CN107375334B - Placenta extract and its preparation method and application - Google Patents
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Abstract
The invention discloses a placenta extract and a preparation method and application thereof, wherein the placenta extract is obtained by freezing placenta tissues, extracting effective components and purifying the effective components. The active ingredients of the placenta extract are prepared by multi-step technical purification, so that the placenta extract not only retains the effective ingredients such as collagen, polypeptide, various growth factors and the like in the placenta, but also does not contain cell ingredients, avoids the problems of safety and immunological rejection of cell preparations, has simple and controllable operation method, can simply realize large-scale preparation, is promoted in a shelf manner, has short time and no preparation period; the active ingredients of the preparation are natural ingredients of human bodies, oral hormone therapy is not needed, hormone dependence is avoided, the preparation is absorbed fully by adopting mucosa transdermal absorption, and the preparation has better safety compared with oral hormone.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a placenta extract, and a preparation method and application thereof.
Background
Premature Ovarian Failure (POF) is a disease caused by multiple causes, such as amenorrhea, decreased estrogen secretion, and increased gonadotropin level in women under 40 years old due to ovarian failure.
The incidence of POF increases year by year, and the proportion of POF in female infertility patients tends to increase year by year. Two major threats to the reproductive health of women from POF are mainly the decrease in estrogen levels and the loss of fertility, which increases the risk of osteoporosis and coronary heart disease in women. And women in the childbearing period close their menstrual flow in advance, and the fertility is lost, which causes a series of psychological and social problems, such as depression, anxiety, difficulty in interpersonal interaction, hostility and other emotions, social psychosanitary problems and marital life quality reduction. Because of the trend of tumor rejuvenation and the characteristics that immune diseases are well developed in young women, premature ovarian failure caused by chemotherapy and immune factors is increasingly emphasized, and the prevention and treatment of premature ovarian failure caused by chemotherapy and immune injury become a hot spot of clinical attention.
At present, the treatment measures of premature ovarian failure mainly comprise psychological treatment, traditional Chinese medicine treatment and western medicine treatment, wherein female and pregnant hormone replacement therapy, ovulation promotion treatment, immunotherapy and ovarian transplantation are commonly used in the western medicine treatment, and although the treatment methods have a certain relieving effect on the clinical symptoms of premature ovarian failure, the damaged ovarian function cannot be fundamentally repaired.
At present, artificial oral hormone is mainly adopted for treating premature ovarian failure, uterus and ovary atrophy can be improved to a certain extent, and the menstrual cycle is recovered, but most patients have artificial hormone dependence, still have no menstruation or have long cycle and small amount after stopping taking medicine. The effective rate of artificial oral hormone for treating premature ovarian failure is only about 20-30%. Hormone replacement therapy may even increase the risk of malignancy, such as breast cancer.
In recent years, the placenta has an obvious effect on treating immune oophoritis and POF. The placenta is a special tissue for providing nutrients for a fetus of a mammal fetus during pregnancy to allow the growth of the fetus, is discharged out of the body during delivery, and is regarded as important for people since ancient times due to the special barrier function and endocrine function of the placenta. Modern biological and medical research proves that the placenta contains immunoglobulin which can enhance the immunity of the organism and inhibit virus from invading cells; contains 17 amino acids including 7 essential amino acids, and the amino acids in the extractive solution exist in polypeptide form, and are easily absorbed by organism; contains various hormones including human chorionic gonadotropin, prolactin, human chorionic thyroid stimulating hormone, corticotropin releasing hormone, estradiol, estrone, progesterone, androgen, etc. In addition, it contains minerals, cytokines, growth factors and various enzymes.
Modern pharmacology proves that the polypeptide extracted from the placenta has the effects of resisting oxidation, preventing aging, enhancing the immunity of the organism, inhibiting bacteria, promoting cell reproduction and the like; the contained multiple hormones can promote the development of mammary gland and female reproductive organs. The contained multienzyme system is involved in the metabolism of steroid hormone and influences the menstrual cycle. Can not only stimulate ovarian tissues directly, but also supplement proper amount of female hormone, cooperate with FSH in vivo to resuscitate follicles. Can be clinically used for treating uterine hypoplasia, uterine atrophy, functional menoxenia, milk deficiency, etc.
