WO2019024250A1 - 胎盘提取物及其制备方法和应用 - Google Patents

胎盘提取物及其制备方法和应用 Download PDF

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WO2019024250A1
WO2019024250A1 PCT/CN2017/105997 CN2017105997W WO2019024250A1 WO 2019024250 A1 WO2019024250 A1 WO 2019024250A1 CN 2017105997 W CN2017105997 W CN 2017105997W WO 2019024250 A1 WO2019024250 A1 WO 2019024250A1
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supernatant
placenta extract
placenta
extract
preparation
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PCT/CN2017/105997
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English (en)
French (fr)
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吴道贫
李文禄
赖小华
陶然
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广州准优生物科技有限公司
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Publication of WO2019024250A1 publication Critical patent/WO2019024250A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells

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  • the present invention belongs to the field of biotechnology, and more particularly, to a placental extract and a preparation method and application thereof.
  • PEF Premature ovarian failure
  • the treatment of premature ovarian failure mainly includes psychotherapy, Chinese medicine treatment and Western medicine treatment.
  • estrogen and progesterone replacement therapy, ovulation induction therapy, immunotherapy and ovarian transplantation are commonly used in Western medicine treatment.
  • these treatment methods have certain clinical symptoms of premature ovarian failure The mitigating effect, but still unable to fundamentally repair the damaged ovarian function.
  • Treatment of premature ovarian failure mainly uses artificial oral hormone therapy, which can improve the uterus and ovarian atrophy to some extent, and restore the menstrual cycle, but most patients have artificial hormone dependence. After stopping the drug, there is still no menstruation or long period, and the amount is small.
  • the effective rate of artificial oral hormone therapy for premature ovarian failure is only about 20-30%. Hormone replacement therapy may even increase the risk of developing malignant tumors such as breast cancer.
  • placenta is a special tissue that provides nutrients to the fetus during pregnancy, and allows the embryo to grow. It is excreted during childbirth. Because of its special barrier function and endocrine function, it has been valued since ancient times. Modern biological and medical research has shown that the placenta contains immunoglobulins that enhance the body's immunity and inhibit the virus from invading cells. It contains 17 kinds of amino acids, including 7 essential amino acids, and the amino acids of the extract are mostly in the form of polypeptides.
  • the peptide extracted from the placenta has anti-oxidation, anti-aging, enhances immunity, inhibits bacteria and promotes cell reproduction. It contains various hormones to promote the development of breast and female reproductive organs. Contains a multi-enzyme system that is involved in the metabolism of steroid hormones and affects the menstrual cycle. In addition to directly stimulating the ovarian tissue, it is also possible to supplement the female hormone in an appropriate amount, and synergize with the FSH in the body to resuscitate the follicle. Clinically, it can be used to treat uterine hypoplasia, uterine atrophy, functional menstruation, and lack of milk.
  • the existing extraction methods for the placenta mainly include a solvent extraction method, a hydrochloric acid hydrolysis method, and an enzyme decomposition method.
  • the extraction efficiency of the solvent method is low, and the dissolved components are mixed with unnecessary substances;
  • the hydrochloric acid hydrolysis method arbitrarily cuts the amino acid chain, and it is difficult to maintain the three-dimensional structure and activity of the polypeptide in the placenta extract;
  • each of the enzyme decomposition methods Enzymes can only catalyze a chemical reaction, specificity is strong, and because the enzyme can mechanically irreversibly cut off specific amino acid binding, the structure of the polypeptide component in the placenta extract changes, physiological activity is lost; therefore, the need for the placenta The extraction method is improved.
  • the present invention provides a placenta extract and a preparation method and application thereof.
  • a method for preparing a placenta extract comprising the steps of:
  • Fresh healthy placental tissue that is thoroughly cleaned and removed from blood vessels and mucosal tissues is homogenized in 20-80 mmoL/L acetic acid-sodium acetate buffer at pH 2.0-6.0, and the homogenate is placed at 4 °C.
  • the mixture was kept in a water bath for 1-4 hours, stirred occasionally, and returned to room temperature, and then centrifuged to obtain a precipitate and a supernatant;
  • step (2) at 4 ° C, slowly add 4-12 times the supernatant volume of -20 ° C pre-cooled ethanol to the supernatant in step (1), continuously stirring during the dropwise addition, freeze centrifugation, and take the supernatant;
  • step (3) rotary evaporation, the supernatant of step (2) is evaporated to obtain an egg yellow viscous substance, which will be treated with physiological saline.
