CN114748490B - Pharmaceutical composition for treating premature ovarian failure, application and preparation method thereof - Google Patents
Pharmaceutical composition for treating premature ovarian failure, application and preparation method thereof Download PDFInfo
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- CN114748490B CN114748490B CN202210451978.4A CN202210451978A CN114748490B CN 114748490 B CN114748490 B CN 114748490B CN 202210451978 A CN202210451978 A CN 202210451978A CN 114748490 B CN114748490 B CN 114748490B
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- astragalus
- saponin
- pharmaceutical composition
- ovarian failure
- premature ovarian
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/481—Astragalus (milkvetch)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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Abstract
The application provides a pharmaceutical composition for treating premature ovarian failure, application and a preparation method thereof. The pharmaceutical composition for treating premature ovarian failure fully utilizes the effects of invigorating qi, consolidating superficial resistance, expelling toxin, promoting pus discharge, healing sore, promoting tissue regeneration, promoting the production of body fluid, nourishing blood, inducing diuresis, relieving edema and the like of astragalus mongholicus serving as Chinese herbal medicines, and one or more of total saponins extract, astragalus mongholicus saponin I, astragalus mongholicus saponin II, astragalus mongholicus saponin III and astragalus mongholicus saponin IV are used as components of the pharmaceutical composition, so that premature ovarian failure symptoms caused by ovarian injury can be effectively relieved and treated, toxic and side effects, cancerogenic risks and treatment cost in the existing treatment method are avoided, and the treatment cost is greatly reduced.
Description
Technical Field
The application belongs to the technical field of medicines, and particularly relates to a pharmaceutical composition for treating premature ovarian failure, application and a preparation method thereof.
Background
Premature Ovarian Failure (POF) refers to the phenomenon of amenorrhea in women before age 40 due to ovarian failure. It is characterized by elevated blood gonadotrophin levels and reduced estrogen levels associated with primary or secondary amenorrhea, and by a range of low estrogen symptoms of varying degrees such as: hot flushes, hyperhidrosis, facial flushing, low sexual desire, etc.
Premature ovarian failure is a syndrome with multiple etiologies, whose etiology is unknown in most cases. The method mainly comprises the following aspects: genetic factors, immune factors, surgery, chemotherapy, radiation therapy, environmental toxins, enzyme deficiency, psychological factors, idiopathic premature ovarian failure, and the like. The average natural menopausal age of women is 50-52 years old, coulom et al summarizes 1858 cases of natural amenorrhea in women, the incidence of POF is 1% below 40 years old and 1% below 30 years old. 10-28% of POF in primary amenorrhea and 4-18% of POF in secondary amenorrhea. Xu Ling and the like found that the occurrence rate of POF in women in Beijing area was 1.8%. It follows that POF is not uncommon clinically.
The existing clinical methods for treating premature ovarian failure include the following methods: 1. supplementary sex hormone treatment: including estrogens and progestogens. However, the use of such drugs does not solve the fertility problem and may increase the risk of recurrence of hormone-sensitive tumors such as ovarian cancer and breast cancer; 2. use of gonadotrophin releasing hormone analogues: including GnRH agonists or GnRH antagonists, the family of drugs still do not meet the fertility needs of the patient; 3. fertilized egg and embryo cryopreservation techniques: although pregnancy rates reach 18%, they are not suitable for use by non-married women and there is a high likelihood of delay in treatment time; 4. oocyte cryopreservation technology and ovarian cortex cryopreservation and transplantation technology: the pregnancy rate of the operation is lower than 3%, the technology is still immature, and the cost is high.
In a word, the existing treatment method cannot fundamentally treat premature ovarian failure, and a new way is urgently required to be opened up, and the problem of fertility of patients is solved.
Disclosure of Invention
To solve the above problems, the present application provides a pharmaceutical composition for treating premature ovarian failure, comprising: one or more of radix astragali total saponin extract, radix astragali saponin I, radix astragali saponin II, radix astragali saponin III and radix astragali saponin IV.