In traditional Chinese medicine, the placenta hominis is called placenta hominis, and ancient people have a habit of taking placenta hominis. The compendium of materia medica states that the medicine has the functions of soothing the nerves and nourishing the blood, tonifying qi, benefiting essence, detoxifying and enriching the blood, has miraculous effect on fatigue, emaciation and weakness, and has the functions of hearing and vision improvement, blackening, prolonging life and taking away from assimilation for long-term use. At present, with the development of biotechnology, the placenta is taken from simple soup supplement to various forms such as capsules and freeze-dried powder, but the placenta is mostly taken orally, a user feels nausea due to the unique fishy smell of the placenta, and the efficacy of the product is influenced to a certain extent by the oral taking way, so that waste is caused.
The existing placenta extraction methods mainly comprise a solvent extraction method, a hydrochloric acid hydrolysis method and an enzyme decomposition method. The solvent method has low extraction efficiency, and the dissolved components are mixed with unwanted substances; the hydrolysis method with hydrochloric acid can arbitrarily cut off amino acid chain, and is difficult to maintain the three-dimensional structure and activity of polypeptide in placenta extract; in the enzyme decomposition method, each enzyme can only catalyze one chemical reaction, the specificity is strong, and the enzyme can mechanically and irreversibly cut off specific amino acid combination, so that the polypeptide component structure in the placenta extract is changed, and the physiological activity is lost; therefore, there is a need for improved methods of extracting placenta.
Disclosure of Invention
Based on the above, in order to overcome the defects of the prior art, the invention provides a placenta extract, a preparation method and application thereof.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method for preparing placenta extract comprises the following steps:
(1) placing fresh healthy placenta tissue which is fully cleaned and is removed with blood vessels and mucosa tissue into 20-80mmoL/L acetic acid-sodium acetate buffer solution with the pH value of 2.0-6.0 for homogenate, placing the homogenate in a water bath with the temperature of 4 ℃ for 1-4 hours, stirring at intervals, recovering to the room temperature, freezing and centrifuging to obtain sediment and supernatant;
(2) slowly dripping 4-12 times of the volume of the supernatant of the step (1) with ethanol precooled at minus 20 ℃ at 4 ℃, continuously stirring in the dripping process, freezing and centrifuging, and taking the supernatant;
(3) evaporating the supernatant obtained in the step (2) by rotary evaporation to obtain yolk viscous substance, dissolving the yellowish viscous substance with physiological saline, centrifuging, and taking the supernatant;
(4) adding 3-5 times of deionized water into the precipitate obtained in the step (1), stirring, adjusting the pH to 7.0-9.0, adding 0.01-20% of trypsin, hydrolyzing for 6-8 hours, freezing, centrifuging, taking the supernatant, and mixing with the supernatant obtained in the step (3) to obtain a crude placenta product;
(5) filtering the placenta crude product of step (4) with 0.1-0.5 μm filter membrane, performing anion exchange chromatography with DE-52, collecting all active peaks, further purifying by HPLC, and preparing normal saline to obtain placenta extract.
In some of these embodiments, the temperature of the rotary evaporation in step (3) is 20-60 ℃.
In some of the examples, the DE-52 anion exchange chromatography described in step (5) uses a column equilibrated with 50mmol/L Tris-HCl buffer pH6.5, which is eluted with the same buffer at a flow rate of 12-20 mL/h.
In some of these embodiments, the conditions of the refrigerated centrifugation in steps (1) and (2) are: 4 ℃ and 6600r/min for 30 min.
In some of these embodiments, the centrifugation conditions in step (3) are: 16000r/min,20 min.
The invention also provides the placenta extract prepared by the preparation method.
The invention also provides application of the placenta extract in preparing a medicament for treating premature ovarian failure.
The biological gel preparation for treating premature ovarian failure comprises the placenta extract and a gel matrix which are used as active ingredients, wherein the placenta extract accounts for 90-99.5% v/v of the biological gel preparation, namely the volume of the placenta extract contained in 1 ml of the biological gel preparation is 0.9-0.995 ml.
