Cholesterol in the inflated abdomen of hippocampus and its extraction method

Abstract

This invention provides a method for extracting cholesterol from the swollen seahorse, belonging to the field of biotechnology. The invention provides a method for separating and purifying cholesterol from the swollen seahorse. Cholesterol was obtained from the swollen seahorse through repeated reflux extraction with 75% ethanol and repeated purification using silica gel column chromatography, achieving a cholesterol purity of over 97%. This invention is simple to operate, low in cost, and yields high-purity cholesterol. It provides a basis and reference for further research on the dose-effect relationship of nutritional functional factors in the swollen seahorse, elucidating metabolic pathways and mechanisms of action, and screening and applying nutritional functional factors. This promotes the transformation, upgrading, and high-quality sustainable development of the entire swollen seahorse industry chain.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to cholesterol in a distended seahorse and its extraction method. Background Technology

[0002] Seahorses are important high-value marine fish in China's aquatic economy, with significant trade volume in the traditional Chinese medicine, marine aquarium, and souvenir markets. Global annual consumption of seahorses exceeds 50 million individuals, generating a value of nearly 30 billion yuan. Seahorses contain various bioactive substances and are known as "Southern Ginseng," possessing the effects of regulating qi and blood, and strengthening the kidneys and tonifying yang. They are widely used in traditional Chinese medicine, primarily in the form of dried products, with 154 prescriptions containing seahorses or their alternative names. Studies have shown that seahorses and their extracts have a blood pressure-lowering effect, possess certain anti-cancer potential, can inhibit tumors, and also show good alleviating effects on certain neurodegenerative diseases.

[0003] Currently, there are 14 main seahorse species commercially farmed worldwide, including the abdominal seahorse (Hippocampus abdominalis), the bargibanti seahorse (Hippocampus bargibanti), and the tiger-tailed seahorse (Hippocampus comes). The abdominal seahorse (Hippocampus abdominalis), also known as the large-bellied seahorse, has gradually become the preferred species for artificial breeding due to its advantages such as high calorie production, strong disease resistance, cold tolerance, and ability to be fed frozen natural food. This provides more options for the industrial development of seahorses and meets the growing demand. The abdominal seahorse has potential development and application value.

[0004] The bloated seahorse is an animal belonging to the class Steichthyes, subclass Actinopteris, order Notacanthiformes, family Syngnathidae, and genus Hippocampus. It can reach a body length of 35 cm and is characterized by its bulging abdomen, extremely long dorsal fin, and large, dark spots around the eyes and gill covers.

[0005] With breakthroughs in the breeding technology of bloated seahorses, the analysis of their nutritional components has become a popular research direction. Studies have found that seahorses are rich in various chemical components such as amino acids, bioactive peptides, steroids, fatty acids, and trace elements, showing good effects in treating sexual dysfunction, delaying aging, anti-fatigue, and anti-tumor effects. Currently, domestic research on bloated seahorses mainly focuses on breakthroughs in breeding technology, with relatively little research on the extraction of active ingredients from seahorses.

[0006] Cholesterol is an essential substance in the human body, mainly involved in the synthesis of cell membranes, various hormones, bile acids, and vitamin D. Its content directly affects human health; excessively high levels can lead to arteriosclerosis and induce a series of complications, while excessively low levels can increase the fragility of blood vessel walls, resulting in decreased immunity and stress response. Seahorses are rich in cholesterol, but currently, there is no simple, low-cost, and high-purity method for extracting cholesterol from seahorses. Summary of the Invention

[0007] The purpose of this invention is to provide a method for extracting cholesterol from bloated seahorse, which is simple to operate, low in cost, and yields cholesterol with high purity.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a method for extracting cholesterol from swollen seahorse, comprising the following steps:

[0010] (1) Grind the dried, bloated seahorse and soak it in ethanol;

[0011] (2) After soaking, reflux extraction is performed, followed by filtration and concentration to obtain an extract;

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

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

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

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

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

[0017] Gradient elution was performed using n-hexane:ethyl acetate:water at volume ratios 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 as follows: select the fraction with the highest content from the first elution fraction, and perform gradient elution with hexane and acetone as eluents at volume ratios of 8.5:1, 8:1, and 4:1, and combine the fractions with the highest content.

[0019] Preferably, in step (3), the two reverse silica gel column chromatography steps are as follows: first, methanol and water are eluted in reverse at a volume ratio of 10:1 to collect the components, and then the mixture is washed with pure methanol.

[0020] The present invention also provides a method for extracting cholesterol from swollen seahorse to prepare cholesterol from swollen seahorse.