  • the yellowish viscous substance is dissolved, centrifuged, and the supernatant is taken;
  • step (3) adding 3-5 times weight of deionized water to the precipitate in step (1), stirring, adjusting pH 7.0-9.0, adding 0.01-20% trypsin, hydrolyzing for 6-8 hours, taking after centrifugation and centrifugation
  • the supernatant, mixed with the supernatant in the step (3), is a crude placenta;
  • step (4) Filtering the crude placenta of step (4) with a 0.1-0.5 ⁇ m filter, DE-52 anion exchange chromatography, collecting all the active peaks, further purifying by HPLC, and preparing the physiological saline to obtain the placenta extract.
  • the temperature of the rotary evaporation in step (3) is from 20 to 60 °C.
  • the column used in the DE-52 anion exchange chromatography described in the step (5) is equilibrated with 50 mmol/L of pH 6.5 Tris-HCl buffer, and the same buffer is used after loading. Elution was carried out at a flow rate of 12-20 mL/h.
  • the conditions of the freeze centrifugation described in steps (1) and (2) are: 4 ° C, 6600 r/min, 30 min.
  • the centrifugation conditions in step (3) are: 16000 r/min, 20 min.
  • the present invention also provides a placenta extract prepared by the above preparation method.
  • the invention also provides the use of the above placental extract in the preparation of a medicament for treating premature ovarian failure.
  • a biogel preparation for treating premature ovarian failure comprising the above-described placental extract and gel matrix as an active ingredient, the placenta extract being 90-99.5% v/ of the biogel preparation v, that is, the volume of the placenta extract contained in the 1 ml biogel preparation is 0.9 to 0.995 ml.
  • the gel matrix is carbomer, sodium carboxymethylcellulose, methylcellulose, hydroxypropylcellulose, sodium alginate, gelatin, or a combination thereof.
  • the gel matrix is carbomer
  • the placental extract is 98-99.5% v/v of the biogel formulation.
  • the present invention has the following beneficial effects:
  • the active ingredient of the placenta extract of the present invention is obtained by multi-step purification, and the unnecessary impurities in the placenta are first dissolved by solvent extraction. In order to improve the utilization rate of the placenta, impurities are also removed by evaporation.
  • the water is removed, and the remaining precipitate is further utilized; then the active ingredient in the placenta product is decomposed into small molecules by trypsin decomposition, and the final product is obtained by filtration and purification; in order to overcome the problem of low extraction efficiency and impurities in the solvent method
  • the applicant removes the impurities in the opposite direction, and then, in order not to destroy the amino acid chain and component structure of the polypeptide in the placenta, it is decomposed into small molecules by trypsin decomposition, thereby retaining their three-dimensional structure and physiology.
  • the impurities in the extraction process are basically removed; therefore, the final placental extract not only retains the collagen, polypeptide, and various growth factors in the placenta.
  • Ingredients and no cellular components avoiding the safety of cell preparations and immune rejection, and easy to operate Controllable, simple to achieve large-scale preparation, shelf-style promotion, short time, no preparation cycle;
  • the placenta extract of the present invention can be made into a bio-gel preparation to promote ovarian function reconstruction in patients with premature ovarian failure, and has a good effect of treating premature ovarian failure.
  • the active ingredient is a natural component of the human body, and does not require oral hormone therapy, and has no hormone. Dependence, transmucosal transdermal absorption, adequate absorption, and better safety than oral hormones.
  • the placental tissue is thoroughly cleaned and cut into small pieces, the blood vessel and mucosal tissue are removed, and the mixture is chopped and placed in 100 mL of 50 mmoL/L acetic acid-sodium acetate buffer (pH 4.0) to homogenize; the homogenate is placed in 4 In a °C water bath for 2.5 hours, stirring from time to time; after returning to room temperature, freeze centrifugation (4 ° C, 6600r / min, 30min), to obtain a precipitate and about 150mL of supernatant;
  • step (3) at 4 ° C, slowly add 6 times the supernatant volume of ethanol (-20 ° C pre-cooling) to the supernatant in step (2), stirring constantly during the addition, freezing and centrifugation (4 ° C, After 6600r/min, 30min), take the supernatant;
  • step (3) using a rotary evaporator to evaporate the supernatant of step (3), water bath temperature of 40 ° C, to obtain egg yellow viscous material, dissolved with 12.0mL of physiological saline, centrifugation (16000r / min, 20min), take the supernatant;
  • step (5) adding 3-5 times weight of deionized water to the precipitate in step (2), stirring, adjusting pH 7.0-9.0, adding 0.1% trypsin, hydrolyzing for 6-8 hours, and freezing and centrifuging (4 ° C, 6600r) After /min, 30min), the supernatant is taken and mixed with the supernatant in step (4), which is a crude product of the placenta extract;
  • the protein content in the placental extract was determined by the biuret method, and the concentration was adjusted to a final concentration of 10 mg/ml, and the final volume was 150 ml, and the final total protein mass was 1500 mg.
