CN107722074B - Calcium-containing compound separated from vinegar egg liquid and application thereof - Google Patents

Calcium-containing compound separated from vinegar egg liquid and application thereof Download PDF

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CN107722074B
CN107722074B CN201710877739.4A CN201710877739A CN107722074B CN 107722074 B CN107722074 B CN 107722074B CN 201710877739 A CN201710877739 A CN 201710877739A CN 107722074 B CN107722074 B CN 107722074B
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calcium
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徐德平
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Jiangnan University
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    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs

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Abstract

The invention discloses a calcium-containing compound separated from vinegar egg liquid and application thereof, belonging to the technical field of food and medicine. The invention extracts the compound with the functions of reducing blood pressure and blood sugar from the vinegar-soaked egg solution, and verifies the effect of the compound on the functions of reducing blood pressure and blood sugar. The result shows that the IC for inhibiting the ACE is 30min after the calcium saccharate compound with the concentration of 50-200 mu g/mL is added50The value is 11.7-34 mu g/mL; according to the results of animal experiments, the fasting blood glucose level of diabetic mice was decreased by 27.67% 2 weeks after administration of the compound of the present invention.

Description

Calcium-containing compound separated from vinegar egg liquid and application thereof
Technical Field
The invention relates to a calcium-containing compound separated from vinegar egg liquid and application thereof, belonging to the technical field of food and medicine.
Background
Vinegar egg liquid is a traditional food therapy product in China, and is recorded in Shang Han dynasty Zhang Zhongjing in Shang Han dynasty. The vinegar egg liquid has effects of softening blood vessel, lowering blood pressure, reducing cholesterol, and reducing blood sugar. It can be used for preventing and treating 70 kinds of diseases such as cardiovascular and cerebrovascular diseases, liver and kidney diseases, etc. However, the specific functional components are not clear, and the mechanism of the effect cannot be clarified. The relevant documents report that the vinegar egg liquid contains a large amount of calcium and exists in the form of calcium acetate, but no report is found that the calcium acetate compound with the above effects is separated from the vinegar egg liquid.
Therefore, the separation and obtaining of the effective components in the vinegar-soaked eggs have very important effects on the prevention of diseases such as cardiovascular and cerebrovascular diseases, liver and kidney diseases and the like. .
Disclosure of Invention
According to the invention, through the component research on the vinegar-pickled egg liquid, the vinegar-pickled egg contains calcium acetate substances, but the substances do not exist in a free form, but exist in a form of combining calcium acetate and oligosaccharide, so that the functional compound is extracted and the applications possibly related to the functional compound are provided.
A first object of the present invention is to provide a compound having the structural formula:
Figure GDA0002222731380000011
wherein R is
Figure GDA0002222731380000012
In one embodiment of the invention, the compound is white amorphous powder, and the melting point is 187-189 ℃.
In one embodiment of the invention, the nmr data for the compound is as shown in the following table:
Figure GDA0002222731380000021
from1H-NMR showed δ 5.11(1H, d, J ═ 3.6), 4.51(1H, d, J ═ 7.6), these two H signals should be terminal signals for sugars, 3.0 to 4.1 for H signals on sugars, 1.79(3H, s), for methyl signals. In that13C-NMR 20 carbon signals in total, delta 181.7 is a carbonyl carbon signal indicating that the compound contains a carboxyl group, delta 98.0, 95.8, 92.0 has three carbon signals, which should be terminal carbon signals of the sugar, indicating that the compound has three sugar residues, and1only two terminal H signals are visible in H-NMR, and from the 135dept spectrum, a quaternary carbon (. delta.98.0) is indicated, which gives compounds containing a ketose, the 135dept spectrum showing that. delta.63.8, 63.3, 62.6, 60.7 are CH2A signal. From the chemical shift values of the sugars and the 135dept information it was determined that the compounds contained two glucose residues and one fructose group. δ 23.3 is the methyl signal, which indicates that the carbonyl group of δ 181.7 should form an acetate group with the methyl group.
The second purpose of the invention is to provide the application of the compound in preparing the medicine for treating hypertension, hyperlipemia, cardiovascular and cerebrovascular diseases, or liver and kidney diseases.
The third purpose of the invention is to provide the application of the compound in preparing health products with the functions of softening blood vessels, reducing blood pressure, reducing cholesterol or reducing blood sugar.
The fourth purpose of the invention is to provide the application of the compound in the non-medical application in the food field.
The compound of the invention is prepared by the following steps:
(1) concentrating the vinegar-soaked egg solution to one fifth volume, adding four times of the concentrated 95% (v/v, mL/mL) ethanol solution while stirring, standing for more than 4 hours, and filtering to obtain precipitate;
(2) dissolving the precipitate obtained in the step (1) with a proper amount of water, loading the precipitate onto an MCI chromatographic column, eluting with deionized water with twice the volume of the column, eluting with an ethanol solution with a volume fraction of 10% with three times the volume of the column, collecting the eluent, and concentrating the eluent to one fifth of the volume;
(3) and (3) separating the eluate concentrated in the step (2) by using an ODS-AQ column, eluting with deionized water, eluting with ethanol with the volume fraction of 6%, collecting the ethanol eluate, continuously separating the ethanol eluate on the ODS-AQ column, and repeating the step (3) for 2-3 times.
Has the advantages that: the invention extracts the compound with the functions of reducing blood pressure and blood sugar from the vinegar-soaked egg solution, and verifies the effect of the compound on the functions of reducing blood pressure and blood sugar. The results show that IC is obtained 30min after adding 50-200. mu.g/mL sugar calcium compound50The value is 11.7-34 mu g/mL; according to the results of animal experiments, after the compound of the invention is administrated for 2 weeks, the fasting blood glucose value of the diabetic mice is reduced by 27.67 percent,
