CN107722074B - A kind of calcium-containing compound separated from vinegar egg liquid and application thereof - Google Patents

Abstract

The invention discloses a calcium-containing compound separated from vinegar and egg liquid and its application, belonging to the technical fields of food and medicine. In the present invention, compounds with functions of lowering blood pressure and blood sugar are extracted from the vinegar foamed egg solution, and the effects of the compounds themselves on the functions of lowering blood pressure and blood sugar are verified. The results showed that after adding 50-200 μg/mL sugar-calcium compound for 30min, the IC ₅₀ value of ACE inhibition reached 11.7-34 μg/mL; The fasting blood glucose value decreased by 27.67%.

Description

A kind of calcium-containing compound separated from vinegar egg liquid and application thereof

technical field

The invention relates to a calcium-containing compound separated from vinegar and egg liquid and its application, and belongs to the technical fields of food and medicine.

Background technique

Vinegar and egg liquid is a traditional dietary product in my country. It was recorded in "Treatise on Febrile Diseases" by Zhang Zhongjing in the late Eastern Han Dynasty. Vinegar and egg liquid has the functions of softening blood vessels, lowering blood pressure, lowering cholesterol and lowering blood sugar. It has a certain preventive effect on more than 70 kinds of diseases such as cardiovascular and cerebrovascular, liver and kidney. However, the specific efficacy components are not clear, and the mechanism of efficacy cannot be clearly defined. Relevant literature report contains a large amount of calcium in the vinegar and egg liquid, and exists in the form of calcium acetate, but there is no report that separates the calcium acetate compound with the above-mentioned effect from the vinegar and egg liquid.

Therefore, separating and obtaining the active ingredients in the vinegar-soaked egg plays a very important role in the prevention of cardiovascular, cerebrovascular, liver and kidney diseases. .

SUMMARY OF THE INVENTION

The present invention finds through the research on the composition of the vinegar-soaked egg liquid that the vinegar-soaked egg contains calcium acetate substances, but such substances do not exist in a free form, but in a form in which calcium acetate and oligosaccharides are combined. Therefore, The present invention extracts the functional compound and provides its possible related applications.

其中，R为

The first object of the present invention is to provide a compound whose structural formula is:

where R is

In one embodiment of the present invention, the compound is a white amorphous powder with a melting point of 187-189°C.

In one embodiment of the present invention, the nuclear magnetic resonance data of the compound are shown in the following table:

It can be seen from ¹ H-NMR that δ5.11 (1H, d, J=3.6), 4.51 (1H, d, J=7.6), these two H should be the end group signal of sugar, 3.0--4.1 is H on sugar Signal, 1.79 (3H, s), should be a methyl signal. There are 20 carbon signals in ¹³ C-NMR, δ181.7 is the carbonyl carbon signal, indicating that the compound contains a carboxyl group, δ98.0, 95.8, 92.0 has three carbon signals, which should be the terminal carbon signal of sugar, indicating that the compound has three sugars residue, and only two terminal H signals were seen in ¹ H-NMR. From the 135dept spectrum, δ98.0 was a quaternary carbon. It can be concluded that the compound contains a ketose, and the 135dept spectrum shows δ63.8, 63.3, 62.6 , 60.7 is the CH ₂ signal. From the chemical shift values of the sugar and the 135dept information, it can be determined that the compound contains two glucose residues and one fructosyl group. δ23.3 is the methyl signal, and it can be concluded that the carbonyl group of δ181.7 should form an acetate group with the methyl group.

The second object of the present invention is to provide the application of the compound in the preparation of a medicament for treating hypertension, hyperlipidemia, cardiovascular and cerebrovascular diseases, or liver and kidney diseases.

The third object of the present invention is to provide the application of the compound in the preparation of health care products with functions of softening blood vessels, lowering blood pressure, lowering cholesterol, or lowering blood sugar.

The fourth object of the present invention is to provide the application of the compound in the non-medical application of the food field.

