CN117899128A - Anti-aging composition containing stem cell components and application thereof - Google Patents
Anti-aging composition containing stem cell components and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-aging composition containing stem cell components and application thereof, and belongs to the technical field of undifferentiated human cells. The invention prepares the anti-aging composition containing stem cell components, the anti-aging composition consists of human adipose tissue-derived stem cell secretion factor freeze-dried powder, cordyceps sinensis extract and spermidine, the data aspect shows a synergistic effect, the data aspect shows that the anti-aging composition has remarkable anti-aging effect on mice for a long time, and the anti-aging composition has practical value on the comprehensive utilization of stem cells, cordyceps sinensis and spermidine, and has important social benefits on exploring the ways of delaying aging, keeping the body health for a long time, improving the living quality of the elderly and relieving the pressure of the aged.
Description
Technical Field
The invention relates to the technical field of undifferentiated human cells, in particular to an anti-aging composition containing stem cell components and application thereof.
Background
Aging is a gradual physiological change in an organism, resulting in a decrease in biological function and the organism's ability to adapt to metabolic stress. Aging occurs in cells, organs, or whole organisms over time. This is a process that extends through the whole adult life cycle of any living being. During the whole life, the body is affected and acted by various substances, energy and information in the external environment, and tissues and organs are inevitably damaged and have functions declined. With the increase of medical technology and living standard, the number of aging population increases, and with aging, physiological functions of human organs are changed, such as hypertension, hyperglycemia, hyperlipidemia, hyperuricemia, high body weight, coronary atherosclerosis and other diseases increase. Delaying aging may reduce the occurrence of these diseases, improving the functional state of the body as a whole, and thus improving the quality of life of the body.
Adipose stem cells (adipose-DERIVED STEM CELLS, ADSCs) are a type of stem cells isolated from adipose tissue that have multipotent differentiation potential. Mainly recovers the repair function of tissue cells, promotes the regeneration of cells, fully improves the body functions while recovering the younger appearance, effectively improves diseases such as sub-health, premature senility and the like, and effectively and truly resists aging from inside to outside.
Spermidine (SPERMIDINE), also known as spermidine tri-hydrochloride, is a polyamine of formula C 7H19N3. Is widely distributed in organisms and is biosynthesized from putrescine (butanediamine) and ademetionine. Spermidine inhibits neuronal synthases, binds to and precipitates DNA; can also be used for purifying DNA binding proteins, stimulating T4 polynucleotide kinase activity.
Cordyceps is a traditional Chinese medicinal material, and is a complex of Cordyceps fungus Cordycepsinesis (BerK.) of Cordyceps of Clavipitaceae of Hypocreaceae, and sacc. Has effects of invigorating kidney, nourishing lung, stopping bleeding, and eliminating phlegm. It is mainly used for treating soreness of waist and knees, cough, asthma, cough, and phlegm and blood. Modern researches have considered that Cordyceps sinensis has effects of tranquilizing, anticonvulsant, cooling, etc. on central nervous system, enhancing humoral immunity, water or alcohol extract of Cordyceps sinensis can obviously inhibit growth of tumor such as mouse sarcoma, etc., cordyceps sinensis fermentation broth can resist ST segment change of rabbit myocardial ischemia, cordyceps sinensis has certain protective effect on rat stress myocardial infarction, and Cordyceps sinensis water extract has obvious protective effect on rat acute renal failure.
The paper "sources and clinical applications of mesenchymal stem cells, wu Qingfa et al, bull ACAD MIL MED SCI", considers that mesenchymal stem cells can be used to develop into surrogate tissue, which may be an ideal source of stem cells for the treatment of congenital or degenerative diseases; patent CN103153293B provides a supramolecular complex of polyanionic polymers and spermidine for tissue maintenance and repair, the components of which are considered to be effective in stimulating fibroblast proliferation, for the nutrition, maintenance, regeneration and repair of connective tissues and mucous membranes in damaged and senescent state; the patent CN1127917C provides a functional nutrient health-care product for delaying senility, which consists of 60-90% of cordyceps sinensis and 10-40% of gynostemma pentaphylla, and is considered to have the functions of delaying senility and protecting chemical liver injury. Although there is a certain knowledge of the efficacy of adipose tissue-derived stem cells, spermidine and Cordyceps sinensis in each field, there has been no report on the co-use of the above three substances for a long time, and the combined efficacy is not known.