Preferably, the pharmaceutical composition for treating premature ovarian failure is astragalus saponin III and astragalus saponin IV.
Preferably, in the pharmaceutical composition for treating premature ovarian failure, the mass fraction of the astragaloside III and the astragaloside IV is 1-3;
preferably, the mass fraction ratio of the astragalus saponin III to the astragalus saponin IV is 1:1.
preferably, the pharmaceutical composition for treating premature ovarian failure is formulated as a unit dose.
Preferably, the pharmaceutical composition for treating premature ovarian failure is formulated as an oral preparation or an injection.
Preferably, the pharmaceutical composition for the treatment of premature ovarian failure is administered at a concentration of 10 μg/mL to 200 μg/mL.
Preferably, the pharmaceutical composition for treating premature ovarian failure is formulated for administration at a concentration of 10 μg/mL.
In addition, in order to solve the problems, the application also provides an application of the pharmaceutical composition for treating premature ovarian failure in preparing a medicament for treating ovarian injury caused by a chemotherapeutic medicament.
Preferably, the chemotherapeutic agent is doxorubicin.
In addition, in order to solve the above problems, the present application also provides a method for preparing a pharmaceutical composition for treating premature ovarian failure, comprising:
extracting radix astragali total saponin, radix astragali saponin I, radix astragali saponin II, radix astragali saponin III and radix astragali saponin IV respectively according to natural product extraction method; one or more of the above materials are combined to obtain the pharmaceutical composition for treating premature ovarian failure;
the natural product extraction method is one or more of decoction, reflux extraction, ultrasonic extraction, microwave extraction, enzyme extraction, supercritical extraction, flash extraction, high-speed countercurrent chromatography and high-performance liquid chromatography.
The application provides a pharmaceutical composition for treating premature ovarian failure, application and a preparation method thereof. Wherein, the pharmaceutical composition for treating premature ovarian failure comprises: one or more of radix astragali total saponin extract, radix astragali saponin I, radix astragali saponin II, radix astragali saponin III and radix astragali saponin IV. The pharmaceutical composition provided by the application fully utilizes the effects of invigorating qi, consolidating superficial resistance, expelling pus, healing sore, promoting granulation, promoting the production of body fluid, nourishing blood, inducing diuresis, removing edema and the like of the astragalus, takes one or more of the total saponins extract of astragalus, the saponins I, the saponins II, the saponins III and the saponins IV of astragalus as the components of the pharmaceutical composition, can effectively relieve and treat premature ovarian failure symptoms caused by ovarian injury, avoids toxic and side effects and cancerogenic risks in the existing treatment method, and greatly reduces the treatment cost.
Drawings
FIG. 1 is a mass spectrum test chart of an astragalus total saponin extract according to an embodiment of the present application;
FIG. 2 is a mass spectrum test chart of Astragalus saponin I in the embodiment of the application;
FIG. 3 is a mass spectrum test chart of Astragalus saponin II in the embodiment of the application;
FIG. 4 is a mass spectrum test chart of Astragalus saponin III in the embodiment of the application;
FIG. 5 is a mass spectrum test chart of Astragalus saponin IV in the embodiment of the application;
FIG. 6 shows the proliferation effect of different groups of the present application on mouse ovarian granulosa cells isolated and cultured in vitro at a concentration of 10. Mu.g/mL (compared to the normal group: P <0.05 mean.+ -. S.E.M, n=6);
FIG. 7 shows the proliferation effect of different proportions of III+IV according to the application on mouse ovarian granulosa cells isolated and cultured in vitro at a concentration of 10. Mu.g/mL (compared to the normal group: P <0.05 mean.+ -. S.E.M, n=6);
