Method for improving postprandial blood sugar regulation activity of phenolic compounds and application of method

Abstract

The invention belongs to the technical field of medicine and food, and particularly relates to a method for improving postprandial blood sugar regulation activity of phenolic compounds and application of the method. The invention provides a method for improving postprandial blood sugar regulation activity of a phenolic compound. The method comprises the following steps: adding a metal salt (forming a mixture of the phenolic compound and the metal salt) into the phenolic compound, wherein the mass ratio of the phenolic compound to the metal salt is 1: (1-4). The invention also provides application of the mixture of the phenolic compound and the metal salt obtained by the method in preparation of a medicine for regulating postprandial blood sugar activity. According to the invention, the common problem that phenolic compounds are generally high in in-vitro alpha-glucosidase inhibitory activity but weak in-vivo postprandial blood sugar lowering effect is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine and food, and relates to a method for improving the postprandial blood sugar regulating activity of phenolic compounds and an application thereof. Background Art

[0002] Excessive fluctuations in blood sugar after a meal can easily induce type 2 diabetes and its complications. Using α-glucosidase inhibitors to delay carbohydrate digestion is one of the key strategies for regulating blood sugar after a meal, but commercially available α-glucosidase inhibitors (such as acarbose, voglibose, and miglitol) often have side effects such as abdominal pain, diarrhea, and gastrointestinal bloating. A large number of studies have shown that many phenolic compounds have few toxic side effects and have in vitro α-glucosidase inhibitory activity that is much stronger than acarbose, but they have a common problem of weak effect in regulating blood sugar after a meal in vivo. For example, the half inhibitory concentration (IC50) of pelargonidin-3-O-rutinoside on α-glucosidase in vitro is 50 , 1.04μg / mL) is much lower than acarbose (230.00μg / mL), but in ICR mice, 150mg / kg pelargonidin-3-O-rutinoside is as effective in improving postprandial blood glucose as 25mg / kg acarbose (Chemical Communications 2019, 55(1), 39–42). Currently, no research has reported the cause of this common problem and its solution.

[0003] α-glucosidase is located in the brush border of the small intestinal mucosa, which is covered by a mucus layer barrier. The mucus layer has the ability to capture foreign substances because it is rich in hydrophobic cysteine and negatively charged oligosaccharides, and it renews itself every 4–6 hours. Therefore, phenolic compounds need to penetrate the mucus layer in time to interact with α-glucosidase. However, Feng et al. found that epigallocatechin gallate (EGCG) and tannic acid, as phenolic compounds, bind to mucin through hydrogen bonds and covalent bonds, resulting in a decrease in the pore size of the mucus layer and an enhancement of the barrier function, but epicatechin does not have this mucin network crosslinking agent (Journal of Agricultural and Food Chemistry 2022, 70(30), 9536–9546). It can be seen that the conjugated structure and high phenolic hydroxyl density of phenolic compounds make it easy for them to undergo a strong complexation with mucin, and this is closely related to their molecular structure. The binding effect of the intestinal mucus network may be the key reason why phenolic compounds have a weak effect in regulating postprandial blood sugar in vivo, which needs further study. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for improving the activity of phenolic compounds in regulating postprandial blood glucose, thereby improving the activity of phenolic compounds in regulating postprandial blood glucose.

[0005] To solve the above problems, the present invention provides a method for improving the activity of phenolic compounds in regulating postprandial blood glucose: adding a metal salt to the phenolic compound (to form a mixture of the phenolic compound and the metal salt), and the mass ratio of the phenolic compound to the metal salt is 1:1 to 4, preferably 1:2 to 4, more preferably 1:2.

[0006] As an improvement to the method for improving the activity of phenolic compounds in regulating postprandial blood glucose according to the present invention:

[0007] The phenolic compound includes any one of the following: gallic acid, chlorogenic acid, caffeic acid, ferulic acid, resveratrol, quercitrin, hesperidin, emodin, rutin, apigenin, piceatannol, p-coumaric acid, EGCG, procyanidin B2, procyanidin C1, and myricetin proanthocyanidins (BLPs).

