CN114306360A - Application of lonicera edulis anthocyanin extract in preparation of medicine and/or health-care product for improving polycystic ovarian syndrome - Google Patents
Application of lonicera edulis anthocyanin extract in preparation of medicine and/or health-care product for improving polycystic ovarian syndrome Download PDFInfo
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- CN114306360A CN114306360A CN202111339915.1A CN202111339915A CN114306360A CN 114306360 A CN114306360 A CN 114306360A CN 202111339915 A CN202111339915 A CN 202111339915A CN 114306360 A CN114306360 A CN 114306360A
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- anthocyanin
- lonicera edulis
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Abstract
The invention discloses an application of a lonicera edulis anthocyanin extract in preparation of a medicine and/or a health-care product for improving polycystic ovarian syndrome; the lonicera edulis anthocyanin extract has obvious effect on improving the follicular development retardation of PCOS mice, has the functions of improving ovary reserve and improving the ovarian oxidation resistance, brings good news to vast PCOS patients, improves the psychological and physiological treatment pressure of the PCOS patients, improves the reproductive health of the vast infertility patients and improves the fertility potential of young women of childbearing age.
Description
Technical Field
The invention relates to the technical field of polycystic ovarian syndrome treatment, and in particular relates to application of a lonicera edulis anthocyanin extract in preparation of a medicine and/or health-care product for improving polycystic ovarian syndrome.
Background
Polycystic ovarian syndrome (PCOS) is the most common reproductive system disease in gynecological endocrine, mainly manifested by the virilized signs of amenorrhea, infertility, easy abortion after pregnancy, excessive obesity, hirsutism and the like, and currently affects the menstruation and fertility of about 5-10% of women in the reproductive age worldwide, and is also closely related to the health of offspring. In recent years, studies have found that metabolic abnormalities in patients with PCOS are mainly manifested by clinical manifestations such as glucose tolerance, dyslipidemia, and insulin resistance. PCOS is a genetic disease, but there are also a number of other factors that can cause disease; although the pathogenesis of PCOS is being studied, the research on the pathogenesis is still unclear, and thus, it is a problem that gynecologists pay attention to the search for effective measures for preventing PCOS by studying the pathogenesis of PCOS.
PCOS is considered a chronic inflammation, and inflammatory factors can induce excessive production of Reactive Oxygen Species (ROS) to cause oxidative damage. The follicular development of patients with PCOS is a retardation phenomenon, and mostly stays in the stage of small follicular development. Physiological levels of ROS contribute to ovarian function maintenance and oogenesis and follicular formation, but excess amounts of ROS adversely affect ovarian function in women. Ovarian oxidative stress not only affects the development of follicles, the growth and maturation of ova, the atresia and ovulation of follicles, etc., but also significantly reduces the quality of ova and the developmental potential of embryos. When the oxidation stress products of the ovum exceed the oxidation resistance of the organism, the phosphorylation of cell membrane lipid is caused and the DNA of the cell is damaged, thereby influencing the division of the cell, the metabolism of substances in the cell and the function of mitochondria and leading to the reduction of the quality of the ovum. Therefore, the occurrence of ovarian oxidative stress is improved, and the ovarian function, the quality of ova and the embryo development are possibly positively influenced.
Anthocyanidin is also called anthocyanidin, and is a water-soluble natural pigment, wherein more than 20 anthocyanidins are known, and 6 anthocyanidins are mainly present in food and are respectively: pelargonidin, cyanidin, delphinidin, peonidin, morning glory pigment, and malvidin. Anthocyanins are extremely unstable and are naturally combined with saccharides such as glucose, galactose, arabinose and the like in the form of glycosides to form anthocyanins. Anthocyanin is a polyphenol substance commonly existing in plants, belongs to a flavonoid compound, and is an important coloring substance in flowers, fruits and vegetables. Plants containing anthocyanin such as fructus Myrtilli, black currant, indigo honeysuckle, Mori fructus, Aronia melanocarpa, fructus Lycii, rhizoma Dioscoreae Esculentae, fructus Zizaniae Caduciflorae, and caulis et folium Brassicae Capitatae. Anthocyanins are generally easily soluble in organic solvents such as water, methanol, and ethanol, and insoluble in organic solvents such as diethyl ether and chloroform. The color of the anthocyanin can change along with the pH value, the pH value is strong acid- > neutral- > strong base, and the color of the anthocyanin changes into red- > purple- > blue. The maximum absorption wavelength of anthocyanin in visible light is within the range of 500-550 nm. Anthocyanin has strong antioxidation effect like vitamin C, and is an important substance for eliminating free radicals, which are harmful substances generated in metabolism of human bodies and can cause cancers, cardiovascular and cerebrovascular diseases, various inflammations and human aging. Anthocyanin is a natural pigment, is safe and nontoxic, has certain nutrition and pharmacological action, and has great application potential in the fields of food, cosmetics, health products and medicines.