  • the placental tissue is thoroughly washed and cut into small pieces, the blood vessel and mucosal tissue are removed, and then chopped and placed in 500 mL 30 mmoL / L acetic acid - sodium acetate buffer (pH 5.0) homogenate; homogenate is placed in 4 Incubate for 3 hours in a °C water bath, stirring occasionally; return to room temperature and freeze centrifugation (4 ° C, 6600r / min, 30min) to obtain a precipitate and about 750mL of supernatant;
  • step (3) at 4 ° C, slowly add 8 times the supernatant volume of ethanol (-20 ° C pre-cooling) to the supernatant in step (2), stirring constantly during the addition, freezing and centrifugation (4 ° C, After 6600r/min, 30min), take the supernatant;
  • step (3) using a rotary evaporator to evaporate the supernatant of step (3), water bath temperature of 40 ° C, to obtain egg yellow viscous material, dissolved with 60.0mL of physiological saline, centrifugation (16000r / min, 20min), take the supernatant;
  • step (5) adding 3-5 times weight of deionized water to the precipitate in step (2), stirring, adjusting pH 7.0-9.0, adding 0.05% trypsin, hydrolyzing for 6-8 hours, and freezing and centrifuging (4 ° C, 6600r) After /min, 30min), the supernatant is taken and mixed with the supernatant in step (4), which is a crude product of the placenta extract;
  • the protein content in the placenta extract was determined by the biuret method, and the concentration was adjusted to a final concentration of 10 mg/ml, and the final volume was 850 ml, and the final total protein mass was 8500 mg.
  • the placental tissue is thoroughly cleaned and cut into small pieces, the blood vessel and mucosal tissue are removed, and the mixture is chopped and placed in 1000 mL of 70 mmoL/L acetic acid-sodium acetate buffer (pH 6.0) to homogenize; the homogenate is placed in 4 In a °C water bath for 2 hours, stirring from time to time; after returning to room temperature, freeze centrifugation (4 ° C, 6600r / min, 30min), to obtain a precipitate and about 1500mL of supernatant;
  • step (3) at 4 ° C, slowly add 10 times the supernatant volume of ethanol (-20 ° C pre-cooling) to the supernatant in step (2), stirring constantly during the dropwise addition, and freezing and centrifuging (4 ° C, After 6600r/min, 30min), take the supernatant;
  • step (3) using a rotary evaporator to evaporate the supernatant of step (3), water bath temperature of 40 ° C, to obtain egg yellow viscous material, dissolved with 120.0mL of physiological saline, centrifugation (16000r / min, 20min), take the supernatant;
  • step (5) adding 3-5 times weight of deionized water to the precipitate in step (2), stirring, adjusting pH 7.0-9.0, adding 10% trypsin, hydrolyzing for 6-8 hours, freezing and centrifuging (4 ° C, 6600r) After /min, 30min), the supernatant is taken and mixed with the supernatant in step (4), which is a crude product of the placenta extract;
  • the protein content in the placental extract was determined by the biuret method, and the concentration was adjusted until the final concentration was 10 mg/ml, and the final volume was 1800 ml, and the total mass of the final protein was 18000 mg.
  • the placenta extract biogel formulation of this example was prepared from the placental extract of Example 1 and 2% carbomer.
  • Carbomer 940 Take 3.06g Carbomer 940, add 100ml placenta extract, fully swell to make water-water colloid; take 0.3g of ethylparaben, dissolve it in 3ml propylene glycol, and then add the solution to carbomer In 940 water-water colloid, stir evenly, add 3.75g of water-soluble azone, add the placenta extract to a volume of 153ml, add triethanolamide dropwise, continue to stir until it forms a beige gel, and then add to the gel.
  • the desired formulation is obtained in a hose or capsule.