drawings
FIG. 1 is an HPLC spectrum of compound calcium saccharate;
FIG. 2 is a mass spectrum of LC-MS of compound calcium saccharate;
FIG. 3 shows the compound of sugar calcium13C-NMR spectrum.
Detailed Description
EXAMPLE 1 extraction of sugar calcium Compound
(1) Preparing vinegar egg liquid: adding 15kg of 9-degree vinegar into 10kg of eggs, soaking for 24h, stirring to break egg skins, soaking for 24h, and filtering to obtain filtrate to obtain vinegar-egg liquid.
(2) Concentrating 10L vinegar egg liquid to 2L volume, adding 8L 95% ethanol under stirring, standing for more than 4 hr, and filtering to obtain precipitate.
(3) Dissolving the precipitate with 0.5L water, filtering to remove impurities, separating the supernatant with MCI column (5 × 150cm), eluting with 6L deionized water, eluting with 9L 10% ethanol, collecting 10% ethanol eluate, and concentrating to 1.8L volume.
(4) And (3) reversely multiplexing the 10% ethanol elution concentrated solution for ODS-AQ column separation, eluting with deionized water, eluting with 6% ethanol, repeatedly separating with the ODS-AQ column for 2-3 times, and detecting by a TLC method until a single point is obtained, namely the single compound. The obtained eluate was dried to obtain 11.7g of white amorphous powder of calcium saccharate, which was named as calcium saccharate. .
EXAMPLE 2 identification of Saccharum compounds
The compound obtained in example 1 was identified by HPLC \ HPLC-MS and nuclear magnetic resonance, respectively.
(1)HPLC
Adopting ODSC18 chromatographic column and methanol: water (6: 94) mobile phase, with flow rate of 0.3mL/min, and detecting wavelength of 228nm chromatogram shown in FIG. 1; the chromatographic peak at a retention time of 1.56min was the compound obtained in step 1.
(2)HPLC-MS
The mass spectrum using ESI + mode at 1800v is shown in FIG. 2.
(3) Nuclear magnetic resonance with D2O as solvent, tetramethylsilane as internal standard, Bruck 500MZNMR measurement was carried out.
TABLE 1 NMR results
Figure GDA0002222731380000041
From1H-NMR showed δ 5.11(1H, d, J ═ 3.6), 4.51(1H, d, J ═ 7.6), these two H signals should be terminal signals for sugars, 3.0 to 4.1 for H signals on sugars, 1.79(3H, s), for methyl signals. In that13C-NMR has a total of 20 carbon signals, a.delta.181.7 ofCarbonyl carbon signal, indicating that the compound contains carboxyl groups, delta 98.0, 95.8, 92.0 has three carbon signals, which should be terminal carbon signals of sugars, indicating that the compound has three sugar residues, and1only two terminal H signals are visible in H-NMR, and from the 135dept spectrum, a quaternary carbon (. delta.98.0) is indicated, which gives compounds containing a ketose, the 135dept spectrum showing that. delta.63.8, 63.3, 62.6, 60.7 are CH2A signal. From the chemical shift values of the sugars and the 135dept information it was determined that the compounds contained two glucose residues and one fructose group. δ 23.3 is the methyl signal, which indicates that the carbonyl group of δ 181.7 should form an acetate group with the methyl group.
From the mass fragment analysis, the m/z 130 fragment is CH3COO-Ca-O-CH2-H, i.e. a Ca bond to acetic acid and a bond to a hydroxyl group on the sugar. The compound contains 3 fragments with m/z of 130, indicating that 3 calcium acetate are attached to different sites of the sugar, from this point on13The abundance of the carboxyl and methyl signals in the C-NMR spectrum can be seen. In nuclear magnetic resonance experiments, calcium has a shielding effect, the connected carbon moves to a high field, and the carbon 3-position chemical shift value of the sugar is relatively small, so that the calcium acetate is determined to be connected with the C3 site of the sugar.
EXAMPLE 3 use of Glycalcium Compounds for lowering blood pressure
The invention utilizes the inhibition effect on angiotensin-converting enzyme (ACE) to test the effect of sugar calcium in lowering blood pressure. Preparing 100 mU L of 50, 100 and 200 mU g/mL calcium saccharate solution, respectively adding 40 mU L of 25mU U of ACE enzyme solution, preserving the temperature at 37 ℃ for 30 minutes, adding 200 mU L of 1mol/L hydrochloric acid, and measuring the absorbance at the wavelength of 228 nm. The deionized water was used as a blank, and the difference ratio between different concentrations of calcium saccharate and the blank was the inhibition ratio, and the results are shown in table 2.
TABLE 2 inhibitory Effect of Saccharum calcium on ACE
Figure GDA0002222731380000051
ACE is angiotensin converting enzyme, which acts through the renin-angiotensin system and kallikrein-kinin system to cause blood pressure elevation, and it can inhibit ACE activity to lower blood pressure. It can be seen from table 2 that calcium saccharate has a better ACE inhibitory activity, indicating that calcium saccharate has a blood pressure lowering effect.
EXAMPLE 4 use of Glycalcium compounds for lowering blood glucose
The invention utilizes the alloxan diabetes mouse to detect the hypoglycemic effect of the calcium saccharate,
taking ICR male mice, and raising the mice in cages in animal laboratories (20 +/-2) DEG C for 12 hours each in light and shade, wherein the experimental animals can freely drink and eat water. Feeding normally for 3 days, after fasting for 18h, injecting alloxan solution at the dose of 60mg/kg, measuring postprandial blood sugar for 72h, and using mice with blood sugar value > 20.0mmol/L as hyperglycemia model.
Animals were randomly divided into 5 groups according to blood glucose level, and each group contained 9 animals, which were a model control group, a positive control group (metformin 240mg/kg), and a calcium-sugar group. Each administration group is administered by intragastric administration at a volume of 10ml/kg once a day with an administration amount of 500 mg/kg-1The model control group was given an equal amount of distilled water. After 2 weeks of continuous administration, postprandial blood glucose and post-administration fasting blood glucose were measured.
TABLE 3 Effect of calcium saccharate on blood glucose in diabetic mice
Figure GDA0002222731380000052
Note: p <0.05, P <0.01
From Table 3, the sugar calcium content was 500 mg/kg-1After 2 weeks of administration, the fasting blood glucose value of the diabetic mice was decreased by 27.67% and showed a significant difference (p < 0.05) compared to the model control group, thus indicating that the calcium gluconate had the effect of decreasing fasting blood glucose in the diabetic mice.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (3)