At present, much research is being conducted on anthocyanins, however, no research on the influence of anthocyanins on follicular development and antioxidant effect of PCOS patients has been reported.
Disclosure of Invention
The invention aims to provide application of a lonicera edulis anthocyanin extract in preparation of a medicine and/or a health-care product for improving polycystic ovarian syndrome.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention provides application of a lonicera edulis anthocyanin extract in preparation of a medicine and/or health-care product for improving polycystic ovarian syndrome.
Preferably, the content of the anthocyanin in the lonicera edulis anthocyanin extract is not lower than 25%.
More preferably, the loniceraedulis anthocyanin mainly exists in forms of cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, paeoniflorin-3-O-rutinoside and cyanidin-3, 5-biglucoside, wherein the ratio of cyanidin-3-O-glucoside is the highest and can reach 90%.
Preferably, the lonicera edulis anthocyanin extract is obtained by the following method:
(a) adjusting the pH value of an ethanol water solution to 3-4 by adopting DL-malic acid and citric acid to obtain an extracting solution;
(b) and extracting and purifying the lonicera edulis pomace by adopting the extracting solution to obtain the lonicera edulis anthocyanin extract.
Preferably, the extracting comprises:
and (3) placing the lonicera edulis pomace into the extracting solution, stirring for a period of time, filtering, collecting filtrate and filter residues, repeating the above process for 2-3 times on the filter residues, and combining the filtrates to obtain the extracting solution.
Preferably, the mass volume ratio of the indigo fruit pomace to the extracting solution is 1: 3-5; the stirring time is not less than 2 h.
Preferably, the purification comprises:
concentrating and diluting the extracting solution, passing through DM21 and polyamide resin column, eluting the column with water solution containing DL-malic acid and citric acid, concentrating the eluate, spraying powder, and drying;
preferably, the concentration of the DL-malic acid is 0.01%, and the concentration of the citric acid is 0.01%.
In a second aspect, the present invention provides a medicament for ameliorating polycystic ovarian syndrome, the medicament comprising an extract of lonicera edulis anthocyanidin.
In a third aspect of the invention, the health care product for improving polycystic ovarian syndrome comprises a lonicera edulis anthocyanin extract.
Compared with the prior art, the invention has the beneficial effects that at least:
the lonicera edulis anthocyanin extract has obvious effect on improving the follicular development retardation of PCOS mice, has the functions of improving ovary reserve and improving the ovarian oxidation resistance, brings good news to vast PCOS patients, improves the psychological and physiological treatment pressure of the PCOS patients, improves the reproductive health of the vast infertility patients and improves the fertility potential of young women of childbearing age.
Drawings
In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.
FIG. 1 shows the PCOS model building and verification results in Experimental example 2 of the present invention;
FIG. 2 is a microscopic observation result of ovarian tissues of each group of mice in Experimental example 2 of the present invention;
FIG. 3 shows AMH expression in each group of mice in Experimental example 2;
FIG. 4 shows the expression of SOD in each group of mice in Experimental example 2;
FIG. 5 shows MDA expression in each group of mice in Experimental example 2 of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.
