Preparation method, product and application of boron-containing dietary supplement

Abstract

The invention provides a preparation method, a product and application of a boron-containing dietary supplement, and the method is as follows: sodium malate or malic acid and boric acid are subjected to chelation reaction to generate boron malate, and the boron-containing dietary supplement is obtained. The preparation method is simple and easy to implement, and the prepared malic acid boron chelate can be applied to treatment of breast cancer or arthritis.

Description

Technical Field

[0001] The present invention belongs to the technical field of nutritional supplements, and particularly relates to a preparation method, product and application of a boron-containing dietary supplement. Background Art

[0002] Boron is one of the trace elements with biological functions in organisms, and is of great significance for maintaining the biological functions of plants, animals, microorganisms and humans, especially playing an important role in the growth and development of animals and humans. For example, supplementing an appropriate amount of boron in the diet can improve the central nervous system, liver metabolism and bone density of humans, and is also beneficial to embryonic development, wound healing and even cancer treatment. Research shows that insufficient boron content in the human body will lead to reduced immune function, increased risk of death, osteoporosis and decline in cognitive ability, and excessive content will cause cell damage. Therefore, supplementing an appropriate amount of boron is of great significance for human health.

[0003] Although more and more studies have found many benefits of boron to humans, boron-containing nutritional supplements have not yet been widely used.

[0004] Common boron-rich preparations include borax, boric acid, sodium tetraborate, etc., but their stability is poor, they are easy to deteriorate, and they are extremely harmful to the human body after taking. The patent application with the publication number CN101129446A discloses a preparation method of a boron-rich nutrient supplement, including the following steps: A. Mix mung beans, water and boric acid in a certain weight ratio and cultivate under suitable conditions; B. Then place the separated solid material under suitable conditions and let it stand; C. Repeat step A by adding a certain amount of water and boric acid to the solid material product in step B and cultivate for a certain time, and separate the solid and liquid materials of the reactants; D. Place the solid material product in step C under suitable temperature and suitable relative humidity conditions and let it stand for a certain time; E. Process the solid material reactant in step D to obtain a finished product.

[0005] At present, most commercial boron-containing dietary supplements first prepare boron-containing chelates by using the chelation of boron with amino acids or polyhydroxy acids, and then are formulated with various nutrients such as vitamin D, calcium, magnesium, soy isoflavones, chondroitin sulfate, glucosamine, etc.

[0006] For example, the patent application with the publication number CN106723065A discloses a vitamin D and boron compound nutritional supplement, including vitamin D and boron. The mass percentage of the boron content is not less than 97%, the mass percentage of the vitamin D content is not less than 0.2%, and the rest are impurities, and the mass percentage of the impurity content is less than 2%; the boron is synthesized in plants and is obtained by nano-sphere filtration using a pure physical extraction technique; the vitamin D is calcitriol.

[0007] However, there are few reports on the exact structure of chelates in boron-containing dietary supplements and their correlation with biological efficacy.

[0008] Research has found that boron mainly exists in the form of boric acid in the human body. Boric acid (H3BO3) is a Lewis acid. Due to the electron-deficient nature of the boron atom, boric acid can undergo an esterification reaction with biomolecules having adjacent hydroxyl group structures in the human body. For example, boric acid can form chelates with biomolecules such as hydroxy acids, amino acids, vitamins, carbohydrates, and nucleotides through electron acceptor-donor interactions to form three configurations of chelates: one is to form an uncharged planar five-membered monocyclic configuration ( Figure 1 compound a in Figure 1 ); the second is to form a negatively charged five-membered monocyclic tetrahedral configuration ( Figure 1 compound b in

[0009] Malic acid (also known as 2-hydroxybutanedioic acid, abbreviated as H2MA in this application) is an organic acid essential to the human body and an ideal food additive with low calories. It also has significant effects in maintaining skin health, improving gastrointestinal problems, reducing blood sugar, regulating blood lipids, antioxidant, relieving muscle fatigue, and improving heart health. Therefore, as a new generation of food acidulant, malic acid is hailed as the "most ideal food acidulant" by the biological and nutritional communities. From the perspective of molecular structure, malic acid belongs to ortho-hydroxy acid and theoretically can chelate with boric acid to form boron-containing chelates. Compared with boron citrate, a boron-containing dietary supplement currently on the market, boron malate has a relatively high boron content, but there is currently no relevant research on using boron malate as a boron supplement. Summary of the Invention

