CN112229990A - Method for researching lactation promoting effect of oyster polysaccharide - Google Patents

Method for researching lactation promoting effect of oyster polysaccharide Download PDF

Info

Publication number
CN112229990A
CN112229990A CN202010898313.9A CN202010898313A CN112229990A CN 112229990 A CN112229990 A CN 112229990A CN 202010898313 A CN202010898313 A CN 202010898313A CN 112229990 A CN112229990 A CN 112229990A
Authority
CN
China
Prior art keywords
ethanol
polysaccharide
oyster
tissue
xylene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202010898313.9A
Other languages
Chinese (zh)
Inventor
秦小明
谌素华
章超桦
曹文红
郑惠娜
林海生
高加龙
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangdong Ocean University
Original Assignee
Guangdong Ocean University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangdong Ocean University filed Critical Guangdong Ocean University
Priority to CN202010898313.9A priority Critical patent/CN112229990A/en
Publication of CN112229990A publication Critical patent/CN112229990A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/44—Sample treatment involving radiation, e.g. heat
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/3563—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing solids; Preparation of samples therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2866—Grinding or homogeneising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/302—Stain compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/305—Fixative compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
    • G01N2021/3155—Measuring in two spectral ranges, e.g. UV and visible
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/3563—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing solids; Preparation of samples therefor
    • G01N2021/3572—Preparation of samples, e.g. salt matrices

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Optics & Photonics (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention discloses a method for researching the lactation promoting effect of oyster polysaccharide, which comprises the following steps: s1, experimental materials (1) oyster, 60 female mice and 30 male mice, the weight is 220-; (2) bromocriptine mesylate tablets, 2.5 mg/tablet; s2, main experimental reagents, absolute ethyl alcohol, concentrated sulfuric acid, concentrated hydrochloric acid, glucose, sodium tetraborate decahydrate, iodine, potassium iodide, Congo red, potassium bromide, trifluoroacetic acid, trichloromethane, n-butanol, redistilled phenol, glucose and potassium sulfate. According to the research method for promoting lactation by using the oyster polysaccharide, the oyster polysaccharide is found to be capable of improving the lactation capacity of a hypogalactia mother mouse, so that the lactation amount of the hypogalactia mother mouse is recovered to be close to the normal production level, and the dosage of the selected polysaccharide is also shown to be correct.

