CN112402480A

CN112402480A - Preparation method and application of eucommia male flower extract with anti-fatigue effect

Info

Publication number: CN112402480A
Application number: CN202011087398.9A
Authority: CN
Inventors: 张东星; 郑艳超; 晏仁义; 李赫宇; 王磊; 胡丹
Original assignee: Tianjin Yibeiyuan Natural Product Technology Co ltd
Current assignee: Tianjin Yibeiyuan Natural Product Technology Co ltd
Priority date: 2020-10-13
Filing date: 2020-10-13
Publication date: 2021-02-26

Abstract

A preparation method and application of eucommia male flower extract with anti-fatigue effect comprises the following steps: taking a certain amount of eucommia male flower, extracting the eucommia male flower with 15 times of water by mass, refluxing for 2 hours at 100 ℃, filtering to obtain filtrate for later use, adding 15 times of water by mass into the eucommia male flower, boiling and refluxing for the second time, filtering and collecting the filtrate, mixing the two filtrates, concentrating and spray-drying to obtain the eucommia male flower extract. The invention extracts eucommia male flower by water, filters and concentrates to obtain extract, and experiments prove that the eucommia male flower extract and the monomer components thereof can promote the secretion of testosterone in testicular interstitial cells and can be used for health-care food with anti-fatigue as the main function.

Description

Preparation method and application of eucommia male flower extract with anti-fatigue effect

Technical Field

The invention relates to the field of health-care food, in particular to a preparation method and application of an eucommia male flower extract with an anti-fatigue effect.

Background

Over 20% of people worldwide are affected by fatigue, which has become a serious problem in modern society affecting human health, work efficiency and quality of life. Fatigue refers to the elimination of physical or psychological manifestations caused by disease or physiological factors, such as lassitude, lack of concentration, dizziness, poor vitality, or palpitation and asthma. Fatigue relief is one of the research directions of health-care functional foods, and has a large proportion in developed products. 15062 nationally approved health foods, wherein 2082 products are used for relieving physical fatigue.

In the future, the anti-fatigue product can be accepted by wide consumers, and the anti-fatigue product with clear mechanism and clear material composition is more popular. Market research finds that although the eucommia male flower anti-fatigue products in different product forms such as solid beverages, candies, chocolates, coffee, biscuits and the like are correspondingly developed in China, the whole body is in the initial stage, and the research is lack of a real functional factor for resisting fatigue of the eucommia male flower. The action mechanism is deeply explored to enable the functions of the raw materials, and corresponding products are developed on the basis, so that the raw materials have better research value and market prospect.

Eucommia male flowers are approved by the national health family planning committee in 6 months of 2014 as a new food raw material. The eucommia male flower contains collagen needed by human body, and the anti-fatigue effect of the ingredients is very obvious. The tea is one of the best tea drinks for people who work for a long time on a table or lack of sports. Since the last 80 s, the eucommia male flowers are widely used in China for tea making due to their anti-fatigue, sedative and anti-aging effects. Therefore, it is necessary to develop a novel anti-fatigue health food having a definite and safe drug effect.

Eucommia Male flowers (Male flower of Eucommia ulmoides Oliv.) are flowers of artificially planted Eucommia Male plant trees (Eucommia ulmoides Oliv.). Eucommia male flowers are approved by the national health family planning committee in 6 months of 2014 as a new food raw material. Eucommia male flowers are approved by the national health family planning committee in 6 months of 2014 as a new food raw material. The inventor finds that the eucommia male flower extract and the monomer components in the eucommia male flower can promote the secretion of testosterone in testicular interstitial cells and can be used for health-care food with the main function of resisting fatigue.

Disclosure of Invention

The invention provides a preparation method and application of an eucommia male flower extract with an anti-fatigue effect for promoting the secretion of testosterone in testicular interstitial cells, and the inventor finds that the eucommia male flower extract can promote the secretion of testosterone in testicular interstitial cells and can be used for health-care food with the main function of anti-fatigue through research.

The technical scheme adopted by the invention is as follows:

a preparation method of eucommia male flower extract with anti-fatigue effect comprises the following steps:

taking a certain amount of eucommia male flower, extracting the eucommia male flower with 15 times of water by mass, refluxing for 2 hours at 100 ℃, filtering to obtain filtrate for later use, adding 15 times of water by mass into the eucommia male flower, boiling and refluxing for the second time, filtering and collecting the filtrate, mixing the two filtrates, concentrating and spray-drying to obtain the eucommia male flower extract.

