CN115287781B - Preparation of fucoxanthin nanofiber and application of fucoxanthin nanofiber in weight losing and lipid lowering - Google Patents

Preparation of fucoxanthin nanofiber and application of fucoxanthin nanofiber in weight losing and lipid lowering Download PDF

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CN115287781B
CN115287781B CN202210971721.1A CN202210971721A CN115287781B CN 115287781 B CN115287781 B CN 115287781B CN 202210971721 A CN202210971721 A CN 202210971721A CN 115287781 B CN115287781 B CN 115287781B
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fucoxanthin
nanofiber
cyclodextrin
fat
weight
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谭明乾
李佳璇
朱蓓薇
苏文涛
彭启辉
宋玉昆
宋爽
吕玥琪
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Jiangsu Blueberry Clinical Nutrition Technology Co ltd
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Abstract

The invention discloses a preparation method of fucoxanthin nanofiber with weight-losing and lipid-lowering effects and application of the fucoxanthin nanofiber to weight-losing and lipid-lowering effects of fat mice induced by high-fat diet, and belongs to the crossing field of food and biological medicine application. The electrostatic spinning technology of the invention prepares fucoxanthin-cyclodextrin nanofiber, and the application result of the nanofiber in weight-losing and lipid-lowering shows that the nanofiber can obviously improve the conditions of liver injury and steatosis caused by obesity, and extremely obviously reduce serum Total Cholesterol (TC), serum Triglyceride (TG), low density lipoprotein (LDL-C) and high density lipoprotein (HDL-C) of a high-fat model mouse, thereby effectively playing the roles of losing weight and lowering lipid. Compared with free fucoxanthin, the prepared nanofiber has the advantages that the solubility of the fucose Huang Zhishui is enhanced, the bioavailability is increased, and the effect is better. Therefore, the health-care food is hopeful to be developed into a new health-care food for treating obesity and reducing blood fat, and achieves the purposes of controlling weight and resisting obesity when the high-fat food is taken.

Description

Preparation of fucoxanthin nanofiber and application of fucoxanthin nanofiber in weight losing and lipid lowering
Technical Field
The invention belongs to the crossing field of food science and biomedical application, and relates to a preparation method of fucoxanthin-loaded hydrophilic nanofiber and application of the fucoxanthin-loaded hydrophilic nanofiber in developing anti-obesity and hypolipidemic health-care foods induced by high-fat diet.
Background
Obesity is an important factor that is harmful to the health of the masses, mainly due to fat accumulation caused by energy metabolism disorder. The national statistical office reports that the overweight rate of the population of China increased from 13% to 30% and the obesity rate increased from 3% to 12% during the 13 years 1992-2015. Obesity has become an important factor for the health of people, and weight loss has become a problem which is urgent to be solved by the public, and weight loss foods have become a hot spot in scientific research, especially the utilization of natural foods has become a serious issue in the research and development fields.
Fucoxanthin (also called brown algae element) is mainly derived from phytoplankton such as brown algae and diatoms and marine shellfish such as oyster, and is a natural carotenoid in nature, and is a substance for brown algae. As natural carotenoids, fucoxanthin appears yellow to brown in color, mostly based on its color properties, which were originally used. In recent years, a great deal of research at home and abroad shows that fucoxanthin has a basic conjugated structure of general carotenoid, namely a multiolefin skeleton, and also contains special groups such as single epoxy group, alkenyl group and the like, so that the fucoxanthin has strong oxidation resistance.
Fucoxanthin is a hot topic in the research field of marine natural active substances, besides the oxidation resistance, and the research on the anti-obesity activity of fucoxanthin is also a hot topic in the research field of marine natural active substances. It has been found that the addition of fucoxanthin to food by means of diet regulation does not cause addiction or any other side effects in the experimental animals (D' orazio n., eugenio g., gammaone m.a., et al, fucoxanthin: a treasure from the sea Drugs,2012,10 (3): 6046-6016). It has also been experimentally observed that fucoxanthin can have therapeutic effects on obese rats, effectively reducing serum triacylglycerol, glucose and leptin levels, and achieving weight and blood lipid lowering effects by regulating expression of enzymes involved in lipid metabolism in white fat (Hu X., li Y., li C., et al, combination of fucoxanthin and conjugated linoleic acid attenuates body weight gain and improves lipid metabolism in high-fat diet-induced obese rats [ J ]. Archives of Biochemistry and Biophysics,2012,519 (1): 59-65.).
