), cleavage from the labelled substrate DEVD-pNA, and p-NA luminescence can then be quantified by microtiter plate reader at 405 nm.

As shown in FIG. 5, Caspase-3 activity was significantly reduced in RIN-M5F cells treated with both cytokine and compound of formula 1 (P <0.01) compared to cytokine-treated cells and Caspase-3 activity was reduced in a concentration-dependent manner in cytokine-treated RIN-M5F cells at 20 μ M concentration of compound of formula 1, Caspase-3 activity was significantly lower in cytokine-treated RIN-M5F cells than in the positive control recombinant human insulin N-nitro-L-arginine methyl ester-treated group, these results indicate that compound of formula 1 inhibits cytokine-induced apoptosis of RIN-M5F cells by limiting Caspase-3 activity.

Example 7

RIN-m5F cells in logarithmic growth phase were cultured at 2 × 10⁵The concentration per well was seeded in 48-well plates. 5% CO at 37 ℃₂The experimental group was added with 5 μ M, 10 μ M and 20 μ M of compound concentration of formula 1, incubated for 1h, I L-1 β (10ng/M L) and IFN- γ (100ng/M L) for 24h, then the well plate was removed, the medium was aspirated and the well bottom was washed 3 times with serum-free culture, 500 μ M L concentration of oxidation-sensitive probe 2',7' -dichlorodihydrofluorescein diacetate (DCFH-DA) was added to each well, the 24 well plate was placed in a FACS cantm Flow cytometer (BD Biosciences, san jose, ca, usa) at 37 ℃ for 20min, the probe and cells were brought into full contact, the cells were washed three times with Phosphate Buffer Solution (PBS) to sufficiently remove DCFH-DA that did not enter the cells, then Flow cytometer was used to examine the ROS level of ROS in cell Flow cytometer by Flow cytometer (joik L on. k software).

Intracellular ROS were analyzed using the oxidation sensitive probe DCFH-DA as shown in FIG. 6. Analysis of cytokine-exposed RIN-m5F cells showed a significant increase in ROS levels (56%). The compound of formula 1 exhibits a concentration-dependent reduction of ROS levels to 53%, 46.67% and 41.67% at concentrations of 5, 10 and 20 μ M, respectively. However, the compound of formula 1 (concentration 20. mu.M) was slightly less able to down-regulate the level of ROS when compared to the positive control Ascorbic acid (concentration 50. mu.M).

Example 8

(1) Adult male Sprague-Dawley (SD) rats 6 weeks old (190-

Advantage, Roche Diagnostics, Mannheim, Germany) measures blood glucose, fasting glucose>Animals at 220mg/d L were considered to have diabetes.

(2) Grouping and administration Male Sprague-Dawley (SD) rats (190-.

(3) And (3) blood sugar detection: fasting and random blood glucose at different dosing times for each group of rats was dynamically monitored using a glucometer, and blood glucose levels were determined after the first dose, 1 week, 2 weeks, 3 weeks, 4 weeks after continuous dosing, 2 hours before dosing and 2 hours after dosing, respectively.

(4) Oral Glucose Tolerance Test (OGTT): after 4 weeks of administration, the rats in each group were fasted for 8 hours without water deprivation, and the rats were gavaged with a glucose solution at a dose of 2g/kg in accordance with body weight, and the blood glucose values of the rats were measured at 0h, 0.5h, 1h, 1.5h, and 2h, and the area under the curve (AUC) was calculated.

(5) Measurement of insulin, hemoglobin, glycated hemoglobin (HbA1C) and C peptide Each group of rats was fasted and not deprived of water for 12 hours after the last administration, rats were anesthetized, and blood was collected from the eyeball.Whole blood was mixed well in a centrifuge tube containing heparin sodium, and plasma insulin levels were measured using rat insulin E L ISA kit (A L PCOCo) according to the kit instructions.

And (3) testing results:

(1) body weight, food intake and food availability

Table 3 shows the changes in Body Weight (BW), food intake and Food Efficiency Ratio (FER) of normal and diabetic rats after oral administration of the compound of formula 1 at various concentrations daily for 4 weeks. All diabetic groups were statistically significant (P <0.05) compared to the non-diabetic group. The type II diabetes control group had the most weight loss and the lowest food intake. In the diabetic group, the FER of Imp2 was higher in the high dose group than in the type II diabetes control group and the positive control group. And shows a concentration-dependent increasing tendency.

TABLE 3 weight, food intake and food utilization Change Table

Note: n: normal, non-diabetic population; c: control group, type II diabetes group; gua: guava, 500mg/kg, used as a positive control; imp 0.08: 0.08: 0.08mg/kg of compound of formula 1; imp 0.4: 0.4mg/kg of compound of formula 1; imp 2: the compound of the formula 1 is 2 mg/kg. Data are presented as mean ± SEM (n ═ 10). The a-i values with different superscripts in the same column are significant at P < 0.05. 1 weight gain/food intake.

(2) Fasting blood glucose level determination

Table 4 shows fasting blood glucose levels over a 4-week period. There was no significant change in fasting blood glucose levels between diabetic groups after STZ injection. After 3 weeks of administration, the blood glucose levels of the positive control group were significantly lower than those of the II diabetic control group (P < 0.05). The blood glucose level of the Imp2 group was significantly lower than that of the positive control group. However, the high dose blood glucose levels in the Imp2 group were significantly higher than in the normal group (P < 0.05). The compound of formula 1 can lower fasting plasma glucose and has better effect than the positive control group. And presents a dose-dependent increasing trend, the optimal dose of which can be further studied.

