CN117323416A - Composition for enhancing effect of stem cells in regulating sub-health state, and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of stem cell therapy, in particular to a composition for enhancing the efficacy of stem cells in regulating sub-health state, a preparation method and application thereof. A composition for enhancing the effect of stem cells in regulating sub-health state comprises epidermal growth factor, resveratrol, guarana leaf extract, human serum albumin, vitamin C and glutathione. The stem cell preparation formed by the composition can improve symptoms of deficiency and fatigue, easy fatigue, insomnia, low work and rest quality, difficult concentration, poor adaptability, poor mental state, low emotion, slow reaction, dreaminess and insomnia, daytime sleepiness, hypomnesis, dysphoria, anxiety, easy convulsion and the like of sub-healthy patients. The composition solves the technical problems of unsatisfactory timeliness and activity and further unsatisfactory initiation effect of stem cells in sub-health state, and has ideal market popularization and application prospect.
Description
Technical Field
The invention relates to the technical field of stem cell therapy, in particular to a composition for enhancing the efficacy of stem cells in regulating sub-health state, a preparation method and application thereof.
Background
In addition to the health and disease states, there is an intermediate state of non-health, non-disease, i.e., sub-health. The main characteristics of the health care product are various symptoms reflected by the sense of inadaptation on the body, such as fatigue, weakness, emotion change and the like, and the condition of the health care product is difficult to be defined in a quite long period of time; various frailty manifestations, microecological imbalance conditions, pre-disease physiopathological changes of certain diseases, which are caused by age-incompatible tissue structures or physiological hypofunction, occur. Sub-health is a constantly changing dynamic process that, if not actively involved, necessarily worsens the disease state. For example, overweight turns into obesity, which in turn causes various diseases; the high value of blood pressure changes into clinical hypertension; diabetes mellitus is converted in the early stage of diabetes mellitus, and even some people are accumulated and become ill, sudden death occurs. Sub-health is the early state of most chronic diseases, and many malignant tumors, cardiovascular diseases, diabetes and the like are converted from the sub-health state, so that if life style intervention can be actively carried out, the state can be reversed, and adverse consequences can be prevented.
The pathological mechanisms underlying sub-health are mainly represented by three types: (1) alteration of neuroendocrine function: studies have shown that various adverse factors act on sub-health states caused by organisms and finally cause functional changes of hypothalamus-pituitary-adrenal axes to cause clinical symptoms, and studies have found that depression can influence the growth and development of nerve cells, and is manifested by reduced dendrites of the nerve cells, and imaging measurement shows that the brain volume is reduced, so that the change of neuroendocrine functions plays a quite important role in the occurrence and development of sub-health states. (2) Alteration of immune system function: various stress factors are very likely to affect the immune system function of the body, leading to the occurrence of various immune-related diseases. (3) Abnormal changes in gene expression: abnormal cell expression occurs in normal tissues under the stimulation of stress factors. When the organism is subjected to bad stimulus, heat Shock Protein (HSP) can be produced, and the protein has the effects of preventing apoptosis and improving organism tolerance. The stress state can also generate the effects of inhibiting the improvement of the expression of a plasminogen activator inhibitor, leading to thrombosis, and inhibiting the mutation of an oncogene P53 under the action of a stress factor and promoting the occurrence of canceration.
According to the report of the 2016 world health organization, the global sub-health population is over 60 hundred million people, accounting for 85% of the global population. Middle age is a sub-healthy high-rise period with an age distribution of between 35 and 55 years. Various physiological functions, physical conditions and immunity tend to be reduced in the middle-aged, and psychological stress is borne, which are adverse health factors. Less attention is given to the sub-health state, and if left to progress, disease symptoms may also occur. The subhealth crowd is continuously younger, the juvenile population is also faced with subhealth threat, heavy rising, employment pressure, unhealthy life style, and problems such as inadaptation of society, environment and interpersonal relationship, and the like, and the problems such as depression, fatigue, obesity and the like are often caused.
The stem cells have the functions of directional homing, multidirectional differentiation, self-proliferation and paracrine, and can recognize in-vivo inflammatory factor signals, automatically homing into tissues needing to be repaired after blood reaches all organs of the whole body, expand and redirect a large number of damaged tissue cells to replace damaged cells, and re-excite organ functions, and simultaneously can release a large number of cytokines, change cell microenvironment, promote repair of damaged aging cells, activate surrounding cells and restore body functions, so that the effect of improving sub-health is achieved. However, the activity state of stem cells has very remarkable influence on the effect of relieving sub-health, and is limited by the existing detection technology and method, the reinfusion time and the long-distance, and the like, so that the cell preparation is difficult to detect and reinfusion in the body in the optimal action time period, and the cell preparation is used as an in-vitro active preparation, so that the influence of timeliness and the activity rate on the functions of the cell preparation is large, and how to further improve the action effect of the stem cells is a problem to be solved in the field.
