CN113230296A - Composition containing stem cell exosomes and used for improving premature ovarian failure and preparation method thereof - Google Patents
Composition containing stem cell exosomes and used for improving premature ovarian failure and preparation method thereof Download PDFInfo
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Abstract
The invention provides a composition containing stem cell exosomes and used for improving premature ovarian failure, which comprises stem cell exosomes, a broccoli extract and pharmaceutically acceptable auxiliary materials, wherein the weight percentage of the stem cell exosomes is 0.1-0.4% in terms of the mass of protein, and the weight percentage of the broccoli extract is 0.06-0.10%. The composition for improving premature ovarian failure containing the stem cell exosomes provided by the invention adopts the stem cell exosomes and the broccoli extract in a specific concentration range to be matched for use, and shows a better improvement effect in the treatment of the premature ovarian failure animal model.
Description
Technical Field
The invention relates to the technical field of biological stem cells, in particular to a composition containing stem cell exosomes and used for improving premature ovarian failure and a preparation method thereof.
Background
Premature ovarian failure as the name suggests refers to premature ovarian failure of women, which is specifically manifested by symptoms of ovarian function decline, such as menstrual cycle disorder, abnormal menstrual flow, inflammation of reproductive tract, and a series of climacteric symptoms, such as skin relaxation, sleep loss, emotional anxiety and the like, which seriously affect the normal life of women. At present, the polycystic ovarian syndrome which is clinically common is a common cause of infertility of women of childbearing age, and the main cause is that ovarian function is weakened to cause follicular dysplasia and hormone metabolism disorder. For the disease, oral administration or hormone injection is often adopted to help the ovary to recover the ovulation function in a short time, but the method treats the symptoms and the root causes but does not achieve the effect of thorough cure, the disease condition is repeated all the time, and psychological and physiological troubles are brought to patients.
Oxidation reactions occur in the human body at all times and free radicals are also produced, and these molecules can cause damage to our cells. Free radicals are necessary to some extent and, while causing some damage, also stimulate repair. When too many free radicals are produced, they disrupt the repair process, resulting in oxidative stress. Oxidative stress gradually causes oxidative damage to human tissues, leading to aging of the body, and inducing chronic diseases. It is considered that oxidative stress may also be one of the causes of premature ovarian failure.
The stem cells are cells with self-replicating multi-differentiation potential, can be differentiated into various functional cells under certain conditions, and can secrete various cytokines, repair tissue damage and regulate the physiological functions of organisms. The stem cells can construct a steady-state tissue repair microenvironment by releasing a large number of exosomes, thereby eliminating inflammatory reactions, promoting wound healing and repairing tissue lesions. Exosomes (exosomes) are vesicular vesicles secreted by living cells, and carry lipids, proteins and genetic material for the transmission of biological information in vivo. Exosomes secreted by different tissue cells carry different biological components, and the exosomes are endowed with unique biological effects, so the exosomes from different cell sources have different characteristics and functions.
Therefore, how to provide a composition capable of effectively improving premature ovarian failure by using stem cell exosomes has become a problem to be solved in the prior art.
Disclosure of Invention
In view of the above, the present invention aims to provide a composition containing stem cell exosomes for improving premature ovarian failure.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a composition containing stem cell exosomes and used for improving premature ovarian failure comprises the stem cell exosomes, a broccoli extract and pharmaceutically acceptable auxiliary materials, wherein the weight percentage of the stem cell exosomes is 0.1-0.4% in terms of the mass of protein, and the weight percentage of the broccoli extract is 0.06-0.10%.
Broccoli contains more vitamin C, is higher than Chinese cabbage, tomato and celery, and has particularly good effect on preventing and treating gastric cancer and breast cancer. The extract of broccoli contains various nutritional components such as protein, sugar, fat, vitamins and carotene, and pharmacological components such as isothiocyanate, Flavonoids (flavanoids) and volatile oil. The broccoli extract can prevent cancers such as breast cancer, rectal cancer and gastric cancer, and has strong ability of eliminating DPPH free radical; ascorbic acid can enhance the detoxifying ability of liver and improve the immunity of organism. The flavonoid substances have effects in regulating and preventing hypertension and heart disease.
