Cholesterol in swelling hippocampus and extraction method thereof

Abstract

The invention provides cholesterols in swelling hippocampus and an extraction method of the cholesterols, and belongs to the technical field of biology. According to the separation and purification method for extracting the cholesteryl from the hippocampus japonicus, the cholesteryl in the hippocampus japonicus is obtained through repeated reflux extraction of 75% ethanol and repeated purification of a silica gel column, and the purity of the cholesteryl reaches 97% or above. The method is simple to operate and low in cost, cholesterol with relatively high purity can be obtained, basis and reference are provided for further researching the dose-effect relationship of the nutritional functional factors of the belly-expanding hippocampus, clarifying the metabolic pathway and the action mechanism and screening and applying the nutritional functional factors, and the method has a wide application prospect. And transformation and upgrading and high-quality sustainable development of the whole industrial chain of the belly-expanding hippocampus are promoted.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to cholesterol in hippocampus bulgingis and an extraction method thereof. Background Art

[0002] Seahorse is an important high-value marine fish in China's aquatic economy. It has a large trade volume in the traditional Chinese medicine, marine aquarium and souvenir markets. The global annual consumption of seahorses is more than 50 million, with an output value of nearly 30 billion. There are many bioactive substances in the seahorse, which is known as the "Southern Ginseng". It has the effect of regulating qi and blood, strengthening the kidney and replenishing yang. It is mainly used in the field of traditional Chinese medicine in the form of dried products. There are 154 prescriptions containing seahorse or its aliases and synonyms. Studies have shown that seahorse and its extracts have the effect of lowering blood pressure, and also have certain anti-cancer potential, can inhibit tumors, and also show good relief for certain neurodegenerative diseases.

[0003] At present, the main seahorse species that are commercially cultivated in the world include 14 species such as Hippocampus abdominalis, Hippocampus bargibanti, and Hippocampus comes. Hippocampus abdominalis, also known as the big-bellied seahorse, has gradually become the preferred species for artificial breeding due to its advantages such as large litter size, strong disease resistance, low temperature resistance, and ability to be fed with frozen natural bait, providing more options for the industrial development of seahorses and the growing demand. Hippocampus abdominalis obtained food use permission from the Korean Food and Drug Safety Administration in February 2016, and countries such as the Philippines have also included it in the food and drug catalog for approval, indicating that this species has potential development and application value.

[0004] The puffy-bellied seahorse is a genus Hippocampus belonging to the phylum Chordata, class Steichthyes, subclass Actinopteri, order Notacanthiformes, family Syngnathidae. It can grow up to 35 cm in length and is characterized by a bulging abdomen, an extremely long dorsal fin, and large black spots around the eyes and on the gill covers.

[0005] With the breakthrough of the breeding technology of puffy seahorses, the analysis of its nutritional components has become a hot research direction. Studies have found that seahorses are rich in amino acids, active peptides, steroids, fatty acids, trace elements and other chemical components, which have shown good effects in treating sexual dysfunction, delaying body aging, anti-fatigue, anti-tumor, etc. At present, domestic research on puffy seahorses mainly focuses on the breakthrough of breeding technology, and there is less research on the extraction of active ingredients in seahorses.

[0006] Cholesterol is an indispensable substance in the human body. It is mainly involved in the synthesis of cell membranes, multiple hormones, bile acids and vitamin D. Its content directly affects the health of the human body. Too high a level can lead to arteriosclerosis and induce some comprehensive complications, while too low a level can easily lead to increased fragility of blood vessel walls, resulting in reduced immunity and emergency response capabilities. The hippocampus is rich in cholesterol, but the existing technology does not have a simple, low-cost, high-purity method for extracting cholesterol from the hippocampus. Summary of the invention

[0007] The object of the present invention is to provide cholesterol in hippocampus distended and a method for extracting the same, which has the advantages of simple operation, low cost and high purity of the obtained cholesterol.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for extracting cholesterol from hippocampus bulgingis, comprising the following steps:

[0010] (1) Grind the dried distended hippocampus and soak it in ethanol;

[0011] (2) after the soaking, reflux extraction is performed, and filtration and concentration are performed to obtain an extract;

[0012] (3) The extract is loaded onto a silica gel column by a dry method, and the first gradient elution and the second gradient elution are performed. After the second gradient elution, the components with higher content are combined, and further separated and purified by two reverse silica gel column chromatography, and finally cholesterol is obtained after concentration.