Example 1
The embodiment is an extraction method of an indigo fruit anthocyanin extract, which comprises the following steps:
(a) adjusting the pH value of 75% ethanol water solution to 3-4 by adopting DL-malic acid and citric acid to obtain an extracting solution;
(b) extracting indigo fruit pomace for 3 times by using an extracting solution, specifically extracting for the first time: placing the indigo fruit pomace in the extracting solution according to the mass-volume ratio of the indigo fruit pomace to the extracting solution of 1: 4, stirring for 4 hours, filtering by adopting a 1000-mesh plate frame, and collecting filtrate and filter residues;
and (3) second extraction: placing the filter residue in the extracting solution according to the mass-volume ratio of the indigo fruit residue to the extracting solution of 1: 3, stirring for 2h, filtering by adopting a 1000-mesh plate frame, and collecting filtrate and filter residue;
and (3) extracting for the third time: placing the filter residue in the extracting solution according to the mass-volume ratio of the indigo fruit residue to the extracting solution of 1: 3, stirring for 2h, filtering by adopting a 1000-mesh plate frame, and collecting filtrate and filter residue;
then, the filtrates obtained in the 3 times are combined to obtain an extracting solution;
concentrating and diluting the extracting solution, passing through DM21 and polyamide resin column, eluting with 0.01% DL-malic acid and 0.01% citric acid water solution, concentrating the eluate, spray drying to obtain the above indigo honeysuckle anthocyanin extract.
Comparative example 1
The comparative example is an extraction method of a blueberry anthocyanin extract, and the extraction method is basically the same as that in example 1, and is different only in that indigo fruit pomace is replaced by blueberry pomace.
Comparative example 2
This comparative example is a method of extracting an anthocyanin extract of blackcurrants, which is substantially the same as the extraction method of example 1 except that the lonicera edulis pomace is replaced with blackcurrant pomace.
Experimental example 1
The anthocyanin extracts obtained in the above example 1 and comparative examples 1-2 were subjected to ultraviolet-visible spectrum analysis, the analysis method is as follows:
1) instruments and appliances: analytical balance, ultrasonic cleaner, ultraviolet visible spectrophotometer.
2) Reagent and solution: methanol, hydrochloric acid, 2% hydrochloric acid-methanol solution and pure water.
3) Method of operation
A. Preparation of test solution
Accurately weighing 15-20 mg (accurate to 0.01mg) of a sample to be tested, putting the sample into a flask, adding 60mL of 2% hydrochloric acid-methanol solution (m/V), carrying out reflux hydrolysis in a water bath at 80 ℃ for 0.5 hour, cooling to room temperature, completely transferring by using the 2% hydrochloric acid-methanol solution (m/V), metering to 100mL, and shaking up to obtain a test solution A. Precisely sucking 5mL of the test solution A into a 50mL volumetric flask, fixing the volume to a scale with a 2% hydrochloric acid-methanol solution (m/V), and shaking up to obtain a solution B to be tested; the solution should be clear and transparent. If turbid, the solution is filtered through a filter paper, about 10mL of the initial filtrate is discarded, and the filtrate is used for detection.
B. Measurement method
2% hydrochloric acid-methanol solution (m/V) as a blank, and performing ultraviolet-visible spectrum analysis by using UV-1700;
and analyzing by ultraviolet visible spectrum to obtain the lonicera edulis anthocyanin content of 25%, the blueberry anthocyanin content of 18% and the blackcurrant anthocyanin content of 25%.
The types and contents of anthocyanin in the anthocyanin extracts obtained in the above example 1 and comparative examples 1-2 were measured by the following method:
after the sample was ultrasonically dissolved, it was measured by High Performance Liquid Chromatography (HPLC).
1) Instruments and appliances: an analytical balance, an ultrasonic cleaning instrument, a high performance liquid chromatograph (with an ultraviolet detector) and a 0.45 mu m microporous filter membrane.
2) Reagent and solution: methanol (chromatographically pure), acetonitrile (chromatographically pure), anhydrous formic acid (chromatographically pure), hydrochloric acid, phosphoric acid, methanol, a 2% hydrochloric acid-methanol solution (V/V), a 10% phosphoric acid aqueous solution (V/V), and pure water.
3) Standard solution: anthocyanin standard Bilberry dry extract CRS (used for determining the content of 15 anthocyanin components and purchased from EDQM); weighing about 125mg of standard substance, placing the standard substance in a 25mL volumetric flask, adding 2% hydrochloric acid-methanol solution, diluting to the scale, and shaking up. 2mL of the above solution was accurately transferred to a 10mL volumetric flask and the volume was determined by 10% phosphoric acid aqueous solution. The concentration of cyanidin-3-glucoside in the prepared standard solution is about 0.035 mg/mL.
4) Sample solution: about 125mg of the sample was weighed and dissolved in 25mL of 2% methanol hydrochloride solution, and 2mL of the above solution was transferred to a 100mL volumetric flask and then the volume was determined by using 10% phosphoric acid aqueous solution. Filtering with 0.45 μm microporous membrane to obtain test solution, and sampling 10 μ L.
