CN113398091A

CN113398091A - Capsule preparation for treating infertility and preparation method thereof

Info

Publication number: CN113398091A
Application number: CN202110879910.1A
Authority: CN
Inventors: 曾令媛
Original assignee: Chongqing Tongliang Yuyuan Hospital Co ltd
Current assignee: Chongqing Tongliang Yuyuan Hospital Co ltd
Priority date: 2021-08-02
Filing date: 2021-08-02
Publication date: 2021-09-17

Abstract

The invention discloses a capsule preparation for treating infertility and a preparation method thereof. The medicine is prepared from Concha Meretricis Seu Cyclinae, Achyranthis radix, fructus Lipuidambaris, cortex Magnolia officinalis, semen Vaccariae, herba Patriniae, caulis Akebiae, caulis et folium Gaultheriae Yunnanensis, fructus Trichosanthis, herba Leonuri, herba Epimedii, fructus Ligustri Lucidi, carapax Trionycis, stigma croci Sativi, fructus Zanthoxyli and probiotic bacteria. The invention discloses a traditional Chinese medicine capsule preparation for treating infertility, which adopts octenyl succinic anhydride modified chitosan and sodium hyaluronate as wall materials, improves the bitter taste of traditional Chinese medicines, and simultaneously improves the water solubility and the drug delivery of the traditional Chinese medicines.

Description

Capsule preparation for treating infertility and preparation method thereof

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to a capsule preparation for treating infertility and a preparation method thereof.

Background

The reasons for infertility are many, and the two main causes are: organic cause and physical function cause. At present, with the rapid development of society, the occurrence of infertility can be caused by mental stress, psychological factors, living environment and the like. At present, various medicines for treating infertility are available in the market, and the clinical effect is not ideal.

Patent CN108042681A provides a Chinese medicinal composition for treating infertility and a preparation method thereof, the raw materials of the Chinese medicinal composition comprise wine-fried white peony root, red peony root, rhizoma corydalis, poria, cistanche deserticola, ligusticum wallichii, vinegar-processed rhizoma cyperi, prepared rhizome of rehmannia, angelica, salvia miltiorrhiza and the like, but the Chinese medicinal composition has poor curative effect, does not solve the ovulation failure infertility symptom of polycystic ovary syndrome, and does not improve the water solubility and the mouthfeel of the Chinese medicinal composition.

Disclosure of Invention

In view of the above-mentioned defects of the prior art, the present invention provides a capsule preparation for treating infertility and a preparation method thereof.

A preparation method of a traditional Chinese medicine composition for treating infertility comprises the following steps:

p1 mixing and crushing 2-4 parts by weight of clam shell, 5-7 parts by weight of radix achyranthis bidentatae, 1-3 parts by weight of fructus liquidambaris, 1-2 parts by weight of mangnolia officinalis, 4-6 parts by weight of cowherb seed, 3-5 parts by weight of patrinia scabiosaefolia, 2-4 parts by weight of caulis akebiae trifoliate, 7-9 parts by weight of garden balsam stem, 3-5 parts by weight of snakegourd fruit, 4-6 parts by weight of motherwort, 2-4 parts by weight of herba epimedii, 5-7 parts by weight of glossy privet fruit, 1-3 parts by weight of turtle shell and 1-3 parts by weight of saffron crocus, and sieving the mixture with a 10-30-mesh sieve to obtain medicinal powder A;

p2 performing steam explosion treatment on the powder A for 140 and 160s under the pressure of 4.8-5.4MPa to obtain powder B;

p3, performing second-order baking treatment on the medicinal powder B to obtain baked medicinal powder; in the second-order baking treatment, the whole process adopts carbon dioxide protective atmosphere and is stirred at the rotating speed of 20-35rpm, the temperature is firstly raised to 178-fold 182 ℃ and is preserved for 210-fold 230s, and then the temperature is raised to 255-fold 265 ℃ and is preserved for 380-fold 420 s;

p4, mixing the baked powder and the synergist in a mass ratio of (22-25):1, sending the mixture into an extraction tank, taking water as an extraction solvent, extracting at 174-177 ℃, under the extraction pressure of 2.1-2.4MPa for 28-35min, and filtering to obtain an extract liquid, wherein the ratio of the baked powder to the water bath is 1kg: 2.8-3.5L;

p5 ultrafiltering and concentrating the extract to 15-20% of the original volume by using a polysulfone ultrafiltration membrane to obtain a concentrated solution; the technological parameters of ultrafiltration concentration are that the cross flow speed is 3-5cm/s, the operation gauge pressure is 0.04-0.08MPa, and the temperature is 30-35 ℃;

p6 spray-drying the concentrated solution with centrifugal spray-dryer to obtain powder, vacuum packaging to obtain Chinese medicinal composition for treating infertility; the technological parameters of the spray powder preparation are that the air inlet pressure is 0.4-0.5MPa, the atomization frequency is 50-65Hz, the striker interval is 5-8s, the air inlet temperature is 185-plus-one 195 ℃, and the feeding speed is 230-plus-one 250 mL/h.

The Concha Meretricis Seu Cyclinae has effects of nourishing yin, promoting fluid production, softening and resolving hard mass; the achyranthes root has the effects of removing blood stasis, stimulating the menstrual flow, tonifying liver and kidney, strengthening bones and muscles, inducing diuresis, treating stranguria and drawing blood downward; the fructus liquidambaris has the effects of dispelling wind, activating collaterals and inducing diuresis; cortex Magnolia officinalis has effects in eliminating dampness, eliminating phlegm, descending qi, and relieving fullness; the snakegourd fruit has the effects of clearing heat and removing phlegm, and relieving chest stuffiness and dissipating stagnation; herba Leonuri has effects of promoting blood circulation, regulating menstruation, promoting diuresis, relieving swelling, clearing away heat and toxic materials, and can be used for treating menoxenia, dysmenorrhea, amenorrhea, lochiorrhea, edema, oliguria, and pyocutaneous disease; herba Epimedii has effects of invigorating kidney yang, strengthening tendons and bones, and dispelling pathogenic wind and dampness; fructus Ligustri Lucidi has effects in invigorating kidney yin, nourishing yin, and prolonging life; carapax Trionycis has effects in nourishing yin, suppressing yang hyperactivity, relieving fever, removing steam, softening and resolving hard mass; stigma croci Sativi has effects of promoting blood circulation for removing blood stasis, resolving stagnation and resolving hard mass, and can be used for treating female amenorrhea and puerperal abdominal pain due to blood stasis; the herbs harmonize yin and yang, reinforce each other, strengthen body resistance to eliminate pathogenic factors, and directly reach the focus.

