CN107375334A - Intacellin and its preparation method and application - Google Patents

Abstract

The invention discloses a kind of intacellin and its preparation method and application, it is by the chilled processing of placenta tissue, effective component extracting, then active ingredient is obtained after purification.The active component of the intacellin of the present invention is purified through multistep techniques and is made, not only remain the active ingredients such as collagen in placenta, polypeptide, a variety of growth factors, and cell component is not contained, avoid the security and immune rejection problems of cell preparation, operating method is simply controllable, can simple realization prepare on a large scale, goods-shelf type promote, time is short, no manufacturing cycle；Biogel preparation can be made, the ovarian function of chain reaction in patients with premature ovarian is promoted to rebuild, effect with good treatment premature ovarian failure, its active component is human body natural component, treated without oral hormone, no hormonal dependent, using mucous membrane Transdermal absorption, absorb fully, security is more preferable compared with oral hormone.

Description

Intacellin and its preparation method and application

Technical field

The invention belongs to biological technical field, more particularly it relates to a kind of intacellin and preparation method thereof And application.

Background technology

Premature ovarian failure, i.e. premature ovarian failure (premature ovarian failure, POF) refer to less than 40 years old woman Female is drawn because of one kind that amenorrhoea occurs in ovarian function failure, estrogen secretion declines, Gonadotropin Level increases by Different types of etiopathogenises The disease risen.

The POF incidence of disease increases year by year, and shared ratio also has the trend risen year by year in female acyesis patient.POF It is mainly estrogen level reduction and loss of fertility to threaten greatly the two of Reproduction Health of Women, and estrogen level reduces increase Women suffers from the danger of osteoporosis and coronary heart disease.And the women in breeding time does sth. in advance amenorrhoea, loss of fertility, can cause A series of psychology and social concerns, such as easily there is the psychology in terms of difficult depression and anxiety, human communication, hostile mood, social activity Hygienic issues and one's wedded life Quality Down.Because the trend and immunity disease of tumour rejuvenation are apt to occur in young woman Feature, the premature ovarian failure caused by chemotherapy and immune factor is increasingly subject to pay attention to, and prevents and treats the ovary caused by chemotherapy and immunologic mjury Early ageing has turned into the focus of clinical concern.

The remedy measures of premature ovarian failure mainly include psychotherapy, Chinese traditional treatment and western medical treatment at present, and its traditional Chinese and western medicine is controlled Estrogen and progestogen alternative medicine, ovulation treatment, immunization therapy and ovarian transplantation are commonly used in treatment, although these treatment methods are to ovary The clinical symptoms of early ageing have certain mitigation, but there is no method fundamentally to repair impaired ovarian function.

Treat premature ovarian failure mainly to treat using artificial oral hormone at present, uterus and ovary can be improved to a certain extent Atrophy, recover the menstrual cycle, but most of patient has artificial hormone dependant, and still amenorrhea or cycle length, amount are few after drug withdrawal.People Work oral hormone treatment premature ovarian failure effective percentage only 20-30% or so.Increase mammary gland possibly even also be present in hormone replacement therapy The risk of the malignant tumours such as cancer.

Positive effect is achieved using placenta treatment immunity oaritis, POF in recent years.Placenta is mammality viviparous animal Nutrient is provided for fetus when pregnant, allows the particular tissues of embryo growth, and it is excreted in childbirth, because of its special barrier Function and endocrine function, just paid attention to since ancient times by people.Modern biology and medical research prove that containing in placenta can Strengthen immunity of organisms, suppress the immunoglobulin of virus infraction cell；, must ammonia including 7 kinds containing 17 kinds of amino acid Base acid, and the more presence in the form of polypeptide of amino acid of extract solution, are easy to be absorbed by organisms utilization；Include people containing a variety of hormones It is human chorionic gonadtropin, prolactin, human chorionic thyrotropic hormone, corticoliberim, estradione, female Ketone, progesterone and androgen etc..In addition mineral matter, cell factor and growth factor and a variety of enzymes are also contained.