The filtrate is concentrated by rotary evaporation at 65 ℃ under vacuum.

The eucommia male flower extract is filtered by a filter screen or filter cloth with at least 200 meshes.

The eucommia male flower extract is applied to preparing anti-fatigue medicines or health-care foods.

The eucommia male flower extract and auxiliary materials approved by medicaments or health-care food are prepared into various dosage forms which mainly comprise the eucommia male flower extract by adopting modern preparation technology, including extractum, tablets, granules, pills, capsules, powder and oral liquid.

The invention has the beneficial effects that: the invention extracts eucommia male flower by water, filters and concentrates to obtain extract, and experiments prove that the eucommia male flower extract and monomer components in the eucommia male flower can promote the secretion of testosterone in testicular interstitial cells, and can be used for health-care food with anti-fatigue as a main function.

Drawings

FIG. 1 is a drawing showing the identification of leydig cells according to the present invention, wherein A in FIG. 1 is a photograph of a band obtained by cell separation after centrifugation; FIG. 1, panel B is a purified leydig cell morphology; FIG. 1, panel C shows the specific staining (adherent cells) of purified leydig cells by 3 β -HSD; in FIG. 1, panel D shows the specific staining (suspension cells) of purified leydig cells by 3 β -HSD.

FIG. 2 is a diagram showing the results of the cell viability test of the leydig cells under different sample interventions.

FIG. 3 is a graph showing the effect of the extract of the male flowers of eucommia ulmoides and the monomer components in the male flowers of eucommia ulmoides on testosterone secretion from leydig cells.

Detailed Description

The invention will be explained in detail below with reference to specific examples and experimental protocols:

example 1

taking a certain amount of eucommia male flower, extracting the eucommia male flower with 15 times of water by mass, refluxing for 2 hours at 100 ℃, filtering to obtain filtrate for later use, adding 15 times of water by mass into the eucommia male flower, boiling and refluxing for the second time, filtering and collecting the filtrate, mixing the two filtrates, concentrating by rotary evaporation at 65 ℃ under vacuum, and then spray-drying to obtain the eucommia male flower extract.

Experimental arrangement is carried out on the secretion of testosterone from testis interstitial cells by the eucommia male flower extract:

an experimental instrument:

electronic analytical balance (MS 204S, mettler-toledo); pipettors (Research plus, Eppendorf, germany); autoclave (MLS-3751L-PC, Sanyo Co., Japan); an electric hot blast drying oven (GZX-9070 MBE, Shanghai Boxun industries, Ltd.); magnetic stirrers (CJB-16, Ci Yuan Hua instruments, Inc.); clean bench (DL-CJ-2N, Tokyo Touhal instruments manufacturing Co., Ltd.); carbon dioxide incubator (BNP-80 CH, Shanghai-Hengchun scientific instruments Co., Ltd.); a fluorescence inverted microscope (IX-73, Olympus, Japan); ultra-low temperature refrigerator (DW-HL 538, Mitsubishi low temperature science and technology, Inc. of China); microplate reader (Bio-Tek Synergy 2, Gene, USA); a micro-shaker (QT-1, Shanghai Qi Te Analyzer Co., Ltd.), a refrigerated Centrifuge (Eppendorf, Centrifuge 5804R, Germany), an electrophoresis apparatus (JUNYIIJY 1000C), a fluorescent quantitative PCR apparatus (LightCycler 480 II, Roche USA), and an ultra-micro ultraviolet spectrophotometer (Thermo Scientific, NanoDrop 2000c, USA).

Experimental reagent:

DME/F-12 medium (AC 10550270), Hyclone; collagenase i, Invitrogen corporation; fetal bovine serum, GIBCO, horse serum, GIBCO, newborn bovine serum, hangzhou biotechnology limited, zhejiang; double antibody, GIBCO, Percoll isolate, BIOSHARP biotechnology; sodium pyruvate, AMRESCO corporation; NBT, BIOSHARP biotechnology; DHEA, shanghai mclin biochemistry science and technology limited; beta-NAD, Shanghai Michelin Biochemical technology, Inc.; nicotinamide, beijing solibao science and technology ltd; 0.4% trypan blue dye solution, Beijing Soilebao Tech Co., Ltd; 22 human chorionic gonadotropin HCG, institute for food and drug testing, china; 2.5% pancreatin, GIBCO; DMSO, Sigma company; testosterone ELISA kit, Nanjing, to build bioengineering research institute; CCK-8 kit, BIOSHARP Biotech.