Description

Method for researching lactation promoting effect of oyster polysaccharide
Technical Field
The invention relates to the technical field of oyster polysaccharide lactation promotion, in particular to a method for researching the oyster polysaccharide lactation promotion effect.
Background
As breast milk is rich in antibody molecules and immune factors necessary in the process of infant development, is easy to digest and absorb and is the best nutrient source in the process of infant growth and development, the public attention of breast feeding is continuously rising, and researches at home and abroad show that 46% of infants can not obtain enough breast milk for postpartum hypogalactia due to puerperal hypogalactia, so that the postpartum hypogalactia is caused by a plurality of reasons, such as nutritional deficiency, insufficient hormone level, emotional tension or poor life style and the like. The traditional Chinese medicine holds that the amount of milk is closely related to the qi and blood of the viscera of the mother, so that the research and the development of the medicine and the high-grade nutriment for treating the hypogalactia of the puerpera have important significance for improving the hypogalactia condition of the puerpera.
The oyster polysaccharide has great significance for the research on the aspects of oxidation resistance, blood fat reduction, spermatogenesis promotion and the like of rich biological activity, and the research on whether the oyster polysaccharide promotes the lactation effect is basically not found after the domestic and foreign documents are consulted, so that the research on the oyster polysaccharide promoting the lactation effect has certain significance for enriching the biological activity content of the oyster polysaccharide. The purpose of the study is to preliminarily discuss whether the oyster polysaccharide can improve the lactation function of the hypogalactia rats, evaluate whether the oyster glycogen can improve the serum prolactin level, the lactation amount, the weight gain of the newborn rats, the organ index and the like of the postpartum hypogalactia mother rats, and observe and compare the morphological changes of mammary tissues of all groups. Lays a theoretical foundation for oyster polysaccharide research and development of related medicaments and high-quality nutrient accumulation data.
Disclosure of Invention
The invention aims to provide a method for researching the lactation promoting effect of oyster polysaccharide, and aims to solve the problems that whether oyster polysaccharide can improve the lactation function of a hypogalactia rat is preliminarily discussed, whether oyster glycogen can improve the serum prolactin level, the lactation amount, the weight gain and the organ index of a newborn rat and the like of a postpartum hypogalactia mother rat is evaluated, and the morphological changes of mammary tissues of all groups are observed and compared.
In order to achieve the purpose, the invention provides the following technical scheme: a method for researching lactation promotion effect of oyster polysaccharide comprises the following steps:
s1 test Material
(1) Oyster, 60 female rats and 30 male rats, the weight of which is 220-;
(2) bromocriptine mesylate tablets, 2.5 mg/tablet.
S2, main experimental reagent
Anhydrous ethanol, concentrated sulfuric acid, concentrated hydrochloric acid, glucose, sodium tetraborate decahydrate, iodine, potassium iodide, congo red, potassium bromide, trifluoroacetic acid, trichloromethane, n-butanol, redistilled phenol, glucose, potassium sulfate, trichloroacetic acid, barium chloride, gelatin, sodium hydroxide, sodium chloride, D-glucuronic acid, xylene, hematoxylin eosin staining agent, chloral hydrate, carbazole, potassium ferrocyanide, zinc acetate, papain, and pepsin.
S2 animal experiment method
(1) Method for extracting oyster crude polysaccharide by enzymolysis
(2) Rat grouping method
The rats are raised in the same cage according to the male-female ratio of 2:1, the rats are combined for three days, female rats with vaginal embolus are marked at the tail ends of the tails by a marking pen, pregnant female rats are selected, the female rats are raised in a single cage, after the female rats are pregnant, 24 female rats with short time difference (about 24 hours) are selected from the female rats after farrowing and serve as experimental objects, the number of the newborn rats in each litter is adjusted to 12, and finally the 24 selected female rats are randomly divided into 3 groups by 8 rats in each group, wherein the groups are respectively a normal group, a model group and an oyster polysaccharide group.
On the day after delivery, the first group of the female mice were gavaged with distilled water only as a normal group, the second group of the female mice were gavaged with bromocriptine solution only (0.45mg/ml) as a model group, and the third group of the treatment test groups were given bromocriptine solution and oyster polysaccharide solution (500mg/kg) simultaneously.
(3) Weighing newborn mouse
(4) One-way analysis of variance of data using SPSS19.0 software
(5) Collecting and preserving serum
(6) Collection of mammary glands
(7) Detection of PRL concentration
(8) Standard curve and concentration calculation
And (3) preparing a standard curve by using Excel, substituting the OD value measured by the sample into the linear regression equation of the lower graph, calculating the concentration of the sample, and multiplying the concentration by the dilution factor to obtain the actual concentration of the sample.
(9) Calculation of mammary index and visceral index
(10) Observation of mammary gland tissue
a. The mammary tissue after 48h fixation was removed, the shape was trimmed, and the fixation solution was washed with running water.
b. The tissue block washed by tap water to remove the fixing liquid is put into alcohol with different gradient concentrations, and the dehydration is carried out by the following steps from low concentration alcohol to high concentration alcohol.