The polypeptide that modern pharmacology proves to extract in placenta has anti-oxidant, anti-aging, enhancing immunity of organisms, suppression Bacterium and promotion cell proliferation and other effects；The a variety of hormones contained can promote mammary gland, the function of female sex organ development.Contain Multienzyme system, participate in the metabolism of steroid hormone, influence the menstrual cycle.It can also be fitted in addition to it directly can stimulate ovary tissue Measure and supplement female sex hormone, FSH collective effects in synergic agent, recovery ovarian follicle.It can be clinically used for treating uterine hypoplasia, uterus Atrophy, functional amenorrhea, agalactosis etc..

Human plactnta after simple Processing methods is referred to as dried human placenta by the traditional Chinese medical science, and ancients just have the custom for taking dried human placenta. 《Compendium of Materia Medica》Claim it that there is " tranquilizing and blood nourishing, tonifying Qi, strengthening the essence, detoxify, enrich blood ", have to " fatigue, becoming thin, languisher " Special effect, " long term usage person, clear-headed and clear-sighted, beard and hair is black, promotes longevity, and has the work(for taking good fortune by force ".Now with the development pair of biotechnology Taking to mend from simple soup and develop into and be made into the various forms such as capsule, freeze-dried powder in placenta, but they are used more Oral mode, because of the unique fishy smell of placenta, user can be allowed to produce certain nausea sense, and by oral mode certain The effect of product is have impact in degree, cause waste.

The existing extracting method to placenta mainly has solvent extraction, hydrochloric acid hydrolyzable method, ferment decomposition method.Solvent method Extraction efficiency is low, and unwanted material is mixed with the composition of dissolution；Hydrochloric acid hydrolyzable method meeting arbitrary cut-off amino acid chain, It is difficult to the three-D D structure and activity for maintaining polypeptide in intacellin；And every kind of ferment can only be respective in ferment decomposition method One chemical reaction of catalysis, selectivity is stronger, and because the mechanical irreversible specific amino acid of cut-out of ferment energy combines, Make polypeptide moiety structural change in intacellin, physiologically active is lost；Therefore, it is necessary to change to the extracting method of placenta Enter.

The content of the invention

Based on this, the defects of in order to overcome above-mentioned prior art, the invention provides a kind of intacellin and its preparation Methods and applications.

In order to realize foregoing invention purpose, this invention takes following technical scheme：

A kind of preparation method of intacellin, comprises the following steps：

(1) the abundant fresh and healthy placenta tissue for cleaning and removing blood vessel and mucosal tissue, is placed in 20-80mmoL/L PH2.0-6.0 acetic acid-sodium acetate buffer solution in be homogenized, homogenate is placed in 4 DEG C of water-baths insulation 1-4 hours, during which not When stir, refrigerated centrifuge after recovering to room temperature is precipitated and supernatant；

(2), in 4 DEG C, the ethanol of -20 DEG C of precoolings of 4-12 times of supernatant volume is slowly added dropwise in the supernatant into step (1), drop It is stirred continuously during adding, refrigerated centrifuge, takes supernatant；

(3), rotary evaporation evaporates the supernatant of step (2), obtains yolk yellow dope, will be faint yellow viscous with physiological saline Thick thing dissolving, centrifugation, takes supernatant；

(4), the precipitation into step (1) adds the deionized water of 3-5 times of weight, stirring, adjusts pH7.0-9.0, adds 0.01-20% trypsase, 6-8 hours are hydrolyzed, supernatant is taken after refrigerated centrifuge, is mixed with the supernatant in step (3), as placenta Semifinished product；

(5), with the placenta semifinished product of 0.1-0.5 μm of membrane filtration step (4), DE-52 anion-exchange chromatographies, institute is collected Active peak, after HPLC is further purified, normal saline, produce intacellin.

In wherein some embodiments, the temperature of rotary evaporation described in step (3) is 20-60 DEG C.