In some of these embodiments, the gel matrix is carbomer, sodium carboxymethylcellulose, methylcellulose, hydroxypropylcellulose, sodium alginate, gelatin, or a combination thereof.
In some of these embodiments, the gel matrix is carbomer and the placental extract is 98-99.5% v/v of the biogel formulation.
Compared with the prior art, the invention has the following beneficial effects:
1. the active ingredients of the placenta extract are prepared by multi-step technology purification, unnecessary impurities in the placenta are dissolved out by adopting a solvent extraction method, in order to improve the utilization rate of the placenta, moisture in the impurities is removed by an evaporation method, and the residual precipitate is further utilized; then decomposing the effective components in the placenta product into small molecules by a pancreatin decomposition method, and filtering and purifying to obtain a final product; in order to overcome the problems of low extraction efficiency and mixed impurities in a solvent method, the applicant removes the impurities in a reverse way, and then decomposes the polypeptides into small molecular substances by a pancreatin decomposition method so as not to damage the amino acid chains and the component structures of the polypeptides in the placenta, thereby keeping the three-dimensional structures and the physiological activities of the polypeptides; after further purification by DE-52 anion exchange chromatography and HPLC, the impurities in the extraction process are substantially removed; therefore, the finally obtained placenta extract not only retains effective components such as collagen, polypeptide, various growth factors and the like in the placenta, but also does not contain cell components, so that the problems of safety and immunological rejection of cell preparations are solved, the operation method is simple and controllable, large-scale preparation can be simply realized, shelf popularization is realized, the time is short, and no preparation period exists;
2. the placenta extract can be prepared into a biological gel preparation, promotes the ovarian function reconstruction of premature ovarian failure patients, has a good effect of treating premature ovarian failure, has natural active ingredients of human bodies, does not need oral hormone treatment, has no hormone dependence, adopts mucosa transdermal absorption, is fully absorbed, and has better safety compared with oral hormone.
Detailed Description
The invention will be further described with reference to specific examples, which are not described herein as being applicable to the prior art. Specific examples of the present invention are given below, but the examples are only for the purpose of further elaborating the present invention and do not limit the claims of the present invention. The reagents and starting materials used in the following examples were all commercially available unless otherwise specified.
Example 1A method for preparing placenta extract
The preparation method of the placenta extract of the present embodiment comprises the following steps:
(1) selecting 100mL of screened fresh healthy placenta tissue;
(2) fully cleaning and cutting the placenta tissue into small pieces, removing blood vessels and mucosa tissue, cutting into pieces, and homogenizing in 100mL of 50mmoL/L acetic acid-sodium acetate buffer solution (pH4.0); placing the homogenate in a water bath at 4 ℃ for heat preservation for 2.5 hours, and stirring at intervals; returning to room temperature, freezing and centrifuging (4 ℃, 6600r/min, 30min) to obtain precipitate and supernatant of about 150 mL;
(3) slowly dripping 6 times of ethanol (precooling at the temperature of minus 20 ℃) in volume of the supernatant into the supernatant obtained in the step (2) at the temperature of 4 ℃, continuously stirring in the dripping process, and taking the supernatant after refrigerated centrifugation (at the temperature of 4 ℃, 6600r/min and 30 min);
(4) evaporating the supernatant obtained in the step (3) by using a rotary evaporator, carrying out water bath at the temperature of 40 ℃ to obtain yolk-colored sticky substances, dissolving the yolk-colored sticky substances by using 12.0mL of physiological saline, centrifuging (16000r/min,20min), and taking the supernatant;
(5) adding 3-5 times of deionized water into the precipitate obtained in the step (2), stirring, adjusting pH to 7.0-9.0, adding 0.1% trypsin, hydrolyzing for 6-8 hours, freezing and centrifuging (4 ℃, 6600r/min, 30min), taking the supernatant, and mixing with the supernatant obtained in the step (4) to obtain a placenta extract crude product;
(6) filtering the crude product of the placenta extract in the step (5) by using a 0.2 mu m filter membrane, further performing DE-52 anion exchange chromatography, balancing a chromatographic column (2.6X40cm) by using 50mmol/L Tris-HCl buffer solution (pH6.5), and eluting by using the same buffer solution after loading, wherein the flow rate is 18 mL/h;
(7) and collecting all active peaks, further purifying by HPLC, and preparing 100ml of normal saline to obtain the placenta extract of the embodiment.