Disclosure of Invention
The invention aims to provide a composition for enhancing the efficacy of stem cells in regulating sub-health states, so as to solve the technical problem of non-ideal activity of stem cells in regulating sub-health states.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a composition for enhancing the effect of stem cells in regulating sub-health state comprises epidermal growth factor, resveratrol, guarana leaf extract, human serum albumin, vitamin C and glutathione.
Further, the composition is for mixing with stem cells in physiological saline.
Further, the working concentrations of the epidermal growth factor, resveratrol, guarana leaf extract, human serum albumin, vitamin C and glutathione are 5-20ng/mL, 0.05-0.2 mu mol/L, 30-60 mu g/mL, 1-3%, 5-15mg/mL and 3-8mg/mL respectively.
Further, the working concentration of the stem cells is 1-10×10 5 And each mL.
Further, the working concentrations of the epidermal growth factor, resveratrol, guarana leaf extract, human serum albumin, vitamin C and glutathione are respectively 10ng/mL, 0.1 mu mol/L, 50 mu g/mL, 2%, 10mg/mL and 5mg/mL; the working concentration of the stem cells is 5×10 5 And each mL.
Further, the guarana leaf extract is prepared by the following method: taking fresh Guarana leaves, drying, crushing, and carrying out hot reflux extraction by using ethanol as an extraction solvent to obtain a crude extract; concentrating and drying the crude extract to obtain the guarana leaf extract.
Further, the stem cells are human umbilical cord mesenchymal stem cells.
The technical scheme also provides a preparation method of the composition for enhancing the effect of stem cells in regulating sub-health state, which is characterized in that: respectively dissolving resveratrol and guarana leaf extract in normal saline to obtain resveratrol solution and guarana leaf extract solution; mixing stem cells, resveratrol solution, guarana leaf extract solution, human serum albumin, epidermal cell growth factor, and vitamin C in physiological saline.
The technical scheme also provides a cell preparation containing the composition for enhancing the effect of regulating the sub-health state of the stem cells, wherein the cell preparation comprises the components of an epidermal cell growth factor, resveratrol, guarana leaf extract, human serum albumin, vitamin C, glutathione, physiological saline and stem cells.
The technical scheme also provides application of the composition for enhancing the effect of stem cells in regulating sub-health state in preparing a cell preparation for regulating sub-health.
The principle of the technical scheme and the beneficial effects are that:
the technical scheme adopts epidermal cell growth factors, resveratrol, guarana leaf extract, vitamin C, glutathione and the like to relieve sub-health state of patients. And the proper formulation composition can also be used for increasing the activity of stem cells, and the survival rate of the stem cells can be kept at a certain level under the condition of long-term storage, so that the efficacy of the preparation for regulating sub-health state is improved, and the requirement on the practical operation time limit is reduced. In the technical field, the general requirement for stem cell preparations is that the survival rate of fresh stem cells stored in an environment of 2-8 ℃ needs to be ensured to be more than 90% within 6 hours, and the survival rate of stem cell preparations when the stem cell preparations are returned to human bodies needs to be ensured to be more than 90%. In order to meet the above requirements, the time required for the whole process of examination, release, transportation to a hospital and administration to a patient after the preparation of stem cell preparation is completed needs to be compressed to within 6 hours. In practice, it is difficult to achieve the ideal state, and therefore, it is necessary to lengthen the time for which the stem cell viability in the stem cell preparation is maintained at 90% or more. In addition, the stem cell preparation of the present embodiment is used for regulating sub-health status, so that the preparation of the present embodiment is different from the general stem cell preparation containing only human serum albumin and physiological saline, and the preparation of the present embodiment further comprises auxiliary substances such as epidermal growth factor, vitamin C, glutathione, etc. How to maintain stem cell activity with new formulation compositions, which is not an answer from the prior art. The technical scheme realizes the mutual promotion effect by screening auxiliary preparations of serial plant extracts and medicines to realize the effects of prolonging the activity maintenance time of the stem cell preparation, regulating the body function and treating diseases, and realizes and satisfies the direction of people to healthy life.
The specific description of each functional component in the composition for enhancing the effect of stem cells on regulating sub-health state in the scheme is as follows:
resveratrol has effects of resisting tumor, resisting oxidation, relieving inflammation, inhibiting platelet aggregation, regulating immunity, etc., is non-flavonoid polyphenol compound beneficial to human health, and has obvious preventing effect on hormone dependent tumor (including breast cancer, prostatic cancer, endometrial cancer, ovarian cancer, etc.). Can also be used for preventing osteoporosis, acne and senile dementia, and has antiviral and immunoregulatory effects. As antitoxin from seed plants, the novel anti-toxin agent has special health care function on human health, has higher popularization and comprehensive utilization value, becomes a natural active ingredient highly valued by scientists, can be widely applied to the fields of medicines, health products, foods, cosmetics and the like, has high medicinal value and wide market prospect, and attracts high importance to biological medical community workers. Resveratrol is a chemopreventive agent for tumor diseases, and also for reducing platelet aggregation, preventing and treating atherosclerosis, and cardiovascular and cerebrovascular diseases. In the 90 s of the 20 th century, the scientific workers in China have conducted intensive studies on resveratrol and revealed its pharmacological actions: inhibiting abnormal aggregation of blood platelet, preventing myocardial hard plug and cerebral embolism, protecting anoxia heart, recovering cardiac output from burn or hemorrhagic shock, dilating arterial vessel and improving microcirculation.