The cowherb seed has the effects of promoting blood circulation, stimulating the menstrual flow, promoting lactation, reducing swelling, inducing diuresis and treating stranguria; herba Patriniae has effects of clearing away heat and toxic materials, removing blood stasis, and expelling pus; the akebia trifoliate has the effects of inducing diuresis for treating stranguria, clearing away heart-fire and relieving restlessness; the garden balsam stem has the effects of dispelling wind and eliminating dampness, relaxing muscles and tendons, promoting blood circulation, removing blood stasis, relieving swelling, removing toxic substances and relieving pain; the three components are jointly used in the specific traditional Chinese medicine formula group of the invention, so that the traditional Chinese medicine formula group can harmonize yin and yang, strengthen body resistance and eliminate pathogenic factors, enhance the efficacy, treat symptoms and consolidate the root.

The cell wall in the adopted medicine is further crushed by a steam explosion treatment mode before extraction, so that the pharmacodynamic ingredients in the medicine can be fully released, the subsequent extraction efficiency is improved, and the promotion of the pharmacodynamic effect is facilitated.

The invention adopts a two-stage heating baking mode to treat the medicinal materials, thereby avoiding the inactivation of the medicinal effect caused by overhigh temperature rise rate.

The synergist is at least one of green pricklyash peel and probiotic freeze-dried powder. Preferably, the synergist is a mixture of green pricklyash peel and probiotic freeze-dried powder in a mass ratio of (1-3) to (1-3).

The green pricklyash peel can treat spleen-kidney yang deficiency, waist cold and weak feet, tooth floating, and edema ascites of dysmenorrheal, knee pain, chronic nephritis and nephrotic syndrome, and can be combined with the probiotic freeze-dried powder prepared by the specific method of the invention to be used as a synergist of the traditional Chinese medicine composition for treating infertility, so that the synergist can eliminate pathogenic factors, strengthen body resistance, consolidate the constitution, and coordinate the functions of viscera and meridians, thereby correcting the excess and partial decline of yin and yang of the organism, restoring the balance of yin and yang, and further improving the curative effect of the traditional Chinese medicine composition.

The probiotic freeze-dried powder prepared by the specific method of the invention is rich in nisin and various globulins, can be harmonized with yin and yang and synergized with active substances such as various plant polysaccharides, plant flavones and the like contained in the components, and enhances the curative effect on patients with polycystic ovarian syndrome ovulation failure infertility. The invention also adopts the modes of electrifying and adding manganese acetate to promote the mass growth and propagation of the mixed strains: the activity of growth enzymes and the like participating in various metabolic activities in the mixed strain is remarkably improved under the electric stimulation, which is beneficial to increasing the yield of nisin; the addition of a proper amount of manganese element can promote the yield of beneficial substances such as nisin and the like.

The preparation method of the probiotic freeze-dried powder comprises the following steps:

d1 mixing the strains to 10³-10⁴Inoculating the inoculation amount of CFU/mL into a seed culture medium, and culturing at 37-40 ℃ for 25-35h to obtain a seed solution;

d2 inoculating the seed liquid into a fermentation tank, wherein the fermentation temperature is 37-40 ℃, and the fermentation period adopts electrification treatment and anaerobic fermentation for 140-160h to obtain fermentation liquid; the mass ratio of the seed liquid to the nutrient solution in the fermentation tank is (1-4) to 100; in the electrifying treatment, direct current with the current of 2.8-3.2mA is adopted, and the electrification is carried out once every 12-18s, wherein the duration of each time is 0.1-0.4 s;

d3, centrifuging the fermentation liquor by adopting a tubular centrifuge, and draining supernatant liquor to obtain bacterial sludge; the rotation speed of the centrifugal treatment is 11000-12000rpm, and the centrifugal time length is 2-4 min;

d4, placing the bacterial sludge in a freeze dryer, and performing freeze drying treatment to obtain the probiotic freeze-dried powder; the technological parameters of the freeze drying treatment are that the lowest temperature value is (-45) - (-40) DEG C, the freezing time is 8-10h, and the total drying time is 42-48 h.

The mixed strain is at least two of lactococcus lactis, Lactobacillus chaff-like bacteria and Lactobacillus Fuji. Preferably, the mixed strain is a mixture of lactococcus lactis, Lactobacillus chaff-like bacteria and Lactobacillus Fuji in a mass ratio of (1-4) to (1-4).

The seed culture medium is composed of the following raw materials in parts by weight: 10-13 parts of peptone, 8-10 parts of beef extract, 5-7 parts of yeast extract, 22-26 parts of glucose, 2-5 parts of dipotassium hydrogen phosphate, 2-5 parts of ammonium citrate, 0.3-0.7 part of magnesium acetate and 28-35 parts of water.

The nutrient solution in the fermentation tank is composed of the following raw materials in parts by weight: 12-18 parts of peptone, 10-15 parts of beef extract, 8-12 parts of yeast extract, 22-28 parts of glucose, 4-6 parts of monopotassium phosphate, 4-6 parts of ammonium citrate, 0.6-1 part of magnesium acetate, 0.7-1.1 part of manganese acetate and 160 parts of water by weight.