[0008] As a further improvement to the method for improving the activity of phenolic compounds in regulating postprandial blood glucose according to the present invention:

[0009] The metal ions in the metal salt include any one of the following: Ca 2+ , Fe 3+ , Mg 2+ , Zn 2+ , Mn 2+ , Co 2+ , Mo 2+ , and Cr 3 + .

[0010] The present invention also simultaneously provides the application of the mixture of the phenolic compound and the metal salt obtained by the above method in the preparation of a drug for regulating postprandial blood glucose activity.

[0011] The present invention has the following beneficial effects:

[0012] The metal ions that can improve the activity of phenolic substances in regulating postprandial blood glucose according to the present invention include Ca 2+ , Fe 3+ , Mg 2+ , Zn 2+ , Mn 2+ , Co 2+ , Mo 2+ , and Cr 3+One of the following substances, which regulates the network structure of the mucus layer to help phenolic substances quickly penetrate the mucus layer and bind to α-glucosidase. Eventually, the postprandial blood glucose-regulating activities of gallic acid, chlorogenic acid, caffeic acid, ferulic acid, resveratrol, quercetin, hesperidin, emodin, rutin, apigenin, piceatannol, p-coumaric acid, EGCG, procyanidin B2, procyanidin C1, and BLPs are improved. This solves the common problem that phenolic compounds generally have strong in vitro α-glucosidase inhibitory activity but weak in vivo postprandial blood glucose-lowering effects.

[0013] In summary, for the mucus layer barrier covering α-glucosidase, the present invention provides a method to weaken the interaction between phenolic compounds and the mucus layer, so as to enhance the mucus layer penetrability of phenolic compounds and promote their effective binding to α-glucosidase.

[0014] In actual use of the present invention, a corresponding medicament or health food can be prepared first according to the mass ratio of phenolic compound to metal salt of 1:(2±0.5), and then taken accordingly with reference to the conventional dosage of the phenolic compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following further elaborates on the specific embodiments of the present invention in conjunction with the drawings.

[0016] Figure 1 is Ca 2+ Effect of BLPs (myricetin proanthocyanidins) on postprandial blood glucose in ICR mice:

[0017] (A) Blood glucose concentration–time curve; (B) AUC value (“ns” indicates no significant difference, “**” indicates a very significant level of P<0.01, “****” indicates a very significant level of P<0.0001);

[0018] Figure 2 is Ca 2+ , BLPs and Ca 2+ –Regulatory activity of BLPs tablets on postprandial blood glucose in ICR mice: (A) Blood glucose concentration–time curve; (B) AUC value (“ns” indicates no significant difference, “**” indicates a very significant level of P<0.01, “****” indicates a very significant level of P<0.0001). SPECIFIC EMBODIMENTS

[0019] To make the technical solutions of the present invention clearer, the following further elaborates on the specific embodiments of the present invention in detail, but does not limit the present invention thereto.

[0020] The reagents, materials, and animals used in the embodiments of the present invention are all commercially available products or self-made products, such as:

[0021] BLPs (bayberry leaf proanthocyanidins) were prepared by the method provided in the reference literature (Yang H, Ye X, Liu D, et al. Characterization of unusual proanthocyanidins in leaves of bayberry (Myrica rubra Sieb. et Zucc.) [J]. Journal of Agricultural and Food Chemistry, 2011, 59(5): 1622–1629).

[0022] Gallic acid, chlorogenic acid, caffeic acid and EGCG were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0023] Caco-2 cells and HT29-MTX-E12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection. DMEM medium, fetal bovine serum, penicillin-streptomycin mixture and HBSS were purchased from Thermo Fisher Scientific, USA.

[0024] D–PBS was purchased from Dalian Meilun Biotechnology Co., Ltd.

[0025] CCK–8 cell viability detection kit and glucose assay kit were purchased from Nanjing Jiancheng Bioengineering Institute.