However, fucoxanthin is a fat-soluble pigment, which has poor water solubility, resulting in low bioavailability and thermal instability, which also limits the range of applications in the food industry. At present, the instability of fucoxanthin is mainly realized by constructing a carrier system to transfer and protect nutritional functional factors. In the study of fucoxanthin encapsulation preparation of nanoparticles using different wall materials, sun et al found that the encapsulation efficiency of fucoxanthin encapsulated with hydroxypropyl-beta-cyclodextrin was as high as 97.06%, but the particle size was large in the micrometer scale (Sun, X., xu, Y., zhao, L., et al, stability and bioaccessibility of fucoxanthin in spray-dried microcapsules based on various biosystems, rsc Advances,2018,8 (61), 35139-35149.). Hydrogel microbeads prepared by Li et al using gelatin, acacia and sodium alginate have a particle size of 1.6 μm and an encapsulation efficiency of 71% (Li Y.,. Dou X., pang J., et al, improvement of fucoxanthin oral efficacy via vehicles based on gum Arabic, gelatin and alginate hydroel. Journal of Functional Foods,2019, 63:103573-). In 2014, xu Liqing et al have studied to obtain a fucoxanthin nanoemulsion which can be reduced in particle size by increasing the homogenization pressure and the homogenization times (Xu Liqing, zhu Xuemei, xiong Hua. The high-pressure microfluidics are used for preparing fucoxanthin nanoemulsion, and its physicochemical analysis [ J ]. Food science, 2014,35 (24): 45-50.). The fucoidan Huang Suke glycan-casein nanoparticle was constructed by Koo et al via an electrospray system to effectively improve the solubility and stability of fucoxanthin in water (Koo s.y., mok i. -k., et al preparation of fucoxanthin-loaded nanoparticles composed of casein and chitosan with improved fucoxanthin bio-availability. Journal of Agricultural and Food Chemistry,2016,64 (49), 9428-9435.). However, the carrier system prepared by using the high-energy technology may damage fucoxanthin, so that the development of a low-energy carrier system is significant for protecting fucoxanthin with low stability, and the development of an active substance carrier system with high encapsulation efficiency and small size, which is beneficial to transfer in human body, has very good research prospect.
Disclosure of Invention
The invention aims at solving the problems of limited application caused by poor water solubility, poor thermal stability and low oral utilization rate of fucoxanthin, preparing hydrophilic nanofiber by utilizing a microfluidic electrospinning technology, and applying the nanofiber to obesity and blood fat reduction of obese mice induced by high-fat diet. The preparation method of the fucoxanthin-loaded hydrophilic nanofiber has the advantages of low cost and simple operation, can improve water solubility and stability, and widens the application of fucoxanthin in resisting obesity and reducing blood fat.
The first aim of the invention is to provide a method for preparing fucoxanthin-loaded hydrophilic nanofiber specifically, which comprises the following steps:
step 1, dissolving cyclodextrin powder with water, and uniformly mixing by stirring, ultrasonic and other physical mixing modes to obtain a cyclodextrin aqueous solution;
step 2, weighing fucoxanthin, adding the fucoxanthin into the cyclodextrin aqueous solution in the step 1, and uniformly mixing to obtain an electrospinning liquid; the mole ratio of fucoxanthin to cyclodextrin is 1:1-2;
and 3, spinning the electrospinning liquid obtained in the step 2 by adopting a microfluidic electrospinning process to obtain a product, namely the fucoxanthin-loaded hydrophilic nanofiber.
In one embodiment of the present invention, the cyclodextrin in step 1 comprises one of beta-cyclodextrin, gamma-cyclodextrin, carboxymethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin and hydroxypropyl-gamma-cyclodextrin.
In one embodiment of the present invention, in step 1, the mass volume fraction of cyclodextrin in the cyclodextrin aqueous solution is 120% to 180%.
In one embodiment of the invention, the electrospinning liquid is uniformly mixed by ultrasonic or stirring.
In one embodiment of the present invention, in step 3, the microfluidic auxiliary device is a channel engraved on a polymethyl methacrylate (PMMA) substrate by computer programming to obtain a spinning solution pipe with a cross-sectional area of 500-50000 μm 2 . The size of the metal tube inserted into the outlet of the microfluidic unit by AB glue seal is as follows
In one embodiment of the present invention, in step 3, the technological parameters of the microfluidic electrospinning are as follows: the voltage is 5-30 kV, the flow rate of the electrospinning liquid is 0.2-1.5 mL/h, and the receiving distance is 10-30 cm.