Guarana is native to the geothermal rainforest of the amazon brazil and its seeds have a high caffeine content. In addition, the leaf of Guarana also contains purine alkaloid, vitamins, tannic acid, protein, etc., and has mild nerve stimulating effect, and can be used for preventing arteriosclerosis, and treating neuralgia, migraine, diarrhea, dysentery, etc. Phenolic acid compounds represented by chlorogenic acids are also reported to be identified in guarana leaves, and the compounds are helpful for preventing and treating cardiovascular and cerebrovascular diseases such as coronary heart disease, atherosclerosis, apoplexy and the like. In addition, research shows that the extract of guarana has certain capacity of regulating stem cell proliferation, and can also increase the activity of cell catalase and the gene expression of corresponding signal channel.
Epidermal Growth Factor (EGF) was found in 1986 by the American scientist, stent Like Endoctor. EGF is a growth factor present in humans and its main function is to promote division of skin cells. Studies have shown that: the trace EGF can strongly stimulate the cell growth, inhibit the expression of senescence genes, prevent skin senescence and keep the components of the skin in an optimal physiological state. In addition, it can stimulate the synthesis and secretion of some macromolecules (such as hyaluronic acid and collagen) outside the cells, moisten the skin, and is a key factor for determining the vitality and health of the skin.
Vitamin C is a vitamin necessary for human body, and has very important meaning and effect on normal functions of human body, functions of viscera and functions of cells. The vitamin C can also support the nutrition of cells, so that the cells are always in an active function. Vitamin C can also prevent arteriosclerosis, improve aging state of cardiovascular and cerebrovascular, improve self-repairing function of vascular endothelium, and also can maintain beauty, keep young and beautify skin. Studies show that the antioxidant effect of Vc can resist the damage of free radicals to cells to prevent the variation of the cells, and the nitrite and the secondary amine are blocked to form strong cancerogenic nitrosamine. Vc protects cells, detoxifies, protects liver and improves the immunity of human body. Vc can enhance chemotaxis and deformability of neutrophils and improve sterilization capability.
Glutathione (GSH) is a tripeptide which is formed by combining glutamic acid, cysteine and glycine and contains sulfhydryl, the glutathione can help to maintain normal immune system functions, and has the functions of antioxidation and integrated detoxification, and the sulfhydryl on the cysteine is used as an active group of the glutathione and is easy to combine with certain medicines, toxins and the like, so that the glutathione has the integrated detoxification function. It can maintain the stability of erythrocyte membrane structure and eliminate the damage of oxidant to erythrocyte membrane structure. The glutathione can be used for medicines and can be used as a base material of functional foods, and can be widely applied to the functional foods such as aging delay, immunity enhancement, sub-health improvement, tumor resistance and the like.
In the technical scheme, resveratrol, guarana leaf extract, epidermal cell growth factor, vitamin C and glutathione all have certain sub-health regulating capacity, and various substances in the formula act synergistically to effectively relieve the sub-health state of a patient. In addition, the proper formulation composition (especially the combination of resveratrol and guarana leaf extract) also effectively maintains the activity and the dryness of the stem cells so as to ensure the action and the effect of reinfusion of the stem cells into human bodies. In particular, the inventor discovers that the guarana leaf extract has a remarkable effect on improving the survival rate of stem cells through screening various plant extracts, and particularly has a remarkable synergistic effect on improving the survival rate of the stem cells when the rest resveratrol is used in combination. This synergistic effect is difficult to achieve with other types of plant extracts. The stem cell preparation prepared by the preparation of the scheme can effectively relieve the sub-health state of a patient and effectively improve the life quality of the patient.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available.