[0026] Transwell membrane inserts (0.4 μm, PET membrane) were purchased from Nanjing Caobenyuan Biotechnology Co., Ltd.

[0027] Sucrose was purchased from Sigma-Aldrich, USA.

[0028] Tablet adjuvants such as microcrystalline cellulose were kindly provided by Shanghai Weihua Trading Co., Ltd.

[0029] Analytical pure reagents such as various metal salts, sodium hydroxide and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0030] Each example of the present invention was repeated 3 times, and the results were expressed as averages.

[0031] Example 1

[0032] The method for using metal ions to improve the postprandial blood glucose-regulating activity of phenolic compounds proposed by the present invention can be applied to different types of metal ions and phenolic compounds.

[0033] To further illustrate the present invention, the present invention will be described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0034] For Ca2+ 、Fe 3+ 、Mg 2+ and Zn 2+ The evaluation of the effect of improving the activities of gallic acid, chlorogenic acid, caffeic acid, EGCG and BLPs in regulating postprandial blood glucose is as follows:

[0035] (1) Caco-2 cells and HT29-MTX-E12 cells were seeded into complete medium and cultured at 37 °C, 5% CO2 and saturated humidity.

[0036] (2) The CCK–8 cell viability detection kit was used to determine the effects of metal ions and phenolic substances on the viability of Caco-2 cells and HT29-MTX-E12 cells. Finally, the maximum dosing dose of metal ions was determined to be 200 μg / mL, and the maximum dosing dose of phenolic substances was 50 μg / mL; that is, metal ions and phenolic substances within the above dosing doses did not affect cell viability.

[0037] (3) After mixing Caco-2 cells and HT29-MTX-E12 cells at a ratio of 3:1, they were seeded into the inner chamber (0.4 μm, PET membrane) of a Transwell insert at a cell density of 3×10 5 cells / cm 2 (both the inner and outer chambers were filled with complete medium), and cultured at 37 °C, 5% CO2 and saturated humidity for 21 days. The medium was changed every 2–3 days.

[0038] Note: The transmembrane resistance value of the co-cultured monolayer membrane of Caco-2 cells and HT29-MTX-E12 cells was greater than 300 Ω·cm 2 , and the apparent permeability coefficient of sodium fluorescein was less than 1× -6 10

[0039] (4) The media in the inner and outer chambers of the Transwell insert in step (3) were discarded and washed 3 times with D–PBS buffer. 0.5 mL of D–PBS buffer containing 100 μg / mL metal ions was added to the inner chamber, and 1.5 mL of D–PBS buffer was added to the outer chamber; incubated at 37 °C, 5% CO2 and saturated humidity for 15 min.

[0040] (5) The solutions in the inner and outer chambers of the Transwell insert in step (4) were discarded, and 0.5 mL of 30 mmol / L sucrose solution containing 50 μg / mL phenolic compounds was added to the inner chamber, and 1.5 mL of D–PBS buffer was added to the outer chamber; after incubating at 37 °C, 5% CO2 and saturated humidity for 40 min, the reaction was terminated by ice bath, serving as the experimental group;

[0041] (6)Collect the solution in the outer chamber of the Transwell insert in step (5), and use a glucose assay kit to measure its glucose content to indirectly indicate the α-glucosidase activity.

[0042] Note: Use a 30 mmol / L sucrose solution as the positive control; that is, relative to the experimental group, use "30 mmol / L sucrose solution" instead of "30 mmol / L sucrose solution dissolved with 50 μg / mL phenolic compound" as the positive control group.

[0043] A negative control was also set; that is, relative to the experimental group, use "D–PBS buffer" instead of "D–PBS buffer with 100 μg / mL metal ions" as the negative control group.

[0044] The phenolic compounds are as described in Table 1 below, and the glucose yields are as shown in Table 1 below.

[0045] Example 2

[0046] Replace the dosage of the metal ions in Example 1 with 200 μg / mL and 50 μg / mL, and the remaining operations are the same as in Example 1.

[0047] The comparison of the glucose yields in Examples 1 and 2 above is as described in Table 1.