  • the prepared biogel preparation is transparent, fine, and the rubber particles are uniformly dispersed without sinking and agglomeration; the gel is kept at normal temperature, does not dry or liquefy, and no foreign matter is visible to the naked eye; the pH is 7.5, the particle size is 165 ⁇ m, The dynamic viscosity is 20 Pa.s.
  • Test Example 4 The therapeutic effect of the placenta extract biogel preparation of Example 4 on premature ovarian failure
  • Example 4 The placenta extract biogel preparation of Example 4 was dispensed into a gel tube and packed in 3 ml for use in patients with premature ovarian failure to observe the therapeutic effect.
  • Table 1 The therapeutic effect of the placenta extract biogel preparation of the present invention on premature ovarian failure
  • the placenta extract of the invention extracts the effective active ingredient of the placenta through the core technology, retains the active components such as collagen, polypeptide and various growth factors in the placenta, does not contain the cellular component, and avoids the safety of the cell preparation and the immune rejection problem; It is prepared into a topical gel preparation, which has the advantages of easier spreading and washing, no greasy feeling, good adhesion, and does not hinder the normal function of the skin and mucous membrane. It can exert therapeutic effect through transmucosal transdermal absorption without injection, and is easy to operate and safe. Strong sex, has a good therapeutic effect on premature ovarian failure.

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Abstract

一种胎盘提取物及其制备方法。制备方法是将胎盘组织经冷冻处理，提取有效成分，再将有效成分进行纯化后得到的。该胎盘提取物能够治疗卵巢早衰。

Description

胎盘提取物及其制备方法和应用 技术领域
本发明属于生物技术领域，更具体地，本发明涉及一种胎盘提取物及其制备方法和应用。
背景技术
卵巢早衰，即卵巢功能早衰(premature ovarian failure，POF)是指40岁以下妇女因卵巢功能衰竭出现闭经、雌激素分泌下降、促性腺激素水平增高的一种由多种病因引起的疾病。