The compounds of the present invention are prepared according to the following steps:

(1) Concentrate the vinegar-soaked egg solution to one-fifth of the volume, add four times the volume of 95% (v/v, mL/mL) ethanol solution after the concentration while stirring, let stand for more than 4 hours, and filter the precipitate. ;

(2) Dissolve the precipitation in step (1) with an appropriate amount of water, put it on the MCI chromatography column, first elute with deionized water with twice the column volume, and then wash with ethanol solution with a volume fraction of 10% for three times the column volume Remove, collect the eluate, and concentrate the eluate to one-fifth volume;

(3) Separating the concentrated eluate in step (2) through an ODS-AQ column, first eluting with deionized water, then eluting with a volume fraction of 6% ethanol, collecting the ethanol eluent, and eluting with ethanol The liquid continued to be separated on the ODS-AQ column, and the step (3) was repeated 2 to 3 times.

Beneficial effects: The present invention extracts compounds with functions of lowering blood pressure and blood sugar from the vinegar-soaked egg solution, and verifies the effects of the compounds themselves on the functions of lowering blood pressure and blood sugar. The results showed that after adding 50-200 μg/mL sugar-calcium compound for 30 minutes, the IC ₅₀ value reached 11.7-34 μg/mL; according to the results of animal experiments, after 2 weeks of administration with the compound of the present invention, the fasting blood glucose value of diabetic mice decreased 27.67%,

Description of drawings

Fig. 1 is the HPLC spectrum of compound sugar calcium;

Fig. 2 is the LC-MS mass spectrum of compound sugar calcium;

Figure 3 is the ¹³ C-NMR spectrum of the compound sugar calcium.

Detailed ways

Example 1 Extraction of sugar and calcium compounds

(1) Preparation of vinegar egg liquid: 10kg eggs are added with 15kg 9° vinegar, soaked for 24h, the egg skin is stirred, soaked for 24h again, and the filtrate is filtered to obtain the vinegar egg liquid.

(2) Concentrate 10L of vinegar-egg solution to 2L volume, add 8L of 95% ethanol by volume while stirring, let stand for more than 4h, filter to get the precipitate.

(3) Add 0.5 liters of water to the precipitate to dissolve, filter to remove impurities, and separate the supernatant with an MCI column (5×150 cm), first eluted with 6 liters of deionized water, and then eluted with 9 liters of 10% ethanol , the 10% ethanol eluate was collected and concentrated to a volume of 1.8 L.

(4) The 10% ethanol eluted concentrate was repeatedly separated by the ODS-AQ column, first eluted with deionized water, then eluted with 6% ethanol, and separated on the ODS-AQ column for 2 to 3 times. TLC method is detected until a single point is obtained, which is a single compound. The obtained eluate was dried to obtain 11.7 g of calcium saccharide white amorphous powder, which was named as calcium saccharide. .

Example 2 Identification of sugar calcium compounds

The compounds obtained in Example 1 were identified by HPLC\HPLC-MS and nuclear magnetic resonance respectively.

(1) HPLC

ODSC18 chromatographic column, methanol:water=6:94 mobile phase, flow rate 0.3mL/min, detection wavelength 228nm chromatogram is shown in Figure 1; the chromatographic peak at retention time 1.56min is the compound obtained in step 1.

(2) HPLC-MS

Using ESI+ mode, the voltage is 1800v, and the mass spectrum is shown in Figure 2.

(3) Nuclear magnetic resonance, using D ₂ O as solvent, tetramethylsilane as internal standard, and Bruck 500M _Z for NMR measurement.