Further preferably, the preparation method of the capsule preparation for treating infertility comprises the following steps:

p1 mixing and crushing 2-4 parts by weight of clam shell, 5-7 parts by weight of radix achyranthis bidentatae, 1-3 parts by weight of fructus liquidambaris, 1-2 parts by weight of mangnolia officinalis, 4-6 parts by weight of cowherb seed, 3-5 parts by weight of patrinia scabiosaefolia, 2-4 parts by weight of caulis akebiae trifoliate, 7-9 parts by weight of garden balsam stem, 3-5 parts by weight of snakegourd fruit, 4-6 parts by weight of motherwort, 2-4 parts by weight of herba epimedii, 5-7 parts by weight of glossy privet fruit, 1-3 parts by weight of turtle shell and 1-3 parts by weight of saffron crocus, and sieving the mixture with a 10-30-mesh;

P7, adding the traditional Chinese medicine composition obtained in the step P6 and tween 80 into water, and uniformly mixing to obtain a core material solution, wherein the mass ratio of the traditional Chinese medicine composition to the tween 80 to the water is 1: (0.2-0.5): (4-6);

p8, adding octenyl succinic anhydride modified chitosan and hyaluronic acid into water, and uniformly stirring to obtain a wall material solution, wherein the mass ratio of the octenyl succinic anhydride modified chitosan to the hyaluronic acid to the water is (0.05-0.1) to (0.02-0.05): 1;

p9, adding the core material solution into the wall material solution, uniformly mixing, and then homogenizing at high pressure of 20-25MPa for 20-40min to obtain a mixed solution, wherein the mass ratio of the core material solution to the wall material solution is 1: (1-3); freeze drying the mixed solution to obtain the capsule preparation for treating infertility.

The preparation method of the octenyl succinic anhydride modified chitosan comprises the following steps:

(1) adding chitosan into a 25-30 wt% dilute hydrochloric acid aqueous solution, and stirring at the rotating speed of 300-: (10-20) mL, after the reaction is finished, centrifuging to obtain a precipitate, washing with water until the precipitate is neutral, and drying; obtaining pretreated chitosan;

(2) adding pretreated chitosan into water, stirring for 3-4h at the temperature of 60-90 ℃ at the speed of 300-: (20-30); adding 2-octenyl succinic anhydride into a pretreated chitosan solution, uniformly mixing, adjusting the pH to 8-9 by adopting 1-2mol/L NaOH aqueous solution, heating to 40-75 ℃ for reaction for 5-7h, stirring at the rotation speed of 400-700r/min for 8-12h at room temperature after the reaction is finished, adjusting the pH to 6-7 by adopting 1-2mol/L hydrochloric acid aqueous solution, performing alcohol precipitation by using 50-60 wt% isopropanol aqueous solution, centrifuging to obtain a precipitate to obtain a crude product, wherein the mass ratio of the 2-octenyl succinic anhydride to the pretreated chitosan solution is 1: (60-100); adding the crude product into water, dialyzing for 20-24h in a dialysis bag with the cut-off molecular weight of 10000-; the mass ratio of the crude product to water is 1: (15-25).

The chitosan is a high-molecular alkaline polysaccharide polymer with cations and has good biocompatibility and antibacterial activity, octenyl succinic anhydride is adopted to modify chitosan to prepare octenyl succinic anhydride modified chitosan, and then the octenyl succinic anhydride modified chitosan and sodium hyaluronate are adopted to prepare the capsule preparation for treating infertility by combining, so that on one hand, the bitter taste of the traditional Chinese medicine is improved, and on the other hand, the water solubility and the drug delivery of the traditional Chinese medicine are improved.

The invention has the beneficial effects that:

1. a Chinese medicinal capsule for treating infertility is prepared from Concha Meretricis Seu Cyclinae, Achyranthis radix, fructus Lipuidambaris, cortex Magnolia officinalis, semen Vaccariae, herba Patriniae, caulis Akebiae, caulis et folium Gaultheriae Yunnanensis, fructus Trichosanthis, herba Leonuri, herba Epimedii, fructus Ligustri Lucidi, carapax Trionycis, stigma croci Sativi, fructus Zanthoxyli and probiotic bacteria, and can be used for effectively improving serum sex hormone level (luteinizing hormone, testosterone, and estradiol); obviously improves the thickness of the endometrium and the pregnancy rate, and has good treatment effect on patients with ovulation failure infertility caused by polycystic ovarian syndrome.

2. The invention discloses a traditional Chinese medicine capsule preparation for treating infertility, which adopts octenyl succinic anhydride modified chitosan and sodium hyaluronate as wall materials, improves the bitter taste of traditional Chinese medicines, and simultaneously improves the water solubility and the drug delivery of the traditional Chinese medicines.

Detailed Description

Clam shell: mactra veneriformis Reeve, from Bozhou, Inc., Yihongtang pharmaceutical Co. The efficacy is as follows: nourish yin, promote the production of body fluid, soften hardness and dissipate nodulation.

Achyranthes root: achyranthes bidentata Bl is a dry root of Achyranthes bidentata belonging to Amaranthaceae, and is from Bozhou city Yihongtang pharmaceutical Co. The efficacy is as follows: dispel blood stasis and dredge channels, tonify liver and kidney, strengthen tendons and bones, induce diuresis and treat stranguria, and draw blood downward.

The road is through: liquidambar formosana Hance, a dry mature infructescence of a Liquidambar formosana tree of Hamamelidaceae, from Yihongtang pharmaceutical industry Co., Ltd, Bozhou city. The efficacy is as follows: dispel wind, activate collaterals, induce diuresis.

Magnolia officinalis: magnolia of finalis Rehd. et wils. var. bioloba Rehd. et wils. is dry bark, root bark and branch bark of Magnolia officinalis or Magnolia officinalis of Magnoliaceae, and is from the billion Hongtong pharmaceutical industry Co., Ltd. The efficacy is as follows: dry dampness and resolve phlegm, descend qi and remove fullness.

Seed of cowherb: vaccaria segetalis (Neck.) Garcke, a dry mature seed of Dianthus chinensis, belonging to Caryophyllaceae, comes from cooperative society of Chinese medicinal plant plantation in heat-treating city in Bozhou city. The efficacy is as follows: promoting blood circulation, dredging channels, promoting lactation, relieving swelling, inducing diuresis, and treating stranguria.