Disclosure of Invention
The present invention is directed to an anti-aging composition containing stem cell ingredients and its use in view of the above prior art. The anti-aging composition consists of human adipose-derived mesenchymal stem cell secretion factor freeze-dried powder, cordyceps sinensis extract and spermidine, is particularly characterized in that the anti-aging composition can maintain the health condition of mice for a long time, has remarkable anti-aging effect on the mice, has practical value on the comprehensive utilization of stem cells, cordyceps sinensis and spermidine, and has important social benefits in exploring the ways of delaying aging, maintaining the health of the living bodies for a long time, improving the living quality of the elderly and relieving the pressure of the aged.
In order to achieve the above purpose, the invention adopts the following technical scheme:
According to the first aspect of the invention, an anti-aging composition containing stem cell components is provided, the anti-aging composition consists of human adipose tissue-derived stem cell secretion factor freeze-dried powder, cordyceps sinensis extract and spermidine, and the mass ratio of the human adipose tissue-derived stem cell secretion factor freeze-dried powder to the Cordyceps sinensis extract to the spermidine is 1:0.1-5:0.1-5.
Further, the human adipose-derived mesenchymal stem cell secretion factor freeze-dried powder is prepared by the following method:
And carrying out subculture on the human adipose-derived mesenchymal stem cells to obtain a culture medium after cell culture, and freeze-drying to obtain the human adipose-derived mesenchymal stem cell secretion factor freeze-dried powder.
Further, during digestion in subculture, the digestion is stopped after the cells retract and become round as observed under an inverted microscope.
Further, the passage number is 3-5 generations.
Further, the Cordyceps sinensis extract is prepared by the following method:
Weighing Cordyceps, extracting with distilled water, removing protein, dialyzing to obtain filtrate, adding ethanol to form precipitate, filtering, washing precipitate with ethanol, and lyophilizing to obtain Cordyceps extract.
Further, the mass volume ratio of the cordyceps sinensis to the distilled water during extraction is 1g: 1-10 mL, and the extraction temperature is 50-80 ℃.
Further, the dialysis temperature is 25-37 ℃ and the dialysis time is 5-12 hours.
And further, adding ethanol into the filtrate, wherein the volume concentration of the ethanol in the solution is 30-80%.
In a second aspect of the invention, there is provided the use of an anti-ageing composition comprising a stem cell component in the manufacture of an anti-ageing medicament.
The invention has the beneficial effects that:
the invention prepares an anti-aging composition containing stem cell components, which consists of stem cell secretion factor freeze-dried powder, cordyceps sinensis extract and spermidine, and can more obviously improve SOD, CAT, GSH-Px activity of corresponding components in a fibroblast experiment, wherein SOD (24.56+/-3.76) KU/L, CAT is (2.78+/-0.86) KU/L, GSH-Px is (20.53+/-2.35) KU/L. The incubation period in the water maze test is (14.1+/-3.3) s, the spleen index and the thymus index are (5.6+/-1.0) mg/g and (2.9+/-0.5) mg/g respectively, the data are obviously superior to those of a control group, compared with a stem cell group, a cordyceps group and a spermidine group, the synergistic effect is greatly improved, the health condition of a mouse is maintained for a long time, the anti-aging effect on the mouse is obvious, the practical value is realized for the comprehensive utilization of the stem cell, the cordyceps and the spermidine, and the method for exploring and delaying aging is also provided with important social benefits in the aspects of long-acting maintenance of body health, improvement of the living quality of the aged and alleviation of the pressure of social care.
Drawings
FIG. 1 is a graph of human dermal fibroblast activity assay.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.
The test materials used in the examples of the present application, which are not specifically described, are all conventional in the art and are commercially available. Spermidine used in the present application was purchased from sigma, human adipose mesenchymal stem cells and complete medium from marsupenamide, DMEM, FBS, human dermal fibroblasts (HDFa) and medium from zemoeid, SOD, CAT and GSH-Px kits from pecan days, SAM P8 aged mice from chinese euphausia.