The broccoli extract has strong capacity of eliminating DPPH free radicals, which indicates that the broccoli contains antioxidant substances capable of eliminating hydroxyl free radicals, alkyl free radicals or peroxy free radicals, and can also block lipid peroxy chain reaction, and the capacity of eliminating DPPH free radicals is in positive correlation with the amount of the broccoli extract. The capability of the water-soluble extract of broccoli for removing DPPH free radicals is stronger than that of the fat-soluble extract, which indicates that the broccoli contains a plurality of antioxidant substances with different polarities.
Further, the stem cell exosomes comprise adipose-derived stem cell exosomes and umbilical cord mesenchymal stem cell exosomes, and the weight ratio of the adipose-derived stem cell exosomes to the umbilical cord mesenchymal stem cell exosomes is (1-4) to (1-2).
Further, the weight ratio of the adipose-derived stem cell exosomes to the umbilical cord mesenchymal stem cell exosomes is 3: 1.
Further, the weight percentage of the stem cell exosomes calculated by the mass of the protein is 0.2%, and the weight percentage of the broccoli extract is 0.08%.
Further, the broccoli extract is prepared by the following steps:
cleaning fresh broccoli, draining, mashing, soaking in 30-40 deg.C water, dynamically extracting for at least two times (each time for 1-2 hr), and treating with ultrasound during extraction process; and finally, centrifuging or filtering the extraction product to obtain the broccoli extract.
The invention also provides a preparation method of the composition containing the stem cell exosomes and used for improving premature ovarian failure, which comprises the following steps:
1) cleaning fresh broccoli, draining, and mashing;
2) adding 10-20 times volume of 30-40 ℃ pure water into 1 part by mass of the mashed broccoli, dynamically extracting for at least two times, wherein the extraction time is 1-2h each time, and simultaneously performing ultrasonic pretreatment by using 200-300W in the extraction process; finally, centrifuging or filtering the extraction product to obtain a broccoli extract;
3) weighing the stem cell exosomes according to the formula amount, and mixing the stem cell exosomes with the broccoli extract according to a proportion;
4) adding a proper amount of pharmaceutically acceptable auxiliary material preparation on the basis of the mixture and forming.
In the present invention, the term "pharmaceutically acceptable excipient" includes pharmaceutically acceptable carriers, excipients, diluents and the like, which are compatible with the pharmaceutically active ingredient. The use of pharmaceutically acceptable excipients for the preparation of pharmaceutical preparations is well known to those skilled in the art.
In the embodiment of the present invention, the ultrasonic extraction conditions can be adjusted within the range given in the present invention according to the ultrasonic effect.
Compared with the prior art, the composition containing the stem cell exosomes and used for improving premature ovarian failure has the following advantages:
the composition for improving premature ovarian failure containing the stem cell exosomes, disclosed by the invention, adopts the stem cell exosomes and the broccoli extract in a specific concentration range to be matched for use, and shows a better improvement effect in the treatment of the premature ovarian failure animal model. When the concentrations of the stem cell exosomes and the broccoli extract exceed the range, the use effect of the stem cell exosomes and the broccoli extract is also obviously reduced.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The present invention will be described in detail with reference to examples.
1. Preparation of broccoli extract
1) Cleaning 1000g of fresh broccoli, draining, and mashing; soaking mashed broccoli in 15000ml (material-liquid ratio of 1:15) of pure water of 35 deg.C for dynamic extraction for 3 times, each time for 1 hr, and treating with 300W ultrasound during extraction; concentrating once every extraction, mixing the 3 concentrated solutions, centrifuging 15000g concentrated solution for 15min to obtain supernatant, concentrating the supernatant under reduced pressure, and lyophilizing to obtain stigma croci Sativi extract C1.
2) Cleaning 1000g of fresh broccoli, draining, and mashing; soaking mashed broccoli in 15000ml (material-liquid ratio of 1:15) ethyl acetate, dynamically extracting for 3 times, each time for 1h, and treating with 300W ultrasound during extraction; concentrating once every extraction, mixing the 3 concentrated solutions, centrifuging 15000g concentrated solution for 15min to obtain supernatant, concentrating the supernatant under reduced pressure, and lyophilizing to obtain stigma croci Sativi extract C2.