Herba patriniae: patrini scabiosaefolia from a mirror flower arrangement in the city of Bozhou, Hongshan, specialty Chinese medicinal plant cooperative. The efficacy is as follows: clear heat and remove toxicity, dispel stasis and expel pus.

Caulis akebiae trifoliate: akebia tri foliata (Thunb.) Koidz, from cooperative society of Chinese medicinal plant for treating heat in the city of Bozhou. The efficacy is as follows: induce diuresis to treat stranguria, clear heart fire and relieve restlessness.

Garden balsam stem: phoma leptostachya l.subsp.asitica (Hara) Kitamura, which is a whole plant of kombu leaves of euphorbiaceae, from medicinal material company, total medicinal material company, ohui, mn city. The efficacy is as follows: dispelling pathogenic wind and removing dampness, relieving rigidity of muscles and promoting blood circulation, removing blood stasis and detumescence, removing toxic substance and relieving pain.

And (3) snakegourd fruit: trichosanthes kirilowii maxim, from tianyang pharmaceutical ltd, bei yi county. The efficacy is as follows: clear heat and remove phlegm, relieve chest stuffiness and dissipate nodulation. The invention adopts the pericarp of snakegourd fruit.

Motherwort: leonurus japonica Houtt, from Bozhou, a hundred million Hongtong pharmaceutical Co., Ltd. The efficacy is as follows: promoting blood circulation to regulate menstruation, inducing diuresis to alleviate edema, and clearing away heat and toxic materials; can be used for treating menoxenia, dysmenorrhea, amenorrhea, lochiorrhea, edema, oliguria, pyocutaneous disease, and toxic swelling.

Herba epimedii: epimedium brevicornu Maxim, a dried leaf of Epimedium brevicornum Maxim, a berberidaceae plant, from Jie Congtang pharmaceutical Co., Ltd. The efficacy is as follows: tonify kidney yang, strengthen tendons and bones, dispel wind-damp.

Glossy privet fruit: fructus Ligustri Lucidi, a fruit of Ligustrum lucidum ait of Oleaceae, is from the Bozhou Renyuyi Chinese medicinal materials marketing Co., Ltd. The efficacy is as follows: tonify kidney yin, nourish yin and prolong life.

Turtle shell: trionyx sinensis Wiegmann, from the general Water agency, Hanshou county, Changde, Hunan. The efficacy is as follows: nourish yin and suppress yang, defervesce and remove steaming, soften hardness and dissipate nodulation.

Saffron: crocus sativus l. from bozhou renyi chinese medicinal material sales limited. The efficacy is as follows: promoting blood circulation, removing blood stasis, resolving stagnation, and treating amenorrhea and puerperal abdominal pain due to blood stasis.

Green pepper: zanthoxylum schinifolium sieb. et Zucc, from qiangyang agricultural development ltd, chongqing. The efficacy is as follows: treating spleen and kidney yang deficiency, waist cold and weak feet, teeth and teeth floating, dysmenorrhea, knee pain, chronic nephritis, and edema ascites of nephrotic syndrome.

Lactococcus lactis: lactococcus lactis provided by China general microbiological culture Collection center, CGMCC: 1.15072.

lactobacillus chaff-like bacteria: lactobacillus parafarraginis provided by China general microbiological culture Collection center (CGMCC): 1.15605.

lactococcus fuscata: lactococcus fujiensis provided by China general microbiological culture Collection center, CGMCC: 1.10453.

peptone, CAS: 73049-73-7, available from Onzhiji technologies, Inc.

Beef extract, CAS: 68990-09-0 from Hubei Shinshun Biotechnology, Inc.

Yeast extract is from Shandong Yubao Biotech GmbH.

Chitosan, CAS: 9012-76-4, product number: c299272, purity: not less than 75%, Shanghai Allatin Biotechnology Co., Ltd.

2-octenyl succinic anhydride, CAS: 26680-54-6, product number: s26964, purity: 99%, Shanghai-sourced leaf Biotech, Inc.

Tween 80, purity: 100%, food grade, Guangdong Runhua chemical Co., Ltd.

Example 1

p1 mixing and crushing 3 parts by weight of clam shell, 6 parts by weight of radix achyranthis bidentatae, 2 parts by weight of fructus liquidambaris, 1 part by weight of cortex magnoliae officinalis, 5 parts by weight of cowherb seed, 4 parts by weight of patrinia scabiosaefolia, 3 parts by weight of akebia trifoliate, 8 parts by weight of garden balsam stem, 4 parts by weight of snakegourd fruit, 5 parts by weight of motherwort, 3 parts by weight of epimedium herb, 6 parts by weight of glossy privet fruit, 2 parts by weight of turtle shell and 1 part by weight of saffron crocus, and sieving by a 20-mesh sieve to obtain medicinal powder A;

p2 subjecting the powder A to steam explosion under 5MPa for 150s to obtain powder B;

p3, performing second-order baking treatment on the medicinal powder B to obtain baked medicinal powder; in the second-order baking treatment, the whole process is stirred at the rotating speed of 30rpm in the carbon dioxide protective atmosphere, the temperature is firstly increased to 180 ℃ and is kept for 220s, and then the temperature is increased to 260 ℃ and is kept for 400 s;

p4, mixing the baked powder and the synergist in a mass ratio of 23:1, sending the mixture into an extraction tank, taking water as an extraction solvent, extracting at 175 ℃, under an extraction pressure of 2.2MPa for 30min, wherein the ratio of the baked powder to water bath is 1kg:3L, filtering, and taking supernatant to obtain extract liquor;

p5 ultrafiltering and concentrating the extract to 18% of the original volume by using a polysulfone ultrafiltration membrane to obtain a concentrated solution; the technological parameters of ultrafiltration concentration are that the cross flow speed is 4cm/s, the operation gauge pressure is 0.06MPa, and the temperature is 30 ℃;

p6 spray-drying the concentrated solution with centrifugal spray-dryer to obtain powder, vacuum packaging to obtain Chinese medicinal composition for treating infertility; the technological parameters of the spray powder preparation are that the air inlet pressure is 0.45MPa, the atomization frequency is 60Hz, the interval of firing pins is 6s, the air inlet temperature is 190 ℃, and the feeding speed is 240 mL/h.