50% ethanol 30 min-75% ethanol 30 min-80% ethanol 30 min-95% ethanol 30min
Anhydrous ethanol I30 min-anhydrous ethanol II 40min
c. The completely dehydrated tissues are respectively placed in xylene: alcohol (1:1), xylene I each 15min, xylene II 40 min.
d. After the paraffin wax is melted, the transparent tissue is waxed according to the following steps, the temperature of the waxed tissue is 60 ℃ (the temperature can be lower or higher)
Paraffin I1 h-Paraffin II 1 h-Paraffin III 1h
(11) Making tissue slices
a. Slicing
The wax block is fixed on the holder, the wax block and the blade form an included angle of 5 degrees, the wax block is sliced, the tissue section in the wax block slice is complete and smooth, the blade is moved leftwards by a distance of about one wax block width, the slice thickness is adjusted to be 5 mu m, and then the slice is sliced, and the slice is continuously banded, complete and uniform.
b. Exhibition piece
Preheating a baking machine at 46 ℃, dripping a small amount of egg white on a clean glass slide, uniformly smearing in the same direction, dripping a few drops of distilled water, carefully clamping a wax sheet on the glass slide, and finally placing the glass slide on the baking machine for drying.
c. Deparaffinization and hydration of tissue sections
The completely dried tissue slices are placed on a slide rack and dewaxed according to the following steps:
xylene I: and 5 min.
Xylene II: and 5 min.
1/2 xylene +1/2 absolute ethanol solution: and 5 min.
After dewaxing was complete, the tissue sections were washed to elute xylene as follows:
anhydrous ethanol: and 2 min.
95% ethanol: and 2 min.
90% ethanol: and 2 min.
80% of ethanol: and 2 min.
70% ethanol: and 2 min.
Washing with distilled water: and 2 min.
Sequentially placing dewaxed tissue slices into gradient ethanol, sequentially removing xylene according to the sequence of anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol, soaking in ethanol of each concentration for 2min, and finally soaking in distilled water for 2 min.
d. Hematoxylin staining of tissue sections
Spin-drying the tissue slices with distilled water, soaking the tissue slices in hematoxylin staining solution for 4min, finally washing with distilled water for 2min, observing the staining result under a low power microscope, and repeating the previous steps until the staining is clear if the cell nucleus staining is not clear.
e. Eosin staining of tissue sections
Decolorizing the tissue section after hematoxylin staining according to the following steps:
70% ethanol: and 2 min.
80% of ethanol: and 2 min.
95% ethanol: and 20 min.
100% ethanol i: and 2 min.
After the decolorization is finished, the tissue section is placed in a mixed solution of 95 percent ethanol and 0.5 percent eosin dye solution for soaking and dyeing for 12min, and then the elution is carried out according to the following steps.
95% ethanol: and 2 min.
Anhydrous ethanol i: and 2 min.
Anhydrous ethanol ii: and 2 min.
1/2 xylene +1/2 ethanol: and 2 min.
Xylene I: for 10 min.
Xylene II: for 10 min.
f. Sealing piece of tissue slice
The stained slide glass is taken out from the xylene, and neutral gum is immediately dropped to cover the cover glass for mounting when the liquid on the surface of the slide glass is not dry.
S3 experiment method for analyzing polysaccharide
(1) Phenol-sulfuric acid method for measuring polysaccharide content
(2) Uronic acid content determination-carbazole colorimetric method
(3) Barium chloride-gelatin method for measuring sulfate group content
(4) Ultraviolet full-wavelength scanning comparison purification separation effect
Adjusting the wavelength range of a double-beam ultraviolet visible light photometer to 200nm-800nm, scanning the oyster crude polysaccharide solution with the concentration of 1mg/ml in the wavelength range, and making an ultraviolet scanning graph by taking an X axis as wavelength change and a Y axis as a light absorption value. Because protein and nucleic acid have maximum absorption peaks under 280nm and 260nm respectively, whether the crude oyster polysaccharide contains two impurities of protein and nucleic acid before and after sevage protein removal can be judged through the change of an absorption value obtained by ultraviolet scanning.
S4 and polysaccharide structure determination method
(1) If the polysaccharide contains multiple helical conformations, congo red will complex with it, increasing the maximum absorption wavelength of the solution. And comparing the maximum absorption wavelength change corresponding to the increase of the concentration of the NaOH solution from low to high to judge whether the polysaccharide contains multi-strand spiral conformation.
(2) Making Congo red curve
(3) Sugar chain structure
Mixing an I-KI reagent with a certain concentration with a polysaccharide solution, and then carrying out ultraviolet scanning, wherein if a maximum absorption peak appears at 350nm, the polysaccharide has more complicated side chains and branches, and if the maximum absorption peak appears at 565nm, the polysaccharide has fewer branched chains.
And (3) sample determination: adjusting the wavelength range of a double-beam ultraviolet visible light photometer to 300nm-700nm, then adding 1.2ml of I-KI reagent into 1mg/ml of crude polysaccharide solution, uniformly mixing, and carrying out ultraviolet scanning in the wavelength range.
(4) Infrared spectroscopy
Grinding potassium bromide, putting the sample and the ground potassium bromide under an infrared lamp for drying for more than 4 hours, pressing the potassium bromide into a semitransparent sheet by a tablet machine after drying, mixing the sample and the potassium bromide according to a proper proportion, tabletting, scanning within the range of 4000 plus 400cm < -1 > [29], and storing related data after scanning.
S5, Experimental results and analysis
(1) Ultraviolet full wave scanning
(2) Sugar chain structure analysis
(3) Conformational analysis
(4) Infrared spectroscopic analysis
(5) Chemical composition analysis