Table 1 NMR results

It can be seen from ¹ H-NMR that δ5.11 (1H, d, J=3.6), 4.51 (1H, d, J=7.6), these two H should be the end group signal of sugar, 3.0--4.1 is H on sugar Signal, 1.79 (3H, s), should be a methyl signal. There are 20 carbon signals in ¹³ C-NMR, δ181.7 is the carbonyl carbon signal, indicating that the compound contains a carboxyl group, δ98.0, 95.8, 92.0 has three carbon signals, which should be the terminal carbon signal of sugar, indicating that the compound has three sugars residue, and only two terminal H signals were seen in ¹ H-NMR. From the 135dept spectrum, δ98.0 was a quaternary carbon. It can be concluded that the compound contains a ketose, and the 135dept spectrum shows δ63.8, 63.3, 62.6 , 60.7 is the CH ₂ signal. From the chemical shift values of the sugar and the 135dept information, it can be determined that the compound contains two glucose residues and one fructosyl group. δ23.3 is the methyl signal, and it can be concluded that the carbonyl group of δ181.7 should form an acetate group with the methyl group.

From the analysis of mass spectrometry fragments, the fragment of m/z 130 is CH ₃ COO-Ca-O-CH ₂ -H, that is, one bond of Ca is connected to acetic acid, and one bond is connected to the hydroxyl group on the sugar. This compound contains three fragments with m/z of 130, indicating that three calcium acetates are attached to different sites of the sugar, which can be seen from the abundance of carboxyl and methyl signals in the ¹³ C-NMR spectrum. In the NMR experiment, calcium has a shielding effect, the connected carbon will move to the high field, and the chemical shift value of the carbon 3 position of the sugar is relatively small, so it is determined that calcium acetate is connected to the sugar C3 site.

The application of embodiment 3 sugar calcium compound in lowering blood pressure

The present invention uses the inhibitory effect on angiotensin-converting enzyme (ACE) to test the blood pressure-lowering effect of sugar and calcium. Prepare 100 μL of 50, 100 and 200 μg/mL sugar calcium solution, add 40 μL of 25mU ACE enzyme solution respectively, incubate at 37°C for 30 minutes, add 200 μL of 1 mol/L hydrochloric acid, and measure the absorbance at 228 nm wavelength. Taking deionized water as the blank, the difference ratio of different concentrations of sugar calcium and blank is the inhibition rate, and the results are shown in Table 2.

Table 2. Inhibitory effect of sugar calcium on ACE

ACE is an angiotensin-converting enzyme that acts through the renin-angiotensin system and the kallikrein-kinin system to increase blood pressure. Inhibiting the activity of ACE can reduce blood pressure. It can be seen from Table 2 that sugar and calcium have a good inhibitory activity against ACE, indicating that sugar and calcium have a hypotensive effect.

Example 4 Application of sugar calcium compound in lowering blood sugar

The present invention utilizes alloxan diabetic mice to detect the hypoglycemic effect of sugar and calcium,

Male mice of ICR species were taken and reared in separate cages in an animal laboratory at (20±2)°C in light and dark for 12 hours. The experimental animals were allowed to drink water and eat freely. After 3 days of normal feeding, after fasting for 18 hours, alloxan solution was injected at a dose of 60 mg/kg, and postprandial blood glucose was measured for 72 hours.

The animals were randomly divided into 5 groups according to the blood glucose value, with 9 animals in each group, namely the model control group, the positive control group (metformin 240 mg/kg), and the sugar and calcium group. Each administration group was intragastrically administered at a volume of 10 ml/kg, once a day, with an administration dose of 500 mg·kg ^-1 , and the model control group was given an equal volume of distilled water. After continuous administration for 2 weeks, postprandial blood glucose and post-administration fasting blood glucose were measured.

Table 3 Effects of sugar and calcium on blood sugar in diabetic mice

Note: *P<0.05,**P<0.01

It can be seen from Table 3 that after 500 mg·kg ^-1 administration for 2 weeks, the fasting blood glucose value of diabetic mice decreased by 27.67% compared with the model control group, and showed a significant difference (p﹤0.05), so it shows that Sugar calcium has the effect of reducing fasting blood sugar in diabetic mice.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (3)

1. a compound is characterized in that, structural formula is shown in formula I:

In formula I, R is

2 . A composition comprising the compound of claim 1 . 3 . 3. The application of the compound of claim 1 in the preparation of a medicine for the treatment of hypertension, hyperlipidemia, cardiovascular and cerebrovascular diseases, or liver and kidney diseases.

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