CN103533944A - Placenta extract and method for manufacturing same - Google Patents
Placenta extract and method for manufacturing same Download PDFInfo
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Abstract
The present invention relates to a placenta extract and a method for safely and effectively manufacturing the same. More particularly, the method for manufacturing the placenta extract of the present invention, includes at least one step selected from the group consisting of: a step of inactivating a virus using a surface active agent and a solvent; a step of irradiating ultraviolet rays to inactivate the virus; and a step of removing the virus using a nano filter. The thus-obtained placenta extract may be used for preventing and treating menopausal disorder symptoms in mammals.
Description
Technical field
The present invention relates to a kind of Placenta Hominis extract and prepare safely and effectively the method for Placenta Hominis extract, more particularly, the preparation method of Placenta Hominis extract of the present invention comprises the above step being selected from the group that the following step forms: use interfacial agent and solvent by the step of inactivation of virus; Irradiation ultraviolet radiation is by the step of inactivation of virus; And use an above step in the group that nanofilter forms the step of virus sweep; The invention still further relates to the prevention of mammals climacteric obstacle symptom or the therapeutic use of the Placenta Hominis extract so obtaining.
Background technology
Placenta Hominis (placenta) is longer organ on the uterus of conceived mammal.Placenta Hominis is discharged from uterus when childbirth, and Placenta Hominis is ejected and is called rear childbirth.Placenta Hominis is considered to the nucleic acid compositions that contains essential amino acids, melatonin, RNA and DNA and so on, as SOD (super oxide dismutase), hyaluronic acid (hyaluronic acid), antioxidant, cytokine and the various somatomedin (growth factor) of antioxidase.
Existing Placenta Hominis extract preparation method comprises: utilize the strong acid of hydrochloric acid and so on or highly basic after Placenta Hominis hydrolysis, obtain its product method, utilize enzyme the method for Placenta Hominis hydrolysis.But, this existing methodical hydrolysising condition and carry out for a long time can allowing in the process of high-temperature process Placenta Hominis contained various biological active substances degeneration or destruction.
On the other hand, do not utilize acid, alkali or enzyme hydrolysis and high-temperature processing method and take gentleness (mild) although can extract when method extracts Placenta Hominis under various biological active substances invariance, non-destructive situation, but because may causing problem, the potential virus coming along with Placenta Hominis must carry out the operation of its deactivation or removing, yet, except heat treatment, do not have so far any deactivation or remove viral Placenta Hominis extract preparation method.
The technical task solving
Basic object of the present invention is to provide a kind of preparation method of Placenta Hominis extract, and it comprises the above step being selected from the group that the following step forms: use interfacial agent and solvent by the step of inactivation of virus; Irradiation ultraviolet radiation is by the step of inactivation of virus; And use nanofilter by the step of virus sweep.
Another object of the present invention is to provide a kind of preparation method of the extract of Placenta Hominis safely and effectively, and it comprises the following steps: that (i) pulverizes the step of obtaining Placenta Hominis solution after placenta tissue in aqueous medium; (ii) above-mentioned Placenta Hominis solution separating is become to the step of liquid phase part and solid phase part; (iii) liquid phase part that obtains in above-mentioned steps (ii) adds interfacial agent and solvent and by the step of inactivation of virus; (iv) the mixed solution obtaining from above-mentioned steps (iii), remove the step of above-mentioned interfacial agent and above-mentioned solvent; (v) the solution irradiation ultraviolet radiation obtaining for above-mentioned steps (iv) is by the step of inactivation of virus; And (vi) use nanofilter and sterilizing filter to having irradiated ultraviolet solution in above-mentioned steps (v) and filtering, to remove the step of virus and antibacterial.
A further object of the present invention is to provide Placenta Hominis extract prepared by a kind of said method, and a kind of prevention of mammals climacteric obstacle symptom or medicine for treatment compositions that comprises the Placenta Hominis extract of being prepared by said method and the carrier pharmaceutically allowing is also provided.
Solve the technical scheme of problem
Basic object of the present invention can be realized by following Placenta Hominis extract preparation method, an above step in the group that the method comprises the following steps to be formed: use interfacial agent and solvent by the step of inactivation of virus; Irradiation ultraviolet radiation is by the step of inactivation of virus; And use nanofilter by the step of virus sweep.
Said method is not used the heat treatment step of prior art can be by deactivation or the removing in addition of the contained virus of Placenta Hominis yet.Above-mentioned placenta tissue in the inventive method can be from the mammal of people, pig, horse or sheep and so on.
Preferably, the above-mentioned interfacial agent that the inventive method is used and solvent energy dissolved cell film but can passivation such as protein etc. be incorporated into the activity of the composition of cell membrane.And above-mentioned interfacial agent can not allow protein denaturation.Above-mentioned interfacial agent can be used nonionic system and zwitterionic surfactant.