[0010] To solve the above technical problems existing in the prior art, the present invention provides a preparation method, product, and application of a boron-containing dietary supplement, which can prepare boron malate chelate used as a boron-containing dietary supplement. The preparation method is simple, and the prepared boron malate chelate can be applied to the treatment of breast cancer or arthritis.

[0011] The present invention provides a preparation method of a boron-containing dietary supplement, which is to carry out a chelation reaction between sodium malate or malic acid and boric acid to generate boron malate, thereby obtaining the boron-containing dietary supplement.

[0012] Chelation mechanism: The boron atom in the boric acid molecule coordinates with the hydroxyl oxygen in the carboxyl group of the malate ion, and one molecule of water is removed to form a boron malate five-membered monocyclic chelate B-MA.

[0013] Since it is actually the malate ion that participates in the chelation reaction, and both sodium malate and malic acid can form malate ions in the solvent, either sodium malate or malic acid can be used as the reaction raw material. When malic acid is used as the reaction raw material, the pH needs to be controlled at 6.5, at which time malic acid mainly exists in the form of malate ions.

[0014] Preferably, the molar ratio of boric acid to sodium malate or malic acid is 0.4∶1 to 1.2∶1.

[0015] Preferably, the pH of the reaction system of the chelation reaction is 5.5 - 8.5.

[0016] More preferably, the molar ratio of sodium malate or malic acid to boric acid is 1∶1, and the pH of the reaction system of the chelation reaction is 6.5. Under these reaction conditions, the thermal stability of the boron malate chelate synthesized is relatively good.

[0017] Preferably, the solvent for the chelation reaction is water, and using water as the solvent is relatively safe.

[0018] Preferably, the reaction temperature of the chelation reaction is 25°C. The reaction temperature has little influence on the chelation reaction between boric acid and o-hydroxy acid, and it is also relatively easy to achieve at room temperature of 25°C.

[0019] Preferably, the reaction time of the chelation reaction is 0.5 - 2.5 h. More preferably, the reaction time of the chelation reaction is 1 h.

[0020] The present invention provides a boron-containing dietary supplement prepared by the above preparation method.

[0021] The present invention also provides the application of the above boron-containing dietary supplement in the treatment of breast cancer or arthritis.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention prepares a boron malate chelate that can be used as a boron-containing dietary supplement through a simple and feasible method, and the prepared boron malate chelate has strong inhibitory activity against human breast cancer cells (MDA-MB-231) and can be applied to the treatment of breast cancer or arthritis. Description of the Drawings

[0024] Figure 1 It is a schematic chemical structure diagram of three configurations of chelates formed by boric acid. Figure 2 It is the Fourier infrared spectrum diagram in Test Example 1.

[0025] Figure 3 It is for B-MA in Test Example 2 11 13C NMR spectrum diagram.

[0026] Figure 4To detect the 11 11B NMR spectrum of B-MA in Example 2.

[0027] Figure 5 To detect the structure optimization diagram of B-MA in Example 3.

[0028] Figure 6 The ionic form of malic acid in aqueous solution.

[0029] Figure 7 The chelation mechanism of boric acid and malic acid at pH 6.5.

[0030] Figure 8 To detect the thermogravimetric (TG) and derivative thermogravimetric (DTG) curves of B-MA in Example 4.

[0031] Figure 9 To detect the in vitro toxicity test results of B-MA at different concentrations on MDA-MB-231 cells in Example 5.

[0032] Figure 10 To detect the correlation between experimental values and theoretical values in Example 3.

[0033] Figure 11 To detect the relative content of B-MA at different pH values in Example 6.

[0034] Figure 12 To detect the relative content of B-MA at different molar ratios in Example 7.