[0048] Table 1 Effects of metal ion concentration on the α-glucosidase inhibitory activity of phenolic compounds

[0049]

[0050]

[0051]

[0052] As can be seen from the above table: The metal ions enhanced the activity of the phenolic compounds, rather than the enhancement of activity caused by the metal ions themselves.

[0053] Metal ions can improve the inhibitory activity of α-glucosidase of phenolic compounds, and the effect is better when the mass ratio of phenolic compound to metal ion is 1:2 or 1:4. Since there is no significant difference between 1:2 and 1:4. Therefore, considering factors such as cost, 1:2 is preferred.

[0054] In summary, phenolic compounds can reduce the metabolism of sucrose into glucose by the co-cultured monolayer membrane of Caco-2 cells and HT29-MTX-E12 cells, that is, inhibit their α-glucosidase activity, and co-treatment with metal ions can further reduce the production of glucose. Among them, the best effect is achieved when the dose ratio of metal ions to phenolic compounds is 2:1. In addition, metal ions themselves have no α-glucosidase inhibitory activity. Therefore, metal ions can enhance the α-glucosidase inhibitory activity of phenolic compounds. In the present invention, metal ions regulate the network structure of the mucus layer (such as an increase in pore size), and competitively bind to mucin, so that phenolic compounds can quickly penetrate the mucus layer and act on α-glucosidase at the mucosa, ultimately improving their postprandial blood glucose lowering effect.

[0055] Example 3

[0056] Taking Ca 2+ and BLPs (mass ratio 2:1) as an example, animal experiments were used to verify the improvement effect of metal ions on the postprandial blood glucose regulation activity of phenolic compounds:

[0057] All gavage volumes were 0.1 mL / 10 g.

[0058] The Ca 2+ specifically refers to CaCl2.

[0059] Thirty-two SPF-grade ICR mice (male, 6 weeks old) were purchased and raised in the Animal Center of Zhejiang Academy of Medical Sciences, and conditions such as 22 ± 1 °C, 12 h day-night cycle, free drinking water, and standard pellet diet were ensured. After one week of adaptation and 16 h of fasting, the mice were randomly divided into 4 groups of 8 mice per group: control group, BLPs group, Ca 2+ group, and BLPs + Ca 2+ group.

[0060] The first two groups were gavaged with sterile water, and the last two groups were gavaged with 100 mg / kg Ca 2+ solution;

[0061] After 15 min, the control group, BLPs group, Ca 2+ group, and BLPs + Ca 2+ group were respectively gavaged with sterile water, 50 mg / kg BLPs, sterile water, and 50 mg / kg BLPs;

[0062] After 15 min, each group was gavaged with 2 g / kg sucrose solution;

[0063] And the tail tip blood glucose was measured at 15, 30, 60, 90, and 120 min after gavage of the sugar. The blood glucose concentration-time curve was plotted, and the area under the curve (AUC) was calculated.

[0064] The results are as Figure 1As shown, 50 mg / kg BLPs has no activity in regulating postprandial blood glucose, while 100 mg / kg Ca 2+ can significantly enhance the hypoglycemic activity of BLPs (the AUC value decreased by 11.01%). In addition, 100 mg / kg Ca 2+ itself has no activity in regulating postprandial blood glucose. The results of this invention show that metal ions can weaken the barrier effect of the mucus layer on phenolic compounds, and thus the activity of phenolic compounds in regulating postprandial blood glucose is significantly enhanced, which solves the common problem that although they have strong α-glucosidase inhibitory activity in vitro, their effect in reducing postprandial blood glucose in vivo is weak.

[0065] Note: Changing BLPs to the phenolic compounds in Table 1 and Ca 2+ to the metal ions in Table 1 all show similar experimental effects.

[0066] Example 4

[0067] This invention also provides health foods or drugs containing the above metal ions and phenolic substances. By adding common ingredients such as excipients, diluents, lubricants, binders, disintegrants, binders, disintegrants, colorants, sweeteners and stabilizers, the above metal ions and phenolic substances can be made into various dosage forms, including tablets, powders, pills, capsules and granules, etc. However, the dosage forms, types and dosages of adjuvants will not limit the scope of this invention in any way.