Example 1: preparation of lyophilized powder of stem cell secretion factor
(1) Taking a cell culture bottle containing human adipose-derived mesenchymal stem cells, sterilizing the bottle body with 75% alcohol, removing the sealing film, placing into a cell culture box with 37 ℃ and 5% CO 2 and saturated humidity, and standing for 3-4h to stabilize the cell state;
(2) Sucking out the culture medium in the cell culture flask, and washing the cells once by using PBS;
(3) Adding 1mL of 0.25% trypsin digestion solution into a culture flask, slightly rotating the culture flask until the digestion solution covers the whole bottom of the culture flask, sucking out the redundant trypsin digestion solution, and carrying out warm bath at 37 ℃ for 1-3 min; observing under an inverted microscope, and adding 5ml of complete culture medium to terminate digestion after the cells retract and become round;
(4) Gently blowing and mixing by a suction tube, inoculating a culture flask according to a passage ratio for passage, then supplementing fresh complete culture medium to 5mL, and placing the culture flask in a cell culture box with 37 ℃ and 5% CO 2 and saturated humidity for static culture;
(5) Culturing and observing after the cells are completely adhered; then replacing fresh complete culture medium according to the liquid replacement frequency;
(6) Subculturing to the fifth generation, observing cell growth, taking 50mL of complete culture medium after culturing cells, subpackaging in culture dish, covering with preservative film with liquid level thickness not higher than 3mm, and freezing in a refrigerator at-86 ℃ for 24h;
(7) Taking out the frozen culture dish, stamping a plurality of small holes on the preservative film by using a fine needle, placing the culture dish in a freeze dryer, and freeze-drying;
(8) Freeze-drying to obtain powder, and packaging.
Example 2: preparation of Cordyceps sinensis extract
Weighing 30g of dried and crushed whole Cordyceps sinensis, and mixing 1g of the dried and crushed whole Cordyceps sinensis: 10 Adding distilled water into the mass volume ratio of mL, extracting for 3 times at 80 ℃, filtering, concentrating the filtrate to about 100mL, adding absolute ethanol to ensure that the volume concentration of the ethanol in the solution is 80%, standing overnight, centrifuging at 5000r/min for 20 min, dissolving the precipitate with water, removing protein 5 times by using a Sevag method (Sevag reagent is n-butanol: chloroform=1:4, V/V), mixing and concentrating the obtained supernatant, placing the supernatant in a dialysis bag for 3kD interception, dialyzing with 30 ℃ water for 12 hours, concentrating the liquid in the bag, adding ethanol to form a precipitate (the volume fraction of the ethanol in the precipitate is 50%), standing overnight, filtering, washing the precipitate with absolute ethanol, and freeze-drying the precipitate, namely the Cordyceps sinensis extract.
Example 3: preparation of anti-aging composition
The stem cell secretion factor lyophilized powder prepared in example 1, the Cordyceps sinensis extract prepared in example 2 and spermidine are mixed according to a mass ratio of 1:1:1 to form an anti-aging composition.
Example 4: preparation of anti-aging composition
The stem cell secretion factor lyophilized powder prepared in example 1, the Cordyceps sinensis extract prepared in example 2 and spermidine are mixed according to a mass ratio of 2:1:1 to form an anti-aging composition.
Example 5: preparation of anti-aging composition
The stem cell secretion factor lyophilized powder prepared in example 1, the Cordyceps sinensis extract prepared in example 2 and spermidine are mixed according to a mass ratio of 1:2:1 to form an anti-aging composition.
Example 6: preparation of anti-aging composition
The stem cell secretion factor lyophilized powder prepared in example 1, the Cordyceps sinensis extract prepared in example 2 and spermidine are mixed according to a mass ratio of 1:1:2 to form an anti-aging composition.
Test example 1
Anti-aging composition selection
(1) Human dermal fibroblasts were inoculated into 6-well plates, six groups of three were placed in parallel, and after adherence, control group and experimental group 1 were cultured using dmem+10% fbs medium, and experimental groups 2 to 5 were cultured using dmem+10% fbs medium containing 100mg/ml of the anti-aging compositions 1 to 4 prepared in examples 3 to 6, respectively;
(2) After 24 hours of culture, the control group is replaced with a new DMEM+10% FBS culture medium, and the experimental groups 1-5 are replaced with a DMEM+10% FBS culture medium with the concentration of 40mmol/L glucose for culture;
(3) Culturing is continued for 3 days, and fresh culture medium is replaced according to the method of the step (2) within 3 days.