The synergist is a mixture of green pricklyash peel and probiotic freeze-dried powder in a mass ratio of 1: 1.

d1 mixing the strains to 10⁴Inoculating the inoculation amount of CFU/mL into a seed culture medium, and culturing at 40 ℃ for 30h to obtain a seed solution;

d2 inoculating the seed liquid into a fermentation tank, fermenting at 40 ℃, and performing electrification treatment and anaerobic fermentation for 150 hours during fermentation to obtain fermentation liquid; the mass ratio of the seed liquid to the nutrient solution in the fermentation tank is 3: 100; in the electrifying treatment, direct current with the current of 3mA is adopted, and the electrification is carried out once every 15s and lasts for 0.3s each time;

d3, centrifuging the fermentation liquor by adopting a tubular centrifuge, and draining supernatant liquor to obtain bacterial sludge; the rotation speed of the centrifugal treatment is 12000rpm, and the centrifugal time is 3 min;

d4, placing the bacterial sludge in a freeze dryer, and performing freeze drying treatment to obtain the probiotic freeze-dried powder; the technological parameters of the freeze drying treatment are that the lowest temperature is-42 ℃, the freezing time is 9h, and the total drying time is 45 h.

The mixed strain is a mixture of lactococcus lactis, lactobacillus chaff-like bacteria and lactobacillus Fuji in a mass ratio of 2:1: 1.

The seed culture medium is composed of the following raw materials in parts by weight: 12 parts by weight of peptone, 9 parts by weight of beef extract, 6 parts by weight of yeast extract, 25 parts by weight of glucose, 3 parts by weight of dipotassium hydrogen phosphate, 3 parts by weight of ammonium citrate, 0.5 part by weight of magnesium acetate and 30 parts by weight of water.

The nutrient solution in the fermentation tank is composed of the following raw materials in parts by weight: 15 parts of peptone, 12 parts of beef extract, 10 parts of yeast extract, 25 parts of glucose, 5 parts of monopotassium phosphate, 5 parts of ammonium citrate, 0.8 part of magnesium acetate, 0.9 part of manganese acetate and 150 parts of water.

Example 2

Essentially the same as example 1, except that: the synergist is green pricklyash peel.

Example 3

Essentially the same as example 1, except that: the synergist is probiotic freeze-dried powder.

Example 4

p4 feeding the baked medicinal powder into an extraction tank, taking water as an extraction solvent, extracting at 175 ℃ under 2.2MPa for 30min, wherein the ratio of the baked medicinal powder to water bath is 1kg:3L, and filtering to obtain an extract;

Example 5

Essentially the same as example 1, except that: p1 contains neither the cowherb nor the Patrinia scabiosaefolia FIq.

Example 6

Essentially the same as example 1, except that: p1 contains herba Patriniae, herba Patriniae and caulis Akebiae.

Example 7

Essentially the same as example 3, except that:

d2 inoculating the seed solution into a fermentation tank, and performing anaerobic fermentation at 40 ℃ for 150h to obtain a fermentation solution; the mass ratio of the seed liquid to the nutrient solution in the fermentation tank is 3: 100;

Example 8

Essentially the same as example 3, except that:

The nutrient solution in the fermentation tank is composed of the following raw materials in parts by weight: 15 parts of peptone, 12 parts of beef extract, 10 parts of yeast extract, 25 parts of glucose, 5 parts of monopotassium phosphate, 5 parts of ammonium citrate, 0.8 part of magnesium acetate and 150 parts of water.

Example 9

Essentially the same as example 1, except that:

in the step P3, performing common baking treatment on the medicinal powder B to obtain baked medicinal powder; in the ordinary baking treatment, the temperature is raised to 260 ℃ and kept for 400s, and the whole process is stirred at the rotating speed of 30rpm under the protective atmosphere of carbon dioxide.

Example 10

p2, performing second-order baking treatment on the medicinal powder A to obtain baked medicinal powder; in the second-order baking treatment, the whole process is stirred at the rotating speed of 30rpm in the carbon dioxide protective atmosphere, the temperature is firstly increased to 180 ℃ and is kept for 220s, and then the temperature is increased to 260 ℃ and is kept for 400 s;

p3, mixing the baked powder and the synergist in a mass ratio of 23:1, sending the mixture into an extraction tank, taking water as an extraction solvent, extracting at 175 ℃, under 2.2MPa for 30min, wherein the ratio of the baked powder to water bath is 1kg:3L, and filtering to obtain an extract;

p4 ultrafiltering and concentrating the extract to 18% of the original volume by using a polysulfone ultrafiltration membrane to obtain a concentrated solution; the technological parameters of ultrafiltration concentration are that the cross flow speed is 4cm/s, the operation gauge pressure is 0.06MPa, and the temperature is 30 ℃;

p5 spray-drying the concentrated solution with centrifugal spray-dryer to obtain powder, vacuum packaging to obtain Chinese medicinal composition for treating infertility; the technological parameters of the spray powder preparation are that the air inlet pressure is 0.45MPa, the atomization frequency is 60Hz, the interval of firing pins is 6s, the air inlet temperature is 190 ℃, and the feeding speed is 240 mL/h.