The protein content of the placenta extract was determined by biuret method, and the concentration was adjusted until the final concentration was 10mg/ml, the final volume was 150ml, and the final total protein mass was 1500 mg.
Example 2A method for preparing placenta extract
The preparation method of the placenta extract of the present embodiment comprises the following steps:
(1) selecting 500mL of screened fresh healthy placenta tissue;
(2) fully cleaning and cutting the placenta tissue into small pieces, removing blood vessels and mucosa tissue, cutting into pieces, and homogenizing in 500mL of 30mmoL/L acetic acid-sodium acetate buffer solution (pH5.0); placing the homogenate in a water bath at 4 ℃ for heat preservation for 3 hours, and stirring at intervals; returning to room temperature, freezing and centrifuging (4 ℃, 6600r/min, 30min) to obtain precipitate and supernatant of about 750 mL;
(3) slowly dropwise adding 8 times of ethanol (precooling at-20 ℃) in volume of the supernatant in the step (2) at 4 ℃, continuously stirring in the dropwise adding process, and taking the supernatant after refrigerated centrifugation (6600 r/min at 4 ℃ for 30 min);
(4) evaporating the supernatant obtained in the step (3) by using a rotary evaporator, carrying out water bath at the temperature of 40 ℃ to obtain yolk-colored sticky substances, dissolving the yolk-colored sticky substances by using 60.0mL of physiological saline, centrifuging (16000r/min,20min), and taking the supernatant;
(5) adding 3-5 times of deionized water into the precipitate obtained in the step (2), stirring, adjusting the pH to 7.0-9.0, adding 0.05% of trypsin, hydrolyzing for 6-8 hours, freezing and centrifuging (4 ℃, 6600r/min, 30min), taking the supernatant, and mixing with the supernatant obtained in the step (4) to obtain a placenta extract crude product;
(6) filtering the crude product of the placenta extract in the step (5) by using a 0.1 mu m filter membrane, further performing DE-52 anion exchange chromatography, balancing a chromatographic column (2.6X40cm) by using 50mmol/L Tris-HCl buffer solution (pH6.5), and eluting by using the same buffer solution after loading, wherein the flow rate is 15 mL/h;
(7) and collecting all active peaks, further purifying by HPLC, and preparing 100ml of normal saline to obtain the placenta extract of the embodiment.
The protein content of the placenta extract is determined by biuret method, and the concentration is adjusted until the final concentration is 10mg/ml, the final volume is 850ml, and the final total protein mass is 8500 mg.
Example 3A method for preparing placenta extract
The preparation method of the placenta extract of the present embodiment comprises the following steps:
(1) selecting 1000mL of screened fresh healthy placenta tissue;
(2) fully cleaning and cutting the placenta tissue into small pieces, removing blood vessels and mucosa tissue, cutting into pieces, and homogenizing in 1000mL of 70mmoL/L acetic acid-sodium acetate buffer solution (pH6.0); placing the homogenate in a water bath at 4 ℃ for heat preservation for 2 hours, and stirring at intervals; returning to room temperature, freezing and centrifuging (4 ℃, 6600r/min, 30min) to obtain precipitate and supernatant of about 1500 mL;
(3) slowly dripping 10 times of ethanol (precooling at the temperature of minus 20 ℃) in volume of the supernatant into the supernatant obtained in the step (2) at the temperature of 4 ℃, continuously stirring in the dripping process, and taking the supernatant after refrigerated centrifugation (at the temperature of 4 ℃, 6600r/min and 30 min);
(4) evaporating the supernatant obtained in the step (3) by using a rotary evaporator, carrying out water bath at the temperature of 40 ℃ to obtain yolk-colored sticky substances, dissolving the yolk-colored sticky substances by using 120.0mL of physiological saline, centrifuging (16000r/min,20min), and taking the supernatant;
(5) adding 3-5 times of deionized water into the precipitate obtained in the step (2), stirring, adjusting the pH to 7.0-9.0, adding 10% trypsin, hydrolyzing for 6-8 hours, freezing and centrifuging (4 ℃, 6600r/min, 30min), taking the supernatant, and mixing with the supernatant obtained in the step (4) to obtain a placenta extract crude product;
(6) filtering the crude product of the placenta extract in the step (5) by using a 0.5 mu m filter membrane, further performing DE-52 anion exchange chromatography, balancing a chromatographic column (2.6X40cm) by using 50mmol/L Tris-HCl buffer solution (pH6.5), and eluting by using the same buffer solution after loading, wherein the flow rate is 20 mL/h;
(7) and collecting all active peaks, further purifying by HPLC, and preparing 100ml of normal saline to obtain the placenta extract of the embodiment.