POF的发病率逐年增高，在女性不孕患者中所占的比例亦有逐年上升的趋势。POF对妇女生殖健康的两大威胁主要是雌激素水平降低及生育能力丧失，雌激素水平降低增加了妇女患骨质疏松和冠心病的危险。而处于生育期的妇女提早闭经，生育能力丧失，会导致一系列心理和社会问题，如易出现抑郁、焦虑、人际交往困难、敌对等情绪、社交方面的心理卫生问题以及婚姻生活质量下降。由于肿瘤年轻化的趋势以及免疫性疾病好发于年轻女性的特征，化疗和免疫因素所致的卵巢早衰日益受到重视，防治化疗和免疫损伤所致的卵巢早衰已成为临床关注的热点。
目前卵巢早衰的治疗措施主要包括心理治疗、中医治疗和西医治疗，其中西医治疗中常用雌孕激素替代疗法、促排卵治疗、免疫治疗及卵巢移植，这些治疗方法虽然对卵巢早衰的临床症状具有一定的缓解作用，但尚无法从根本上修复受损的卵巢功能。
治疗卵巢早衰目前主要采用人工口服激素治疗，可在一定程度上改善子宫和卵巢萎缩，恢复月经周期，但大部分患者有人工激素依赖，停药后依然无月经或周期长，量少。人工口服激素治疗卵巢早衰有效率仅20-30％左右。激素替代治疗甚至可能还存在增加乳腺癌等恶性肿瘤的患病风险。
近年来采用胎盘治疗免疫性卵巢炎、POF取得了明显效果。胎盘是哺乳类胎生动物在怀胎时为胎儿提供养分，让胚胎生长的特殊组织，它在分娩时排出体外，因其特殊的屏障功能和内分泌功能，自古以来就为人们所重视。现代生物学和医学研究证明，胎盘中含有可增强机体免疫力，抑制病毒侵害细胞的免疫球蛋白；含有17种氨基酸，其中包括7种必须氨基酸，且提取液的氨基酸多以多肽的形式存在，易于被机体吸收利用；含有多种激素包括人绒毛膜促性腺激素、催乳素、人绒毛促甲状腺激素、促肾上腺皮质激素释放激素、雌二酮、雌酮、孕酮及雄激素等。除此之外还含有矿物质，细胞因子和生长因子及多种酶。
现代药理学证明胎盘中提取出来的多肽具有抗氧化、防衰老、增强机体免疫力、抑菌和促进细胞繁殖等功效；含有的多种激素可以促进乳腺、女性生殖器官发育的功能。含有的多酶系统，参与甾体激素的代谢，影响月经周期。除了可以直接刺激卵巢组织外还可以适量补充女性激素，协同体内FSH共同作用，复苏卵泡。临床上可用于治疗子宫发育不全、子宫萎缩、机能性无月经、乳汁缺乏等。
中医将经过简单加工炮制后的人胎盘称为紫河车，古人就有服用紫河车的习惯。《本草纲目》称其具有“安神养血、补气、益精、解毒、补血”的作用，对“疲劳、消瘦、衰弱者”有奇效，“久服者，耳聪目明，须发黑，延年益寿， 有夺造化之功”。现在随着生物技术的发展对于胎盘的服用已经从简单的汤补发展到将其制成胶囊、冻干粉等各种形态，但它们多采用口服的方式，因胎盘独特的腥味，会让服用者产生一定的恶心感，且通过口服的方式在一定程度上影响了产品的功效，造成了浪费。
现有对胎盘的提取方法主要有溶媒提取法，盐酸加水分解法，酵素分解法。溶媒法提取效率低，而且溶出的成分中混有不需要的物质；盐酸加水分解法会任意切断氨基酸链，难以维持胎盘提取物中多肽的三次元立体构造和活性；而酵素分解法中每种酵素只能各自催化一个化学反应，专一性较强，且由于酵素能机械性的不可逆的切断特定的氨基酸结合，使胎盘提取物中多肽成分构造改变，生理活性丧失；因此，需要对胎盘的提取方法进行改进。
发明内容
基于此，为了克服上述现有技术的缺陷，本发明提供了一种胎盘提取物及其制备方法和应用。
为了实现上述发明目的，本发明采取了以下技术方案：
一种胎盘提取物的制备方法，包括以下步骤：
(1)、将充分清洗、并去除血管和粘膜组织的新鲜健康胎盘组织置于20-80mmoL/L的pH2.0-6.0的乙酸-乙酸钠缓冲液中匀浆，将匀浆液置于4℃水浴中保温1-4小时，期间不时搅拌，恢复至室温后冷冻离心，得到沉淀和上清；
(2)、于4℃，向步骤(1)中的上清缓慢滴加4-12倍上清体积的-20℃预冷的乙醇，滴加过程中不断搅拌，冷冻离心，取上清；
(3)、旋转蒸发将步骤(2)的上清蒸发，得到蛋黄色粘稠物，用生理盐水将 淡黄色粘稠物溶解，离心，取上清；
(4)、向步骤(1)中的沉淀加入3-5倍重量的去离子水，搅拌，调pH7.0-9.0，加入0.01-20％胰蛋白酶，水解6-8小时，冷冻离心后取上清，与步骤(3)中的上清混合，即为胎盘粗制品；