1. A compound having the formula i:
Figure FDA0002279222290000011
in the formula I, R is
Figure FDA0002279222290000012
2. A composition comprising a compound of claim 1.
3. The use of a compound according to claim 1 for the manufacture of a medicament for the treatment of hypertension, hyperlipidemia, cardiovascular and cerebrovascular diseases, or liver and kidney diseases.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008290995A (en) * 2007-05-28 2008-12-04 Otaka Koso Kk New oligosaccharide and its manufacturing method
CN103588827A (en) * 2012-08-15 2014-02-19 天津天士力现代中药资源有限公司 Separation and purification method for oligomeric trisaccharide in compound red sage root extract

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008290995A (en) * 2007-05-28 2008-12-04 Otaka Koso Kk New oligosaccharide and its manufacturing method
CN103588827A (en) * 2012-08-15 2014-02-19 天津天士力现代中药资源有限公司 Separation and purification method for oligomeric trisaccharide in compound red sage root extract

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Structural Analysis of Two Trisaccharides Isolated from Fermented Beverage of Plant Extract.;Naoki Kawazoe, et al.,;《Open Glycoscience》;20081231;第1卷;第25-30页. *
分离纯化醋蛋水解物制备降血压肽的研究.;陈黎斌等,;《食品科技》;20111231;第36卷(第4期);第78-80、88页. *
醋蛋液的成分及保健功效研究进展.;王学英等,;《中国调味品》;20131231;第38卷(第5期);第7-11页. *

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