In wherein some embodiments, the chromatographic column that DE-52 anion-exchange chromatographies described in step (5) use passes through 50mmol/L pH6.5 Tris-HCl buffer solutions balance, and are eluted after loading with same buffer solution, flow velocity is 12- 20mL/h。

In wherein some embodiments, the condition of refrigerated centrifuge is described in step (1) and (2)：4 DEG C, 6600r/min, 30min。

In wherein some embodiments, centrifugal condition is described in step (3)：16000r/min,20min.

Present invention also offers the intacellin that above-mentioned preparation method is prepared.

Present invention also offers above-mentioned intacellin to prepare the application in treating premature ovarian failure medicine.

A kind of biogel preparation for treating premature ovarian failure, the biogel preparation are included as the above-mentioned of active component Intacellin and gel-type vehicle, the intacellin be the biogel preparation 90-99.5%v/v, that is, 1 milli It is 0.9~0.995 milliliter to rise the volume containing intacellin in biogel preparation.

In wherein some embodiments, the gel-type vehicle is carbomer, sodium carboxymethylcellulose, methylcellulose, hydroxyl Propyl cellulose, sodium alginate, gelatin or its composition.

In wherein some embodiments, the gel-type vehicle is carbomer, and the intacellin is the biogel The 98-99.5%v/v of preparation.

Compared with prior art, the invention has the advantages that：

1st, the active component of intacellin of the invention be purify through multistep techniques made from, first using solvent extraction Unwanted impurity in placenta is dissolved out, in order to improve the utilization rate of placenta, also removed the moisture in impurity with evaporation Remove, remaining precipitation is further utilized；The active ingredient in placental article is then resolved into small point using pancreatin decomposition method Son, Purification by filtration obtain whole product；In order to overcome extraction efficiency in solvent method low, and the problem of be mixed with impurity, applicant it is anti-its Road and the removing impurity of row, then in order to not destroy the amino acid chain and constituent structure of polypeptide in placenta, are decomposed using pancreatin Method, small-molecule substance is broken down into, so as to retain their stereochemical structure and physiologically active；Handed over by DE-52 anion Change chromatography and after HPLC is further purified, the impurity in extraction process is substantially removed；Therefore, the placenta extraction finally given Thing not only remains the active ingredients such as collagen in placenta, polypeptide, a variety of growth factors, and does not contain cell component, avoids The security and immune rejection problems of cell preparation, operating method is simply controllable, can simple realization prepare on a large scale, shelf Formula is promoted, and the time is short, no manufacturing cycle；

2nd, biogel preparation can be made in intacellin of the invention, promote the ovarian function weight of chain reaction in patients with premature ovarian Build, there is the effect for the treatment of premature ovarian failure well, its active component is human body natural component, is treated without oral hormone, nothing Hormonal dependent, using mucous membrane Transdermal absorption, absorb fully, security is more preferable compared with oral hormone.

Embodiment

The present invention is further discussed below with reference to specific embodiment, the present invention does not address part and is applied to prior art.Under Face provides the specific embodiment of the present invention, but embodiment is not intended to limit the present invention merely to this explanation is described in further detail Claim.Reagent or raw material used in following examples, unless otherwise specified, derive from commercially available.

A kind of preparation method of 1 intacellin of embodiment

The preparation method of the intacellin of the present embodiment, comprises the following steps：

(1), from the fresh and healthy placenta tissue 100mL by screening；

(2), placenta tissue is fully cleaned and is cut into small pieces, blood vessel and mucosal tissue is removed, 100mL is placed in after chopping It is homogenized in 50mmoL/L acetic acid-sodium acetate buffer solutions (pH4.0)；Homogenate is placed in 4 DEG C of water-baths and is incubated 2.5 hours, during which not When stir；Recover to refrigerated centrifuge after room temperature (4 DEG C, 6600r/min, 30min), precipitated and about 150mL supernatants；

(3), at 4 DEG C, the ethanol (- 20 DEG C of precoolings) of 6 times of supernatant volumes is slowly added dropwise in the supernatant into step (2), It is stirred continuously during dropwise addition, supernatant is taken after refrigerated centrifuge (4 DEG C, 6600r/min, 30min)；