The invention discloses a fucoxanthin-loaded hydrophilic nanofiber which is obtained by the method.
The second purpose of the invention is to apply the fucoxanthin-loaded hydrophilic nanofiber to the field of health-care foods.
A third object of the present invention is to apply the above-mentioned fucoxanthin-loaded hydrophilic nanofiber to the fields of anti-obesity and hypolipidemic induced by high-fat diet.
[ advantageous effects ]
(1) The microfluidic electrostatic spinning method is adopted in the preparation of the fiber, the condition is mild, the adverse effect on active substances is avoided, the operation of the preparation process is simple, the energy consumption is low, the environment is protected, and the prepared nanofiber has controllable and uniform size;
(2) The cyclodextrin is adopted to wrap the fucoxanthin which is a hydrophobic active substance, so that the problems of poor water solubility and poor thermal stability of the fucoxanthin are remarkably improved, the hydrophilic nanofiber fucoxanthin loaded with the fucoxanthin, which is prepared through microfluidic electrostatic spinning, has high loading quantity and improves the bioavailability;
(3) The fucoxanthin-loaded hydrophilic nanofiber prepared by the invention can effectively regulate the blood lipid level of obese mice induced by high-fat diet, and obviously improve the conditions of liver injury and adipose tissue degeneration caused by obesity.
Drawings
FIG. 1 is a scanning electron microscope image of fucoxanthin-loaded hydrophilic nanofibers prepared in an example of the present invention.
FIG. 2 is an infrared spectrum of a fucoxanthin-loaded nanosystem in an embodiment of the present invention.
Figure 3 is an X-ray diffraction pattern of fucoxanthin-loaded nanosystems in an embodiment of the invention.
FIG. 4 shows the results of measurement of the thermal stability of the nanofiber carrying the nanofibers prepared in the examples of the present invention.
FIG. 5 is a graph showing the comparison of the change in TC of serum total cholesterol in mice of each group in the examples of the present invention.
FIG. 6 is a graph showing the comparison of the changes in the total triglyceride TG in serum of mice of each group in the examples of the present invention.
FIG. 7 is a graph showing the comparison of changes in LDL-C in serum of mice in each group in the examples of the present invention.
FIG. 8 is a graph showing the comparison of changes in HDL-C of serum of mice in each group in the examples of the present invention.
FIG. 9 is a comparison of H & E staining of liver tissue of mice of each group in the examples of the present invention.
FIG. 10 is a comparative graph of H & E staining of white adipose tissue of each group of mice in the example of the present invention.
FIG. 11 is a graph showing the liver index change of each group of mice in the example of the present invention.
FIG. 12 is a graph showing the comparison of testis index change in each group of mice in the example of the present invention.
In fig. 5 to 12, in comparison with the normal control group, # is P <0.05, # is P <0.01, # is P <0.001, # is P <0.0001; p <0.05, P <0.01, P <0.001, P <0.0001, compared to the model control group.
Detailed Description
The present application describes in detail preferred embodiments of the present invention and is not limited to the specific conditions and details of the following embodiments. The various specific features may be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the invention is not described in any way in which it is possible. Any person skilled in the art can make simple variants and substitutions within the technical scope of the description of the invention, depending on the circumstances, which are all within the scope of protection of the invention. The following detailed description of embodiments of the invention is provided for purposes of illustration and description of the invention, and is not intended to limit the invention to the reagents or apparatus used in the invention.
Scanning electron microscope image: shearing the prepared fucoxanthin-loaded hydrophilic nanofiber for 1cm 2 The size is adhered on a sample stage by conductive adhesive, and microscopic morphology is observed by a scanning electron microscope after vacuum metal spraying treatment.
Interaction between fucoxanthin and hydroxypropyl-beta-cyclodextrin: the Fourier infrared transformation spectrum is adopted to carry out the wavelength of 4000 cm to 400cm -1 Where a scan was performed to determine the interaction between fucoxanthin and hydroxypropyl-beta-cyclodextrin.
X-ray diffraction: with Cu-K alpha radiation detection, the instrument parameters were set as: the scanning range is 10-70 degrees, and the scanning speed is 5 degrees/min.
Thermal stability: the thermal stability of the nanofibers was analyzed by using a Q500 thermogravimetric analyzer from TA company, U.S. and heated from 30℃to 550℃under the protection of 40mL/min of nitrogen at a heating rate of 20℃per minute, and the thermal stability of the nanofibers was measured.