More particularly, above-mentioned interfacial agent is Triton-X100 (Polyethylene Glycol p-(1, 1, 3, 3-tetramethyl butyl)-phenyl ether), Triton-X114 (Polyethylene Glycol p-(1, 1, 3, 3-tetramethyl butyl)-phenyl ether), Tween20 (PEG (20) sorbitol anhydride monolaurate), Tween80 (PEG (20) sorbitol anhydride list oleic acid), Nonidet P-40 (Triton X-100), Brij-35 (polyoxyethylene (23) lauryl alcohol), Brij-58 (polyoxyethylene (20) cetyl), CHAPS (3-[(3-gallbladder amido propyl) dimethylamino]-1-propane sulfonic acid inner salt) or CHAPSO (3-[(3-gallbladder amido propyl) dimethylamino]-2-hydroxyl-1-propane sulfonic acid inner salt) or their combination.Preferably, take the volume of above-mentioned liquid phase part adds 0.1vol% to the above-mentioned interfacial agent about 2vol% as benchmark.And above-mentioned solvent is better with TNBP (tri-n-butyl phosphate).For example, above-mentioned interfacial agent is that Triton-X100, above-mentioned solvent are TNBP, and its content is respectively 0.3% (v/v) and 1% (v/v).
In the method for the invention, above-mentioned irradiation ultraviolet radiation is may be present in the virus in Placenta Hominis extract for deactivation.Rely on ultraviolet radiation people is originated to virus and model virus HIV, HAV, BHV, BVDV, PPV deactivation and further reduced biological substance and pollute.Preferably, above-mentioned ultraviolet energy is 1,000J/m
2to 3,000J/m
2.
In the method for the invention, use the nanofiltration operation of above-mentioned nanofilter to carry out efficiency checking for removing and the deactivation operation with the virus of possibility of pollution and model virus HIV, HAV, BHV, BVDV, PPV.The filtering accuracy of above-mentioned nanofilter is better to 30nm with 15nm.
Another object of the present invention can be realized by a better example of aforementioned Placenta Hominis extract preparation method of the present invention, and it comprises the following steps: that (i) pulverizes the step of obtaining Placenta Hominis solution after placenta tissue in aqueous medium; (ii) above-mentioned Placenta Hominis solution separating is become to the step of liquid phase part and solid phase part; (iii) liquid phase part that obtains in above-mentioned steps (ii) adds interfacial agent and solvent and by the step of inactivation of virus; (iv) the mixed solution obtaining from above-mentioned steps (iii), remove the step of above-mentioned interfacial agent and above-mentioned solvent; (v) the solution irradiation ultraviolet radiation obtaining for above-mentioned steps (iv) is by the step of inactivation of virus; And (vi) use nanofilter and sterilizing filter to having irradiated ultraviolet solution in above-mentioned steps (v) and filtering, to remove the step of virus and antibacterial.
In the methods of the invention, above-mentioned placenta tissue can be from the mammal of people, pig, horse or sheep and so on.And the aqueous medium in the present invention represents water or take the medium that water is solvent.Above-mentioned aqueous medium is better with water or PBS (phosphate buffer).
Above-mentioned pulverizing can be used blender (blender) or homogenizer to realize.When making small-particle placenta tissue, above-mentioned pulverizing can also carry out physical dissolution to the cell in placenta tissue.Above-mentioned pulverizing can be made placenta tissue such as the size below 3mm.This pulverizing is made small-particle placenta tissue and is increased and is dissolved into the surface area in medium such as the useful component in the placenta tissues such as protein.The solution of above-mentioned pulverizing can be homogeneous phase solution (homogenous solution).Above-mentioned pulverizing is carried out better at 2 ℃ to 8 ℃.
And, after also above-mentioned placenta tissue can being frittered, in aqueous medium, pulverize.Frittering of placenta tissue can be used chipper (mincer) or fritter machine (chopper) and carry out.This fritters and can at 2 ℃ to 8 ℃, carry out.
Preferably, use the above-mentioned placenta tissue that relies on cleaning and removed blood.When removing blood, placenta tissue can use the realizations such as erythrocyte cracked liquid (RBC lysis buffer).Above-mentioned erythrocyte cracked liquid (RBC lysis buffer) can be to contain NH
4cl, KHCO
3and the aqueous solution of EDTA, can be for example the NH that contains 8.3g in 1,000ml water
4the KHCO of Cl, 1.0g
3and the aqueous solution of 5%EDTA1.8ml.
In the method for the invention, can after above-mentioned (i) step, optionally carry out the pre-filtration step of removing the contained solid of above-mentioned liquid phase part.Above-mentioned pre-filtering can realize by centrifugalize.
Above-mentioned liquid phase part is the part of having dissolved the aqueous medium of dissolubility composition, and above-mentioned solid phase is partly the part beyond liquid phase part.Above-mentioned solid phase part can comprise cell debris (cell debris) and organelle official rank.Above-mentioned centrifugalize can be such as 10, and 000rpm to 15, carries out under 000rpm.Above-mentioned liquid phase part can be to separate from the granule of the test portion of centrifugalize (pellet).Select the liquid phase part obtaining after separation and get rid of solid phase part.Above-mentioned pre-filtration step can be used 0.4 μ m to carry out to the filter of 0.6 μ m size.Can rely on this pre-filtering to remove the impurity that above-mentioned liquid phase part may contain.