(4) SA- β -Gal staining was performed: cells were fixed in G/F fixative (PBS containing 50% glutaraldehyde and 37% formaldehyde) for 5min at room temperature. After washing twice in PBS, the cells were left in a staining solution [ citrate/sodium phosphate buffer (containing 0.1M citric acid, 0.2M sodium phosphate solution) containing 200mM potassium ferricyanide, 200mM potassium ferrocyanide, 200mM MgCl 2, 6M NaCl and 1mg/ml X-gal ] at 37℃for 2 to 4 hours in a CO 2 -free humidification chamber, observed under a microscope and photographed.
TABLE 1 cell death rate results (%)
As can be seen from table 1, the mortality of the cells in the experimental groups 1 to 5 after the cells were treated with high sugar was significantly increased compared to the control group, and the mortality in the experimental group 1 to 5 was the lowest in the experimental group 2, so that the ratio compound used in the experimental group 2 was selected for the subsequent experiments, i.e., the three substances were mixed in a mass ratio of 1:1:1 as described in example 3.
Test example 2
SOD, CAT, GSH-Px Activity assay
The human dermal fibroblast is subcultured, and the 4 th generation cell is selected for experiment. The packet settings are as follows: a stem cell group, adding the lyophilized powder of the stem cell secretion factor prepared in example 1; cordyceps sinensis group, adding Cordyceps sinensis extract prepared in example 2; spermidine group, adding spermidine; a complex set to which the anti-aging composition prepared in example 3 was added; control group, PBS added as control; the final concentration of the additives in the cell culture solution is 5, 10, 20 and 30mg/L respectively, 3 additives are arranged in parallel in each treatment, and after the cells are treated for 72 hours, the proliferation of the cells is detected by a tetramethyl azoazole salt (MTT) colorimetric method; the results are shown in FIG. 1, wherein each concentration is a control group, a stem cell group, a Cordyceps sinensis group, a spermidine group, and a complex group in this order from left to right.
As can be seen from the results of FIG. 1, after the corresponding drugs are added to the cells of each group in the culture for 72 hours, the effect of promoting the cell growth is obtained compared with the condition of the control group (the equivalent PBS is added), the absorbance of 600nm can reach 0.81 after the compound acts for 72 hours at the concentration of 20mg/L, and the compound has a better effect of promoting the proliferation of aging cells.
The cells cultured for 72 hours were subjected to an antioxidant enzyme activity assay for each group of cells treated at a drug concentration of 20 mg/L: each group of cells was collected separately and sonicated. Supernatant was collected by centrifugation and SOD, CAT, GSH-Px activity was measured according to the method described in the kit. The results are shown in Table 2.
TABLE 2 SOD, CAT and GSH-Px detection results
As can be seen from the results in Table 2, the stem cell group, the Cordyceps sinensis group, the spermidine group and the complex group can all improve the activity of SOD, CAT, GSH-Px, and compared with the control group, the complex group can more obviously improve the activity of SOD, CAT, GSH-Px of the corresponding components, the SOD is (24.56+/-3.76) KU/L, CAT and the KU/L, GSH-Px is (2.78+/-0.86) KU/L, GSH-Px, and the substances in the complex group show synergistic effect.
SAM P8 aged mouse model study
SAMP8 of 3 month old rapid aging model mice is taken, the male and female mice are randomly grouped, 18 mice in each group are arranged in three parallel, and the group is as follows: the stem cell group comprises the stem cell secretion factor lyophilized powder prepared in the example 1; cordyceps sinensis group, wherein the medicine is Cordyceps sinensis extract prepared in example 2; spermidine group, the drug is spermidine; a complex group, the drug being the anti-aging composition prepared in example 3; each administration group was administered by gavage at an administration dose of 2mg/kg, the control group was administered with an equivalent amount of physiological saline, and the administration was performed once daily until the test was completed, and the following test was performed on surviving mice after 6 months of feeding under conventional conditions:
1. Water maze test: the mice were placed in a circular pool containing a circular platform with a water depth of 30cm, and if the mice were unable to find the platform for more than 90s, the testers placed the mice in a environment familiar with the platform for 10s, and after continuous training for 3 days, the time for each mouse to find the platform was recorded.