Anti-obesity and hypolipidemic experiments: after the male BALB/c mice are adapted to the environment for one week, the normal control group is given basic maintenance feed, the other mice are given high-fat feed, and after two weeks, the establishment of the weight gain rate judgment model is successful, and the mice are randomly divided into three groups: model control group, fucoxanthin group and fucoxanthin-cyclodextrin nanofiber group. After grouping, the normal control group was given maintenance feed, 200 μl of purified water was filled per day, the remaining groups were continued to be given high fat feed, the model control group was filled with an equal amount of purified water, the fucoxanthin group was filled with an equal amount of 250 μM fucoxanthin Huang Zhishui solution per day, and the fucoxanthin-cyclodextrin nanofiber group was filled with an equal amount of fucoxanthin-cyclodextrin nanofiber aqueous solution with a fucoxanthin concentration of 250 μM per day.
After the experiment is finished, the eyeballs of the mice are subjected to blood sampling to obtain blood samples, the blood samples are centrifuged, and the blood samples are taken and stored in an ultralow temperature refrigerator. And taking the preserved serum sample out of the ultralow temperature refrigerator, and placing the serum sample in an ice box for waiting detection. Total Cholesterol (TC), total Triglycerides (TG), high density lipoprotein (HDL-C), low density lipoprotein (LDL-C) in serum were assayed as required by the kit instructions.
Liver injury: after dissection of the mice, liver tissue and inguinal adipose tissue were removed, and a portion was cut and fixed in 4% paraformaldehyde. Paraffin embedding, slicing, dehydration, fixation, staining with hematoxylin and eosin (H & E), and histopathological analysis under a microscope.
Adipose tissue morphology:
organ index: the liver and testis of the mice were weighed, and the organ index was obtained by a proportional relationship with the body weight, and organ index=organ mass/body weight of the mice.
Example 1
Preparation of fucoxanthin-loaded hydrophilic nanofiber: 7.5g of hydroxypropyl-beta-cyclodextrin is weighed and added into 5mL of purified water in sequence until the hydroxypropyl-beta-cyclodextrin is completely dissolved, 1.6g of fucoxanthin is weighed and added into cyclodextrin water solution, and the mixture is fully mixed for 2 hours at the room temperature with the rotating speed of 200 rpm/min. Adding the metal tube into a microfluidic device, and selecting the size of an outlet metal tube of a microfluidic channel as the inner diameterOuter diameter->And the positive electrode of the high-voltage power supply is connected, the aluminum foil paper is used as a receiving plane to be connected with the negative electrode, and the injection pump is used for controlling the injection flow rate of the electrospinning liquid. The high-voltage power supply was turned on, the flow rate of the syringe was adjusted to 0.3mL/h, the receiving distance was 15cm, and the voltage was 20kV. To-be-electrospun liquidAfter spraying, the electrospun fibers on the aluminum foil paper were collected.
Example 2
Referring to example 1, electrospun fibers were prepared with fucoxanthin and cyclodextrin in a molar ratio of 1:1 and 1:2, with the remaining conditions unchanged.
As shown in figure 1, the prepared fucoxanthin-loaded hydrophilic nanofiber has uniform morphology, no adhesion and no beading, and has a fiber diameter of about 500nm in nanometer scale.
The result is shown in FIG. 2, 3600-3200cm -1 There are stretching peaks of O-H and N-H, and strong absorption of the stretching peaks indicates that hydroxyl and carboxyl groups are still present in the nanofibers. Furthermore, at 1920cm -1 The stretching vibration peak at the position can be attributed to the unique allene characteristic peak of fucoxanthin, and the peak disappears in fucoxanthin-cyclodextrin nanofiber, and fucoxanthin is found at 1715 cm and 1241cm -1 The stretching vibration peak at the site migrates in the nanofiber, indicating that fucoxanthin and hydroxypropyl-beta-cyclodextrin were successfully bound by hydrogen bonding.
The X-ray diffraction results of fig. 3 show irregular crystallization peaks, whereas fucoxanthin molecules in fucoxanthin-cyclodextrin nanofibers are separated from each other by cyclodextrin cavities and cannot form crystalline aggregates, and show an amorphous broad peak shaped like hydroxypropyl-beta-cyclodextrin in a spectrogram, thereby proving that fucoxanthin and cyclodextrin form inclusion compounds.