(5)、用0.1-0.5μm滤膜过滤步骤(4)的胎盘粗制品，DE-52阴离子交换层析，收集所有活性峰，经HPLC进一步纯化后，生理盐水配制，即得胎盘提取物。
在其中一些实施例中，步骤(3)中所述旋转蒸发的温度为20-60℃。
在其中一些实施例中，步骤(5)中所述DE-52阴离子交换层析采用的层析柱经50mmol/L的pH6.5的Tris-HCl缓冲液平衡，上样后用同样的缓冲液进行洗脱，流速均为12-20mL/h。
在其中一些实施例中，步骤(1)和(2)中所述冷冻离心的条件为：4℃，6600r/min，30min。
在其中一些实施例中，步骤(3)中所述离心条件为：16000r/min,20min。
本发明还提供了上述制备方法制备得到的胎盘提取物。
本发明还提供了上述胎盘提取物在制备治疗卵巢早衰药物中的应用。
一种治疗卵巢早衰的生物凝胶制剂，所述生物凝胶制剂包括作为活性成分的上述胎盘提取物和凝胶基质，所述胎盘提取物为所述生物凝胶制剂的90-99.5％v/v，也就是1毫升生物凝胶制剂中含有胎盘提取物的体积为0.9～0.995毫升。
在其中一些实施例中，所述凝胶基质为卡波姆、羧甲基纤维素钠、甲基纤维素、羟丙基纤维素、海藻酸钠、明胶或其组合物。
在其中一些实施例中，所述凝胶基质为卡波姆，所述胎盘提取物为所述生物凝胶制剂的98-99.5％v/v。
与现有技术相比，本发明具有以下有益效果：
1、本发明的胎盘提取物的活性成分是经多步技术纯化制得的，先采用溶媒提取法将胎盘中不需要的杂质溶解出来，为了提高胎盘的利用率，还用蒸发法将杂质中的水分去除，将剩余的沉淀进一步利用；而后采用胰酶分解法将胎盘制品中的有效成分分解成小分子，过滤纯化得到终制品；为了克服溶媒法中提取效率低，且混有杂质的问题，申请人反其道而行之将杂质去除，而后为了不破坏胎盘中多肽的氨基酸链和成分结构，采用胰酶分解法，将其分解成小分子物质，从而能保留它们的立体结构和生理活性；通过DE-52阴离子交换层析和HPLC进一步纯化后，提取过程中的杂质基本上被去除；因此，最终得到的胎盘提取物不仅保留了胎盘中的胶原、多肽、多种生长因子等有效成分，并且不含有细胞成分，避免了细胞制剂的安全性和免疫排斥问题，操作方法简单可控，能够简单实现大规模制备，货架式推广，时间短，无制备周期；
2、本发明的胎盘提取物可以制成生物凝胶制剂，促进卵巢早衰患者的卵巢功能重建，具有很好的治疗卵巢早衰的效果，其活性成分为人体天然成分，无需口服激素治疗，无激素依赖性，采用粘膜透皮吸收，吸收充分，与口服激素相比安全性更好。
具体实施方式
下面结合具体实施例进一步叙述本发明，本发明未述及之处适用于现有技术。下面给出本发明的具体实施例，但实施例仅是为了进一步详细叙述本说明，并不限制本发明的权利要求。以下实施例中所使用的试剂或原料，如无特殊说明，均来源于市售。
实施例1 一种胎盘提取物的制备方法
本实施例的胎盘提取物的制备方法，包括以下步骤：
(1)、选用经过筛选的新鲜健康胎盘组织100mL；
(2)、将胎盘组织充分清洗并切成小块，去除血管和粘膜组织，切碎后置于100mL 50mmoL/L乙酸-乙酸钠缓冲液(pH4.0)中匀浆；匀浆液置于4℃水浴中保温2.5小时，期间不时搅拌；恢复至室温后冷冻离心(4℃，6600r/min，30min)，得到沉淀和约150mL上清；
(3)、在4℃，向步骤(2)中的上清中缓慢滴加6倍于上清体积的乙醇(-20℃预冷)，滴加过程中不断搅拌，冷冻离心(4℃，6600r/min，30min)后取上清；
(4)、用旋转蒸发器将步骤(3)的上清蒸发，水浴温度40℃，得蛋黄色粘稠物，用12.0mL生理盐水溶解，离心(16000r/min,20min)，取上清；
(5)、向步骤(2)中的沉淀加入3-5倍重量去离子水，搅拌，调pH7.0-9.0，加入0.1％胰蛋白酶，水解6-8小时，冷冻离心(4℃，6600r/min，30min)后取上清，与步骤(4)中的上清混合，即为胎盘提取物粗制品；
(6)、用0.2μm滤膜过滤步骤(5)中胎盘提取物粗制品，进一步用DE-52阴离子交换层析，层析柱(2.6X40cm)先用50mmol/L Tris-HCl缓冲液(pH6.5)平衡，上样后用同样的缓冲液进行洗脱，流速均为18mL/h；
(7)、收集所有活性峰，经HPLC进一步纯化后，100ml生理盐水配制，即得本实施例的胎盘提取物。