2. Preparation of the composition
The adipose-derived stem cell exosome A and the umbilical cord mesenchymal stem cell exosome B are purchased from Liaoning Runky Biotech limited.
TABLE 1 ingredient Table of compositions of examples 1-10
| Stem cell exosomes/g | Broccoli extract/g | Physiological saline/ml | |
| Example 1 | 0.1, weight ratio of A to B is 1:1 | 0.06，C1 | 99.84 |
| Example 2 | 0.2, A: B (weight ratio) 3:1 | 0.08，C1 | 99.72 |
| Example 3 | 0.3, A: B (weight ratio) 1:2 | 0.09，C1 | 99.61 |
| Example 4 | 0.4, A: B (weight ratio) 2:1 | 0.10，C1 | 99.50 |
| Example 5 | 0.2, A: B (weight ratio) 3:1 | 0.06，C1 | 99.74 |
| Example 6 | 0.2, A: B (weight ratio) 3:1 | 0.09，C1 | 99.71 |
| Example 7 | 0.1, A: B (weight ratio) 3:1 | 0.08，C1 | 99.82 |
| Example 8 | 0.3, A: B (by weight)Ratio) 3:1 | 0.08，C1 | 99.62 |
| Example 9 | 0.2, A: B (weight ratio) 1:1 | 0.08，C1 | 99.72 |
| Example 10 | 0.2, A: B (weight ratio) 1:2 | 0.08，C1 | 99.72 |
TABLE 2 ingredient tables of compositions for comparative examples 1 to 11
The composition preparation was prepared by dissolving the stem cell exosomes and the broccoli extract in physiological saline according to the above table.
3. Pharmacological experiment-ovary presenility mouse model
3.1 Molding
Female ICR mice of 6 weeks of age were screened and injected intraperitoneally with 50 mg/kg/day Cyclophosphamide (CTX) for 15 consecutive days, at the same time each day, rewarding the POF mouse model. Ovarian reserve function is assessed by indicators such as follicle number, hormone levels, oestrus cycle and fertility tests.
After successful modeling, 220 modeling mice are selected and randomly divided into 22 groups, 10 mice in each group are selected, 10 groups are selected as No. 1-10 experimental groups, and 1.5ml of the composition preparation prepared in the examples 1-10 is intraperitoneally injected every day; 11 groups were randomly selected from the remaining groups as experiment groups No. 11-21, and were intraperitoneally injected with 1.5ml of the composition preparations prepared in comparative examples 1-11 each day, respectively, and the remaining group was used as a control group, and was intraperitoneally injected with 1.5ml of physiological saline each day. The formulation and saline were injected once every 2 days for 2 weeks.
In addition, 10 same batches of 6-week-old female ICR mice were selected, and the 10 mice were not treated at all and were normally bred together with the model mice as a normal group.
After the end of the last injection, the animals were raised to day 28.
3.2 detection of the levels of estradiol (E2), Follicle Stimulating Hormone (FSH), anti-Mullerian hormone (AMH)
Blood was collected from the orbit of each group of mice, serum was separated, and stored at-20 ℃. Enzyme-linked immunosorbent assay (ELISA) analyzed the levels of estradiol (E2), Follitropin (FSH), anti-Mullerian hormone (AMH).
TABLE 3 table of physicochemical parameters of serum test
3.3 follicle count of ovarian tissue in mice
The mice are anesthetized and killed, the left ovary tissues of the mice are fixed in 4% paraformaldehyde, the fixed tissues are dehydrated by series of alcohol, xylene is transparent, paraffin is embedded, the tissues are continuously sliced, the slice thickness is 5 mu m, HE staining is carried out, and the tissues are observed under a microscope.
TABLE 4 follicular Change Table
The data in tables 3 and 4 show that the compositions prepared in examples 1-10 have obviously increased E2 and AMH, obviously decreased FSH, obviously recovered primary follicle, secondary follicle and mature follicle, and obviously decreased atretic follicle number, which is close to the level of normal mice, and show that the formula of the invention can effectively improve the premature ovarian failure symptom. Also, the comparison of examples 1 to 10 shows that the effect of example 2 is the best.