5) Chromatographic conditions
A chromatographic column: zorbax extended-C18, 4.6X 250mm,5 μm or the same type of column; mobile phase: phase A: formic acid-water solution (8.5:91.5, V/V), phase B acetonitrile-methanol-formic acid-water solution (22.5:22.5:8.5:41.5, V/V/V/V); detection wavelength: 535 nm; flow rate: 1.0 mL/min.
6) Calculation of the content of each anthocyanin in the small berry extract:
in the formula:
wi-mass fraction of anthocyanin in the test sample,%;
Ai-peak area of anthocyanin in test solution;
Astda-peak area of cyanidin-3-glucoside in the standard solution;
Cstda-the concentration of cyanidin-3-glucoside in the standard solution in milligrams per milliliter (mg/mL);
m is the sample weight of the test sample in milligrams (mg);
v is the volume of the test sample with constant volume, and the unit is milliliter (mL);
p is the purity of cyanidin-3-glucoside in the standard solution,%;
MWi-the molecular weight of each anthocyanin in the test solution;
MWstda-molecular weight of cyanidin-3-glucoside.
The results are as follows:
there are three main types of lonicera edulis anthocyanins: the anthocyanin forms mainly comprise cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, paeoniflorin-3-O-rutinoside and cyanidin-3, 5-biglucoside, wherein the cyanidin-3-O-glucoside has the highest proportion of about 90%. The blueberry anthocyanin extract contains more kinds of anthocyanin, including 15 kinds, wherein anthocyanin mainly derived from cyanidin, delphinidin, petunidin, paeonia lactiflora and malvidin is taken as a main component; wherein the delphinidin derivative, the petunidin derivative and the malvidin derivative account for the highest proportion, and are respectively 35%, 28% and 25%. Blackcurrant anthocyanin mainly exists in two forms, namely cyanidin and delphinidin, and anthocyanin of the blackcurrant anthocyanin mainly exists in the following 4 forms: cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, delphinidin-3-O-glucoside and delphinidin-3-O-rutinoside, wherein the content of each anthocyanin is similar to that of the delphinidin-3-O-rutinoside, and the proportion is 92%.
Experimental example 2
Study on the effect of anthocyanin extracts obtained in example 1 and comparative examples 1 to 2 on PCOS:
(1) establishment and model verification of PCOS mouse model
25 days old C57BL/6 female mice were injected continuously with DHEA (6mg/100g, 0.2 mL. d)-1Sesame oil dissolved) while continuously feeding 60% high fat diet for 20 days; feeding control group normally; after the experiment is finished, respectively taking blood and ovaries on two sides of each group of mice, fixing one side of the ovaries on the ovaries for pathological detection, and carrying out subsequent detection on the other side of the ovaries; the verification method is as follows:
1) vaginal smear: and collecting vaginal exfoliated cell smears from the 11 th day of administration, continuously collecting the mouse vaginal exfoliated cells at the speed of 5: 00-6: 00 every afternoon for 10 days, and observing the smears. (3 were randomly selected for the normal and model groups).
2) Polycystic ovarian pathology: HE staining, procedure was as follows:
A. making slices
Fixing: fixing in 4% formaldehyde solution for 3-5 days;
block repairing: taking out the tissue from the fixing solution, and trimming the tissue into a proper shape and thickness;
and (3) dehydrating: dehydrating the tissue blocks by 80%, 90%, 95%, 100% ethanol I, 100% ethanol II and 100% ethanol III;
and (3) transparency: xylene I for 30min and xylene II for 30 min;
wax dipping: paraffin I1 h and paraffin II 5 h;
embedding: embedding the tissue with paraffin according to the principle that the material is taken downwards, and refrigerating at-20 ℃ after the wax block is cooled and solidified;
slicing: the slice thickness is 5 μm;
baking slices: putting the slices into a thermostat at 65 ℃ for 6-12 h;
and (3) storage: and (5) boxing and storing at normal temperature.