Preferably, the above-mentioned interfacial agent that the inventive method is used and solvent energy dissolved cell film but can passivation be incorporated into the activity of the composition of cell membrane such as protein.And above-mentioned interfacial agent can not allow protein denaturation.Above-mentioned interfacial agent can be used nonionic system and zwitterionic surfactant.
More particularly, above-mentioned interfacial agent is Triton-X100 (Polyethylene Glycol p-(1, 1, 3, 3-tetramethyl butyl)-phenyl ether), Triton-X114 (Polyethylene Glycol p-(1, 1, 3, 3-tetramethyl butyl)-phenyl ether), Tween20 (PEG (20) sorbitol anhydride monolaurate), Tween80 (PEG (20) sorbitol anhydride list oleic acid), Nonidet P-40 (Triton X-100), Brij-35 (polyoxyethylene (23) lauryl alcohol), Brij-58 (polyoxyethylene (20) cetyl), CHAPS (3-[(3-gallbladder amido propyl) dimethylamino]-1-propane sulfonic acid inner salt) or CHAPSO (3-[(3-gallbladder amido propyl) dimethylamino]-2-hydroxyl-1-propane sulfonic acid inner salt) or their combination.The volume of above-mentioned liquid phase part of take adds 0.1vol% to the above-mentioned interfacial agent in 2vol% left and right as benchmark.And above-mentioned solvent is better with TNBP (tri-n-butyl phosphate).For example, above-mentioned interfacial agent is that Triton-X100, above-mentioned solvent are TNBP, and its content is respectively 0.3% (v/v) and 1% (v/v).
In the step (iv) of the inventive method, can use the resin that is incorporated into special component to be removed in order to remove the contained interfacial agent of above-mentioned liquid phase part and solvent, can also obtain the extract of removing erythrocyte (RBC) and having improved purity.Preferably, above-mentioned steps (iv) can be used SDR resin (solvent/detergent removal resin) to carry out.
In the step (v) of the inventive method, irradiation ultraviolet radiation can be by above-mentioned steps (iv) inactivation of virus that may contain in solution that obtains.Rely on ultraviolet radiation people is originated to virus and model virus HIV, HAV, BHV, BVDV, PPV deactivation and further reduced biological substance and pollute.Preferably, above-mentioned ultraviolet energy is with 1,000J/m
2to 3,000J/m
2better.
In (vi) of the inventive method step, in order to carry out efficiency checking and to carry out nanofiltration (nano-filtration) operation for removing and the deactivation operation with the virus of possibility of pollution and model virus HIV, HAV, BHV, BVDV, PPV.Can rely on ultrafilter (nanofilter) from potential viral infection, to guarantee the safety of Placenta Hominis extract.And, can rely on ultrafilter to remove the bacterium that may contain in refining Placenta Hominis extract.The filtering accuracy of above-mentioned nanofilter is better to 30nm with 15nm.The filtering accuracy of above-mentioned sterilizing filter is better to 0.3 μ m size with 0.2 μ m.
A further object of the present invention can rely on provides the Placenta Hominis of being prepared by said method extract, the mammals climacteric obstacle symptom prevention that comprises the Placenta Hominis extract of being prepared by said method and the carrier pharmaceutically allowing or medicine for treatment compositions and realize.The final protein concentration of above-mentioned Placenta Hominis extract is better to 20pg/ml with 2pg/ml.
Above-mentioned " carrier pharmaceutically allowing " can be selected from the group that diluent, polishing agent, bonding agent, disintegrating agent, sweeting agent, stabilizing agent and antiseptic form.Aforementioned pharmaceutical compositions can also comprise additive.Above-mentioned additive can comprise spice, vitamin and antioxidant.Above-mentioned carrier is so long as allow on pharmaceutics, for example, diluent can be lactose, corn starch, soybean oil, microcrystalline Cellulose or mannitol, and polishing agent can be magnesium stearate or Talcum (talc), and bonding agent can be polyvinylpyrrolidone or hydroxypropyl cellulose.And, disintegrating agent can be carboxymethylcellulose calcium, carboxymethyl starch sodium, Po Lakelin potassium (polacrillin potassium) or crospovidone (crospovidone), sweeting agent can be white sugar, fructose, sorbitol or aspartame (Aspartame), stabilizing agent can be sodium carboxymethyl cellulose, beta-schardinger dextrin-, white beeswax or xanthan gum, and antiseptic can be hydroxymethyl-benzoic acid, hydroxypropyl benzoic acid or potassium sorbate.
Aforementioned pharmaceutical compositions can be made the dosage form on the known general pharmaceutics of the art, and aforementioned pharmaceutical compositions can be made administration after the dosage form of oral agents, injection, seat agent, preparation capable of permeating skin and preparation capable of permeating skin.