S6 animal results and analysis
(1) Comparison of milk yield of the female mouse
(2) Comparison of mammary gland indices
(3) Comparison of visceral indices
(4) Comparison of serum prolactin content
(5) Comparison of body weight gain in mice
(6) Comparison of mammary gland structures
S7, Experimental results and analysis
The viscera index of rats in each group shows that the viscera index values of the oyster crude polysaccharide group except the liver index are higher than those of the normal group and the model group, wherein the spleen index and the thymus index of the oyster polysaccharide group are obviously higher than those of the model group, and have significant difference (P < 0.05); the experimental results show that the model building of the rat hypogalactia model is successful, and the oyster polysaccharide can improve the lactation capacity of the mother rat
Compared with the prior art, the invention has the beneficial effects that: the oyster polysaccharide has effect in promoting lactation;
1. in the experiment, SD rats are taken as research objects, and whether the oyster polysaccharide has the lactation promoting effect is researched by manufacturing rat hypogalactia models. The research result shows that the milk secretion of the normal group is remarkably increased compared with the milk secretion of the model group on 5 th, 8 th, 11 th, 12 th and 15 th days, and has a very significant difference (P < 0.01); the milk secretion of the oyster polysaccharide group is remarkably increased on the 7 th day, the 5 th day, the 9 th day, the 10 th day, the 13 th day, the 11 th day, the 12 th day, the 15 th day and the 16 th day compared with the milk secretion of the model group, and has remarkable difference (P <0.05), while the milk secretion of the oyster polysaccharide group is not remarkably different compared with the milk secretion of the normal group; the Prolactin (PRL) level of the model group is reduced by 14.3 percent compared with that of the normal group, and has a significant difference (P < 0.05); the differences of the oyster crude polysaccharide group and the normal group and the model group are very significant (P is less than 0.01), wherein the oyster crude polysaccharide group is increased by 35.7 percent compared with the model group and 18.8 percent compared with the normal group, which indicates that the oyster crude polysaccharide can improve the serum prolactin level;
2. under an optical microscope, compared with a normal group, the model group has the advantages that small lobular lobules of mammary glands are not obvious, acinus are irregular in shape, acinus cavities are small, and acinus tissues are rare and scattered; compared with the model group, the oyster polysaccharide group has the advantages that the number of mammary glands is obviously increased, the arrangement is neat, the cavity of the mammary gland is large, and the mammary lobules are basically recovered to be normal; the weight gain of the newborn mouse is reduced by 33.62 percent compared with that of the normal group, the difference is very significant (P <0.01), the weight gain of the oyster polysaccharide group is increased by 38.21 percent compared with that of the newborn mouse of the model group, and the difference is very significant (P < 0.01); all groups of rat visceral organ indexes show that except the liver index, other visceral organ index values of the oyster crude polysaccharide group are higher than those of the normal group and the model group, wherein the spleen index and the thymus index of the oyster polysaccharide group are obviously higher than those of the model group, and have obvious difference (P <0.05), the experimental results show that the model building of the rat hypogalactia model is successful, and the oyster polysaccharide can improve the lactation capacity of a mother rat;
3. the research finds that the oyster polysaccharide can improve the lactation capacity of the hypogalactia mother mouse, so that the lactation amount of the hypogalactia mother mouse is recovered to be close to the normal yield level, the dosage of the selected polysaccharide is correct, and the research lays a theoretical foundation for researching and developing postpartum hypogalactia medicines and high-quality nutrient accumulation data of the oyster polysaccharide later.
Drawings
FIG. 1 is a schematic view of the process for extracting crude oyster polysaccharide according to the present invention;
FIG. 2 is a schematic view of a structural prolactin assay according to the present invention;
FIG. 3 is a schematic diagram of a prolactin standard curve structure according to the present invention;
FIG. 4 is a schematic diagram of the structure of the mammary gland index and the organ index according to the present invention;
FIG. 5 is a schematic diagram of a glucose standard curve according to the present invention;
FIG. 6 is a schematic diagram of a glucose standard curve according to the present invention;
FIG. 7 is a schematic diagram of the structure of a standard sulfate curve according to the present invention;
FIG. 8 is a schematic diagram of a Congo red curve structure according to the present invention;
FIG. 9 is a schematic diagram of a comparative structure of ultraviolet full-wave scanning before and after deproteinization of sevage reagent of the present invention;
FIG. 10 is a schematic structural diagram of an I-KI ultraviolet scan before and after deproteinization of the sevage reagent of the present invention;
FIG. 11 is a structural diagram of a curve showing the variation of the maximum absorption wavelength before and after deproteinization of Congo red and sevage reagent according to the present invention;
FIG. 12 is a schematic representation of the IR spectrum of sevage of the present invention before deproteinization;
FIG. 13 is a schematic representation of the structure of the infrared spectrum of sevage of the present invention after protein removal;
FIG. 14 is a schematic diagram of an absorption wavenumber structure according to the present invention;