Test example 1

Testing the pregnancy rate of the hamster: for each test group, 50 male mice and 50 female mice of Zhonghua hamster (five weeks old, provided by the university of Chongqing medical laboratory animal center) were used, and the male mice and the female mice of each test group were fed with the Chinese medicinal composition, respectively, and a control group fed with physiological saline alone was provided. Each group was given 20mg/kg of the composition intraperitoneally once a week for a total of five times. After the injection of cyclophosphamide in the fourth week, the gerbils are grouped and labeled according to the weight of the gerbils, and the dosage is calculated according to the weight before each administration and is administered once a day until the end of the experiment. At 6-8 weeks, the rats of the model and the normal rats were in the same cage, and thereafter only the Chinese medicinal composition was administered, and the physiological changes (mental, activity, secretion, etc.) during estrus of the rats were observed and whether the rats were pregnant or not was observed. At the end of the tenth week, mice were sacrificed by dislocation and pregnant mice were dissected and counted. And (3) pregnancy judgment: the red beaded particles can be seen in the oviduct of the female mouse; newborn mice: it was born ten weeks ago. Detection indexes are as follows: the pregnancy rate (%) of the Chinese hamster is the number of pregnant mice/the number of female mice in the group; compared with the traditional Chinese medicine composition only, the traditional Chinese medicine composition has statistical significance.

The test results are shown in table 1.

TABLE 1 pregnancy rates in female mice

Test example 2

Human clinical test:

200 patients with ovulation disorder infertility caused by polycystic ovarian syndrome are divided into 50 cases of treatment groups (1-3) and control groups by using a random number table method. The age distribution of the treatment group and the control group is 25-40 years, and the average (29.17 +/-6.45) years; the disease courses are respectively 3-10 years, and the average (6.46 +/-2.02) years; the infertility time is 1-5 years respectively, and the average (3.87 +/-1.76) years. The comparison of the general conditions of age, disease course, duration of infertility, etc. of the patients in each group did not show statistical differences (P > 0.05), indicating that the two groups are comparable.

Diagnostic criteria:

(1) western diagnostic criteria: according to the diagnosis standard of polycystic ovarian syndrome established by 2003 world conference of rotterdan: firstly, dilute ovulation or no ovulation; abnormal clinical manifestation and/or biochemical indexes of hyperandrogenism; ③ the ovarian polycystic variant, which accords with at least 2 of the above 3 items and eliminates related diseases such as cushing's syndrome, androgen-secreting tumor, and the like.

(2) The traditional Chinese medicine diagnosis standard is as follows: meets the diagnosis standard of the kidney yin deficiency infertility in the traditional Chinese medicine gynecology: the main symptoms are: prolonged infertility, premature menstruation, scanty menstruation or amenorrhea, and bright red menstruation; or even collapse or leak with prolonged menstruation time. Second symptoms are emaciation, dizziness, tinnitus, soreness and weakness of waist and knees, dysphoria with smothery sensation in chest, insomnia, dreaminess, dim eyesight, palpitation, loss of moisture in skin and dryness in yin. A slightly red and dry tongue with little coating and a thready or thready and rapid pulse. The diagnosis can be made by referring to tongue pulse conditions with the above main symptoms and 1-2 secondary symptoms.

(3) Inclusion criteria were: meeting the Chinese and Western diagnosis standard; ② 20-40 years old; third, voluntarily receive the treatment of the subject and sign the informed consent.

(4) Exclusion criteria: firstly, infertility (such as physiological defect, deformity and genetic factor) is caused by congenital factors; ② infertility caused by male factors; ③ diseases of the gravity, liver, kidney, hematopoietic system, etc. are combined; fourthly, the used medicines have allergic history; use of hormone-like drugs within 3 months.

The treatment method comprises the following steps:

control group: the clomiphene citrate tablet is taken orally at 50mg from 5 days of menstrual cycle or progesterone withdrawal bleeding, 1 time per day for 5 consecutive days, and is increased by 50mg/d per cycle if no ovulation occurs, but the maximum dose is not more than 150mg per day.

Treatment groups: the Chinese medicinal composition obtained in examples 1-3 of the present invention (treatment groups 1-3) was added to the control group, and the patients were asked to make a re-visit once a week to show the specific symptoms and to add or subtract the amount of the medicament. The preparation is decocted with water twice for a total of 400ml, and is taken orally in the morning and evening. Stopping treatment during menstrual period.

The four groups of patients are treated continuously for 3 months, and the curative effect is observed after the period, or until the pregnancy is confirmed, and the follow-up visit is 3 months.

And (3) observation of curative effect:

observation indexes are as follows: the clinical curative effects of two groups of patients are shown; ② two groups of patients serum sex hormone level changes: collecting 5ml of morning fasting blood from patients before treatment and on day 5 of 1 month period, and detecting Luteinizing Hormone (LH), testosterone (T) and estradiol (E2) levels; comparing the thickness of the endometrium of two groups of patients: the two groups adopt B ultrasonic dynamic monitoring on the thickness of the endometrium and the development of the follicles from the 10 th day of the menstrual cycle, and compare the thickness of the endometrium when the maximum diameter of the follicles in the two groups is more than 18 mm; clinical pregnancy rates were compared between the two groups: pregnancy rate (number of pregnancies/number of cases) × 100%; two groups were scored for the occurrence of ovarian hyperstimulation syndrome (OHSS).

Therapeutic efficacy judgment criteria clinical efficacy judgment criteria refer to the previous clinical study and are combined with clinical practice as follows: firstly, curing: the serum sex hormone of the patient is improved to a normal level, the menstruation rule is to be affected, and the pregnancy is to be performed; secondly, effect is displayed: the serum sex hormones and the menstrual cycle are basically restored to normal, and ovulation occurs, but pregnancy does not occur; ③ effective: the menstruation, color and quality of the menstruation are better, but the serum sex hormone is not obviously improved; fourthly, invalidation: neither serum sex hormone levels nor (or) menstruation improved or even aggravated, no ovulation. Effective rate (cure + significant effect + effective)/total number of cases.

The statistical method comprises the following steps: the database was built using SPSS17.0 software and statistical analysis was performed. The comparison between the counting data adopts X2 test, the comparison between the metering data adopts t test, and P is less than 0.05 to indicate that the difference has statistical significance.

And (3) testing results:

the clinical effects of two groups of patients are as follows: 25 cases are cured in a treatment group, 10 cases are obviously effective, 12 cases are effective, and the total effective rate is 94%; the control group heals 5 cases, has obvious effect of 11 cases and 7 cases, and has total effective rate of 46 percent. The total effective rate of the treatment group is higher than that of the control group, and the difference has statistical significance (P is less than 0.05).