用双缩脲法测定胎盘提取物中的蛋白质含量，调整浓度直至终浓度为10mg/ml，最终体积为150ml，最终蛋白质总质量是1500mg。
实施例2 一种胎盘提取物的制备方法
本实施例的胎盘提取物的制备方法，包括以下步骤：
(1)、选用经过筛选的新鲜健康胎盘组织500mL；
(2)、将胎盘组织充分清洗并切成小块，去除血管和粘膜组织，切碎后置于500mL 30mmoL/L乙酸-乙酸钠缓冲液(pH5.0)中匀浆；匀浆液置于4℃水浴中保温3小时，期间不时搅拌；恢复至室温后冷冻离心(4℃，6600r/min，30min)，得到沉淀和约750mL上清；
(3)、在4℃，向步骤(2)中的上清中缓慢滴加8倍于上清体积的乙醇(-20℃预冷)，滴加过程中不断搅拌，冷冻离心(4℃，6600r/min，30min)后取上清；
(4)、用旋转蒸发器将步骤(3)的上清蒸发，水浴温度40℃，得蛋黄色粘稠物，用60.0mL生理盐水溶解，离心(16000r/min,20min)，取上清；
(5)、向步骤(2)中的沉淀加入3-5倍重量去离子水，搅拌，调pH7.0-9.0，加入0.05％胰蛋白酶，水解6-8小时，冷冻离心(4℃，6600r/min，30min)后取上清，与步骤(4)中的上清混合，即为胎盘提取物粗制品；
(6)、用0.1μm滤膜过滤步骤(5)中胎盘提取物粗制品，进一步用DE-52阴离子交换层析，层析柱(2.6X40cm)先用50mmol/L Tris-HCl缓冲液(pH6.5)平衡，上样后用同样的缓冲液进行洗脱，流速均为15mL/h；
(7)、收集所有活性峰，经HPLC进一步纯化后，100ml生理盐水配制，即得本实施例的胎盘提取物。
用双缩脲法测定胎盘提取物中的蛋白质含量，调整浓度直至终浓度为10mg/ml，最终体积为850ml，最终蛋白质总质量是8500mg。
实施例3 一种胎盘提取物的制备方法
本实施例的胎盘提取物的制备方法，包括以下步骤：
(1)、选用经过筛选的新鲜健康胎盘组织1000mL；
(2)、将胎盘组织充分清洗并切成小块，去除血管和粘膜组织，切碎后置于1000mL 70mmoL/L乙酸-乙酸钠缓冲液(pH6.0)中匀浆；匀浆液置于4℃水浴中保温2小时，期间不时搅拌；恢复至室温后冷冻离心(4℃，6600r/min，30min)，得到沉淀和约1500mL上清；
(3)、在4℃，向步骤(2)中的上清中缓慢滴加10倍于上清体积的乙醇(-20℃预冷)，滴加过程中不断搅拌，冷冻离心(4℃，6600r/min，30min)后取上清；
(4)、用旋转蒸发器将步骤(3)的上清蒸发，水浴温度40℃，得蛋黄色粘稠物，用120.0mL生理盐水溶解，离心(16000r/min,20min)，取上清；
(5)、向步骤(2)中的沉淀加入3-5倍重量去离子水，搅拌，调pH7.0-9.0，加入10％胰蛋白酶，水解6-8小时，冷冻离心(4℃，6600r/min，30min)后取上清，与步骤(4)中的上清混合，即为胎盘提取物粗制品；
(6)、用0.5μm滤膜过滤步骤(5)中胎盘提取物粗制品，进一步用DE-52阴离子交换层析，层析柱(2.6X40cm)先用50mmol/L Tris-HCl缓冲液(pH6.5)平衡，上样后用同样的缓冲液进行洗脱，流速均为20mL/h；
(7)、收集所有活性峰，经HPLC进一步纯化后，100ml生理盐水配制，即得本实施例的胎盘提取物。
用双缩脲法测定胎盘提取物中的蛋白质含量，调整浓度直至终浓度为10mg/ml，最终体积为1800ml，最终蛋白质的总质量是18000mg。
实施例4 一种胎盘提取物生物凝胶制剂
本实施例的胎盘提取物生物凝胶制剂，由实施例1的胎盘提取物和2％的卡波姆制备而得。
取3.06g卡波姆940，加入100ml胎盘提取物，充分溶胀后制成水-水胶体；取0.3g的尼泊金乙酯，将其溶于3ml丙二醇中，而后将溶解液加入卡波姆940水-水胶体中，搅拌均匀后加入3.75g水溶性氮酮，再加入胎盘提取物定容至153ml，逐滴加入三乙醇酰胺，不断搅拌至其形成米黄色凝胶，而后分装于凝胶管或胶囊中即得到所需的制剂。
制备的生物凝胶制剂透明、细腻、胶粒分散均匀，不出现下沉、结块等现象；常温时保持胶状，不干涸或液化，无肉眼可见外来杂质；pH值为7.5，粒度165μm，动力粘度为20Pa.S。