2. Spleen index and thymus index determination: the spleen and thymus of the mice were taken and weighed, and the ratio of the weight of spleen and thymus to the weight of mice was calculated.
Table 3 shows the results of the water maze test for each group of mice, which shows that the incubation period of each experimental group of mice is shortened compared with the control group. The latency is an important index of the water maze positioning navigation stage, and is the time required for the animal to successfully find the platform for the first time after each water entry. The length of the animal also represents the quality of the learning and memory capacity of the animal space, and the incubation period is short, which indicates that the learning and memory capacity of the animal is good. Wherein, the compound group performs optimally, and the incubation period of mice in the compound group is (24.1+/-3.3) s. It can be seen that the health condition of the mice is improved after the treatment of the compound group, the compound group treatment shows a synergistic effect, and the health condition of the mice can be maintained for a long time and the aging process is delayed.
TABLE 3 Water maze test
Table 4 shows the results of spleen index and thymus index of each group of mice, and it can be seen that the spleen index and thymus index of each experimental group of mice are increased as compared with the control group. The thymus and spleen index levels depend on the degree of lymphocyte proliferation therein, and can be estimated as to the intensity of immune function. Wherein, the compound group performs optimally, and the spleen index and thymus index of the mice in the compound group are (5.6+/-1.0) mg/g and (2.9+/-0.5) mg/g respectively. After the treatment of the compound group, the mice have stronger immune function, better health condition maintenance and aging process delay.
TABLE 4 spleen index and thymus index
In conclusion, the compound group has optimal performance, SOD (24.56+/-3.76) KU/L, CAT (2.78+/-0.86) KU/L, GSH-Px (20.53+/-2.35) KU/L, latency in a water maze test is (14.1+/-3.3) s, spleen index and thymus index are (5.6+/-1.0) mg/g and (2.9+/-0.5) mg/g respectively, and the compound group has remarkable advantages in data compared with a control group, has larger promotion compared with a stem cell group, a Cordyceps sinensis group and an spermidine group, shows a synergistic effect, and is particularly effective in maintaining the health condition of mice for a long time and has remarkable anti-aging effect on the mice.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (9)
1. The anti-aging composition containing the stem cell component is characterized by comprising human adipose-derived stem cell secretion factor freeze-dried powder, cordyceps sinensis extract and spermidine, wherein the mass ratio of the human adipose-derived stem cell secretion factor freeze-dried powder to the cordyceps sinensis extract to the spermidine is 1 (0.1-5): 0.1-5.
2. The anti-aging composition according to claim 1, wherein the human adipose-derived mesenchymal stem cell secretion factor lyophilized powder is prepared by the following method:
And carrying out subculture on the human adipose-derived mesenchymal stem cells to obtain a culture medium after cell culture, and freeze-drying to obtain the human adipose-derived mesenchymal stem cell secretion factor freeze-dried powder.
3. The anti-aging composition of claim 2, wherein digestion is terminated after cell retraction and rounding as observed under an inverted microscope during digestion in subculture.
4. The anti-aging composition according to claim 2, wherein the number of passages is 3 to 5.
5. The anti-aging composition according to claim 1, wherein the Cordyceps sinensis extract is prepared by the following method:
Weighing Cordyceps, extracting with distilled water, removing protein, dialyzing to obtain filtrate, adding ethanol to form precipitate, filtering, washing precipitate with ethanol, and lyophilizing to obtain Cordyceps extract.
6. The anti-aging composition according to claim 5, wherein the mass to volume ratio of Cordyceps sinensis to distilled water is 1g: 1-10 mL, and the extraction temperature is 50-80 ℃.
7. The anti-aging composition according to claim 5, wherein the dialysis temperature is 25-37 ℃ and the time is 5-12 hours.
8. The anti-aging composition according to claim 5, wherein the volume concentration of ethanol in the solution after adding ethanol to the filtrate is 30-80%.
9. Use of an anti-aging composition comprising stem cell ingredients according to any one of claims 1 to 8 in the manufacture of an anti-aging medicament.
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