[0068] (1) Preparation of tablets of metal ions and phenolic substances, taking Ca 2+ and BLPs (mass ratio 2:1) as an example: Mix 40 wt% Ca 2+ , 20 wt% BLPs, 25 wt% microcrystalline cellulose, 5 wt% cross-linked carboxymethyl cellulose sodium, 5 wt% talc powder and 5 wt% colloidal silica, and then use the direct compression method to prepare Ca 2+ –BLPs tablets.

[0069] Note: Using tablets without Ca 2+ or BLPs as controls, they are BLPs tablets and Ca 2+ tablets respectively. The Ca 2+ can specifically be CaCl2.

[0070] (2) Grind the tablets obtained in step (1) into powder, and use animal experiments to verify their activity in regulating postprandial blood glucose: 32 SPF-grade ICR mice (male, 6 weeks old) are purchased and raised in the Animal Center of Zhejiang Academy of Medical Sciences, and conditions such as 22 ± 1°C, 12 h day-night cycle, free drinking water and standard pellet diet are ensured. After adapting for one week and fasting for 16 h, the mice are randomly divided into 4 groups at 8 mice / group, and are respectively intragastrically administered with sterile water, Ca 2+ tablets (100 mg / kg, calculated by Ca2+ mass), BLPs tablets (50 mg / kg, based on BLPs mass), and Ca 2+ BLPs tablets (50 mg / kg, based on BLPs mass) were administered. 15 minutes later, each group was gavaged with a 2 g / kg sucrose solution. Tail tip blood glucose was measured 15, 30, 60, 90, and 120 minutes after gavage. Blood glucose concentration-time curves were plotted, and the area under the blood glucose curve (AUC) was calculated.

[0071] All gavage volumes were 0.1 mL / 10 g.

[0072] The results are as follows Figure 2 As shown in the figure, BLPs tablets (50 mg / kg, based on BLPs mass) had no activity in regulating postprandial blood glucose, while Ca 2+ –BLPs tablets (50 mg / kg, based on BLPs mass) can significantly enhance the hypoglycemic activity of BLPs (AUC value decreased by 13.28%). 2+ Tablets (100 mg / kg, Ca 2+ The results of this study are consistent with those of Example 3. Therefore, the method of using metal ions to enhance the postprandial blood glucose regulation activity of phenolic compounds can be successfully applied to tablets.

[0073] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A method for improving the postprandial blood glucose-regulating activity of phenolic compounds, characterized in that: A metal salt is added to the phenolic compound, and the mass ratio of the phenolic compound to the metal salt is 1:1 to 4.

2. The method for enhancing the activity of phenolic compounds in regulating postprandial blood glucose according to claim 1, characterized in that: The mass ratio of the phenolic compound to the metal salt is 1:2 to 4.

3. The method for enhancing the postprandial blood glucose-regulating activity of phenolic compounds according to claim 2, wherein: The mass ratio of the phenolic compound to the metal salt is 1:

2.

4. The method for improving the activity of phenolic compounds in regulating postprandial blood glucose according to any one of claims 1 to 3, characterized in that: The phenolic compound includes any one of the following: gallic acid, chlorogenic acid, caffeic acid, ferulic acid, resveratrol, quercitrin, hesperidin, emodin, rutin, apigenin, piceatannol, p-coumaric acid, EGCG, procyanidin B2, procyanidin C1, and myricetin.

5. The method for improving the activity of phenolic compounds in regulating postprandial blood glucose according to claim 4, characterized in that: The metal ions in the metal salt include any one of the following: Ca 2+ , Fe 3+ , Mg 2+ , Zn 2+ , Mn 2+ , Co 2+ , Mo 2+ and Cr 3+ .

6. Use of the mixture of the phenolic compound and the metal salt obtained by any one of the methods of claims 1 to 5 in the preparation of a drug for regulating postprandial blood glucose activity.

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