Example 1:
a composition for enhancing the efficacy of stem cells in modulating sub-health states comprising: epidermal Growth Factor (EGF)Resveratrol, human serum albumin, vitamin C (Vc) and glutathione. The composition is used for maintaining and/or enhancing the activity of stem cells so as to promote the effect of the stem cells on relieving sub-health. In the following specific embodiments, human umbilical cord mesenchymal stem cells will be described as an example. Human umbilical cord mesenchymal stem cells can be obtained by the following method: taking fresh umbilical cord of about 10cm in caesarean section in a sterile way, washing off residual blood of the umbilical cord with normal saline, cutting into small sections of 2-3 cm, rinsing again, splitting the umbilical cord in longitudinal rows, removing 1 umbilical vein and two umbilical arteries, and stripping the Huatong glue. Shearing Huatong gel into 1mm pieces by using ophthalmic scissors 2 Transferring to a cell culture flask, adding complete culture medium of DMEM/F12, and adding 5% CO 2 And (3) standing and culturing in an incubator at 37 ℃. After 1 week, the liquid is changed for the first time, and after 3-4 days, the liquid is changed for 1 time. And when the cells grow to 80% -90% of fusion, digesting and passaging by using trypsin/EDTA digestive juice, and controlling the digestion time under a microscope. And taking 4-5 generations of purified and stable human umbilical cord mesenchymal stem cells for use after the cells are qualified for inspection. The stem cells used in the experiment (the object of action of the composition of the present embodiment) may be isolated by conventional means of the prior art or purchased commercially. The human umbilical cord mesenchymal stem cells can be purchased in a public cell bank, the public cell bank is a preferred seed cell, the characteristics after amplification are stable, the quality is good, the screening is strict, and the public can obtain and use the umbilical cord mesenchymal stem cells. The technical scheme does not relate to any operation process for obtaining stem cells, and the composition plays a role in increasing the activity of the stem cells. In the subsequent experiments, the human umbilical cord mesenchymal stem cells are directly purchased in a public cell bank and are used for preparing stem cell preparations after 4-5 generations of conventional subculture.
Dissolving resveratrol with normal saline, and sterilizing by conventional filtration. Taking isolated culture to prepare qualified human umbilical mesenchymal stem cells, resveratrol solution (resveratrol, merck, CAS#: 501-36-0), 20% human serum albumin (amborine, cat# P100463599), epidermal cell growth factor (Homopoly, cat# GMP-TL 613), vitamin C (vitamin C for injection, jiangsu ripple water, cat# 20220225), guarana leaf extract solution and glutathione (injection)Glutathione, drug friends pharmacy, goods number: 22231970 Adding into normal saline, blowing uniformly, and filling into an infusion bag to a volume of 100ml to form the preparation for relieving sub-health. Wherein, resveratrol, 20% of human serum albumin, epidermal cell growth factor and vitamin C can be purchased through commercial means, and the human serum albumin, the epidermal cell growth factor, the vitamin C and physiological saline are sterile preparations, and are not described in detail herein. The guarana leaf extract is self-made, and the preparation method is described in detail later. In the preparation in the infusion bag (stem cell preparation), the concentration of human serum albumin was 2%, the concentration of resveratrol was 0.1. Mu. Mol/L, EGF was 10ng/mL, the concentration of vitamin C was 10mg/mL, the concentration of glutathione was 5mg/mL, and the concentration of guarana leaf extract was 50. Mu.g/mL. Each 100mL of the preparation contains 5×10 7 Human umbilical cord mesenchymal stem cells. The cell viability is counted by sampling, and the bacteria, fungi, mycoplasma and endotoxin are detected by sampling and inspection, and the qualified sample can be used in the subsequent treatment process. The treatment method using the preparation comprises the following steps: intravenous injection is carried out by using a disposable blood transfusion device with a filter screen, the infusion is completed within 1 hour (100 ml), and the patient is at rest and observed for 2 hours after the infusion is completed.
Wherein, the guarana leaf extract is prepared by the following method:
fresh leaves of Guarana (Paullina cupana Kunth), a plant of the genus Hibiscus of the family Sapindaceae, are taken. Placing fresh leaves in a 50 ℃ oven, drying to constant weight, then crushing and sieving with a 60-mesh sieve to obtain medicinal powder. 60% ethanol was used as extraction solvent, according to 1g: and (3) carrying out hot reflux extraction on the medicinal powder for 2h according to the feed liquid ratio of 10mL, extracting twice, collecting the filtrate obtained by the two extraction and combining the filtrate to obtain a crude extract. Concentrating the crude extract into extract with relative density of about 1.2 by conventional rotary evaporation, and lyophilizing (80deg.C) to obtain Guarana leaf extract (water content < 8%). Prior to use, the guarana leaf extract is formulated as a stock solution of 0.1g/mL (using normal saline as the solvent), and the stock solution is subjected to conventional wet heat sterilization and 0.22 μm filtration for later use (if a 0.22 μm filter is difficult to filter, prefiltration can be performed with a filter having a larger pore size, followed by 0.22 μm filtration).
The medicinal plant extract can be used as a supplement to activate stem cell proliferation and maintain stem cell stem properties. The technical scheme uses resveratrol and guarana leaf extract as stem cell maintenance and enhancement supplements, is used for stem cell treatment of public health, improves treatment effect and reduces side effects.