B.HE staining
Dewaxing and rehydration: xylene I15 min, xylene II 15min, anhydrous ethanol I5 min, anhydrous ethanol II 5min, 95% ethanol 5min, 80% ethanol 5min, and tap water soaking for 1 min;
dyeing: immersing the slices in hematoxylin staining solution, staining for 5min at normal temperature, and washing for 1min with tap water; immersing the slices in 1% hydrochloric acid alcohol solution for several seconds until the tissue turns blue; immersing the section into eosin dye solution for dyeing for 3-5min, and washing away the floating color on the slide with tap water;
dehydrating, transparent and sealing: 80% ethanol 0.5min, 95% ethanol I0.5 min, 95% ethanol II 0.5min, anhydrous ethanol I0.5 min, anhydrous ethanol II 0.5min, xylene I transparent 3min, xylene II transparent 3min, taking out, and sealing with neutral gum; in the dyeing condition, the cell nucleus is blue or violet blue, the cytoplasm is pink, and the red cell is brighter red;
the PCOS model building and verification results are shown in fig. 1; in fig. 1, a is a picture of intraperitoneal drug injection of mice; b is a drawing picture of mouse ovary; c is the comparison of the ovary size of the control group mouse and the ovary size of the PCOS group mouse, and the picture shows that the diameter of the PCOS mouse ovary is obviously larger than that of the control group mouse ovary; d and E are PCOS mouse ovary HE staining, E is D red frame magnification picture. F and G are the ovarian HE staining condition of the control group mice, and G is an F red frame enlarged picture. D and F, compared with the control group, a large number of small follicles are observed in the ovary of the PCOS mouse compared with the ovary of the control group mouse. H is the last 7 days of vaginal smear given to control mice. I is a negative tract smear of 7 days after the administration of PCOS mice;
as can be seen from FIG. 1, a PCOS mouse model was successfully established by DHEA injection and high-fat diet feeding. HE staining observation shows that the ovarian cortex and medulla structure of the control group are clear and visible, the structure is complete, all levels of follicles with normal development, regular structure and different sizes can be seen, the oocyte cytoplasm is rich, the number of the granular cells in the layer is large, the granular cells are arranged in order, and the corpus luteum can be seen (figure 1, F/G). The number of follicles in the model group is obviously increased, most of the follicles are dysplastic, granulosa cells are disorganized and loose, and a large number of atretic follicles are visible (FIG. 1, D/E).
(2) Grouping and feeding of PCOS mice
Adult PCOS mice were randomly divided into 7 groups of 10 mice each, which were PCOS control group, blueberry anthocyanin low dose group, blueberry anthocyanin high dose group, indigo honeysuckle anthocyanin low dose group, indigo honeysuckle anthocyanin high dose group, blackcurrant anthocyanin low dose group, blackcurrant anthocyanin high dose group, respectively. Another 10 sexually mature BALB/c female mice served as normal control group.
76 25-day-old mice (6 of them were model-verified) were continuously fed with DHEA (6mg/100g) while 60% high-fat diet for 20 days; then, the two groups were randomly divided into 7 groups, 1 group was used as a control group (10), and 6 groups were respectively a low dose group (10/group, 150 mg/kg/d) and a high dose group (10/group, 300mg/kg/d) fed with each baccatin extract, and the two groups were administered in a gavage volume of 0.1 mL.10 g < -1 >, and were administered in separate cages for 30 days with free water and free food intake (basal diet) at a temperature of 22-24 ℃ and a relative humidity of 55-65%.
After the experiment is finished, blood and ovaries on both sides of each group of mice are respectively taken, one side of the ovaries is fixed for pathological detection, and the other side of the ovaries is frozen for standby. Each group of mice was bled and the AMH, SOD and MDA contents were measured.
(3) PCOS mouse treatment and index determination
After the last gastric lavage, fasting the mice of each group without water supply, weighing the body mass of the mice of each group after 24h, taking blood from the orbit, killing the mice by a neck-breaking method, quickly taking out ovarian tissues, preparing paraffin sections by fixing, dehydrating and the like, observing the development level of follicles under a microscope, wherein the observation result is shown in figure 2; the plasma was centrifuged at 4 ℃ and the serum was separated. The contents of AMH, SOD and MDA in serum are respectively determined according to the kit instruction.
Measurement of AMH:
A. experimental materials: MIS, AMH ELISA kit (Jianglai organism, cat # JL 20476).
B. An experimental instrument: full-wavelength microplate reader (SpectraMax Plus 384, USA MD), low-speed automatic balance centrifuge (TDZ4-WS model, Hunan Xiang apparatus).