(4), the supernatant of step (3) is evaporated with rotary evaporator, 40 DEG C of bath temperature, obtains yolk yellow dope, is used 12.0mL physiological saline solutions, centrifuge (16000r/min, 20min), take supernatant；

(5), the precipitation into step (2) adds 3-5 times of weight deionized water, stirring, adjusts pH7.0-9.0, adds 0.1% Trypsase, 6-8 hours are hydrolyzed, supernatant are taken after refrigerated centrifuge (4 DEG C, 6600r/min, 30min), with the supernatant in step (4) Mixing, as intacellin semifinished product；

(6), with intacellin semifinished product in 0.2 μm of membrane filtration step (5), further with DE-52 anion exchanges Chromatograph, chromatographic column (2.6X40cm) is first balanced with 50mmol/L Tris-HCl buffer solutions (pH6.5), is delayed after loading with same Fliud flushing is eluted, and flow velocity is 18mL/h；

(7) all Peak Activities, are collected, after HPLC is further purified, 100ml normal salines, produce the present embodiment Intacellin.

The protein content in intacellin is determined with biuret method, adjustment concentration is until final concentration of 10mg/ml, most Final volume is 150ml, and final protein gross mass is 1500mg.

A kind of preparation method of 2 intacellin of embodiment

The preparation method of the intacellin of the present embodiment, comprises the following steps：

(1), from the fresh and healthy placenta tissue 500mL by screening；

(2), placenta tissue is fully cleaned and is cut into small pieces, blood vessel and mucosal tissue is removed, 500mL is placed in after chopping It is homogenized in 30mmoL/L acetic acid-sodium acetate buffer solutions (pH5.0)；Homogenate is placed in 4 DEG C of water-baths and is incubated 3 hours, during which frequently Stirring；Recover to refrigerated centrifuge after room temperature (4 DEG C, 6600r/min, 30min), precipitated and about 750mL supernatants；

(3), at 4 DEG C, the ethanol (- 20 DEG C of precoolings) of 8 times of supernatant volumes is slowly added dropwise in the supernatant into step (2), It is stirred continuously during dropwise addition, supernatant is taken after refrigerated centrifuge (4 DEG C, 6600r/min, 30min)；

(4), the supernatant of step (3) is evaporated with rotary evaporator, 40 DEG C of bath temperature, obtains yolk yellow dope, is used 60.0mL physiological saline solutions, centrifuge (16000r/min, 20min), take supernatant；

(5), the precipitation into step (2) adds 3-5 times of weight deionized water, stirring, adjusts pH7.0-9.0, adds 0.05% trypsase, hydrolyzes 6-8 hours, takes supernatant after refrigerated centrifuge (4 DEG C, 6600r/min, 30min), and in step (4) Supernatant mixing, as intacellin semifinished product；

(6), with intacellin semifinished product in 0.1 μm of membrane filtration step (5), further with DE-52 anion exchanges Chromatograph, chromatographic column (2.6X40cm) is first balanced with 50mmol/L Tris-HCl buffer solutions (pH6.5), is delayed after loading with same Fliud flushing is eluted, and flow velocity is 15mL/h；

(7) all Peak Activities, are collected, after HPLC is further purified, 100ml normal salines, produce the present embodiment Intacellin.

The protein content in intacellin is determined with biuret method, adjustment concentration is until final concentration of 10mg/ml, most Final volume is 850ml, and final protein gross mass is 8500mg.