[0035] Figure 13 To detect the relative content of B-MA at different reaction times in Example 8. Detailed implementation method

[0036] Example 1

[0037] First, add 10 mL of deionized water to a polytetrafluoroethylene beaker, then add 1.52 g of sodium malate (Na2MA), stir and dissolve at room temperature, and then slowly add 0.48 g of boric acid (H3BO3) powder according to the molar ratio of boric acid to sodium malate of 1:1, mix well and dissolve. At this time, the pH of the reaction system is 6.5. After reacting for 1 h under magnetic stirring at room temperature, the reaction solution is heated and concentrated at 80 °C until it becomes viscous, then stop heating, cool and crystallize, filter by suction to obtain the sample, wash it 3 times with a small amount of cold acetone, and store it in a desiccator for standby, denoted as B-MA.

[0038] Example 2

[0039] First, add 10 mL of deionized water into 5 PTFE beakers respectively, then add 1.52 g of sodium malate into each beaker, stir and dissolve at room temperature. Then, slowly add 0.48 g of boric acid in powder form according to the molar ratio of n(boric acid)∶n(sodium malate) = 1∶1, stir under magnetic force at room temperature, and then adjust the pH of the reaction system to 5.5, 6.5, 7.0, 7.5, and 8.5 respectively. The subsequent steps are the same as those in Example 1.

[0040] Example 3

[0041] First, add 10 mL of deionized water into 5 PTFE beakers respectively, then add 1.52 g of sodium malate into each beaker, stir and dissolve at room temperature. Then, add boric acid according to the molar ratio of n 硼酸 ∶n 苹果酸钠 of 0.4∶1, 0.6∶1, 0.8∶1, 1∶1, and 1.2∶1 in powder form, stir under magnetic force at room temperature, and then adjust the pH of the reaction system to the optimal pH obtained in Example 2 respectively. The subsequent steps are the same as those in Example 1.

[0042] Example 4

[0043] First, add 10 mL of deionized water into 5 PTFE beakers respectively, then add 1.52 g of sodium malate, stir and dissolve at room temperature. Add boric acid according to the optimal molar ratio obtained in Section 1.3.2 in powder form, stir under magnetic force at room temperature, then adjust the pH of the reaction system to the optimal pH obtained in Example 2, and then react for 0.5 h, 1.0 h, 1.5 h, 2.0 h, and 2.5 h respectively. The subsequent steps are the same as those in Example 1.

[0044] Detection Example 1

[0045] Take boric acid (H3BO3), sodium malate (Na2MA), and B-MA prepared in Example 1, grind and press tablets with KBr at a ratio of 1∶200 respectively, and perform infrared spectrum measurement in the wavenumber range of 400 - 4000 cm -1 under the transmittance measurement mode, with 16 scanning times and a resolution of 4.

[0046] The test results are shown in Figure 2 , and the vibration frequencies of important groups are summarized in Table 1. The absorption peaks of all test samples in the range of 3500 - 3000 cm -1 are attributed to the stretching vibration of the O-H bond, which is related to the bound water and borate anions in the samples.

[0047] Table 1

[0048]

[0049] Figure 2 In, the absorption peak at 1397 cm-1 The peak at [X] cm⁻¹ is attributed to the bending vibration of the -CH₂- bond in the malate ion; the peak at 1708 cm -1 The peak at [X] cm⁻¹ is attributed to the asymmetric stretching vibration of the C=O bond. Compared with the C=O vibration peak of Na₂MA at 1686 cm -1 The C=O vibration peak in B-MA is shifted, which is analyzed to be caused by the coordination of boron atoms. The peak of Na₂MA at 1320 cm -1 ~1260 cm -1 is attributed to the stretching vibration of the C-O bond of the carboxyl group, while the vibration peak of B-MA in this region weakens or disappears, indicating that H₃BO₃ reacts with Na₂MA. Compared with the O-H stretching vibration of Na₂MA at about 3461 cm -1 The O-H vibration peak in B-MA becomes broader because the O-H adjacent to the carboxyl group participates in the reaction. Compared with the characteristic vibration peak of H₃BO₃ at 1189 cm -1 belonging to the B-O-H bond, the absorption peak of B-MA in this region disappears, indicating that H₃BO₃ has completely participated in the reaction. In addition, compared with the spectrum of H₃BO₃, in the fingerprint region of 1200 - 800 cm -1 Two new peaks appear in B-MA. The peak at 1054 cm -1 is attributed to the asymmetric stretching vibration of the B-O bond in the tetrahedral BO₄, and the peak at 890 cm -1 is attributed to the symmetric stretching vibration of the B-O bond in the tetrahedral BO₄. It can be speculated that H₃BO₃ chelates with the malate ion in a tetrahedral configuration.