② two groups of patients serum sex hormone level changes: the hormone levels before and after two groups of treatment are compared with the hormone levels before two groups of treatment, the hormone levels before two groups of treatment are compared without statistical difference (P is more than 0.05), and the hormone levels before two groups of treatment are comparable. The hormone levels in the two groups after treatment are improved compared with those before treatment (P is less than 0.05); wherein, the improvement range of the treatment group is larger than that of the control group, and the difference has statistical significance (P is less than 0.05), which is shown in table 2.

TABLE 2 serum sex hormone level changes in two groups of patients

Comparing the thickness and ovulation condition of the endometrium of two groups of patients: the ovulation promotion curative effect indexes of the two groups are obviously superior to those of a control group (P is less than 0.05) in comparison of the ovulation promotion curative effect indexes of mature follicles and the ovulation improvement conditions of the endometrium of the treatment group; the clinical pregnancy rate of the treatment group is 36.7 percent, which is higher than 16.7 percent of the control group, and the difference has statistical significance (P is less than 0.05). During the treatment process, no moderate or severe ovarian hyperstimulation syndrome occurs in the two groups. Specifically, the results are shown in Table 3.

TABLE 3 comparison of the two ovulation-promoting efficacy indexes

The Concha Meretricis Seu Cyclinae has effects of nourishing yin, promoting fluid production, softening and resolving hard mass; the achyranthes root has the effects of removing blood stasis, stimulating the menstrual flow, tonifying liver and kidney, strengthening bones and muscles, inducing diuresis, treating stranguria and drawing blood downward; the fructus liquidambaris has the effects of dispelling wind, activating collaterals and inducing diuresis; cortex Magnolia officinalis has effects in eliminating dampness, eliminating phlegm, descending qi, and relieving fullness; the cowherb seed has the effects of promoting blood circulation, stimulating the menstrual flow, promoting lactation, reducing swelling, inducing diuresis and treating stranguria; herba Patriniae has effects of clearing away heat and toxic materials, removing blood stasis, and expelling pus; the akebia trifoliate has the effects of inducing diuresis for treating stranguria, clearing away heart-fire and relieving restlessness; the garden balsam stem has the effects of dispelling wind and eliminating dampness, relaxing muscles and tendons, promoting blood circulation, removing blood stasis, relieving swelling, removing toxic substances and relieving pain; the snakegourd fruit has the effects of clearing heat and removing phlegm, and relieving chest stuffiness and dissipating stagnation; herba Leonuri has effects of promoting blood circulation, regulating menstruation, promoting diuresis, relieving swelling, clearing away heat and toxic materials, and can be used for treating menoxenia, dysmenorrhea, amenorrhea, lochiorrhea, edema, oliguria, and pyocutaneous disease; herba Epimedii has effects of invigorating kidney yang, strengthening tendons and bones, and dispelling pathogenic wind and dampness; fructus Ligustri Lucidi has effects in invigorating kidney yin, nourishing yin, and prolonging life; carapax Trionycis has effects in nourishing yin, suppressing yang hyperactivity, relieving fever, removing steam, softening and resolving hard mass; stigma croci Sativi has effects of promoting blood circulation for removing blood stasis, resolving stagnation and resolving hard mass, and can be used for treating female amenorrhea and puerperal abdominal pain due to blood stasis; the herbs harmonize yin and yang, reinforce each other, strengthen body resistance to eliminate pathogenic factors, and directly reach the focus.

Example 11

A preparation method of a capsule preparation for treating infertility comprises the following steps:

P7, adding the traditional Chinese medicine composition obtained in the step P6 and tween 80 into water, and uniformly mixing to obtain a core material solution, wherein the mass ratio of the traditional Chinese medicine composition to the tween 80 to the water is 1:0.2: 5;

p8, adding chitosan and hyaluronic acid into water, and uniformly stirring to obtain a wall material solution, wherein the mass ratio of the chitosan to the hyaluronic acid to the water is 0.1:0.02: 1;

p9, adding the core material solution into the wall material solution, uniformly mixing, and then homogenizing at high pressure for 40min, wherein the homogenizing pressure is 25MPa, so as to obtain a mixed solution, wherein the mass ratio of the core material solution to the wall material solution is 1: 2; freeze drying the mixed solution to obtain the capsule preparation for treating infertility.

Example 12

Essentially the same as example 11, except that: and replacing chitosan with octenyl succinic anhydride modified chitosan.

(1) adding chitosan into a 30 wt% dilute hydrochloric acid aqueous solution, and stirring at the rotating speed of 500r/min for 4 hours at room temperature, wherein the bath ratio of the chitosan to the dilute hydrochloric acid aqueous solution is 1 g: 15mL, after the reaction is finished, centrifuging to obtain a precipitate, washing the precipitate with water until the precipitate is neutral, and drying the precipitate; obtaining pretreated chitosan;

(2) adding pretreated chitosan into water, and stirring for 4 hours at the temperature of 75 ℃ at the speed of 500r/min to obtain a pretreated chitosan solution, wherein the mass ratio of the pretreated chitosan to the water is 1: 30, of a nitrogen-containing gas; adding 2-octenyl succinic anhydride into a pretreated chitosan solution, uniformly mixing, adjusting the pH to 9 by using a 1.5mol/L NaOH aqueous solution, heating to 65 ℃ for reaction for 7 hours, stirring at the room temperature at the rotating speed of 700r/min for 12 hours after the reaction is finished, adjusting the pH to 7 by using a 1.5mol/L hydrochloric acid aqueous solution, performing alcohol precipitation by using a 60 wt% isopropanol aqueous solution, centrifuging to obtain a precipitate, and thus obtaining a crude product, wherein the mass ratio of the 2-octenyl succinic anhydride to the pretreated chitosan solution is 1: 80; adding the crude product into water, dialyzing for 24h in a dialysis bag with the cut-off molecular weight of 13000, and finally freeze-drying the solution in the dialysis bag to obtain octenyl succinic anhydride modified chitosan; the mass ratio of the crude product to water is 1: 20.

test example 3

The obtained capsule preparations for treating infertility prepared in examples 11 to 12 were subjected to hygroscopicity and solubility tests.