FIG. 15 is a schematic structural diagram of the protein-removing component of sevage according to the present invention;
FIG. 16 is a schematic view showing the comparison of days in lactation of the female mouse according to the present invention;
FIG. 17 is a structural diagram of the average lactation yield of the mother mouse according to the present invention;
FIG. 18 is a graph showing the comparison of the average values of the total amount of lactation per hour for a mother mouse lactating for 16 days according to the present invention;
FIG. 19 is a schematic diagram of a comparative breast index structure according to the present invention;
FIG. 20 is a schematic view showing a comparative structure of organ indexes according to the present invention;
FIG. 21 is a schematic diagram showing a structure of comparison of prolactin content in serum according to the present invention;
FIG. 22 is a schematic diagram showing a comparison of the prolactin concentration in serum of each group of female mice according to the present invention;
FIG. 23 is a structural diagram of comparison of body weight gain of a baby mouse according to the present invention;
FIG. 24 is a structural diagram showing the comparison of the average values of the total body weight gains of 16 days for the groups of newborn mice according to the present invention;
FIG. 25 is a schematic structural view of HE staining (X100) of various groups of mammary tissues according to the present invention;
FIG. 26 is a schematic structural view of HE staining (X200) of various groups of mammary tissues according to the present invention;
FIG. 27 is a structural schematic diagram of HE staining (X300) of various breast tissues in accordance with the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a method for researching lactation promotion effect of oyster polysaccharide comprises the following steps:
s1 test Material
(1) Oyster, 60 female rats and 30 male rats, the weight of which is 220-;
(2) bromocriptine mesylate tablets, 2.5 mg/tablet.
S2, main experimental reagent
Anhydrous ethanol, concentrated sulfuric acid, concentrated hydrochloric acid, glucose, sodium tetraborate decahydrate, iodine, potassium iodide, congo red, potassium bromide, trifluoroacetic acid, trichloromethane, n-butanol, redistilled phenol, glucose, potassium sulfate, trichloroacetic acid, barium chloride, gelatin, sodium hydroxide, sodium chloride, D-glucuronic acid, xylene, hematoxylin eosin staining agent, chloral hydrate, carbazole, potassium ferrocyanide, zinc acetate, papain, and pepsin.
S2 animal experiment method
(1) Extracting Concha Ostreae crude polysaccharide by enzymolysis (see figure 1)
(2) Rat grouping method
The rats are raised in the same cage according to the male-female ratio of 2:1, the rats are combined for three days, female rats with vaginal embolus are marked at the tail ends of the tails by a marking pen, pregnant female rats are selected, the female rats are raised in a single cage, after the female rats are pregnant, 24 female rats with short time difference (about 24 hours) are selected from the female rats after farrowing and serve as experimental objects, the number of the newborn rats in each litter is adjusted to 12, and finally the 24 selected female rats are randomly divided into 3 groups by 8 rats in each group, wherein the groups are respectively a normal group, a model group and an oyster polysaccharide group.
On the day after delivery, the first group of the female mice were gavaged with distilled water only as a normal group, the second group of the female mice were gavaged with bromocriptine solution only (0.45mg/ml) as a model group, and the third group of the treatment test groups were given bromocriptine solution and oyster polysaccharide solution (500mg/kg) simultaneously.
(3) Weighing newborn mouse
(4) One-way analysis of variance of data using SPSS19.0 software
(5) Collecting and preserving serum
(6) Collection of mammary glands
(7) Detection of PRL concentration (see FIG. 2)
(8) Standard curve and concentration calculation
And (3) preparing a standard curve by using Excel, substituting the OD value measured by the sample into the linear regression equation of the lower graph, calculating the concentration of the sample, and multiplying the concentration by the dilution factor to obtain the actual concentration of the sample. (see FIG. 3)
(9) Calculation of mammary index and organ index (see FIG. 4)
(10) Observation of mammary gland tissue
a. The mammary tissue after 48h fixation was removed, the shape was trimmed, and the fixation solution was washed with running water.
b. The tissue block washed by tap water to remove the fixing liquid is put into alcohol with different gradient concentrations, and the dehydration is carried out by the following steps from low concentration alcohol to high concentration alcohol.
50% ethanol 30 min-75% ethanol 30 min-80% ethanol 30 min-95% ethanol 30min
Anhydrous ethanol I30 min-anhydrous ethanol II 40min
c. The completely dehydrated tissues are respectively placed in xylene: alcohol (1:1), xylene I each 15min, xylene II 40 min.
d. After the paraffin wax is melted, the transparent tissue is waxed according to the following steps, the temperature of the waxed tissue is 60 ℃ (the temperature can be lower or higher)
Paraffin I1 h-Paraffin II 1 h-Paraffin III 1h
(11) Making tissue slices
a. Slicing
The wax block is fixed on the holder, the wax block and the blade form an included angle of 5 degrees, the wax block is sliced, the tissue section in the wax block slice is complete and smooth, the blade is moved leftwards by a distance of about one wax block width, the slice thickness is adjusted to be 5 mu m, and then the slice is sliced, and the slice is continuously banded, complete and uniform.
b. Exhibition piece
Preheating a baking machine at 46 ℃, dripping a small amount of egg white on a clean glass slide, uniformly smearing in the same direction, dripping a few drops of distilled water, carefully clamping a wax sheet on the glass slide, and finally placing the glass slide on the baking machine for drying.