For example, above-mentioned dosage form can be liquor, suspending agent, powder, granule, tablet, capsule, pill, or the dosage form for oral use of extracting solution and so on.
In order to obtain improvement, prevention or the therapeutic effect of climacteric obstacle, aforementioned pharmaceutical compositions can be used as effective ingredient and take adult be as the criterion every day total dosage as above-mentioned extract 0.1g/kg to 100g/kg, 0.1g/kg to 50g/kg or 0.1g/kg to 30g/kg administration.Above-mentioned dosage can treat as required or prevent climacteric obstacle the factors such as degree, administration path, sex, age, body weight of carrying out of improving type, disease suitably increase and decrease.The improvement of obstacle of above-mentioned climacteric can be the improvement for the obstacle before climacteric.Above-mentioned mammal can be people.
Beneficial effect
Placenta Hominis extract preparation method of the present invention can be prepared the Placenta Hominis extract of the physiologically active ingredient comprising in placenta tissue safely and effectively.Active skull cap components ground extraction in can not passivation placenta tissue, can also get rid of potential virus completely and pollute.
Placenta Hominis extract of the present invention can comprise the natural physiological active composition in placenta tissue.
Pharmaceutical composition of the present invention can prevent or treat mammiferous menopause syndrome.
Accompanying drawing explanation
Fig. 1 is the uterus of nude mice and the weight change of vagina during administration Placenta Hominis extract of the present invention in embodiments of the invention 3.
Fig. 2 is the T-CHOL of nude mice and the variation of IP during administration Placenta Hominis extract of the present invention in embodiments of the invention 3.
Fig. 3 is the uterus of nude mice and the varied in thickness of skin during administration Placenta Hominis extract of the present invention in embodiments of the invention 3.
Fig. 4 is the variation of the vagina epidermis of nude mice during administration Placenta Hominis extract of the present invention in embodiments of the invention 3.
The specific embodiment
Below in conjunction with the following example, illustrate the present invention.But the following example just describes with specific embodiment of the invention form, can not therefore interest field of the present invention be defined in to record in this explanation interior and perhaps restrictively explain.
Embodiment 1. prepares Placenta Hominis extract from human placenta
At 2 ℃ to 8 ℃, freezing human placenta is thawed.Utilize the water for injection of 50L to clean the placenta tissue 5kg after thawing.Rely on this cleaning that the blood that is exposed to placenta tissue outside has been removed to a part.And umbilical cord and amniotic membrane have been removed from placenta tissue.Then, use blender to fritter placenta tissue.Fritter result, having obtained diameter is the placenta tissue that the particle below 3mm forms.
At 2 ℃ to 8 ℃, in its volume, be equivalent to the PBS buffer of 3 times (4g NaCl, 0.1g KCl, the 0.575g Na in water 1L of the placenta tissue volume that above-mentioned process fritters
2hPO
4and 0.1gKH
2pO
4) in utilize homogenizer (IKA company) the placenta tissue solution that obtains pulverizing after the placenta tissue that fritters and clean is pulverized.The solution so obtaining is in fact homogeneous phase solution.
Then, use whizzer (Hanil science industry), at 12,000rpm, 4 ℃, the solution of above-mentioned pulverizing is carried out to centrifugalize 20 minutes.After centrifugalize, reclaim supernatant and keeping at 2 ℃ to 8 ℃, eliminating particle.
Use 0.45 μ m filter (Sartorius company) to carry out pre-filtering and obtain refined liquid the about 12L of above-mentioned supernatant.Protein mixing water after pre-filtering becomes 1vol%Triton X-100 and 0.3vol%TNBP and adds each interfacial agent and solvent by concentration, then at 2 ℃ to 8 ℃ standing 30 minutes.Afterwards, in order to remove interfacial agent and solvent, use SDR resin (Solvent-Detergent Removal Resin) to carry out S/D clearing process, also carried out removing RBC and put forward highly purified operation.
Use the UVC equipment of UVivatech company with 3000J/m
2energy by ultraviolet (UV-C irradiation) by the mixing water so obtaining, for the solution passing through, for for thering is the viral removing of possibility of pollution and deactivation operation is carried out efficiency checking and utilize nanofilter (Pall company) to filter and refine.
After above-mentioned filter progress, use 0.22 μ m filter (Millipore company) take to remove bacterium and refine and obtain final Placenta Hominis extract as object.
Embodiment 2. results of animals
(1) effectiveness analysis of Placenta Hominis extract
The present embodiment is extractd (Ovariectomy; OVX) ovary of female nude mice (Mouse/CanN.Cg-Foxn1 nu/CrljOri) brought out after climacteric, the substances of repeatedly Placenta Hominis being originated gives subcutaneous administration, for the climacteric of evaluation test material improves effect and carried out internal organs weight measurement, serum biochemistry analysis and histopathological analysis.