[0013] Preferably, in step (1), the ethanol is 70 to 80% ethanol by volume, and the soaking time is 2 to 4 hours.

[0014] Preferably, in step (2), the reflux extraction is performed 2 to 4 times, and the time for each reflux extraction is 0.5 to 2 hours.

[0015] Preferably, in step (3), the amount of silica gel in the silica gel column is 1000-2000 g, the mobile phase for the first gradient elution is n-hexane, ethyl acetate and water; and the mobile phase for the second gradient elution is n-hexane and acetone.

[0016] Preferably, in step (3), the first gradient elution step is:

[0017] Gradient elution was performed using n-hexane:ethyl acetate:water at a volume ratio of 7:1:0.5, 6:1:0.5, 4:1:0.5, 3:1:0.5, and 2:1:0.5;

[0018] The second gradient elution step is: select the part with large content in the first elution component, use n-hexane and acetone as eluents to perform gradient elution in a volume ratio of 8.5:1, 8:1, and 4:1, and combine the components with large content.

[0019] Preferably, in step (3), the steps of the two reverse silica gel column chromatography are: first, reverse elution is performed with methanol: water in a volume ratio of 10:1, the components are collected, and then rinsed with pure methanol.

[0020] The invention also provides cholesterol in hippocampus bulging prepared by a method for extracting cholesterol in hippocampus bulging.

[0021] Preferably, the structure of cholesterol in the puffy hippocampus is as shown in formula (1):

[0022]

[0023] The beneficial effects of the present invention compared with the prior art are:

[0024] (1) The present invention provides a separation and purification method for extracting cholesterol from hippocampus bulging, wherein the cholesterol in hippocampus bulging is obtained by multiple reflux extraction with 75% ethanol and repeated purification with a silica gel column, and the purity of the cholesterol reaches more than 97%.

[0025] (2) The present invention is simple to operate and low in cost, and can obtain cholesterol with high purity. It provides a basis and reference for further studying the dose-effect relationship of the nutritional functional factors of Hippocampus inflatus, clarifying the metabolic pathways and mechanisms of action, and screening and applying nutritional functional factors, thereby promoting the transformation and upgrading of the entire Hippocampus inflatus industry chain and high-quality sustainable development. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0027] Figure 1 This is a liquid phase analysis spectrum of cholesterol prepared in Example 1 of the present invention;

[0028] Figure 2 The cholesterol prepared in Example 1 of the present invention 1 HNMR spectrum;

[0029] Figure 3 The cholesterol prepared in Example 1 of the present invention 13 CNMR spectrum. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example 1

[0036] Example 1 of the present invention provides a method for extracting cholesterol from hippocampus bulgingis, and the specific steps are as follows:

[0037] (1) About 945 g of dried hippocampus bulgingus sample was collected, ground with a grinder, soaked in 75% ethanol for 3 hours, and refluxed for 3 hours each time for 3 times. The extract was filtered while hot, and the filtrate was collected for concentration and recovery to obtain 217.4 g of concentrated extract.

[0038] (2) The extract was dissolved in 100 mL of ethanol, and 100 g of silica gel was added for stirring. The extract was dry-loaded onto a silica gel column. The amount of silica gel was 1600 g. The silica gel column was eluted using an eluent of n-hexane, ethyl acetate and water in a volume ratio of 7:1:0.5, 6:1:0.5, 4:1:0.5, 3:1:0.5, and 2:1:0.5. After the elution, 7 components (H1 to H7) were obtained. Among them, the H3 component with a higher content (3:1:0.5) was further eluted using n-hexane and acetone in a volume ratio of 8.5:1, 8:1, and 4:1 to obtain 10 components (M1-10). The M3-M9 components were combined and eluted using a C-18 reverse silica gel column. The elution solvents were methanol and water (volume ratio of 10:1) and methanol. The white powder was finally obtained after recovery and concentration. The powder was cholesterol.