The experimental method comprises the following steps:

cell separation and purification: namely, the acquisition of primary cells:

killing SPF-grade healthy adult male rats after cervical spine removal (provided by research institute of disease control center in Hubei province), rinsing in 75% ethanol for 30 s, dissecting abdominal cavity with sterile scissors, peeling and taking out testis, placing in a culture dish containing PBS, removing peripheral fat, rinsing with PBS for 2 times, removing membrane on ice, cutting, adding a small amount of DMEM/F12 culture medium, soaking, shaking at 34 deg.C for 15 min, taking out, gently dispersing testis with forceps, adding appropriate amount of 0.05% collagenase, digesting in 37 deg.C water bath constant temperature oscillator (120 rpm) for 15 min, taking out, adding 3 times volume of DMEM/F12 complete culture solution (containing 9% fetal calf serum, 1% horse serum, 0.5% sodium pyruvate, and 1% double antibody), stopping digestion, standing for 2 min, decomposing undispersed tissue, filtering with 70 μ M nylon filter screen, centrifuge at 4 ℃ for 10 min in a 230 g centrifuge, wash the cells twice with DMEM/F12 complete medium for 5 min each time, discard the supernatant and resuspend in 2 mL of complete medium. For the remaining tissue filtered through the filter, the enzymatic lysate is added, and then the digestion is stopped and the cells are resuspended as above.

The resuspended cells were applied to a preformed Percoll density gradient centrifugation (5% gradient up and down, 30%, 58%,70%, 2 mL each gradient), centrifuged at 3000 rpm at 4 ℃ for 30 min, followed by careful pipetting of the second strip and centrifugation twice at 1000 rpm with complete medium for 5 min, and the cells were pelleted. Then adding proper amount of complete culture medium, inoculating in cell bottle, and culturing at 37 deg.C with 5% CO₂Culturing in an incubator, changing the culture solution for 24 h, and changing the culture solution every 2 days.

And (3) observing cell morphology:

collecting cells in logarithmic phase, adjusting the concentration of cell suspension, adding 100 mu L of the cell suspension into each well, inoculating the cells into a 6-well culture plate according to the volume of 1 × 106/mL, and observing the morphological change of the cells in different time after inoculation by using an inverted microscope.

3 beta-HSD enzyme staining solution method detection purity:

NBT and DHEA were dissolved together in DMSO to give a final concentration of 10 mg/mL^-1The solution A is the solution A; NAD was dissolved in PBS buffer to a final concentration of 10 mg/mL^-1The solution B is solution B; nicotinamide was dissolved in PBS buffer to give a final concentration of 1 mg. multidot.mL^-1This is solution C. And preparing 1mL of 3 beta-HSD coloring agent from 10 muL of A solution, 100 muL of LB solution, 100 muL of L C solution and 790 muL of PBS solution. And (3) placing the primary testicular interstitial cells into a 96-well plate for culturing for 24 hours, sucking out culture solution in a culture well, and adding a newly-prepared 3 beta-HSD staining agent into each well with the volume of 200 muL. The 96-well plate was placed in an incubator and incubated for 1 h before microscopic observation. 10 pieces of the testis were selected, 20 high power lens fields were counted per piece, and the average value was taken as the purity of the leydig cells. The formula for calculating the purity of cells: number of positively stained cells/total number of cells × 100%.

Cell viability detection by trypan blue staining:

the cell survival rate is detected by trypan blue staining exclusion method, and the live cells are colorless and transparent, and the dead cells are blue. Preparing primary cultured testis cells into cell suspension with density of 1 × 106. mL-1 with DMEM-F12 culture solution, mixing the cell suspension and 0.4% trypan blue solution, standing for 3 min, and counting live cells and dead cells with cell counting plate. Survival = live cells/(live + dead cells) × 100%.