(2) administration Placenta Hominis extract
Consider the characteristic that Mouse/CanN.Cg-Foxn1nu/CrljOri nude mice immunity reduces, raising environment, make aseptic condition and individually measure body weight after quarantine and purification, select during purification in body weight increase and general symptom do not have to have carried out after abnormal healthy animal component from.Being constructed as follows shown in list 1 of each group.
Table 1
G1 is matched group and carried out non-ovariectomy virtual operation, and G2 carries out ovariectomy operation to G6 group have been brought out climacteric.After the recoveries of 3 weeks and climacteric inductive phase, for G2 group administration normal saline (saline), for G3 group administration low capacity substances, for capacity test material in the administration of G4 group, for G5 group administration high power capacity substances, G6 group is positive controls and the menopause syndrome treatment of Japanese import is given to subcutaneous administration with Placenta Hominis pharmaceuticals Melsmon.During administration, what after administration pre-test Mus body weight, be converted into each individuality gives amount of liquid medicine (0.15ml/kg), in the mode of 3 times weekly common administration in 4 weeks 12 times.During off-test, use O
2gas inhalation makes its euthanasia, then gets blood and carries out serum biochemistry measurement, carries out obduction and has carried out naked eyes opinion record, internal organs weight measurement, tissue pathology checking.
(3) internal organs weight and Clinical and Pathological Analysis
All individualities are not all observed dead individuality, and body weight also demonstrates normal growth curve.The body weight observed value of having removed ovary group and having removed the group that gives substances and positive control substance after ovary is higher than normal group.
Internal organs weight measurement, compares with G2 (OVX+saline), and uterus and the vagina of observing substances administration group are the growth tendency that capacity is relevant, at G6 (OVX+melsmon), also observe and increase tendency.Compare with G1 (Sham+saline), the uterus of viewed G2 and vagina weight is (p < 0.01) low (referring to Fig. 1) significantly.As for bladder and stomach fat, between group, do not observe significant increase and decrease.
Clinical pathology measurement result, compare with G2 (OVX+saline), G1 (sham+saline), G5 (OVX+ substances 13.33ug/kg) and G6 (OVX+melsmon) observe T-CHOL (total cholesterol) significantly (p < 0.05) low, in substances administration group, observe capacity and reduce relatively tendency.Compare with G2, at G1, observe IP and ALP project low (p < 0.05) significantly, substances administration group is not observed significance statistically, but observe its capacity, reduces relatively tendency (referring to Fig. 2).
(4) tissue pathology checking analyzes
For carrying out after Autopsied animal use microscope takes uterus and skin photo, carry out image (Image) analysis.Compare with G2 (OVX+saline), the thickness in the uterus of substances administration group is capacity and increases relatively tendency, at G5 (OVX+ substances 13.33ug/kg) and G6 (OVX+melsmon), observes significant variation (p < 0.05).Compare with G1 (Sham+saline), the uterus thickness of observing G2 is (p < 0.01) low (referring to Fig. 3) significantly.
Aspect skin, compare with G2 (OVX+saline), the subcutaneous tissue thickness (subcutis thickness) of observing substances administration group is capacity and increases relatively tendency, at G5 (OVX+ substances 13.33ug/kg), observes significant variation (p < 0.05).Compare with G1 (Sham+saline), the subcutaneous tissue thickness of G2 is not observed larger difference (referring to Fig. 3).
Cancellous bone tissue volume (trabecular bone volume) is in surpassing the model of 3 months, to observe the project of effect at climacteric inductive phase, when our company tests because inductive phase compared with short and do not observe the significant difference between each group, but in substances administration group, there is the tendency of cancellous bone tissue volume growth.
Aspect vagina, at G2 (OVX+saline), observe significantly the vagina epithelium atrophy that causes epithelium attenuation due to the horny layer disappearance of vagina epithelium, in substances administration group and melsmon administration group, observe slighter degree (referring to Fig. 4).
Inactivation of virus and the removing of embodiment 3. checking Placenta Hominis extract preparation sections
(1) inactivation of virus of checking solvent (Solvent/Detergent) treatment process
Viral storage solutions (stock solution) is melted and carries out immediately titration (titration), by its called after Con.Titer.Virus at people's Placenta Hominis extract 45ml admixture (spiking) 5ml.At admixture viral test portion take 5ml after titration immediately by the titer of its called after admixture (spiked titer).At remaining 45ml test portion, add 10X Solvent/Detergent[3%tri (n-butyl) phosphate, 10%Triton X-100] 5ml and relief tributyl phosphate (tri (n-butyl) phosphate) becomes 0.3% and 1% with the concentration of Triton X-100.Stirs on one side allow added tributyl phosphate and Triton X-100 with admixture viral test portion mix, one side after 5 minutes, 10 minutes, 30 minutes, 60 minutes, obtain separately titration immediately after the test portion of 5ml.The result of so carrying out of take is basis, has calculated the inactivation of virus degree of solvent (Solvent/Detergent) treatment process according to exposure meter.