[0039] According to liquid phase analysis ( Figure 1 ), it was determined that the powder was a monomer compound with a purity of 97.33%. 1 HNMR spectrum ( Figure 2 )and 13 CNMR spectrum ( Figure 3 ), the compound was identified as cholesterol, which was extracted from hippocampus bulging for the first time, and its molecular formula was C 27 H 46 O, with a molecular weight of 386, and its structure is shown in formula (1):

[0040]

[0041] (3) Structural identification

[0042] The monomer compound was identified as cholesterol by NMR spectrum. This compound was extracted from hippocampus bulgingus for the first time. About 20 mg of powder was dissolved in deuterated chloroform and structural analysis was performed using a Bruker 700M NMR spectrometer (AVANCEⅢ700MHz). The carbon spectrum 13CNMR data was basically consistent with the literature. δ140.76 (C-5), 121.75 (C-6), 71.83 (C-3), 56.77 (C-14), 56.15 (C-17), 50.13 (C-9), 42.31 (C-4), 42.29 (C-13), 39.78 (C-16), 39.52 (C-24), 37.26 (C-1), 36.50 (C-10), 36.19 (C-22), 35.79 ( C-20), 31.91(C-7), 31.79(C-8), 29.74(C-2), 28.12(C-12), 28.02(C-25), 24.30(C-23), 23.8 3(C-15), 22.83(C-27), 22.57(C-26), 21.08(C-11), 19.40(C-19), 18.72(C-21), 11.85(C-18).

[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting cholesterol from hippocampus bulging, characterized in that: The steps include: (1) Grind the dried distended hippocampus and soak it in ethanol; (2) after the soaking, reflux extraction is performed, and filtration and concentration are performed to obtain an extract; (3) The extract is loaded onto a silica gel column by a dry method and subjected to gradient elution, the first gradient elution and the second gradient elution are performed, and the components with higher content are combined after the second gradient elution and further separated and purified by two reverse silica gel column chromatography, and finally cholesterol is obtained after concentration.

2. The method for extracting cholesterol from hippocampus bulgingis according to claim 1, characterized in that: In step (1), the ethanol has a volume fraction of 70 to 80%, and the soaking time is 2 to 4 hours.

3. The method for extracting cholesterol from the sea cucumber according to claim 1, characterized in that: In step (2), the reflux extraction is performed 2 to 4 times, and the time for each reflux extraction is 0.5 to 2 hours.

4. The method for extracting cholesterol from hippocampus bulgingis according to claim 1, characterized in that: In step (3), the amount of silica gel in the silica gel column is 1000-2000 g, the mobile phase for the first gradient elution is n-hexane, ethyl acetate and water; the mobile phase for the second gradient elution is n-hexane and acetone.

5. The method for extracting cholesterol from hippocampus bulgingis according to claim 4, characterized in that: In step (3), the first gradient elution step is: Gradient elution was performed using n-hexane:ethyl acetate:water at a volume ratio of 7:1:0.5, 6:1:0.5, 4:1:0.5, 3:1:0.5, and 2:1:0.5; The second gradient elution step is: select the part with large content in the first elution component, use n-hexane and acetone as eluents to perform gradient elution in a volume ratio of 8.5:1, 8:1, and 4:1, and combine the components with large content.

6. The method for extracting cholesterol from hippocampus bulgingis according to claim 1, characterized in that: In step (3), the steps of the two reverse silica gel column chromatography are: first, reverse elution is performed with methanol: water at a volume ratio of 10:1, the components are collected, and then rinsed with pure methanol.

7. Cholesterol in hippocampus bloated prepared according to the method for extracting cholesterol in hippocampus bloated according to any one of claims 1 to 6.

8. The cholesterol in the distended hippocampus according to claim 7, characterized in that: The structure of cholesterol in the puffy hippocampus is shown in formula (1):

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