Experimental example 1: optimal action concentration screening of resveratrol
The activity of stem cells in stem cell preparations is critical to the performance of their effects, and therefore the timeliness of such preparations is important. After stem cells are generally prepared into stem cell preparations, the stem cells are required to be input into a human body within 8 hours, and the phenomenon that a large number of cells die out after the time is exceeded influences the regulation effect, so that the requirements on the time limit and the activity rate of the stem cell preparations are very high. However, in many cases, after the stem cell preparation is completed, due to limitations of the existing detection techniques and methods, the feedback time and transportation, the operator may not be able to complete various preparation tasks (e.g., various relevant detection of the stem cell preparation, data checking, infusion preparation, etc.) for the subsequent treatment in such a short time, resulting in a decrease in the activity of the stem cell preparation and an unsatisfactory therapeutic effect. Therefore, how to effectively maintain the activity of stem cells in a stem cell preparation is an important point of study for those skilled in the art, and if the maintenance time of the activity of stem cells in a stem cell preparation (preferably, survival rate > 90%) can be increased to 48 hours, then a sufficient time can be allowed for intermediate operations. In addition, although stem cells are prepared as stem cell preparations, the stem cells themselves need to be subjected to related assays, such as: survival rate, bacteria, fungi, mycoplasma, viruses, endotoxin, etc. However, the above-described tests are still required to ensure safety after preparation into stem cell preparations, in accordance with relevant regulations. Since the above detection processes are all conventional, and the focus of the study of the present technical solution is on how to increase (or maintain) the activity of stem cells in the stem cell preparation, the following data is focused on the stem cell survival rate (activity) of the stem cell preparation. If the cell viability is too low, this means that there are too many dead cells, which also greatly detracts from the efficacy of the cell preparation, and therefore the activity of the stem cells plays a decisive role.
In the prior art, stem cells are mainly formed by mixing stem cells with physiological saline containing human serum albumin, in order to ensure the activity of the stem cell preparation, it is generally considered that the stem cells need to complete each detection within 6 hours after the preparation is completed, and the survival rate of the fresh stem cells needs to be ensured to be more than 90% within 6 hours when the fresh stem cells are stored in an environment of 2-8 ℃. However, the above-mentioned requirement for the timeliness of the viability of the stem cell preparation is too high, and it is difficult to complete each test for 6 hours in the actual operation process, and complete the subsequent whole process of release, transportation to a hospital, and administration to a patient. In addition, in order to enhance the regulation effect of stem cells on sub-health diseases, the proposal also adds components such as epidermal cell growth factors, vitamin C, glutathione and the like, which are different from the common stem cell preparations in the prior art. Unlike other chemical additives, the active state of stem cells plays an important role in the efficacy of the preparation, and the inventor focuses on how to maintain the survival rate of stem cells in the stem cell preparation to be more than 90% under the condition of exceeding 6 hours of storage time, so that the stem cells fully play a role in sub-health regulation. Therefore, in the subsequent experiments, the inventors studied mainly the stem cell preparation state 12 hours after completion of the preparation.
In the process of researching how to improve the activity of a stem cell preparation, the inventor firstly tries to add resveratrol into a basic formula (containing human serum albumin, vitamin C, glutathione and epidermal cell growth factor) so as to utilize the excellent properties of the resveratrol such as antioxidation and the like, improve the activity of the stem cell preparation, and research the optimal action concentration of the resveratrol. In this experimental example, the stem cell preparation comprises the following components: resveratrol (see Table 1), 2% human serum albumin, 10ng/ml EGF, 10mg/ml vitamin C, 5mg/ml glutathione, 5×10 7 Personal umbilical cord mesenchymal stem cells/100 mL. The stem cell preparation was placed in a refrigerator at 4-8 c (conventional storage temperature of stem cell preparation) for 24 hours, and then the stem cell viability was measured. At the same time, a blank is arranged, and the composition of the stem cell preparation of the blank is (i.e. no white addedVeratrole): 2% human serum albumin, 10ng/ml EGF, 10mg/ml vitamin C, 5mg/ml glutathione, 5X 10 7 Personal umbilical cord mesenchymal stem cells/100 mL. See table 1 for experimental groupings and experimental results. From the data in table 1, it is clear that the addition of resveratrol in a certain concentration range can exert a certain effect on maintaining the activity of stem cells, but its effect is limited and needs to be further enhanced. And taking resveratrol with optimal action concentration of 0.1 mu mol/L for subsequent experimental study. As can be seen from the data in table 1, even under the optimal action concentration condition of resveratrol, the survival rate of stem cells can be maintained to be about 85.6% in 24 hours, and the survival rate of stem cells in 48 hours is only about 68.8%, but the application requirement of the stem cell preparation cannot be met.