A kind of preparation method of 3 intacellin of embodiment

The preparation method of the intacellin of the present embodiment, comprises the following steps：

(1), from the fresh and healthy placenta tissue 1000mL by screening；

(2), placenta tissue is fully cleaned and is cut into small pieces, blood vessel and mucosal tissue is removed, 1000mL is placed in after chopping It is homogenized in 70mmoL/L acetic acid-sodium acetate buffer solutions (pH6.0)；Homogenate is placed in 4 DEG C of water-baths and is incubated 2 hours, during which frequently Stirring；Recover to refrigerated centrifuge after room temperature (4 DEG C, 6600r/min, 30min), precipitated and about 1500mL supernatants；

(3), at 4 DEG C, the ethanol (- 20 DEG C of precoolings) of 10 times of supernatant volumes is slowly added dropwise in the supernatant into step (2), It is stirred continuously during dropwise addition, supernatant is taken after refrigerated centrifuge (4 DEG C, 6600r/min, 30min)；

(4), the supernatant of step (3) is evaporated with rotary evaporator, 40 DEG C of bath temperature, obtains yolk yellow dope, is used 120.0mL physiological saline solutions, centrifuge (16000r/min, 20min), take supernatant；

(5), the precipitation into step (2) adds 3-5 times of weight deionized water, stirring, adjusts pH7.0-9.0, adds 10% Trypsase, 6-8 hours are hydrolyzed, supernatant are taken after refrigerated centrifuge (4 DEG C, 6600r/min, 30min), with the supernatant in step (4) Mixing, as intacellin semifinished product；

(6), with intacellin semifinished product in 0.5 μm of membrane filtration step (5), further with DE-52 anion exchanges Chromatograph, chromatographic column (2.6X40cm) is first balanced with 50mmol/L Tris-HCl buffer solutions (pH6.5), is delayed after loading with same Fliud flushing is eluted, and flow velocity is 20mL/h；

(7) all Peak Activities, are collected, after HPLC is further purified, 100ml normal salines, produce the present embodiment Intacellin.

The protein content in intacellin is determined with biuret method, adjustment concentration is until final concentration of 10mg/ml, most Final volume is 1800ml, and the gross mass of final protein is 18000mg.

A kind of intacellin biogel preparation of embodiment 4

The intacellin biogel preparation of the present embodiment, by the intacellin and 2% carbomer system of embodiment 1 It is standby and obtain.

3.06g Acritamer 940s are taken, 100ml intacellins is added, water-hydrocolloid fully is made after swelling；Take 0.3g's Ethylparaben, it is dissolved in 3ml propane diols, then lysate is added in Acritamer 940 water-hydrocolloid, stirred 3.75g water-solubleazones are added afterwards, are added intacellin and are settled to 153ml, triglycolyl amine is added dropwise, is stirred continuously Ecru gel is formed to it, is then sub-packed in gel tube or capsule and obtains required preparation.

The biogel preparation of preparation is transparent, fine and smooth, micelle is uniformly dispersed, phenomena such as occurring without sinking, caking；During normal temperature Keep gluey, not dry or liquefaction, be visible by naked eyes exogenous impurity；PH value is 7.5,165 μm of granularity, and dynamic viscosity is 20Pa.S。

Therapeutic effect of the intacellin biogel preparation of test example embodiment 4 to premature ovarian failure

The intacellin biogel preparation of Example 4 is dispensed into gel tube, 3ml dresses, is suffered from for premature ovarian failure Person, observe its curative effect.

20 chain reaction in patients with premature ovarian are recruited, are randomly assigned the above-mentioned intacellin biogel preparation for treating of 10 use, often The moon 10,3 months are 1 course for the treatment of, using vagina mucosa Transdermal absorption, are treated in the non-menstrual period, and patient carries out vaginal cleaning Afterwards, intacellin gel preparation is slowly injected into, patient lies down 10min after injection.Other 10 patients are with conventional hormone Medicine is treated.

After 1 course for the treatment of, therapeutic effect is observed, as a result as shown in table 1.

Therapeutic effect of the intacellin biogel preparation of the present invention of table 1 to premature ovarian failure

Packet	Intacellin gel preparation	Conventional Hormone therapy
			Patient	10	10
It is efficient	90%	30%
			Sleep improvement	Well	Unobvious
Endocrine improves	Well	Unobvious
			Color spot improves	Well	Unobvious
Climacteric improves	Well	Unobvious
			Infertile improvement	Well	Unobvious

As can be known from the results of Table 1, after the intacellin biogel preparation for treating of embodiment 4, swash with traditional oral Plain therapy is compared, and most of patient uterine's ovarian atrophy is effectively improved, and menstruation recovery is good, climacteric malaise symptoms are bright Aobvious to take a turn for the better, color spot improves, and sleep quality significantly improves.