试验例 实施例4的胎盘提取物生物凝胶制剂对卵巢早衰的治疗效果
取实施例4的胎盘提取物生物凝胶制剂分装到凝胶管中，3ml装，用于卵巢早衰患者，观察其疗效。
招募20名卵巢早衰患者，随机分配10名用上述胎盘提取物生物凝胶制剂治疗，每月10支，3个月为1疗程，采用阴道粘膜透皮吸收，在非月经期进行治疗，患者进行阴道清洁后，缓慢注入胎盘提取物凝胶制剂，注射完毕后患者平卧10min。其他10名患者用常规激素药物进行治疗。
1个疗程后，观察治疗效果，结果如表1所示。
表1本发明的胎盘提取物生物凝胶制剂对卵巢早衰的治疗效果
分组 胎盘提取物凝胶制剂 常规激素周期治疗
患者 10名 10名
有效率 90％ 30％
睡眠改善 良好 不明显
内分泌改善 良好 不明显
色斑改善 良好 不明显
更年期改善 良好 不明显
不孕不育改善 良好 不明显
从表1的结果可知，经实施例4的胎盘提取物生物凝胶制剂治疗后，与常规口服激素疗法相比，大部分患者子宫卵巢萎缩得到了有效改善，月经恢复良好、更年期不适症状明显好转，色斑改善，睡眠质量明显提高。
本发明的胎盘提取物是通过核心技术提取胎盘有效活性成分，保留了胎盘中的胶原、多肽、多种生长因子等有效成分，不含有细胞成分，避免了细胞制剂的安全性和免疫排斥问题；制备成外用凝胶制剂，具有更易于涂展和洗除、无油腻感、粘附性好、不妨碍皮肤粘膜正常功能等优点，通过粘膜透皮吸收发挥治疗作用，无需注射，操作简便，安全性强，对卵巢早衰有较好治疗效果。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种胎盘提取物的制备方法，其特征在于，包括以下步骤：
    (1)、将充分清洗、并去除血管和粘膜组织的新鲜健康胎盘组织置于20-80mmoL/L的pH2.0-6.0的乙酸-乙酸钠缓冲液中匀浆，将匀浆液置于4℃水浴中保温1-4小时，期间不时搅拌，恢复至室温后冷冻离心，得到沉淀和上清；
    (2)、于4℃，向步骤(1)中的上清缓慢滴加4-12倍上清体积的-20℃预冷的乙醇，滴加过程中不断搅拌，冷冻离心，取上清；
    (3)、旋转蒸发将步骤(2)的上清蒸发，得到蛋黄色粘稠物，用生理盐水将淡黄色粘稠物溶解，离心，取上清；
    (4)、向步骤(1)中的沉淀加入3-5倍重量的去离子水，搅拌，调pH7.0-9.0，加入0.01-20％胰蛋白酶，水解6-8小时，冷冻离心后取上清，与步骤(3)中的上清混合，即为胎盘提取物粗制品；
    (5)、用0.1-0.5μm滤膜过滤步骤(4)的胎盘提取物粗制品，DE-52阴离子交换层析，收集所有活性峰，经HPLC进一步纯化后，生理盐水配制，即得胎盘提取物。
  2. 根据权利要求1所述的胎盘提取物的制备方法，其特征在于，步骤(3)中所述旋转蒸发的温度为20-60℃。
  3. 根据权利要求1所述的胎盘提取物的制备方法，其特征在于，步骤(5)中所述DE-52阴离子交换层析采用的层析柱经50mmol/L的pH6.5的Tris-HCl缓冲液平衡，上样后用同样的缓冲液进行洗脱，流速均为12-20mL/h。
  4. 根据权利要求1所述的胎盘提取物的制备方法，其特征在于，步骤(1)和(2)中所述冷冻离心的条件为：4℃，6600r/min，30min。
  5. 根据权利要求1所述的胎盘提取物的制备方法，其特征在于，步骤(3)中所述离心条件为：16000r/min,20min。
  6. 权利要求1～5任一项所述的制备方法制备得到的胎盘提取物。
  7. 权利要求6所述的胎盘提取物在制备治疗卵巢早衰药物中的应用。
  8. 一种治疗卵巢早衰的生物凝胶制剂，其特征在于，所述生物凝胶制剂包括作为活性成分的上述胎盘提取物和凝胶基质，所述胎盘提取物为所述生物凝胶制剂的90-99.5％v/v。
  9. 根据权利要求8所述的治疗卵巢早衰的生物凝胶制剂，其特征在于，所述凝胶基质为卡波姆、羧甲基纤维素钠、甲基纤维素、羟丙基纤维素、海藻酸钠、明胶或其组合物。
  10. 根据权利要求9所述的治疗卵巢早衰的生物凝胶制剂，其特征在于，所述凝胶基质为卡波姆，所述胎盘提取物为所述生物凝胶制剂的98-99.5％v/v。
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