Table 1: optimum effect concentration study results of resveratrol (data in table are presented as mean ± standard deviation, n=5)
Experimental example 2: screening of plant extracts with maintained Stem cell Activity
Based on experimental example 2, resveratrol alone was able to maintain higher stem cell activity for about 24 hours, but the activity decay was very strong at 24-48 hours, indicating that the stem cell activity maintenance time of resveratrol alone was still limited. Therefore, the inventors have further explored and screened for stem cell activity maintenance and enhancement in an effort to find additional substances that could enhance and maintain the aging and effects of stem cell preparations. Since the medicinal plant extract can be used as a supplement for cell culture, activates proliferation of stem cells and maintains stem cell stem property, the inventors have further tried some plant extracts having antioxidant and proliferation promoting properties (see table 2). The stem cell preparation was tested according to the method of experimental example 1, and the stem cell preparation was prepared in the following manner: 2% human serum albumin, 10ng/ml EGF, 10mg/ml, vitamin C, 5mg/ml glutathione, 5X 10 7 Individual umbilical cord mesenchymal stem cells/100 mL and different concentrations of plant extracts (see table 2 for specific types and concentration gradients, without resveratrol added to the formulation). The composition of the stem cell preparation of the blank control was: 2% human serum albumin, 10ng/ml EGF, 10mg/ml vitamin C, 5mg/ml glutathione, 5X 10 7 Personal umbilical cord mesenchymal stem cells/100 mL (without other plant extracts). The inventors have screened a large number of plant extracts, three plant extracts being described herein as examples. The research shows that the guarana leaf extract, the thyme leaf extract and the basil leaf extract all have certain effect of maintaining the survival rate of stem cells, but the difference is not too large compared with the effect of resveratrol alone. Wherein, the optimal action concentration of the Guarana leaf extract is 50 mug/mL, the optimal action concentration of the thyme leaf extract is 20 mug/mL, and the optimal action concentration of the basil leaf extract is 10 mug/mL. At the optimal action concentration, the action effect of guarana leaf extract is not very different from that of thyme leaf extract and basil leaf extract.
Table 2: effect screening test results of plant extracts (data in the table are presented as mean ± standard deviation, n=5)
In table 2, thyme leaf extract and basil leaf extract were prepared in the same manner as guarana leaf extract (see example 1 in particular), except that the extraction subjects were replaced accordingly.
Experimental example 3: research on combined action effect of plant extracts and resveratrol
The experimental example researches the combined action effect of resveratrol and plant extracts, and selects the optimal action concentration confirmed in experimental examples 1 and 2 aiming at the plant extracts and the resveratrol, and the experimental groups are as follows:
the formulation of the stem cell preparation of No. 1 is: 2% human serum albumin, 10ng/ml EGF, 10mg/ml vitamin C, 5mg/ml glutathione, 5X 10 7 Personal umbilical cord mesenchymal stem cells/100 mL; the formulation is free of resveratrol and any other plant extracts. The experiment of this experimental example No. 1 was identical to the experiment of experimental example 2 without any other plant extracts and resveratrol, so the data of table 2 No. 1 were used in this experiment.
The formulation of the stem cell preparation of No. 2 is: 0.1 mu mol/L resveratrol, 2% human serum albumin, 10ng/ml EGF, 10mg/ml vitamin C, 5mg/ml glutathione, 5X 10 7 Personal umbilical cord mesenchymal stem cells/100 mL; the formulation does not contain any other plant extracts, but contains resveratrol.
The formulation of the stem cell preparation of No. 3 is: 2% human serum albumin, 10ng/ml EGF, 10mg/ml vitamin C, 5mg/ml glutathione, 5X 10 7 Individual umbilical cord mesenchymal stem cells/100 mL, 50 μg/mL guarana leaf extract; the formulation does not contain resveratrol but contains guarana leaf extract.
The formulation of the stem cell preparation of No. 4 is: 0.1 mu mol/L resveratrol, 2% human serum albumin, 10ng/ml EGF, 10mg/ml vitamin C, 5mg/ml glutathione, 5X 10 7 Individual umbilical cord mesenchymal stem cells/100 mL, 50 μg/mL guarana leaf extract; the formulation contains both resveratrol and guarana leaf extract.
The formulation of the stem cell preparation of No. 5 is: 2% human serum albumin, 10ng/ml EGF, 10mg/ml vitamin C, 5mg/ml glutathione, 5X 10 7 Individual umbilical cord mesenchymal stem cells/100 mL, 20 μg/mL thyme leaf extract; the formulation does not contain resveratrol but contains thyme leaf extract.
The formulation of the stem cell preparation of No. 6 is: 0.1 mu mol/L resveratrol, 2% human serum albumin, 10ng/ml EGF, 10mg/ml vitamin C, 5mg/ml glutathione, 5X 10 7 Individual umbilical cord mesenchymal stem cells/100 mL, 20 μg/mL thyme leaf extract; the formula contains resveratrolAlcohol and thyme leaf extract.