c. Deparaffinization and hydration of tissue sections
The completely dried tissue slices are placed on a slide rack and dewaxed according to the following steps:
xylene I: and 5 min.
Xylene II: and 5 min.
1/2 xylene +1/2 absolute ethanol solution: and 5 min.
After dewaxing was complete, the tissue sections were washed to elute xylene as follows:
anhydrous ethanol: and 2 min.
95% ethanol: and 2 min.
90% ethanol: and 2 min.
80% of ethanol: and 2 min.
70% ethanol: and 2 min.
Washing with distilled water: and 2 min.
Sequentially placing dewaxed tissue slices into gradient ethanol, sequentially removing xylene according to the sequence of anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol, soaking in ethanol of each concentration for 2min, and finally soaking in distilled water for 2 min.
d. Hematoxylin staining of tissue sections
Spin-drying the tissue slices with distilled water, soaking the tissue slices in hematoxylin staining solution for 4min, finally washing with distilled water for 2min, observing the staining result under a low power microscope, and repeating the previous steps until the staining is clear if the cell nucleus staining is not clear.
e. Eosin staining of tissue sections
Decolorizing the tissue section after hematoxylin staining according to the following steps:
70% ethanol: and 2 min.
80% of ethanol: and 2 min.
95% ethanol: and 20 min.
100% ethanol i: and 2 min.
After the decolorization is finished, the tissue section is placed in a mixed solution of 95 percent ethanol and 0.5 percent eosin dye solution for soaking and dyeing for 12min, and then the elution is carried out according to the following steps.
95% ethanol: and 2 min.
Anhydrous ethanol i: and 2 min.
Anhydrous ethanol ii: and 2 min.
1/2 xylene +1/2 ethanol: and 2 min.
Xylene I: for 10 min.
Xylene II: for 10 min.
f. Sealing piece of tissue slice
The stained slide glass is taken out from the xylene, and neutral gum is immediately dropped to cover the cover glass for mounting when the liquid on the surface of the slide glass is not dry.
S3 experiment method for analyzing polysaccharide
(1) Polysaccharide content determination-phenol sulfuric acid method (see FIG. 5)
(2) Uronic acid content determination-carbazole colorimetry (see FIG. 6)
(3) Sulfate group content measurement-barium chloride-gelatin method (see FIG. 7)
(4) Ultraviolet full-wavelength scanning comparison purification separation effect
Adjusting the wavelength range of a double-beam ultraviolet visible light photometer to 200nm-800nm, scanning the oyster crude polysaccharide solution with the concentration of 1mg/ml in the wavelength range, and making an ultraviolet scanning graph by taking an X axis as wavelength change and a Y axis as a light absorption value. Because protein and nucleic acid have maximum absorption peaks under 280nm and 260nm respectively, whether the crude oyster polysaccharide contains two impurities of protein and nucleic acid before and after sevage protein removal can be judged through the change of an absorption value obtained by ultraviolet scanning.
S4 and polysaccharide structure determination method
(1) If the polysaccharide contains multiple helical conformations, congo red will complex with it, increasing the maximum absorption wavelength of the solution. And comparing the maximum absorption wavelength change corresponding to the increase of the concentration of the NaOH solution from low to high to judge whether the polysaccharide contains multi-strand spiral conformation.
(2) Making congo red curve (see fig. 8)
(3) Sugar chain structure
Mixing an I-KI reagent with a certain concentration with a polysaccharide solution, and then carrying out ultraviolet scanning, wherein if a maximum absorption peak appears at 350nm, the polysaccharide has more complicated side chains and branches, and if the maximum absorption peak appears at 565nm, the polysaccharide has fewer branched chains.
And (3) sample determination: adjusting the wavelength range of a double-beam ultraviolet visible light photometer to 300nm-700nm, then adding 1.2ml of I-KI reagent into 1mg/ml of crude polysaccharide solution, uniformly mixing, and carrying out ultraviolet scanning in the wavelength range.
(4) Infrared spectroscopy
Grinding potassium bromide, putting the sample and the ground potassium bromide under an infrared lamp for drying for more than 4 hours, pressing the potassium bromide into a semitransparent sheet by a tablet machine after drying, mixing the sample and the potassium bromide according to a proper proportion, tabletting, scanning within the range of 4000 plus 400cm < -1 > [29], and storing related data after scanning.
S5, Experimental results and analysis
(1) Ultraviolet full wave scan (see FIG. 9)
(2) Sugar chain Structure analysis (see FIG. 10)
(3) Conformation analysis (see FIG. 11)
(4) Infrared spectroscopic analysis (see FIGS. 12-14)
(5) Chemical composition analysis (see FIG. 15)
S6 animal results and analysis
(1) Comparison of lactation yield of female mouse (see FIG. 16)
(2) Comparison of mammary gland indices (see FIG. 17)
(3) Comparison of organ indexes (see FIG. 18)
(4) Comparison of serum prolactin content (see FIG. 19)
(5) Comparison of body weight gain of mice (see FIG. 20)
(6) Mammary gland structure comparison (see FIG. 21-FIG. 23)
S7, Experimental results and analysis
The viscera index of rats in each group shows that the viscera index values of the oyster crude polysaccharide group except the liver index are higher than those of the normal group and the model group, wherein the spleen index and the thymus index of the oyster polysaccharide group are obviously higher than those of the model group, and have significant difference (P < 0.05); the experimental results show that the model building of the rat hypogalactia model is successful, and the oyster polysaccharide can improve the lactation capacity of the mother rat.
Those not described in detail in this specification are within the skill of the art.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (1)