In order to verify the Viral safety of Placenta Hominis extract, in Placenta Hominis extract preparation section, in solvent (Solvent/Detergent) treatment process, verified people originate virus and model virus HIV, BHV, BVDV inactivating efficacy,
In solvent processing operation, do not detect HIV, BHV and BVDV and can learn and be all inactivated on earth in detecting boundary.In twice independent experiment, relying on the HIV logarithm clearing factor (log clearance factor) of solvent processing operation is >=2.73, and it is (log reduction factor) >=2.44 that logarithm reduces coefficient.BHV logarithm clearing factor >=3.47, logarithm reduces coefficient >=3.05.BVDV logarithm clearing factor >=3.63, logarithm reduces coefficient >=3.50.
From the above results can judge Placenta Hominis extract preparation section rely on solvent processing operation effectively deactivation virus.
Table 2
Virus sweep factorial experiment result
Table 3
Virus reduces coefficient experimental result
(2) inactivation of virus of checking UVC treatment process
UVC irradiation process utilizes UVivatec Lab System to carry out.According to UVC handbook, install after machinery, according to installation, cleaning and storage process (Installation, Cleaning; Storage procedure) filling distilled water.The distilled water that utilizes 2.5L was with the speed wash machine device of 15L/hr 10 minutes.The 1M NaOH that utilizes 2.5L sterilizes with the speed wash machine device of 15L/hr for 10 minutes, then, allows 1M NaOH circulation 20 minutes.After the 1M NaOH solution removal being filled in machinery, the PBS that utilizes 2.5L was with the speed wash machine device of 15L/hr 10 minutes.Activate after UV lamp, UV intensity (intensity) is set as to 1,000J/m
2, 2,000J/m
2or 3,000J/m
2.After PBS circulation machinery device with 100ml, after 15 minutes, UV intensity has reached the value setting greatly.The intensity of UV reaches after the value setting, and the test portion ascending pipe (inlet tube) that is contained in PBS is moved on to admixture the has been housed container (vessel) of operation test portion of 100ml virus.After inactivation of virus operation pattern starts, the operation test portion 100ml of institute's admixture moves on to test portion ascending pipe the container of the PBS that 100ml is housed after flowing in machinery at once again.By UVC machinery viral operation test portion 100ml, the PBS100ml that flowed out successively PBS100ml, admixture.Abandon after the PBS50ml flowing out first, connect the test portion of lower 200ml left and right and carry out immediately titration.
After being melted, viral storage solutions carries out immediately titrimetry (titration assay) and by its called after Con.Titer.At UV irradiation process parent material, add 10% viral storage solutions.Now, sampling is carried out immediately titrimetry after (sampling) part and is referred to as the titer (spiked titer) of admixture.According to the treatment conditions of manufacturer added viral test portion 100ml carry out UVC operation and sample after carry out immediately titration.The result of so carrying out of take is basis, has calculated the inactivation of virus degree of UVC treatment process according to exposure meter.
In order to verify the Viral safety of Placenta Hominis extract, in Placenta Hominis extract preparation section, in UVC irradiation process, people originate virus and model virus HIV, HAV, BHV, BVDV, PPV inactivating efficacy have been verified.
At UVC irradiation process, do not detect HIV, HAV, BVDV and PPV and can learn and be inactivated on earth in detecting boundary.Aspect BHV, can confirm to irradiate UVC after titer (titer) sharply decline.Virus that aforementioned result represents to have relied on ideally deactivation of UVC.In twice independent experiment, relying on the HIV logarithm clearing factor of UVC irradiation process is >=4.18, and it is >=3.89 that logarithm reduces coefficient.BHV logarithm clearing factor 5.31, logarithm reduces coefficient 5.30.BVDV logarithm clearing factor >=6.02, logarithm reduces coefficient >=5.96.PPV logarithm clearing factor >=4.50, logarithm reduces coefficient >=4.18.HAV logarithm clearing factor >=5.34, logarithm reduces coefficient >=5.27.
From the above results can judge Placenta Hominis extract preparation section rely on UVC irradiation process effectively deactivation virus.
Table 4
Virus sweep factorial experiment result
Table 5
(3) virus sweep of checking nanofiltration (Nano-filtration) operation
Nanofiltration operation virus sweep and inactivating efficacy in checking Placenta Hominis extract preparation section.
After being melted, viral storage solutions carries out immediately titrimetry and by its called after Con.Titer.At specimen solution, suitably after virus dilution raw material (stock), make diluent.Admixture a sampling part carry out immediately titrimetry immediately after virus, by the titer of its called after admixture (spiked titer).For admixture viral sample carry out operation during, virus is quantitative after the titer (titer) that compares the specimen (Hold sample) of test is placed under room temperature.Virus filter utilizes buffer (buffer) to smear (Coating) virus filter surface to prepare to carry out the filter progress of virus filter after prewet in advance (pre-wetting).In the virus filter chuck (cartridge) that has completed pre-treatment operation be written into (loading) admixture viral specimen and rely on the pressure filtration solution of manufacturer's operation.Measure the volume of the solution through filtering, the titration of virus amount of filtrate after measurement nanofiltration operation.