C. The detection method comprises the following steps:
a. the kit is balanced for 60min at room temperature, and required reagents are prepared;
b. setting standard substance holes and sample holes, wherein 50 mu L of standard substances with different concentrations are added into the standard substance holes respectively;
c. adding 50 mu L of sample to be detected into the sample hole, and adding 50 mu L of the diluent into the blank hole;
d. adding 100 mu L of detection antibody marked by horseradish peroxidase (HRP) into each of the standard wells and the sample wells except for blank wells, sealing the plates by using sealing plate membranes, and incubating at the constant temperature of 37 ℃ for 60 min;
e. discarding liquid, patting dry on absorbent paper, filling each hole with cleaning solution, standing for 1min, discarding cleaning solution, patting dry, and repeating for 5 times. Adding 50 μ L of substrate A, B into each well, and incubating at 37 deg.C in dark for 15 min;
f. stop solution was added in an amount of 50. mu.L per well, and the OD value of each well was measured at a wavelength of 450nm within 15 min.
② measuring the content of total superoxide dismutase (T-SOD):
A. experimental materials: total superoxide dismutase (T-SOD) determination kit (Nanjing Kangjing, cat # A001-1-2)
B. An experimental instrument: full-wavelength microplate reader (SpectraMax Plus 384, USA MD), low-speed automatic balance centrifuge (TDZ4-WS model, Hunan Xiang apparatus).
C. The detection method comprises the following steps:
a. preparing all required reagents;
b. the operation table is as follows:
mixing, standing at room temperature for 10min, adjusting the wavelength to 550nm with 1cm light path cuvette and distilled water, and measuring OD value of each tube.
Calculating the formula: total SOD activity (U/ml) ═ control OD value-measured OD value)/control OD value/50%. dilution of reaction system times fold of sample before test.
Thirdly, detecting the content of Malondialdehyde (MDA):
A. experimental materials: malondialdehyde (MDA) assay kit (Nanjing Kangkui, cat # A003-1-1).
B. An experimental instrument: full-wavelength microplate reader (SpectraMax Plus 384, USA MD), low-speed automatic balance centrifuge (TDZ4-WS model, Hunan Xiang apparatus).
C. The detection method comprises the following steps:
a. preparing all required reagents;
b. the operation table is as follows:
covering the centrifuge tube with a cover, pricking a small hole on the cover with a needle, mixing with a vortex mixer, taking out in water bath at 95 deg.C for 40 min, cooling with running water, centrifuging at 3500 rpm for 10 min. The supernatant was collected at 200pl at 532nm with an optical path of 1cm, and adjusted to zero with distilled water, and the absorbance of each tube was measured.
Calculating the formula: serum MDA content (measured OD value-control OD value)/(standard OD value-blank OD value) × standard concentration (10nmol/mL) × dilution factor before sample test.
As a result:
the microscopic observation results of the ovarian tissues of each group of mice are shown in FIG. 2; the AMH expression of each group of mice is shown in figure 3; SOD expression of each group of mice is shown in figure 4; MDA expression of each group of mice is shown in FIG. 5; in fig. 3, a vs b, c, d, P < 0.01; b vs c, P < 0.05; c vs d, P < 0.01; in FIG. 4 a, b, c, d vs e, P < 0.01; c vs e, P < 0.01; b vs d, P < 0.01; b vs c, P < 0.05; d vs e, P < 0.05; b, c, d, e, fvsa, P <0.01 in fig. 5; b vs d, P < 0.01; c vs e, P < 0.01; b vs c, P < 0.05; d vs e, P < 0.05.
As can be seen from fig. 2: the number of follicles in the model group is obviously increased, most of follicles are dysplastic, granulosa cells are disorganized and loose, and atretic follicles are observed. The indigo honeysuckle, blueberry and blackcurrant anthocyanin extracts fed to the low-dose atresia follicles are reduced compared with those fed to the model group, the granular cells are arranged neatly, the number of the atresia follicles in the high-dose group is least, most of the atresia follicles are normal, and the granular cells are arranged neatly.