As shown in fig. 4, initial thermal decomposition of non-embedded fucoxanthin occurs at 44 ℃, and the thermal stability of the hydrophilic nanofiber prepared in the example is significantly improved, the initial thermal decomposition temperatures are 157.9 ℃, which illustrates that the thermal stability of Gao Yanzao yellow can be effectively improved by preparing the nanofiber after wrapping with cyclodextrin.
5-8, the serum TC, TG, LDL-C was significantly increased and HDL-C was significantly decreased in the model control mice compared to the normal control mice; compared with a model control group, the fucoxanthin group and the fucoxanthin-cyclodextrin nanofiber group effectively improve the phenomenon of dyslipidemia in serum, and the fucoxanthin-cyclodextrin nanofiber group has better effect than the fucoxanthin group, which indicates that the nanofiber improves the water solubility of fucoxanthin and improves the bioavailability of fucoxanthin.
As shown in fig. 9, compared with the normal control group, the pathological section of the liver tissue of the mouse in the model control group obviously observes the occurrence of fatty vacuoles, and compared with the model control group, the fucoxanthin group and the fucoxanthin-cyclodextrin nanofiber group can reduce the quantity and the volume of the fatty vacuoles, and the cells of the fucoxanthin-cyclodextrin nanofiber group are orderly arranged and approach to the cell arrangement state of the normal control group, which indicates that the fucoxanthin and the fucoxanthin-loaded hydrophilic nanofiber can improve the liver injury induced by high-fat diet.
As shown in fig. 10, the normal control group had regular alignment of adipocytes, a small cell volume, a substantially uniform cell size, and a thick cell wall. Compared with the normal control group, the model control group has obviously reduced fat cell number, larger cell volume, irregular arrangement and thinned cell wall under the visual field, and obviously improves the situation after being treated by fucoxanthin and fucoxanthin-cyclodextrin nanofiber, which indicates that the fucoxanthin and the hydrophilic nanofiber loaded with fucoxanthin can improve the steatosis caused by high-fat diet.
As shown in fig. 11, the liver of the high-fat model group tended to be larger than that of the normal control group, and the morphological observation volume was larger and the color was blunted, and after treatment, the liver index was significantly decreased to approach that of the normal group; in contrast, as shown in fig. 12, the testis index of the mice in the high-fat model group changed extremely significantly, and abnormality in testis index change after treatment was improved. It is explained that fucoxanthin and fucoxanthin-loaded hydrophilic nanofibers can improve abnormal visceral changes induced by high-fat diet.

Claims (7)

1. A preparation method of fucoxanthin-loaded hydrophilic nanofiber comprises the following steps:
step 1, dissolving cyclodextrin powder, and uniformly mixing the cyclodextrin powder by stirring, ultrasonic treatment and other physical mixing modes to obtain a cyclodextrin aqueous solution; the mass of cyclodextrin in the cyclodextrin aqueous solution is 7.5g, and the volume of water is 5ml; the cyclodextrin is hydroxypropyl-beta-cyclodextrin;
step 2, weighing fucoxanthin, adding the fucoxanthin into the cyclodextrin aqueous solution in the step 1, and uniformly mixing to obtain an electrospinning liquid; wherein the mole ratio of fucoxanthin to cyclodextrin is 1:1-2;
and 3, spinning the electrospinning liquid obtained in the step 2 by adopting a microfluidic electrospinning process to obtain a product, namely the fucoxanthin-loaded hydrophilic nanofiber.
2. The method of claim 1, wherein the electrospinning liquid is mixed by ultrasonic or agitation.
3. The method according to claim 1, wherein in step 3, the microfluidic auxiliary device is a channel engraving on a polymethyl methacrylate (PMMA) substrate by computer programming to obtain a spinning solution pipe with a cross-sectional area of 500-50000 μm 2 。
4. A method according to claim 3, characterized in that the size of the inserted metal tube sealed with AB glue at the outlet of the microfluidic unit is phi 100-1450 μm, phi 240-1820 μm.
5. The method according to claim 1, wherein in step 3, the technological parameters of the microfluidic electrospinning are as follows: the voltage is 5-30 kV, the flow rate of the electrospinning liquid is 0.2-1.5 mL/h, and the receiving distance is 10-30 cm.
6. The fucoxanthin-loaded hydrophilic nanofiber prepared by the method according to any one of claims 1-5.
7. The use of the fucoxanthin-loaded hydrophilic nanofiber as set forth in claim 6 in the field of health food.
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Citations (3)

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