Cytotoxicity assay:

adopting CCK-8 method, collecting testis interstitial cells in logarithmic growth phase, digesting with 0.25% pancreatin, gently blowing to obtain single cell suspension, adding fresh culture solution for cell counting, inoculating 100 μ L/well and 5 × 103 cells into 96-well culture plate (blank group is replaced with PBS with equal volume), placing at 37 deg.C and 5% CO₂After 24 h incubation in the incubator, fresh 100. mu.L of culture medium was replaced and different samples were added for intervention. Control group and blank group (zero setting hole) are set, each group is set with 3 parallel holes, wherein, the control group is added with culture solution with equal volume of sample for inoculating cells, and the blank group is added with culture solution without inoculating cells. Adding medicine, incubating 96-well plate in incubator for 72 hr, adding 100 μ L of CCK-8 solution into each well, incubating for 4 hr, adding the mixture into incubator, incubatingMeasuring absorbance at 450 nm, calculating corresponding inhibition rate and half inhibition concentration (IC 50 value), wherein the inhibition rate = (control group OD-addition group OD)/(control group OD-blank group OD) × 100%

ELISA detection of Testosterone secretion in rat Leydig cells:

cells were collected in log phase and the cell suspension concentration was adjusted to 3X 104. multidot.mL^-1Inoculating 300 muL per well to a 24-well culture plate with 5% CO₂Incubating and culturing at 37 ℃ for 24 h until cell monolayers are paved on the bottom of the hole, replacing 270 mu L of fresh culture solution and adding 30 mu L of medicine to enable the final concentration to be 25 mu g-mL respectively^-1、5 μg·mL^-1、1 μg·mL^-1And 0.1% DMSO was included as a control. 37 ℃ and 5% CO₂Culturing for 24 h, collecting cell supernatant, centrifuging at 4 deg.C and 2600 rpm for 20 min, and collecting supernatant for detecting testosterone content according to kit instructions.

The experimental results are as follows:

and (3) identifying testis interstitial cells:

FIG. 1 shows the bands of cell separation after centrifugation as shown in FIG. 1A, and 4 mL of the bands are leydig cells. The morphology of the purified leydig cells is shown in B of figure 1. Leydig cells were primary cultured in vitro for 24 h, stained specifically with 3 β -HSD, as shown in FIG. 1, panel C, and in FIG. 1, panel D: it can be seen that most of the cytoplasm of the cells is bluish black, the cell bodies have elongated tentacles protruding, and a small part of the cytoplasm of the cells is slightly stained and grayish blue. The purity of the cells is more than 90%. The trypan blue staining method is used for staining, and the calculated activity can reach more than 75%.

Leydig cell morphology:

FIG. 1A shows a testis-mesenchymal cell separation band, in which the lowest part is blood cells, 4 mL of the band is testis-mesenchymal cells, and 6 mL of the band is tissue fragments and epithelial cells; FIG. 1 panel B shows the purified leydig cell morphology; the leydig cells purified in panel C of fig. 1 were stained specifically for 3 β -HSD (adherent cells); the leydig cells purified in panel D of FIG. 1 were stained specifically for 3 β -HSD (adherent cells).

Cell viability assay:

from FIG. 2 canIt is found that the concentration of the compound is 10. mu.g.mL by CCK-8 detection^-1The cell survival rate of the eucommia male flower extract is more than 85 percent below, and no obvious cytotoxic effect is seen. The cell survival rate of the monomer components in the eucommia male flower is more than 85% under 100 mu M, and no obvious cytotoxic effect is seen, which indicates that the eucommia male flower extract and the monomer components in the eucommia male flower have no toxicity to cells.

The influence of the eucommia male flower extract and monomer components in the eucommia male flower on the testosterone secretion of the testis interstitial cells of the rat:

as can be seen from figure 3, eucommia male flower extract 0.361 μ g/mL, caffeic acid, deacetyl asperuloside, naringenin, Acantoside B, geniposide, rutin and quercetin all can promote testosterone secretion by leydig cells at 50 μ M, and can be used as main functional and health food for resisting fatigue.

The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.

Claims

1. A preparation method of eucommia male flower extract with anti-fatigue effect comprises the following steps:

2. The method for preparing eucommia ulmoides male flower extract with anti-fatigue effect according to claim 1, wherein the filtrate is concentrated by rotary evaporation at 65 ℃ under vacuum.

3. The method for preparing an eucommia male flower extract with an anti-fatigue effect according to claim 1, wherein the eucommia male flower extract is filtered by a filter screen or a filter cloth of at least 200 meshes.

4. Use of the eucommia male flower extract of claim 1 in the preparation of a medicine or health food having an anti-fatigue effect.

5. The use as claimed in claim 4, wherein the eucommia male flower extract and the pharmaceutical or health food approved auxiliary materials are prepared into various dosage forms including extract, tablet, granule, pill, capsule, powder and oral liquid by modern preparation technology.