[0050] Detection Example 2

[0051] Take appropriate amounts of Na₂MA and B-MA prepared in Example 1, dissolve them in heavy water (D₂O) respectively, and then transfer them into a 5 mm quartz NMR tube, and perform liquid 13 ¹³C NMR and 11 ¹¹B NMR detections.

[0052] The results of liquid 13 ¹³C NMR detection are shown in Figure 3 , and the chemical shifts of the respective carbon atoms measured are summarized in Table 2. Compared with the chemical shifts of carbon in Na₂MA, the chemical shifts of carbon at 4 positions in B-MA are all shifted, and the chemical shifts at the C-1 and C-2 positions are shifted more significantly. Analyzing the reasons, the change at the C-1 position may be due to the coordination of boron with the oxygen in the carboxyl group, and the change at the C-2 position is due to the participation of the hydroxyl group on the carbon in the reaction, which can be mutually confirmed with the FT-IR analysis results of Detection Example 1.

[0053] Table 2

[0054] (Note: In the above translation, [X] represents the specific wavenumber value that is not clearly given in the original text and needs to be filled in according to the actual situation.)

[0055] 11 The results of B NMR detection are shown in Figure 4 . According to the literature, the NMR chemical shift of boron-containing chelates is related to the number of rings in the chelate. The chemical shift of the five-membered monocyclic chelate formed by boric acid and hydroxy acid is around δ5-6, and that of the five-membered bicyclic chelate is between δ9-10; the chemical shift of free H3BO3 is near δ19; the tetrahedral B(OH)4 - has a chemical shift in the high field of δ1.9, which is due to the strong magnetic shielding effect of the negatively charged B(OH)4 - .

[0056] In the boron spectrum of B-MA ( Figure 4 ), the peak at δ19.13 can be attributed to the free H3BO3 molecule; the peak at δ6.44 is attributed to the five-membered monocyclic chelate. In addition, it can also be seen from Figure 4 that there are free boric acid molecules in the reaction system, which may come from the partial decomposition of B-MA. This is not contradictory to the fact that the characteristic peak of H3BO3 was not found in the FT-IR spectrum of the B-MA sample in Test Example 1, because the FT-IR test was on a dry solid sample, so B-MA was relatively stable and did not decompose.

[0057] Test Example 3

[0058] The theoretical chelate was subjected to DFT calculations using the Gaussian09 program. Under the 6-311G+(2d, p) basis set, the B3LYP method was used to optimize the structure of the chelate (see Figure 5 ) and calculate the frequencies. At the same basis set level, the shielding constants of the optimized chelate were calculated using the gauge-independent atomic orbital method, and the polarizable continuum model (IEF-PCM) was used for the solvation reaction in water. Theoretical NMR chemical shift calculations were performed on the optimized structure, and the results are as Figure 10 shown. The experimental values and theoretical values show good correlation, so the above 11 assignment of the B NMR signals is reasonable. Just as the results of the peak assignment in Test Example 2, boric acid and sodium malate will form a five-membered monocyclic chelate at a molar ratio of 1:1 and a pH of 6.5.

[0059] Based on the results characterized by FT-IR, NMR and DFT calculations, the present application proposes a chelation mechanism of boric acid and sodium malate, as Figure 7 shown.