Compared with experimental groups 11-12, experimental group 2 can find that the effect of treating with two kinds of stem cell exosomes is better than that of treating with only one kind of stem cell exosomes, probably because the two kinds of stem cell exosomes contain different components, synergistic effect is achieved when the two kinds of stem cell exosomes are used simultaneously, and therefore better improvement effect is achieved on premature ovarian failure. Comparing the experimental group 2 with the experimental groups 13-16 shows that the stem cell exosomes and the broccoli extract have to be used at the same time to achieve better effect, and the stem cell exosomes may have a repairing function, and the broccoli extract has an anti-oxidation function, and when the stem cell exosomes and the broccoli extract exist at the same time, the stem cell exosomes and the broccoli extract mutually play a mutual promoting role, so that the using effect is enhanced. However, the experimental groups 17 to 20 show that when the application range of the stem cell exosomes and the broccoli extract exceeds the range specified in the application, the application effect is obviously reduced, which indicates that the stem cell exosomes and the broccoli extract in the specific range can be used together to achieve better improvement effect. The experiment group 21 adopts ethyl acetate to extract the broccoli, and compared with the experiment group 2, the improvement effect is slightly worse, which indicates that the water extract is more beneficial to the repair of premature ovarian failure.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. A composition for improving premature ovarian failure, which contains a stem cell exosome, and is characterized in that: the feed additive comprises stem cell exosomes, a broccoli extract and pharmaceutically acceptable auxiliary materials, wherein the weight percentage of the stem cell exosomes is 0.1-0.4% in terms of the mass of protein, and the weight percentage of the broccoli extract is 0.06-0.10%.
2. The composition for improving premature ovarian failure containing a stem cell exosome according to claim 1, characterized in that: the stem cell exosomes comprise adipose-derived stem cell exosomes and umbilical cord mesenchymal stem cell exosomes, and the weight ratio of the adipose-derived stem cell exosomes to the umbilical cord mesenchymal stem cell exosomes is (1-4) to (1-2).
3. The composition for improving premature ovarian failure containing a stem cell exosome according to claim 2, characterized in that: the weight ratio of the adipose-derived stem cell exosomes to the umbilical cord mesenchymal stem cell exosomes is 3: 1.
4. The composition for improving premature ovarian failure containing a stem cell exosome according to claim 1, characterized in that: the weight percentage of the stem cell exosomes calculated by the mass of the protein is 0.2%, and the weight percentage of the broccoli extract is 0.08%.
5. The composition for improving premature ovarian failure containing a stem cell exosome according to claim 1, characterized in that: the broccoli extract is prepared by the following steps:
cleaning fresh broccoli, draining, mashing, soaking in 30-40 deg.C water, dynamically extracting for at least two times (each time for 1-2 hr), and treating with ultrasound during extraction process; and finally, centrifuging or filtering the extraction product to obtain the broccoli extract.
6. A preparation method of a composition containing stem cell exosomes and used for improving premature ovarian failure is characterized in that: the method comprises the following steps:
1) cleaning fresh broccoli, draining, and mashing;
2) adding 10-20 times volume of 30-40 ℃ pure water into 1 part by mass of the mashed broccoli, dynamically extracting for at least two times, wherein the extraction time is 1-2h each time, and simultaneously performing ultrasonic pretreatment by using 200-300W in the extraction process; finally, centrifuging or filtering the extraction product to obtain a broccoli extract;
3) weighing the stem cell exosomes according to the formula amount, and mixing the stem cell exosomes with the broccoli extract according to a proportion;
4) adding a proper amount of pharmaceutically acceptable auxiliary material preparation on the basis of the mixture and forming.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023040938A1 (en) * | 2021-09-15 | 2023-03-23 | 青岛卓云海智医疗科技有限公司 | Method for treating ilk signaling pathway related diseases using exosome derived from mesenchymal stem cells, and pharmaceutical composition |
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2021
- 2021-05-11 CN CN202110510185.0A patent/CN113230296A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023040938A1 (en) * | 2021-09-15 | 2023-03-23 | 青岛卓云海智医疗科技有限公司 | Method for treating ilk signaling pathway related diseases using exosome derived from mesenchymal stem cells, and pharmaceutical composition |
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