fig. 8 shows the effect of total saponins of astragalus on ovariectomy granulosa cells after ovariectomy injury (P <0.05, P <0.01, P <0.001 compared to the normal group; # P <0.01, # P <0.001mean ± s.e.m, n=6 compared to the doxorubicin model group);
FIG. 9 shows the effect of total saponins of Astragalus membranaceus extract on Ovarian size and wet weight of ovaries after doxorubicin-induced Ovarian injury (Ovarian weight/g; compared with normal group: P <0.05, mean.+ -. S.E.M, n=6);
FIG. 10 shows the effect of total saponins of Astragalus on ovaries size and wet weight after ovaries injury with doxorubicin in accordance with the present application (Ovarian weight/body weight; compared to normal group: P <0.05, mean.+ -. S.E.M, n=6);
FIG. 11 shows the effect of Astragalus total saponins extract on follicular release after ovarian injury with doxorubicin according to the present application 1 (Number of primordial primary follicles; FIGS. 10-13: P <0.05, <0.01; compared to the normal group; compared to the doxorubicin model group: # P <0.01 mean.+ -. S.E.M, n=6);
FIG. 12 shows the effect of Astragalus total saponins extract on follicular release after ovarian injury with doxorubicin in accordance with the present application 2 (Number of secondary follicles; FIGS. 10-13: P <0.05, <0.01; compared to the doxorubicin model group: # P <0.01 mean.+ -. S.E.M, n=6);
FIG. 13 shows the effect of Astragalus total saponins extract on follicular release after ovarian injury with doxorubicin according to the present application 3 (Number of mature follicles; FIGS. 10-13: P <0.05, <0.01; compared to the normal group; compared to the doxorubicin model group: # P <0.01 mean.+ -. S.E.M, n=6);
fig. 14 shows the effect of total saponins of astragalus extract on follicular release after ovarian injury with doxorubicin according to the present application 4 (Number of total follicles; fig. 10-13: P <0.05, < P <0.01 compared to the normal group; # P <0.01mean ± s.e.m, n=6 compared to the doxorubicin model group);
FIG. 15 shows the results of reducing FSH hormone release and promoting E2 hormone synthesis of Astragalus total saponins extract according to the present application FIG. 1 (FSH IU/L; compared to normal group: P <0.05, <0.01; compared to doxorubicin model group: P <0.05 mean.+ -. S.E.M, n=6);
fig. 16 shows the results of reducing FSH hormone release and promoting E2 hormone synthesis of astragalus total saponin extract according to the present application fig. 2 (E2 ng/mL; P <0.05, P <0.01 compared to the normal group; P <0.05mean±s.e.m, n=6 compared to the doxorubicin model group).
The achievement of the objects, functional features and advantages of the present application will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
The technical solutions of the present application will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The technical solution of the present application is further described in detail below with reference to specific embodiments, but the present application is not limited thereto, and any modifications made by anyone within the scope of the claims of the present application are still within the scope of the claims of the present application.
The application aims to solve the technical problems of avoiding toxic and side effects and cancerogenic risks existing in the existing treatment method for premature ovarian failure, reducing the treatment cost and further providing a pharmaceutical composition for treating premature ovarian failure, which comprises the following components: one or more of radix astragali total saponin extract, radix astragali saponin I, radix astragali saponin II, radix astragali saponin III and radix astragali saponin IV.
The four compounds of the astragalus saponin I, the astragalus saponin II, the astragalus saponin III and the astragalus saponin IV all have the following mother nucleus structural formulas:
wherein R in astragaloside I 1 ＝Glc、R 2 ＝H、R 3 ＝Ac、R 4 =h and R 5 ＝Ac；
R in astragalus saponin II 1 ＝Glc、R 2 ＝H、R 3 ＝H、R 4 =h and R 5 ＝Ac；
R in astragalus saponin III 1 ＝H、R 2 ＝H、R 3 ＝H、R 4 =h and R 5 ＝Glc；
R in astragaloside IV 1 ＝Glc、R 2 ＝H、R 3 ＝H、R 4 =h and R 5 ＝Glc；
Wherein Xyl is xylose and Glc is glucosyl.