The formulation of the stem cell preparation of No. 7 is: 2% human serum albumin, 10ng/ml EGF, 10mg/ml vitamin C, 5mg/ml glutathione, 5X 10 7 Individual umbilical cord mesenchymal stem cells/100 mL, 10 μg/mL basil leaf extract; the formulation does not contain resveratrol but contains basil leaf extract.
The formulation of the stem cell preparation of No. 8 is: 0.1 mu mol/L resveratrol, 2% human serum albumin, 10ng/ml EGF, 10mg/ml vitamin C, 5mg/ml glutathione, 5X 10 7 Individual umbilical cord mesenchymal stem cells/100 mL, 10 μg/mL thyme leaf extract; the formulation contains both resveratrol and basil leaf extract.
The stem cell preparation was placed in a refrigerator at 4 c (normal storage temperature of stem cell preparation) for 12, 24, 36, 48 hours, and then the stem cell viability was measured. See table 3 for experimental groupings and experimental results.
Table 3: combined action experimental data of plant extracts and resveratrol
From the above table data, it is clear that the effect of the various treatment methods on the maintenance of the viability of the stem cells is similar in this experimental example after 12 hours of standing, but the difference of the maintenance effect of the various treatment methods on the viability starts to be displayed with the increase of time, and especially the synergy between resveratrol and guarana leaf extract is more remarkable at 36 hours and 48 hours. And the synergistic effect between the basil leaf extract and the resveratrol is not shown between the thyme leaf extract and the resveratrol.
More specifically, the number 1, 2, 3, 4 experiments in table 3 demonstrate the effect of resveratrol and guarana leaf extract on stem cell viability. In the initial storage period (12 h) of the cell preparation, whether resveratrol or guarana leaf extract is added into the preparation alone or simultaneously does not have a significant influence on the survival rate of stem cells. However, the viability of stem cells was very significantly reduced with increasing storage time, especially at 48h storage, with no resveratrol and guarana leaf extract No. 1, to 39.8±2.8%. While the survival rate of stem cells can be improved to 62.5+/-2.8% when resveratrol is singly added for 48 hours; the stem cell survival rate can be improved to 64.2+/-3.1% by independently adding the Guarana leaf extract. Under the optimal action concentration, the action effects of resveratrol and the guarana leaf extract are similar, and compared with a blank control without adding the two substances, the survival rate of stem cells can be improved by more than 20 percent. However, the viability of stem cells of more than 60% at 48 hours is insufficient for the application, resulting in an unsatisfactory therapeutic effect of the stem cell preparation. The inventors tried to combine resveratrol and guarana leaf extract (number 4) to increase the stem cell survival rate to 90.3±2.7% and to 50% more than that of the guarana leaf extract (blank) without resveratrol, which was unexpected by the inventors before the experiment, and unexpected technical effects were obtained. Experiments numbered 1, 2, 3 and 4 show that the combined use of resveratrol and guarana leaf extract produces a synergistic effect in improving the survival rate of stem cells in the stem cell preparation.
The number 1, 2, 5, 6 experiments in table 3 demonstrate the effect of resveratrol and thyme leaf extract on stem cell viability. The stem cell survival rate can be improved to 63.8+/-2.3% by independently adding thyme leaf extract for 48 hours. The inventors tried to combine resveratrol and guarana leaf extract (number 6), which can increase the survival rate of stem cells to 78.9+ -2.6%, which is less than 40% compared with the blank control number 1, which is poor compared with the combination of resveratrol and guarana leaf extract (number 4), and which does not show the synergistic effect of both substances.
The number 1, 2, 7, 8 experiments in table 3 demonstrate the effect of resveratrol and basil leaf extract on stem cell viability. The stem cell survival rate can be improved to 65.7+/-2.7% by adding the basil leaf extract alone at 48 hours. The inventors tried to combine resveratrol and basil leaf extract (number 8) to increase the stem cell survival rate to 81.3±2.6%, compared with the blank control number 1, the stem cell survival rate was increased by more than 40%, compared with the combination of resveratrol and basil leaf extract (number 4), the combination of resveratrol and basil leaf extract was poor, and no synergistic effect was shown.