The protein content of the placenta extract was determined by biuret method, and the concentration was adjusted until the final concentration was 10mg/ml, the final volume was 1800ml, and the total mass of the final protein was 18000 mg.
Example 4A placental extract biogel formulation
The placental extract biogel formulation of this example was prepared from the placental extract of example 1 and 2% carbomer.
Adding 3.06g carbomer 940 into 100ml placenta extract, swelling completely, and making into water-hydrocolloid; dissolving 0.3g of ethylparaben in 3ml of propylene glycol, adding the dissolved solution into carbomer 940 water-hydrocolloid, stirring uniformly, adding 3.75g of water-soluble azone, adding the placenta extract to a constant volume of 153ml, dropwise adding triethanolamine, continuously stirring to form beige gel, and subpackaging in gel tubes or capsules to obtain the required preparation.
The prepared biological gel preparation is transparent and fine, the colloidal particles are uniformly dispersed, and the phenomena of sinking, caking and the like do not occur; the glue is kept at normal temperature, does not dry or liquefy, and has no visible foreign impurities; the pH value was 7.5, the particle size was 165 μm, and the dynamic viscosity was 20 Pa.S.
Test examples the therapeutic effect of the placental extract biogel preparation of example 4 on premature ovarian failure
The placenta extract bio-gel preparation of example 4 was dispensed into gel tubes, 3ml of which was filled, and used for premature ovarian failure patients to observe the therapeutic effects.
20 premature ovarian failure patients are recruited, 10 patients are randomly distributed and treated by the placenta extract biological gel preparation, 10 patients are treated 10 times per month, 3 months are 1 course of treatment, the vaginal mucosa is adopted for transdermal absorption, the treatment is carried out in the non-menstrual period, the placenta extract gel preparation is slowly injected into the patients after the vagina is cleaned, and the patients lie flat for 10min after the injection. The other 10 patients were treated with conventional hormonal drugs.
After 1 treatment period, the treatment effect was observed, and the results are shown in table 1.
TABLE 1 therapeutic Effect of the placenta extract biogel preparation of the present invention on premature ovarian failure
| Grouping | Placenta extract gel preparation | Conventional hormonal cycle therapy |
| Patient's health | 10 name of | 10 name of |
| High efficiency | 90％ | 30％ |
| Sleep improvement | Good effect | Is not obvious |
| Endocrine improvement | Good effect | Is not obvious |
| Color spot improvement | Good effect | Is not obvious |
| Improvement in menopause | Good effect | Is not obvious |
| Improvement of infertility | Good effect | Is not obvious |
As can be seen from the results in table 1, after treatment with the placenta extract bio-gel preparation of example 4, uterine ovarian atrophy was effectively improved in most patients, menstruation was recovered well, climacteric discomfort was significantly ameliorated, color spots were improved, and sleep quality was significantly improved, as compared to conventional oral hormone therapy.