The intacellin of the present invention is to extract placenta effective active composition by core technology, remains the glue in placenta The active ingredients such as original, polypeptide, a variety of growth factors, do not contain cell component, avoid the security of cell preparation and immune row Reprimand problem；Be prepared into external-use gel preparation, have be easier to apply exhibition and remove, without greasy feeling, adhesiveness is good, is glued without prejudice to skin The advantages that film normal function, therapeutic action is played by mucous membrane Transdermal absorption, without injection, easy to operate, high safety, to ovum Nest early ageing has preferable therapeutic effect.

Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, the scope that this specification is recorded all is considered to be.

Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that come for one of ordinary skill in the art Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

Claims (10)

1. a kind of preparation method of intacellin, it is characterised in that comprise the following steps：

(1) the abundant fresh and healthy placenta tissue for cleaning and removing blood vessel and mucosal tissue, is placed in 20-80mmoL/L's It is homogenized in pH2.0-6.0 acetic acid-sodium acetate buffer solution, homogenate is placed in insulation 1-4 hours in 4 DEG C of water-baths, during which frequently Stirring, refrigerated centrifuge after recovering to room temperature are precipitated and supernatant；

(2), in 4 DEG C, the ethanol of -20 DEG C of precoolings of 4-12 times of supernatant volume is slowly added dropwise in the supernatant into step (1), is added dropwise It is stirred continuously in journey, refrigerated centrifuge, takes supernatant；

(3), rotary evaporation evaporates the supernatant of step (2), obtains yolk yellow dope, with physiological saline by faint yellow dope Dissolving, centrifugation, takes supernatant；

(4), the precipitation into step (1) adds the deionized water of 3-5 times of weight, stirring, adjusts pH7.0-9.0, adds 0.01- 20% trypsase, 6-8 hours are hydrolyzed, supernatant is taken after refrigerated centrifuge, is mixed with the supernatant in step (3), as placenta extracts Thing semifinished product；

(5), with the intacellin semifinished product of 0.1-0.5 μm of membrane filtration step (4), DE-52 anion-exchange chromatographies, collect All Peak Activities, after HPLC is further purified, normal saline, produce intacellin.

2. the preparation method of intacellin according to claim 1, it is characterised in that rotate and steam described in step (3) The temperature of hair is 20-60 DEG C.

3. the preparation method of intacellin according to claim 1, it is characterised in that DE-52 described in step (5) is cloudy The Tris-HCl buffer solutions for pH6.5 of the chromatographic column through 50mmol/L that ion-exchange chromatography uses balance, and are used equally after loading Buffer solution is eluted, and flow velocity is 12-20mL/h.

4. the preparation method of intacellin according to claim 1, it is characterised in that cold described in step (1) and (2) Freezing the condition centrifuged is：4 DEG C, 6600r/min, 30min.

5. the preparation method of intacellin according to claim 1, it is characterised in that step centrifuges bar described in (3) Part is：16000r/min,20min.

6. the intacellin that the preparation method described in any one of Claims 1 to 5 is prepared.

7. the intacellin described in claim 6 is preparing the application in treating premature ovarian failure medicine.

8. a kind of biogel preparation for treating premature ovarian failure, it is characterised in that the biogel preparation is included as activity The above-mentioned intacellin and gel-type vehicle of composition, the intacellin are the 90-99.5%v/ of the biogel preparation v。

9. the biogel preparation for the treatment of premature ovarian failure according to claim 8, it is characterised in that the gel-type vehicle is Carbomer, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl cellulose, sodium alginate, gelatin or its composition.

10. the biogel preparation for the treatment of premature ovarian failure according to claim 9, it is characterised in that the gel-type vehicle For carbomer, the intacellin is the 98-99.5%v/v of the biogel preparation.