TABLE 4 measurement of fasting blood glucose level

Note: n: normal, non-diabetic population; c: control group, type II diabetes group; gua: guava, 500mg/kg, used as a positive control; imp 0.08: 0.08: 0.08mg/kg of compound of formula 1; imp 0.4: 0.4mg/kg of compound of formula 1; imp 2: the compound of the formula 1 is 2 mg/kg. Data are presented as mean ± SEM (n ═ 10). The a-i values with different superscripts in the same column are significant at P < 0.05.

(3) Results of Oral Glucose Tolerance Test (OGTT):

table 5 shows the blood glucose levels of the normal group and the experimental group of type II diabetic rats after oral glucose administration. The blood glucose levels in the normal group rose to a peak 1 hour after glucose loading and dropped to near normal levels at 2 hours. In the diabetic control group, the blood glucose concentration reached a peak after 1 hour and remained high for the next 1 hour. Even though the blood glucose level of the positive control group decreased after 1 hour, it did not approach the normal level after 2 hours. In the Imp2 group, the blood glucose concentration dropped significantly to a level of 0h (P <0.05) at 2h compared to 1 h. But not reduced to normal levels compared to the normal group.

TABLE 5 results of Oral Glucose Tolerance Test (OGTT)

Note: n: normal, non-diabetic population; c: control group, type II diabetes group; gua: guava, 500mg/kg, used as a positive control; imp 0.08: 0.08: 0.08mg/kg of compound of formula 1; imp 0.4: 0.4mg/kg of compound of formula 1; imp 2: the compound of the formula 1 is 2 mg/kg. Data are presented as mean ± SEM (n ═ 10).

(4) Results of measurements of plasma insulin, hemoglobin, HbA1C and serum C-peptide levels:

table 6 shows plasma insulin, hemoglobin, HbA1C and serum C-peptide levels in normal and experimental groups in type II diabetic rats. HbA1C was significantly increased in hemoglobin and plasma, while plasma insulin, C-peptide, was decreased in the type II diabetes group compared to the normal group (P < 0.05). The Imp 0.4 group has similar levels of plasma insulin, hemoglobin and HbA1c compared with the positive control group; the plasma insulin level of the Imp2 group is higher than that of the positive control group; the HbA1c level of the Imp2 group is lower than that of the positive control group; the C-peptide level of the Imp2 group is higher than that of the positive control group. But the Imp2 group has not reached the normal group level.

TABLE 6 measurement of plasma insulin, hemoglobin, HbA1C and serum C-peptide levels

As can be seen from the above examples, the compound expressed acid (3 α, 11 α -dihydroxy-lupin-20 (29) -ene-28-acid) in the formula 1 has obvious hypoglycemic activity and has wide application prospect in preparing medicines for preventing and treating diabetes.

Claims

1.3 application of 1.3 α, 11 α -dihydroxy-lupin-20 (29) -alkene-28-acid in preparing hypoglycemic medicine.

2. The application of 3 α, 11 α -dihydroxy-lupin-20 (29) -ene-28-acid in the preparation of hypoglycemic drugs according to claim 1, wherein the application of 3 α, 11 α -dihydroxy-lupin-20 (29) -ene-28-acid in the preparation of drugs for preventing and treating diabetes is provided.

3. The use of 3 α, 11 α -dihydroxy-lupin-20 (29) -en-28-oic acid in the preparation of a hypoglycemic medicament according to claim 2, wherein the medicament for preventing and treating diabetes is a medicament capable of inhibiting the activities of α -glucosidase, α -amylase and PTP 1B.

4. The use of 3 α, 11 α -dihydroxy-lupin-20 (29) -en-28-oic acid in the preparation of a hypoglycemic medicament according to claim 2, wherein the medicament for preventing and treating diabetes is a medicament capable of promoting insulin secretion under the stimulation of glucose.

5. The use of 3 α, 11 α -dihydroxy-lupin-20 (29) -en-28-oic acid in the preparation of a medicament for the reduction of blood glucose according to claim 2, wherein the medicament for the prevention or treatment of diabetes is a medicament capable of partially restoring the cell viability of the RIN-m5F cells treated with the cytokines I L-1 β and IFN- γ -induced RIN-m5F apoptosis and reducing the Caspase-3 activity of the RIN-m5F cells treated with the cytokines I L-1 β and IFN- γ -induced RIN-m5F apoptosis in a concentration-dependent manner.

6. The use of 3 α, 11 α -dihydroxy-lupin-20 (29) -en-28-oic acid in the preparation of a hypoglycemic agent according to claim 2, wherein the antidiabetic agent is one that can regulate the levels of NO and ROS in RIN-m5F cells treated with cytokine I L-1 β and IFN- γ -induced apoptosis of RIN-m5F cells in a concentration-dependent manner.

7. The application of 3 α, 11 α -dihydroxy-lupin-20 (29) -ene-28-acid in the preparation of the hypoglycemic drug according to any one of claims 1 to 6, wherein the mass percentage of 3 α, 11 α -dihydroxy-lupin-20 (29) -ene-28-acid in the drug for preventing and treating diabetes is 0.1-100%.

8. The use of 3 α, 11 α -dihydroxy-lupin-20 (29) -en-28-oic acid according to any one of claims 1 to 6 in the preparation of a medicament for lowering blood glucose, wherein the medicament for preventing and treating diabetes further comprises a pharmaceutically acceptable adjuvant or carrier.

9. The use of 3 α, 11 α -dihydroxy-lupin-20 (29) -en-28-oic acid as claimed in any one of claims 1 to 6 in the preparation of a hypoglycemic medicament, wherein the medicament for preventing and treating diabetes is in the form of tablet, granule, capsule, dripping pill, oral liquid or injection.