In order to verify the Viral safety of Placenta Hominis extract, in Placenta Hominis extract preparation section in nanofiltration process validation the originate removing effect of virus and model virus HIV, BHV, BVDV, HAV and PPV of people.In nanofiltration operation, do not detect HIV, BHV, BVDV, HAV and PPV and can learn that above-mentioned virus is scavenged into the end in detecting boundary.
In above-mentioned twice independent experiment, relying on the HIV logarithm clearing factor of nanofiltration operation is >=3.66, and it is >=3.96 that logarithm reduces coefficient.BHV logarithm clearing factor >=4.45, logarithm reduces coefficient >=4.59.BVDV logarithm clearing factor >=5.86, logarithm reduces coefficient >=5.49.HAV logarithm clearing factor >=5.63, logarithm reduces coefficient >=5.46.PPV logarithm clearing factor >=5.19, logarithm reduces coefficient >=4.98.
From the above results, can judge Placenta Hominis extract preparation section and rely on nanofiltration operation effectively to remove virus.
Table 6
Virus sweep factorial experiment result
Table 7
Virus reduces coefficient experimental result
Claims (15)
1. a preparation method for Placenta Hominis extract, is characterized in that,
It comprises the above step being selected from the group that the following step forms: use interfacial agent and solvent by the step of inactivation of virus; Irradiation ultraviolet radiation is by the step of inactivation of virus; And use nanofilter by the step of virus sweep.
2. the preparation method of Placenta Hominis extract according to claim 1, is characterized in that,
Above-mentioned interfacial agent is selected from Triton-X100 (Polyethylene Glycol p-(1, 1, 3, 3-tetramethyl butyl)-phenyl ether), Triton-X114 (Polyethylene Glycol p-(1, 1, 3, 3-tetramethyl butyl)-phenyl ether), Tween20 (PEG (20) sorbitol anhydride monolaurate), Tween80 (PEG (20) sorbitol anhydride list oleic acid), Nonidet P-40 (Triton X-100), Brij-35 (polyoxyethylene (23) lauryl alcohol), Brij-58 (polyoxyethylene (20) cetyl), CHAPS (3-[(3-gallbladder amido propyl) dimethylamino]-1-propane sulfonic acid inner salt) and CHAPSO (3-[(3-gallbladder amido propyl) dimethylamino]-2-hydroxyl-1-propane sulfonic acid inner salt) form group at least one.
3. the preparation method of Placenta Hominis extract according to claim 1, is characterized in that,
Above-mentioned solvent is TNBP (tri-N-butyl phosphate).
4. the preparation method of Placenta Hominis extract according to claim 1, is characterized in that,
Above-mentioned ultraviolet energy is 1,000J/m
2to 3,000J/m
2.
5. the preparation method of Placenta Hominis extract according to claim 1, is characterized in that,
The filtering accuracy of above-mentioned nanofilter is that 15nm is to 30nm size.
6. a preparation method for Placenta Hominis extract, is characterized in that, comprises the following steps:
(i) in aqueous medium, pulverize the step of obtaining Placenta Hominis solution after placenta tissue; (ii) above-mentioned Placenta Hominis solution separating is become to the step of liquid phase part and solid phase part; (iii) liquid phase part that obtains in above-mentioned steps (ii) adds interfacial agent and solvent and by the step of inactivation of virus; (iv) the mixed solution obtaining from above-mentioned steps (iii), remove the step of above-mentioned interfacial agent and above-mentioned solvent; (v) the solution irradiation ultraviolet radiation obtaining for above-mentioned steps (iv) is by the step of inactivation of virus; And (vi) use nanofilter and sterilizing filter to having irradiated ultraviolet solution in above-mentioned steps (v) and filtering, to remove the step of virus and antibacterial.
7. the preparation method of Placenta Hominis extract according to claim 6, is characterized in that,
Above-mentioned interfacial agent is selected from Triton-X100 (Polyethylene Glycol p-(1, 1, 3, 3-tetramethyl butyl)-phenyl ether), Triton-X114 (Polyethylene Glycol p-(1, 1, 3, 3-tetramethyl butyl)-phenyl ether), Tween20 (PEG (20) sorbitol anhydride monolaurate), Tween80 (PEG (20) sorbitol anhydride list oleic acid), Nonidet P-40 (Triton X-100), Brij-35 (polyoxyethylene (23) lauryl alcohol), Brij-58 (polyoxyethylene (20) cetyl), CHAPS (3-[(3-gallbladder amido propyl) dimethylamino]-1-propane sulfonic acid inner salt) and CHAPSO (3-[(3-gallbladder amido propyl) dimethylamino]-2-hydroxyl-1-propane sulfonic acid inner salt) form group at least one.