As can be seen from fig. 3:
the AMH expression levels of the control group, PCOS group, each low dose anthocyanin-fed group, and each high dose anthocyanin-fed group were determined and calculated by plotting a standard curve with the standard (fig. 3, a). The results showed that the PCOS group had an increased expression level of AMH compared to the control group (fig. 3B, P < 0.01); the levels of AMH were significantly reduced in the anthocyanin-fed group and PCOS group (fig. 3B, P < 0.01); at the same time, the level of AMH was also significantly down-regulated for the high dose anthocyanins compared to the low dose anthocyanins (figure 3B, P < 0.05). In addition, the expression level of AMH in each group of the indigo fruit is higher than that in each group of blackcurrant and blueberry. The results show that the various anthocyanin extracts can positively regulate the ovarian reserve function of PCOS mice after being fed, and the effect of the lonicera edulis anthocyanin is most obvious.
As can be seen from fig. 4:
the expression level of SOD in the PCOS group is obviously lower than that in the control group (P <0.01), the low-dose anthocyanin group and the high-dose anthocyanin group (P <0.01, figure 4). The SOD expression levels of the high-dose group of the indigo honeysuckle are not different from those of the control group, and meanwhile, the SOD expression levels of each group of the indigo honeysuckle are higher than those of each group of blueberry and blackcurrant (figure 4). The result shows that the anthocyanin extract can obviously improve the antioxidant capacity in PCOS mice after being fed, and the lonicera edulis anthocyanin has the most obvious effect and has obvious difference.
As can be seen from fig. 5:
the PCOS group showed significantly higher MDA expression levels compared to the control group (P <0.01, fig. 5); in the anthocyanin group, the expression level of MDA was significantly reduced compared to the PCOS group (P <0.01, fig. 5), and the expression level of MDA in the high dose anthocyanin group was also significantly lower than that in the low dose anthocyanin group (P <0.05, fig. 5). In addition, the expression levels of the MDA in the low-dose group and the high-dose group of the lonicera edulis are lower than the expression levels of the MDA in each group corresponding to the blackcurrant group and the blueberry group. The result shows that the anthocyanin extract can reduce the peroxidation level in mice and reduce the damage of cells after being fed, and the indigo fruit anthocyanin has the most obvious effect and has obvious difference.
In conclusion, the anthocyanin extracts of each group can obviously reduce the peroxidation level of the ovary tissue of the PCOS mouse, improve the oxidation resistance of the ovary tissue, improve the follicular development level of the PCOS mouse and improve the ovarian reserve capacity. The lonicera edulis anthocyanin extract has better using effect and dose dependence compared with blueberry and blackcurrant anthocyanin extracts. The main existing form of the loniceraedulis anthocyanin is cyanidin-3-O-glucoside, which can be determined to play an important role and lay a foundation for further research.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.
Claims (9)
1. Application of indigo honeysuckle anthocyanin extract in preparation of medicine and/or health product for improving polycystic ovarian syndrome.
2. The use as claimed in claim 1, wherein the lonicera edulis anthocyanin extract contains not less than 25% anthocyanins.
3. The use as claimed in claim 1, wherein the lonicera edulis anthocyanin extract is obtained by the following method:
(a) adjusting the pH value of an ethanol water solution to 3-4 by adopting DL-malic acid and citric acid to obtain an extracting solution;
(b) and extracting and purifying the lonicera edulis pomace by adopting the extracting solution to obtain the lonicera edulis anthocyanin extract.
4. The use according to claim 3, wherein said extracting comprises:
and (3) placing the lonicera edulis pomace into the extracting solution, stirring for a period of time, filtering, collecting filtrate and filter residues, repeating the above process for 2-3 times on the filter residues, and combining the filtrates to obtain the extracting solution.
5. The use of claim 4, wherein the mass-volume ratio of the indigo fruit pomace to the extracting solution is 1: 3-5; the stirring time is not less than 2 h.
6. Use according to claim 3, wherein the purification comprises:
concentrating and diluting the extract, passing through DM21 and polyamide resin column, eluting with water solution containing DL-malic acid and citric acid, concentrating the eluate, spraying powder, and drying.
7. The use according to claim 6, wherein the concentration of DL-malic acid is 0.01% and the concentration of citric acid is 0.01%.
8. A medicament for ameliorating polycystic ovarian syndrome, comprising an extract of lonicera edulis anthocyanidin.
9. A health product for improving polycystic ovarian syndrome, which is characterized by comprising a lonicera edulis anthocyanin extract.
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| CN115887439A (en) * | 2022-12-06 | 2023-04-04 | 武汉大学 | Application of compound in preparation of polycystic ovarian syndrome ovarian protection medicine |
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