The placenta extract of the invention extracts effective active ingredients of the placenta by a core technology, reserves effective ingredients of collagen, polypeptide, various growth factors and the like in the placenta, does not contain cell ingredients, and avoids the problems of safety and immunological rejection of cell preparations; the external gel preparation prepared by the method has the advantages of easy spreading and washing, no greasy feeling, good adhesiveness, no interference to normal functions of skin mucosa and the like, plays a role in treatment through mucosal transdermal absorption, does not need injection, is simple and convenient to operate and high in safety, and has a good treatment effect on premature ovarian failure.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (1)
1. The application of placenta extract in preparing medicine for treating premature ovarian failure;
the preparation method of the placenta extract comprises the following steps:
(1) placing fresh healthy placenta tissue which is fully cleaned and is removed with blood vessels and mucosa tissue into 20-80mmoL/L acetic acid-sodium acetate buffer solution with the pH value of 2.0-6.0 for homogenate, placing the homogenate in a water bath with the temperature of 4 ℃ for 1-4 hours, stirring at intervals, recovering to the room temperature, freezing and centrifuging to obtain sediment and supernatant;
(2) slowly dripping 4-12 times of the volume of the supernatant of the step (1) with ethanol precooled at minus 20 ℃ at 4 ℃, continuously stirring in the dripping process, freezing and centrifuging, and taking the supernatant;
the conditions of the refrigerated centrifugation in the steps (1) and (2) are as follows: at 4 ℃, 6600r/min, 30 min;
(3) evaporating the supernatant obtained in the step (2) by using a rotary evaporator to obtain yolk sticky matter, dissolving the yolk sticky matter by using normal saline, centrifuging, and taking the supernatant; the temperature of the rotary evaporation is 20-60 ℃;
the centrifugation conditions in the step (3) are as follows: 16000r/min,20 min;
(4) adding 3-5 times of deionized water into the precipitate obtained in the step (1), stirring, adjusting the pH to 7.0-9.0, adding 0.01-20% of trypsin, hydrolyzing for 6-8 hours, freezing, centrifuging, taking the supernatant, and mixing with the supernatant obtained in the step (3) to obtain a crude placenta extract product;
(5) filtering the placenta extract crude product of step (4) with 0.1-0.5 μm filter membrane, performing anion exchange chromatography in DE-52, collecting all active peaks, further purifying by HPLC, and preparing normal saline to obtain placenta extract;
the chromatographic column adopted by the DE-52 anion exchange chromatography is balanced by 50mmol/L Tris-HCl buffer solution with pH6.5, and the same buffer solution is used for elution after the sample is loaded, wherein the flow rate is 12-20 mL/h.
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| CN201710652265.3A CN107375334B (en) | 2017-08-02 | 2017-08-02 | Placenta extract and its preparation method and application |
| PCT/CN2017/105997 WO2019024250A1 (en) | 2017-08-02 | 2017-10-13 | Placental extract, preparation method therefor, and use thereof |
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| CN108578264A (en) * | 2018-05-24 | 2018-09-28 | 广州准优生物科技有限公司 | Placental hormone and its preparation, application |
| CN112057409B (en) * | 2020-09-18 | 2022-09-06 | 成都清科生物科技有限公司 | Sheep placenta extracting solution with anti-aging function and preparation method and application thereof |
| CN116139329B (en) * | 2022-09-09 | 2024-06-21 | 河南省银丰生物工程技术有限公司 | Placenta-derived matrix gel and method for preparing same |
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| US4597899A (en) * | 1984-05-25 | 1986-07-01 | Behringwerke Aktiengesellschaft | Process for obtaining a factor XIII preparation, and its use |
| CN1687107A (en) * | 2005-04-21 | 2005-10-26 | 石家庄三鹿集团股份有限公司 | Method for separating purified immuno regulation active polypeptide from placenta of cattle |
| CN102925523A (en) * | 2012-11-15 | 2013-02-13 | 辽宁大学 | Enzyme hydrolysis method for preparing fetus cervi active peptide |
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| US4597899A (en) * | 1984-05-25 | 1986-07-01 | Behringwerke Aktiengesellschaft | Process for obtaining a factor XIII preparation, and its use |
| CN1687107A (en) * | 2005-04-21 | 2005-10-26 | 石家庄三鹿集团股份有限公司 | Method for separating purified immuno regulation active polypeptide from placenta of cattle |
| CN102925523A (en) * | 2012-11-15 | 2013-02-13 | 辽宁大学 | Enzyme hydrolysis method for preparing fetus cervi active peptide |
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| "紫河车粉"在妇产科内分泌的临床应用;马文芝;《黄河医学》;19941231;第3卷(第1期);第26页左栏第1-2段 * |
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