1. A method for researching the lactation promoting effect of oyster polysaccharide is characterized by comprising the following steps: the method comprises the following steps:
s1 test Material
(1) Oyster, 60 female rats and 30 male rats, the weight of which is 220-;
(2) bromocriptine mesylate tablets, 2.5 mg/tablet.
S2, main experimental reagent
Anhydrous ethanol, concentrated sulfuric acid, concentrated hydrochloric acid, glucose, sodium tetraborate decahydrate, iodine, potassium iodide, congo red, potassium bromide, trifluoroacetic acid, trichloromethane, n-butanol, redistilled phenol, glucose, potassium sulfate, trichloroacetic acid, barium chloride, gelatin, sodium hydroxide, sodium chloride, D-glucuronic acid, xylene, hematoxylin eosin staining agent, chloral hydrate, carbazole, potassium ferrocyanide, zinc acetate, papain, and pepsin.
S2 animal experiment method
(1) Method for extracting oyster crude polysaccharide by enzymolysis
(2) Rat grouping method
The rats are raised in the same cage according to the male-female ratio of 2:1, the rats are combined for three days, female rats with vaginal embolus are marked at the tail ends of the tails by a marking pen, pregnant female rats are selected, the female rats are raised in a single cage, after the female rats are pregnant, 24 female rats with short time difference (about 24 hours) are selected from the female rats after farrowing and serve as experimental objects, the number of the newborn rats in each litter is adjusted to 12, and finally the 24 selected female rats are randomly divided into 3 groups by 8 rats in each group, wherein the groups are respectively a normal group, a model group and an oyster polysaccharide group.
On the day after delivery, the first group of the female mice were gavaged with distilled water only as a normal group, the second group of the female mice were gavaged with bromocriptine solution only (0.45mg/ml) as a model group, and the third group of the treatment test groups were given bromocriptine solution and oyster polysaccharide solution (500mg/kg) simultaneously.
(3) Weighing newborn mouse
(4) One-way analysis of variance of data using SPSS19.0 software
(5) Collecting and preserving serum
(6) Collection of mammary glands
(7) Detection of PRL concentration
(8) Standard curve and concentration calculation
And (3) preparing a standard curve by using Excel, substituting the OD value measured by the sample into the linear regression equation of the lower graph, calculating the concentration of the sample, and multiplying the concentration by the dilution factor to obtain the actual concentration of the sample.
(9) Calculation of mammary index and visceral index
(10) Observation of mammary gland tissue
a. The mammary tissue after 48h fixation was removed, the shape was trimmed, and the fixation solution was washed with running water.
b. The tissue block washed by tap water to remove the fixing liquid is put into alcohol with different gradient concentrations, and the dehydration is carried out by the following steps from low concentration alcohol to high concentration alcohol.
50% ethanol 30 min-75% ethanol 30 min-80% ethanol 30 min-95% ethanol 30min
Anhydrous ethanol I30 min-anhydrous ethanol II 40min
c. The completely dehydrated tissues are respectively placed in xylene: alcohol (1:1), xylene I each 15min, xylene II 40 min.
d. After the paraffin wax is melted, the transparent tissue is waxed according to the following steps, the temperature of the waxed tissue is 60 ℃ (the temperature can be lower or higher)
Paraffin I1 h-Paraffin II 1 h-Paraffin III 1h
(11) Making tissue slices
a. Slicing
The wax block is fixed on the holder, the wax block and the blade form an included angle of 5 degrees, the wax block is sliced, the tissue section in the wax block slice is complete and smooth, the blade is moved leftwards by a distance of about one wax block width, the slice thickness is adjusted to be 5 mu m, and then the slice is sliced, and the slice is continuously banded, complete and uniform.
b. Exhibition piece
Preheating a baking machine at 46 ℃, dripping a small amount of egg white on a clean glass slide, uniformly smearing in the same direction, dripping a few drops of distilled water, carefully clamping a wax sheet on the glass slide, and finally placing the glass slide on the baking machine for drying.
c. Deparaffinization and hydration of tissue sections
The completely dried tissue slices are placed on a slide rack and dewaxed according to the following steps:
xylene I: and 5 min.
Xylene II: and 5 min.
1/2 xylene +1/2 absolute ethanol solution: and 5 min.
After dewaxing was complete, the tissue sections were washed to elute xylene as follows:
anhydrous ethanol: and 2 min.
95% ethanol: and 2 min.
90% ethanol: and 2 min.
80% of ethanol: and 2 min.
70% ethanol: and 2 min.
Washing with distilled water: and 2 min.
Sequentially placing dewaxed tissue slices into gradient ethanol, sequentially removing xylene according to the sequence of anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol, soaking in ethanol of each concentration for 2min, and finally soaking in distilled water for 2 min.
d. Hematoxylin staining of tissue sections
Spin-drying the tissue slices with distilled water, soaking the tissue slices in hematoxylin staining solution for 4min, finally washing with distilled water for 2min, observing the staining result under a low power microscope, and repeating the previous steps until the staining is clear if the cell nucleus staining is not clear.
e. Eosin staining of tissue sections
Decolorizing the tissue section after hematoxylin staining according to the following steps:
70% ethanol: and 2 min.
80% of ethanol: and 2 min.
95% ethanol: and 20 min.
100% ethanol i: and 2 min.
After the decolorization is finished, the tissue section is placed in a mixed solution of 95 percent ethanol and 0.5 percent eosin dye solution for soaking and dyeing for 12min, and then the elution is carried out according to the following steps.
95% ethanol: and 2 min.
Anhydrous ethanol i: and 2 min.
Anhydrous ethanol ii: and 2 min.
1/2 xylene +1/2 ethanol: and 2 min.
Xylene I: for 10 min.
Xylene II: for 10 min.
f. Sealing piece of tissue slice
The stained slide glass is taken out from the xylene, and neutral gum is immediately dropped to cover the cover glass for mounting when the liquid on the surface of the slide glass is not dry.
S3 experiment method for analyzing polysaccharide
(1) Phenol-sulfuric acid method for measuring polysaccharide content
(2) Uronic acid content determination-carbazole colorimetric method
(3) Barium chloride-gelatin method for measuring sulfate group content
(4) Ultraviolet full-wavelength scanning comparison purification separation effect
Adjusting the wavelength range of a double-beam ultraviolet visible light photometer to 200nm-800nm, scanning the oyster crude polysaccharide solution with the concentration of 1mg/ml in the wavelength range, and making an ultraviolet scanning graph by taking an X axis as wavelength change and a Y axis as a light absorption value. Because protein and nucleic acid have maximum absorption peaks under 280nm and 260nm respectively, whether the crude oyster polysaccharide contains two impurities of protein and nucleic acid before and after sevage protein removal can be judged through the change of an absorption value obtained by ultraviolet scanning.
S4 and polysaccharide structure determination method
(1) If the polysaccharide contains multiple helical conformations, congo red will complex with it, increasing the maximum absorption wavelength of the solution. And comparing the maximum absorption wavelength change corresponding to the increase of the concentration of the NaOH solution from low to high to judge whether the polysaccharide contains multi-strand spiral conformation.
(2) Making Congo red curve
(3) Sugar chain structure
Mixing an I-KI reagent with a certain concentration with a polysaccharide solution, and then carrying out ultraviolet scanning, wherein if a maximum absorption peak appears at 350nm, the polysaccharide has more complicated side chains and branches, and if the maximum absorption peak appears at 565nm, the polysaccharide has fewer branched chains.
And (3) sample determination: adjusting the wavelength range of a double-beam ultraviolet visible light photometer to 300nm-700nm, then adding 1.2ml of I-KI reagent into 1mg/ml of crude polysaccharide solution, uniformly mixing, and carrying out ultraviolet scanning in the wavelength range.
(4) Infrared spectroscopy
Grinding potassium bromide, putting the sample and the ground potassium bromide under an infrared lamp for drying for more than 4 hours, pressing the potassium bromide into a semitransparent sheet by a tablet machine after drying, mixing the sample and the potassium bromide according to a proper proportion, tabletting, scanning within the range of 4000 plus 400cm < -1 > [29], and storing related data after scanning.
S5, Experimental results and analysis
(1) Ultraviolet full wave scanning
(2) Sugar chain structure analysis
(3) Conformational analysis
(4) Infrared spectroscopic analysis
(5) Chemical composition analysis
S6 animal results and analysis
(1) Comparison of milk yield of the female mouse
(2) Comparison of mammary gland indices
(3) Comparison of visceral indices
(4) Comparison of serum prolactin content
(5) Comparison of body weight gain in mice
(6) Comparison of mammary gland structures
S7, Experimental results and analysis
The viscera index of rats in each group shows that the viscera index values of the oyster crude polysaccharide group except the liver index are higher than those of the normal group and the model group, wherein the spleen index and the thymus index of the oyster polysaccharide group are obviously higher than those of the model group, and have significant difference (P < 0.05); the experimental results show that the model building of the rat hypogalactia model is successful, and the oyster polysaccharide can improve the lactation capacity of the mother rat.
CN202010898313.9A 2020-08-31 2020-08-31 Method for researching lactation promoting effect of oyster polysaccharide Pending CN112229990A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010898313.9A CN112229990A (en) 2020-08-31 2020-08-31 Method for researching lactation promoting effect of oyster polysaccharide