[0060] Figure 6shows the ionic forms of malic acid in aqueous solution. According to the preparation method of Example 1 (when malic acid is used as the reaction raw material, the pH of the reaction system is adjusted to 6.5 with sodium hydroxide), the molar ratio of boric acid to malic acid (sodium) is 1:1, and the pH of the system is 6.5. At this time, malic acid mainly exists in the form of MA 2- type (see Figure 6 for compound c). In the reaction, the boron atom in the boric acid molecule coordinates with the hydroxyl oxygen in the carboxyl group of the malate, and one molecule of water is removed to form a five-membered monocyclic chelate B-MA of boron malate.

[0061] Detection Example 4

[0062] Take about 5 mg of B-MA prepared in Example 1 and place it in a platinum crucible. Perform measurements on a synchronous thermal analyzer (TG-DSC) under a dynamic nitrogen atmosphere (100 mL / min) and a heating rate of 10 °C / min. Record the thermogravimetry (TG) and derivative thermogravimetry (DTG) curves within 0 - 1000 °C.

[0063] The results are as Figure 8 shown. The mass loss is 5.15% between 35 and 220 °C, and the first endothermic degradation peak appears at 84.2 °C, which is the removal of a small amount of adsorbed water in the sample. Subsequently, between 228 and 505 °C, the mass loss is 43.97%, and two endothermic degradation peaks appear at 272 °C and 398 °C, which are the degradations of the organic ligand malate in the chelate. Finally, between 540 and 1000 °C, the pyrolysis products of the chelate further thermally degrade, the mass first gradually decreases and then stabilizes. After the degradation ends, a gray-black residue remains in the crucible, which is formed by the deposition of pyrolytic carbon on the final product Na2O–B2O3 of metal borate. The initial decomposition temperature of the B-MA ligand is 228 °C, indicating that B-MA has good thermal stability.

[0064] Detection Example 5

[0065] Take the B-MA prepared in Example 1 and perform a 24-hour toxicity evaluation experiment on MDA-MB-231 cells (human breast cancer cells) in vitro using a cell counting reagent (Cell Counting Kit-8, CCK8). Inoculate the cells in the MDA-MB-231 cell culture flask into a 96-well plate. After culturing for 24 hours, add B-MA solutions with concentration gradients respectively, and set 8 concentrations (0.78, 1.56, 3.13, 6.25, 12.50, 25.00, 50 and 100 μg·μL -1) For each concentration, 6 parallel wells were set up. After adding the samples, the 96-well plate was placed in a cell incubator at 37 °C and 5% CO2 for 24 h. After the incubation, 10 μL of CCK-8 solution was added to each well, and then it was incubated in the cell incubator for another 1 h. Finally, the optical density (OD) value was measured at 450 nm, and the cell inhibition rate and IC50 (half maximal inhibitory concentration) were calculated.

[0066] Cell inhibition rate = [(A c -A s ) / (A c -A b )]×100% (1)

[0067] In the formula, A s is the absorbance of the experimental well (containing cells, medium, CCK-8 solution and drug solution); A c is the absorbance of the control well (containing cells, medium, CCK-8 solution, without drug); A b is the absorbance of the blank well (containing medium, CCK-8 solution, without cells and drug).

[0068] The results are as Figure 9 shown. B-MA was applied to MDA-MB-231 cells at concentrations of 0.78, 1.56, 3.12, 6.25, 12.50, 25.00, 50.00, 100.00 μg·μL -1 . After 24 h, the inhibition rates of B-MA on the cells were 11.2%, 24.7%, 36.8%, 62.2%, 81.4%, 91.1%, 96.6% and 96.4% respectively. The experimental results also showed that when the concentration of B-MA was greater than 12.50 μg·μL -1 , the cell inhibition rate was greater than 80%, and it had a lethal effect on MDA-MB-231 cells; when the concentration of B-MA was less than 6.25 μg·μL -1 , the cell inhibition rate was less than 40%, and it had no anti-proliferative effect on the cells. Nonlinear regression analysis was performed on the experimental data, and the IC50 value of B-MA was calculated to be 5.52 μg·μL -1 . It can be seen that B-MA has a significant inhibitory effect on MDA-MB-231 breast cancer cells.