8. the preparation method of Placenta Hominis extract according to claim 6, is characterized in that,
Above-mentioned solvent is TNBP (tri-N-butyl phosphate).
9. the preparation method of Placenta Hominis extract according to claim 6, is characterized in that,
Above-mentioned steps (iv) is used SDR resin (solvent/detergent removal resin) to realize.
10. the preparation method of Placenta Hominis extract according to claim 6, is characterized in that,
Above-mentioned ultraviolet energy is 1,000J/m
2to 3,000J/m
2.
11. the preparation method of Placenta Hominis extract according to claim 6, is characterized in that,
The filtering accuracy of above-mentioned nanofilter is that 15nm is to 30nm size.
12. the preparation method of Placenta Hominis extract according to claim 6, is characterized in that,
The filtering accuracy of above-mentioned sterilizing filter is that 0.2 μ m is to 0.3 μ m size.
13. 1 kinds of Placenta Hominis extracts, is characterized in that,
Utilize claim 1 to make to the preparation method of the Placenta Hominis extract described in any one of claim 12.
Prevention or the medicine for treatment compositions of 14. 1 kinds of mammals climacteric obstacle symptoms, is characterized in that,
Comprise and utilize the Placenta Hominis extract that claim 1 makes to the preparation method of the Placenta Hominis extract described in any one of claim 12 and the carrier pharmaceutically allowing.
Prevention or the medicine for treatment compositions of 15. mammals climacteric obstacle symptoms according to claim 14, is characterized in that,
Comprise 2pg/ml to the above-mentioned Placenta Hominis extract of 20pg/ml.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020110017995A KR20120098202A (en) | 2011-02-28 | 2011-02-28 | Placenta extract and process for producing the same |
| KR10-2011-0017995 | 2011-02-28 | ||
| PCT/KR2012/001494 WO2012118321A2 (en) | 2011-02-28 | 2012-02-28 | Placenta extract and method for manufacturing same |
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| RU2710015C1 (en) * | 2019-03-29 | 2019-12-24 | Инна Анатольевна Аполихина | Method of treating genitourinary syndrome in menopausal women by means of placental therapy |
| IT202200003230A1 (en) * | 2022-02-22 | 2023-08-22 | Bruno Silvestrini | Sublingual pharmaceutical composition as antiviral vaccine. |
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| EP1661914A1 (en) * | 2004-11-18 | 2006-05-31 | Ortho-Clinical Diagnostics, Inc. | Optimal placement of a robust solvent/detergent process post viral ultrafiltration of an immune gamma globulin |
| CN101721458A (en) * | 2009-10-30 | 2010-06-09 | 葛龙海 | Method for preparing human placenta tissue fluid |
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| US6096872A (en) * | 1997-10-14 | 2000-08-01 | Ortho Diagnostic Systems, Inc. | Viral clearance process |
| KR20020063597A (en) * | 1999-12-20 | 2002-08-03 | 미츠비시 웰파마 가부시키가이샤 | Virus-free plasma protein compositions treated with porous membrane and process for producing the same |
| JP2002020299A (en) * | 2000-07-04 | 2002-01-23 | Fine Chemical Laboratory Co Ltd | Placenta-containing sublingual tablet |
| EP3246396B1 (en) * | 2001-02-14 | 2020-01-29 | Celularity, Inc. | Renovation and repopulation of decellularized tissues and cadaveric organs by stem cells |
| US20080090222A1 (en) * | 2004-06-30 | 2008-04-17 | Terumo Kabushiki Kaisha | Virus-Inactivated Hemoglobin And Method Of Producing The Same |
| JP2006158214A (en) * | 2004-12-02 | 2006-06-22 | Happy Shoji Kk | Placental extract powder-formulated food |
| JP2007106760A (en) * | 2005-09-16 | 2007-04-26 | Kenji Yoshida | Hematopoietic stem cell proliferator |
| KR100642080B1 (en) * | 2006-02-11 | 2006-11-13 | 케이에이취팜 주식회사 | A food composition containing swine placenta extraction for improving menopausal disorder, health foods comprising of it and preparation method thereof |
| KR100932183B1 (en) * | 2008-10-22 | 2009-12-16 | 주식회사 한국천연물사이언스 | Extraction Method of Horse Placenta and Food Composition Using the Same |
-
2011
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- 2012-02-28 WO PCT/KR2012/001494 patent/WO2012118321A2/en active Application Filing
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1661914A1 (en) * | 2004-11-18 | 2006-05-31 | Ortho-Clinical Diagnostics, Inc. | Optimal placement of a robust solvent/detergent process post viral ultrafiltration of an immune gamma globulin |
| CN101721458A (en) * | 2009-10-30 | 2010-06-09 | 葛龙海 | Method for preparing human placenta tissue fluid |
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| WO2012118321A3 (en) | 2012-12-20 |
| KR20120098202A (en) | 2012-09-05 |
| JP2014506921A (en) | 2014-03-20 |
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