Experimental example 4
20 (experimental group) +20 (control group) study groups aged 39-49 years were screened according to sub-health related criteria. Experimental group: the cell preparation (example 1) is prepared according to the proportion and is subjected to three intravenous feedback (the feedback time is required to be ensured to be within 48 hours after the stem cell preparation is prepared, the cell survival rate is more than 90 percent in the experimental process), and 100mL of the stem cell preparation is taken once a week; control group: 100ml of 0.9% physiological saline was used for reinfusion. The first six months of feedback were subjected to a follow-up record (see Table 4 for results) and a test of biochemical indicators of related functions (see Table 5 for results), which were body mass index BMI, body temperature, blood pressure, respiration, heart rate, blood general indicators, urine general indicators, liver function (TB serum bilirubin, TP total protein, LDH lactate dehydrogenase, ALP alkaline phosphatase, AST glutamate aspartate aminotransferase, ALT glutamate pyruvate aminotransferase), kidney function indicators (BUN urea nitrogen, CR creatinine), blood glucose (GLU sugar), immune function lymphocyte subpopulation test, gout test (blood uric acid), pancreatic assay (pancreatic amylase), tumor markers (CEA carcinoembryonic antigen, AFP alpha fetoprotein), fasting blood glucose, glucose tolerance, total cholesterol, triglycerides, rheumatism, inflammatory disease factors, and the like. The stem cell obtaining method is mature, a large number of stem cells can be obtained in a standardized mode, and the stem cells can be purchased through a commercial means to finally form a standardized stem cell preparation and be used for treating sub-health states of a large number of people. The stem cells adopted in the technical scheme are stem cells purchased through commercial means.
Table 4: table of symptom change trend records for the first six months after feedback:
table 5: biochemical function index detection result (data of one person of the experimental group selected from the subjects is displayed)
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The experimental results are shown in tables 4 and 5, and the symptoms of deficiency and fatigue, easy fatigue, insomnia, low work and rest quality, difficult concentration, poor adaptability, poor mental state, low emotion, slow reaction, dreaminess and insomnia, drowsiness in daytime, hypomnesis, dysphoria, anxiety, easy convulsion and the like of sub-healthy patients are obviously improved from the change of the first 6 months after the stem cell preparation is infused back, and the life and working quality are obviously improved. From the detection of related biochemical function indexes, the respiratory rate, blood pressure, heart rate, liver and kidney functions, urine tests, blood tests, pancreas functions, blood sugar, blood fat, gout, immune functions, rheumatism, inflammatory disease treatment factors and the like of patients are improved, which indicates that the preparation has good treatment effect on sub-healthy people.
The foregoing is merely exemplary of the present invention, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present invention, and these should also be regarded as the protection scope of the present invention, which does not affect the effect of the implementation of the present invention and the practical applicability of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (10)
1. A composition for enhancing the efficacy of stem cells in regulating sub-health conditions, comprising: the composition consists of an epidermal cell growth factor, resveratrol, guarana leaf extract, human serum albumin, vitamin C and glutathione.
2. A composition for enhancing the efficacy of stem cells in modulating sub-health status according to claim 1, wherein: the composition is for mixing with stem cells in physiological saline.
3. A composition for enhancing the efficacy of stem cells in modulating sub-health status according to claim 2, wherein: the working concentrations of the epidermal growth factor, the resveratrol, the guarana leaf extract, the human serum albumin, the vitamin C and the glutathione are 5-20ng/mL, 0.05-0.2 mu mol/L, 30-60 mu g/mL, 1-3%, 5-15mg/mL and 3-8mg/mL respectively.
4. A composition for enhancing the efficacy of stem cells in modulating sub-health status according to claim 3, wherein: the working concentration of the stem cells is 1-10×10 5 And each mL.
5. The composition for enhancing the efficacy of stem cells in regulating sub-health according to claim 4, which comprisesIs characterized in that: the working concentrations of the epidermal growth factor, resveratrol, guarana leaf extract, human serum albumin, vitamin C and glutathione are respectively 10ng/mL, 0.1 mu mol/L, 50 mu g/mL, 2%, 10mg/mL and 5mg/mL; the working concentration of the stem cells is as follows: 5X 10 5 And each mL.
6. A composition for enhancing the efficacy of stem cells in modulating a sub-health state according to claim 5, wherein: the guarana leaf extract is prepared by the following method: taking fresh Guarana leaves, drying, crushing, and carrying out hot reflux extraction by using ethanol as an extraction solvent to obtain a crude extract; concentrating and drying the crude extract to obtain the guarana leaf extract.
7. A composition for enhancing the efficacy of stem cells in modulating sub-health status according to claim 6, wherein: the stem cells are human umbilical cord mesenchymal stem cells.
8. A method of preparing a composition for enhancing the efficacy of stem cells in modulating sub-health status according to any one of claims 1-7, wherein: respectively dissolving resveratrol and guarana leaf extract in normal saline to obtain resveratrol solution and guarana leaf extract solution; mixing stem cells, resveratrol solution, guarana leaf extract solution, human serum albumin, epidermal cell growth factor, and vitamin C in physiological saline.
9. A cell preparation comprising a composition for enhancing the efficacy of stem cells in modulating sub-health conditions according to any one of claims 1 to 7, characterized in that: the components of the composition comprise epidermal cell growth factor, resveratrol, guarana leaf extract, human serum albumin, vitamin C, glutathione, physiological saline and stem cells.
10. Use of a composition for enhancing the efficacy of stem cells in modulating a sub-health state according to any of claims 1-7 for the preparation of a cell preparation for modulating sub-health.
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