[0069] Detection Example 6

[0070] According to the method of Example 2, the changing trend of the relative content of B-MA with the pH value was investigated, and the results are as Figure 11As shown, it can be seen that in the pH range of 5.5 to 6.5, the relative content of B-MA shows a gradually increasing trend; while when the pH value is in the range of 6.5 to 8.5, its relative content shows a gradually decreasing trend. Among them, when the pH value is 6.5, the relative content of B-MA 1:1 reaches the maximum, and the specific value is 64%.

[0071] Relative quantitative analysis

[0072] Referring to the quantitative nuclear magnetic resonance analysis method provided by John C. Edwards et al., the signal peaks of the corresponding components in the B NMR spectrum of the sample 11 were integrated, and relative quantitative analysis was carried out on the residual free boric acid molecules and complexes in the product. The calculation method is as follows:

[0073] ω1 = [(A1 / n1) / (A1 / n1 + A2 / n2)] × 100% (1)

[0074] In the formula:

[0075] ω1 - the relative molar concentration of the specified component in the sample;

[0076] A1 - the integrated area of a certain proton in the specified component;

[0077] A2 - the integrated area of a certain proton in another component of the sample;

[0078] n1, n2 - the number of protons of this proton.

[0079] Test Example 7

[0080] According to the method of Example 3, the effect of different molar ratios on the relative content of B-MA was investigated. The results are as Figure 12 shown. It can be clearly seen that when n 硼酸 ∶n 苹果酸钠 is in the range of 0.4:1 to 0.8:1, the relative content of B-MA shows an upward trend, and the relative content in this range always remains below 60%. When n 硼酸 ∶n 苹果酸钠 reaches 1:1, the relative content of B-MA reaches the maximum value of 65%, and this molar ratio is just the theoretical molar ratio of the B-MA synthesis reaction. However, when the molar ratio increases to 1.2, the relative content of B-MA shows a slight decrease. This may be because the molar ratio at this time has exceeded the theoretical molar ratio, resulting in an excess of boric acid, thus reducing the relative content of B-MA. Therefore, n 硼酸 ∶n 苹果酸钠 of 1.0 was selected as the optimal molar ratio.

[0081] Test Example 8

[0082] The influence of reaction time on the relative content of B-MA was investigated according to the method of Example 4, and the results are as Figure 13 shown. It can be observed that in the time period of 0.5 h to 1.0 h, the relative content of B-MA gradually increased and reached 65%. However, in the subsequent period of 1 h to 2.5 h, the relative content of B-MA showed a gradually decreasing trend, which may be due to the decomposition of some chelates in the aqueous solution as the reaction time extended.

Claims

1. A preparation method of a boron-containing dietary supplement, characterized in that, The method is to carry out a chelation reaction between sodium malate or malic acid and boric acid to generate boron malate, and thus obtain the boron-containing dietary supplement.

2. The preparation method of the boron-containing dietary supplement according to claim 1, characterized in that, The molar ratio of boric acid to sodium malate or malic acid is 0.4∶1 to 1.2∶1.

3. The preparation method of the boron-containing dietary supplement according to claim 1, characterized in that, The pH of the system for the chelation reaction is 5.5 to 8.

5.

4. The preparation method of the boron-containing dietary supplement according to claim 1, characterized in that, The molar ratio of sodium malate or malic acid to boric acid is 1∶1, and the pH of the system for the chelation reaction is 6.

5.

5. The preparation method of the boron-containing dietary supplement according to claim 1, characterized in that, The solvent for the chelation reaction is water.

6. The preparation method of the boron-containing dietary supplement according to claim 1, characterized in that, The reaction time for the chelation reaction is 0.5 to 2.5 h.

7. The preparation method of the boron-containing dietary supplement according to claim 1, characterized in that, The reaction temperature for the chelation reaction is 25 °C, and the reaction time for the chelation reaction is 1 h.

8. A boron-containing dietary supplement prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the boron-containing dietary supplement according to claim 8 in the preparation of a drug for treating breast cancer or arthritis.

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