And (3) measuring the hygroscopicity: placing a glass dryer with a supersaturated solution of sodium chloride at the bottom at room temperature for 48 hours to enable the glass dryer to reach equilibrium, wherein the relative humidity in the dryer is 75%; 1g of the capsule preparation for treating infertility, prepared according to examples 11 to 12, was put into the bottom of a weighing bottle having been dried to a constant weight, and after shaking gently to make it uniformly distributed and accurately weighed, the capsule preparation was put into a dryer containing a supersaturated solution of sodium chloride (the weighing bottle cap was opened), and after 48 hours, the capsule preparation was weighed and the moisture absorption percentage was calculated. Percent moisture absorption (% after moisture absorption weight-before moisture absorption weight)/before moisture absorption weight × 100%.

The solubility was measured: 10g of the capsule preparation for treating infertility prepared in examples 11 to 12 was added with 200mL of 80 ℃ hot water, and stirred at 300 rpm for 5 minutes to observe the dissolution.

TABLE 4 results of hygroscopicity and solubility test of capsule preparation for treating infertility

	48h moisture absorption/%)	Solubility in water
			Example 11	5.78	Full dissolution
Example 12	5.24	Full dissolution

Claims

1. A preparation method of a capsule preparation for treating infertility is characterized by comprising the following steps:

p1 mixing Concha Meretricis Seu Cyclinae, radix Achyranthis bidentatae, fructus Liquidambaris, cortex Magnoliae officinalis, semen Vaccariae, herba Patriniae, caulis Akebiae, herba speranskiae tuberculatae, fructus Trichosanthis, herba Leonuri, herba Epimedii, fructus Ligustri Lucidi, carapax Trionycis, and stigma croci, pulverizing, and sieving to obtain powder A;

p2 subjecting the powder A to steam explosion to obtain powder B;

p3 baking the medicinal powder B to obtain baked medicinal powder;

p4, mixing the baked powder and the synergist, feeding the mixture into an extraction tank, taking water as an extraction solvent, and filtering to obtain an extract liquid;

p5 ultrafiltering and concentrating the extract by using a polysulfone ultrafiltration membrane to obtain a concentrated solution;

p6 spray the concentrated solution to get powder, and vacuum packaging to obtain Chinese medicinal composition for treating infertility;

p7 adding the traditional Chinese medicine composition obtained in the step P6 and Tween 80 into water, and uniformly mixing to obtain a core material solution;

p8 adding octenyl succinic anhydride modified chitosan and hyaluronic acid into water, and uniformly stirring to obtain a wall material solution;

p9 adding the core material solution into the wall material solution, mixing uniformly, homogenizing under high pressure to obtain a mixed solution, and freeze-drying the mixed solution to obtain the capsule preparation for treating infertility.

2. The method for preparing the capsule preparation for treating infertility according to claim 1, wherein the method for preparing the octenyl succinic anhydride modified chitosan comprises the steps of: (1) adding chitosan into a 25-30 wt% dilute hydrochloric acid aqueous solution, and stirring at the rotating speed of 300-: (10-20) mL, after the reaction is finished, centrifuging to obtain a precipitate, washing with water until the precipitate is neutral, and drying; obtaining pretreated chitosan;

3. The method for preparing a capsule formulation for the treatment of infertility according to claim 1, wherein the step P1 comprises: mixing and crushing 2-4 parts of clam shell, 5-7 parts of achyranthes root, 1-3 parts of beautiful sweetgum fruit, 1-2 parts of officinal magnolia bark, 4-6 parts of cowherb seed, 3-5 parts of patrinia scabiosaefolia, 2-4 parts of akebia trifoliate, 7-9 parts of garden balsam stem, 3-5 parts of snakegourd fruit, 4-6 parts of motherwort, 2-4 parts of epimedium herb, 5-7 parts of glossy privet fruit, 1-3 parts of turtle shell and 1-3 parts of saffron crocus, and sieving the mixture by a 10-30-mesh sieve to obtain medicinal powder A.

4. The method for preparing the capsule preparation for treating infertility according to claim 1, wherein the synergist is at least one of zanthoxylum schinifolium and probiotic lyophilized powder.

5. The method for preparing the capsule preparation for treating infertility according to claim 4, wherein the method for preparing the probiotic freeze-dried powder comprises the following steps:

d1, inoculating the mixed strains to a seed culture medium, and culturing to obtain a seed solution;

d2 inoculating the seed liquid into a fermentation tank, and performing electrifying treatment and anaerobic fermentation during fermentation to obtain fermentation liquid; in the electrifying treatment, direct current is adopted;

d3, carrying out centrifugal treatment on the fermentation liquor, and draining supernatant liquor to obtain bacterial sludge;

d4, placing the bacterial sludge in a freeze dryer, and performing freeze drying treatment to obtain the probiotic freeze-dried powder.

6. The process for preparing a capsule preparation for the treatment of infertility according to claim 5, wherein: the mixed strain is at least two of lactococcus lactis, Lactobacillus chaff-like bacteria and Lactobacillus Fuji.

7. The process for preparing a capsule preparation for the treatment of infertility according to claim 5, wherein: the seed culture medium consists of the following raw materials: peptone, beef extract, yeast extract, glucose, dipotassium hydrogen phosphate, ammonium citrate, magnesium acetate and water.

8. The process for preparing a capsule preparation for the treatment of infertility according to claim 5, wherein: the nutrient solution in the fermentation tank consists of the following raw materials: peptone, beef extract, yeast extract, glucose, potassium dihydrogen phosphate, ammonium citrate, magnesium acetate, manganese acetate and water.

9. A capsule preparation for treating infertility is characterized in that: the method for preparing the capsule for treating infertility according to any of claims 1 to 8.