[0021] Preferably, the structure of cholesterol in the bloated seahorse is shown in formula (1):

[0022]

[0023] The beneficial effects of this invention compared to the prior art are as follows:

[0024] (1) This invention provides a method for separating and purifying cholesterol extracted from bloated seahorse. Cholesterol in bloated seahorse was obtained by repeated reflux extraction with 75% ethanol and repeated purification with silica gel column. The cholesterol purity reached more than 97%.

[0025] (2) This invention is simple to operate, low in cost, and can obtain cholesterol with high purity. It provides a basis and reference for further research on the dose-effect relationship of the nutritional functional factors of the bloated seahorse, to clarify the metabolic pathway and mechanism of action, and to provide a basis for the screening and application of nutritional functional factors. It promotes the transformation and upgrading of the entire industrial chain of the bloated seahorse and its high-quality and sustainable development. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The liquid phase analysis spectrum of cholesterol prepared in Example 1 of this invention;

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

[0029] Figure 3 The cholesterol prepared in Example 1 of this invention 13 CNMR spectrum. Detailed Implementation

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

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0035] Example 1

[0036] Example 1 of this invention provides a method for extracting cholesterol from swollen seahorse, the specific steps of which are as follows:

[0037] (1) A sample of dried, bloated seahorse was collected and ground. The sample was then soaked in 75% ethanol for 3 hours and extracted by reflux for 3 hours each time. The extraction was repeated 3 times. The extract was filtered while hot and the filtrate was collected and concentrated to obtain 217.4g of concentrated extract.

[0038] (2) The extract was dissolved in 100 mL of ethanol, and 100 mL of silica gel was added and stirred. The mixture was then loaded onto a silica gel column using a dry method with a silica gel weight of 1600 g. The silica gel column was eluted using a volume ratio of hexane, ethyl acetate and water (7:1:0.5, 6:1:0.5, 4:1:0.5, 3:1:0.5, 2:1:0.5). After elution, 7 fractions (H1 to H7) were obtained. The fraction H3 (3:1:0.5), which had a higher content, was eluted again using a volume ratio of hexane and acetone (8.5:1, 8:1, 4:1) to obtain 10 fractions (M1-10). Fractions M3 to M9 were combined and eluted using a C-18 reverse silica gel column with methanol and water (volume ratio 10:1) and methanol as the elution solvent. After recovery and concentration, a white powder was finally obtained, which was cholesterol.

[0039] According to liquid phase analysis ( Figure 1 The powder was determined to be a monomeric compound with a purity of 97.33%. 1 HNMR spectrum ( Figure 2 )and 13 CNMR spectrum ( Figure 3 The compound was identified as cholesterol, and this is the first time it has been extracted from the bloated seahorse. Its molecular formula is C6H2O. 27 H 46 O, with a molecular weight of 386, has the structure shown in formula (1):

[0040]

[0041] (3) Structural identification

[0042] The monomeric compound was identified as cholesterol by NMR spectroscopy. This compound is the first to be extracted from the swollen seahorse. Approximately 20 mg of the powder was dissolved in deuterated chloroform, and structural analysis was performed using a Bruker 700M NMR spectrometer (AVANCEⅢ 700MHz). The 13C NMR data were largely consistent with those in the literature. δ 140.76 (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 description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting cholesterol from swollen seahorse, characterized in that, Includes the following steps: (1) Grind the dried, bloated seahorse and soak it in ethanol; (2) After soaking, reflux extraction is performed, followed by filtration and concentration to obtain an extract; (3) The extract was loaded onto a silica gel column by dry method and subjected to gradient elution. The first gradient elution and the second gradient elution were performed. After the second gradient elution, the components with higher content were combined and further separated and purified by two reverse silica gel column chromatography. After concentration, cholesterol was finally obtained. In step (1), the ethanol is 70-80% ethanol by volume, and the soaking time is 2-4 hours; In step (2), the reflux extraction is performed 2 to 4 times, and the reflux extraction time for each time is 0.5 to 2 hours; In step (3), the amount of silica gel in the silica gel column is 1000-2000g, the mobile phase of the first gradient elution is n-hexane, ethyl acetate and water; the mobile phase of the second gradient elution is n-hexane and acetone. In step (3), the first gradient elution step is as follows: Gradient elution was performed using hexane:ethyl acetate:water at volume ratios 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 as follows: select the fraction with the highest content from the first elution fraction, and perform gradient elution with hexane and acetone as eluents at volume ratios of 8.5:1, 8:1, and 4:1, and combine the fractions with the highest content.

2. The method for extracting cholesterol from swollen seahorse according to claim 1, characterized in that, In step (3), the two reverse silica gel column chromatography steps are as follows: first, methanol and water are eluted in reverse at a volume ratio of 10:1 to collect the components, and then the mixture is washed with pure methanol.

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