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010898313.9A CN112229990A (en) 2020-08-31 2020-08-31 Method for researching lactation promoting effect of oyster polysaccharide

Publications (1)

Publication Number Publication Date
CN112229990A true CN112229990A (en) 2021-01-15

Family

ID=74116638

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010898313.9A Pending CN112229990A (en) 2020-08-31 2020-08-31 Method for researching lactation promoting effect of oyster polysaccharide

Country Status (1)

Country Link
CN (1) CN112229990A (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060008512A1 (en) * 2004-07-07 2006-01-12 Hooge Danny M Composition and methods for improved animal performance
CN108936277A (en) * 2018-05-29 2018-12-07 佛山市三水区嘉信农业技术研究院（普通合伙） A kind of noodles promoting postpartum milk secretion and recovery

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060008512A1 (en) * 2004-07-07 2006-01-12 Hooge Danny M Composition and methods for improved animal performance
CN108936277A (en) * 2018-05-29 2018-12-07 佛山市三水区嘉信农业技术研究院（普通合伙） A kind of noodles promoting postpartum milk secretion and recovery

Similar Documents

Publication Publication Date Title
Pearce et al. The mucopolysaccharides of human skin
CN106138541B (en) Processing technology of fresh rhizoma pinelliae preparata in producing area
CN103284153B (en) Cod liver oil soft capsule and preparation method thereof
CN112229990A (en) Method for researching lactation promoting effect of oyster polysaccharide
CN109540823B (en) Method for detecting aflatoxin in corn feed stored in feedlot
CN105211794A (en) Ultralow temperature extracts the method for the pancebrin of potato activity
KR101190575B1 (en) Manufacturing method of honey containg kitosan and the honey obtained thereby
CN117462467A (en) Purslane extract with moisturizing and soothing effects, and preparation method and application thereof
CN105462769A (en) Preparation method for cistanche fermented wine and low wine prepared from cistanche fermented wine
CN103509711A (en) Compound type biological tissue and cell chip
CN115894729B (en) Porphyra haitanensis polysaccharide degradation product and preparation method and application thereof
CN1931181A (en) New use of tremella heteropolysaccharide or its extract
CN102871070B (en) Pumpkin silk noodles and preparation method thereof
CN113788904B (en) Laminarin compound with intestinal barrier repairing effect and preparation method thereof
CN111973635A (en) Method for researching lactation promoting effect of oysters
CN103555527B (en) Snake peptide healthcare wine and preparation method thereof
CN102613142A (en) Method for circulating collection and brewing of honey by taking honey as honey source in isolated space
CN115201189A (en) Influence of feeding growing-finishing pigs with pepper seeds on small intestine tissues
CN113640245A (en) Method for detecting beta-carotene, astaxanthin and starch in haematococcus pluvialis under nitrogen stress
CN106805236A (en) Extracting method and its application of mushroom mucus are slided with inoxidizability
CN111249317A (en) Preparation method of myxomycete fermentation liquor, myxomycete fermentation liquor and application
CN112121181A (en) Method for evaluating immunity enhancing effect of health product
CN113559114B (en) Application of enteromorpha polysaccharide in reducing foam cell lipid accumulation
CN117919150B (en) Preparation method and application of small molecular plant water base
WO2024139063A1 (en) Method for fast measurement of crude polysaccharide in ganoderma lucidum spore powder

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20210115

RJ01 Rejection of invention patent application after publication