The astragalus total saponin extract, namely astragalus total saponin, is also astragalus total saponin, is obtained by extracting roots of astragalus mongholicus of leguminous plants, and is a mixture of multiple saponin compounds and other compounds in the astragalus mongholicus. The astragalus total saponins are a mixture of multiple components with saponin components, and the components can include: contains flavonoid component calycosin, 3-hydroxy-9, 10-dimethoxy pterocarpan, and Astragaloside I, V, III, and effective components detected by high resolution mass spectrum mainly comprise Astragaloside III and Astragaloside IV. At present, the existing reports mainly aim at the astragalus total saponins for regulating blood sugar in vivo, enhancing the immunity of organisms, promoting growth, improving the oxidation resistance of organisms, and in addition, the astragalus total saponins are beneficial to inducing diuresis to alleviate edema, supporting sore and promoting granulation, and are used for chronic nephritis proteinuria, diabetes and the like.
The astragalus saponin I, the astragalus saponin II, the astragalus saponin III and the astragalus saponin IV are all saponin compounds.
The above-mentioned pharmaceutical composition for treating premature ovarian failure may be one of the monomeric compounds, or an extract of total saponins of astragalus, or a combination of a plurality of monomeric compounds, or even a combination of total saponins of astragalus and one or more monomeric compounds.
The astragalus root and the Chinese medicinal material are named. The product is root of Astragalus mongholicus bge of Leguminosae. Collected in spring and autumn, removed with soil, fibrous root and root head, sun dried to six seven times, bundled and dried. The main functions are as follows: tonify qi, strengthen superficies, expel pus, promote urination and promote tissue regeneration. Can be used for treating qi deficiency, debilitation, chronic diarrhea, rectocele, spontaneous perspiration, edema, uterine prolapse, chronic nephritis, albuminuria, diabetes, and chronic open sore. The main active ingredient is astragalus saponin compounds.
At present, most of products containing astragalus are traditional Chinese medicine compounds, and although clinical verification shows that the products have a certain treatment effect, effective treatment components are not clear due to complex components. Therefore, the astragalus root needs to be deeply researched to develop a modern Chinese medicinal preparation with clear effective components, clear action mechanism and easily controlled quality. In recent years, astragalus saponins have attracted attention due to their good natural biological activity. The total saponins of Astragalus have high antioxidant potential, such as the extract can reduce active oxygen release and free fatty acid injury. Although the literature reports that the astragalus total saponins have various pharmacological activities, how to prevent and treat premature ovarian failure is not reported in China.
The pharmacological activities of astragalus mongholicus currently known mainly include effects on renal function, mainly in three aspects: the first aspect is to improve kidney function, and the effect of reducing blood creatinine is obvious (P is less than 0.01) compared with the effect of single medicament, and the medicament has obvious improvement effect on renal parenchymal cell metabolism. In the second aspect, the regulation of the immune function of the organism is proved by the prior literature report, and the astragalus can regulate the content of plasma cyclic nucleotide and correct the unbalance of the cyclic nucleotide system, thereby prompting the astragalus to have the anti-aging effect. The astragalus has high selenium content, can participate in the synthesis and activation of various enzymes, and protects cells from damage in the biological oxidation process. The third aspect is the effect on hematopoietic system, mainly astragalus root has the functions of promoting hematopoietic function of bone marrow and promoting bone marrow growth, and also has the functions of heart strengthening, blood pressure reducing, blood sugar reducing, diuresis, anti-aging, anti-fatigue and the like. However, at present, pharmacological activity of astragalus and main components thereof for treating or preventing premature ovarian failure is not reported.
The medicine composition for treating premature ovarian failure provided in the embodiment fully utilizes the effects of invigorating qi, strengthening exterior, expelling toxin, expelling pus, promoting wound healing, promoting granulation, promoting the production of body fluid, nourishing blood, inducing diuresis, relieving swelling and the like of astragalus as Chinese herbal medicines, and takes the most main active ingredients in the astragalus as follows: the composition of one or more of astragalus total saponin extract, astragalus saponin I, astragalus saponin II, astragalus saponin III and astragalus saponin IV as components of the pharmaceutical composition can effectively relieve and treat premature ovarian failure symptoms caused by ovarian injury, avoids toxic and side effects and cancerogenic risks in the existing treatment method, and greatly reduces the treatment cost.
Further, the pharmaceutical composition for treating premature ovarian failure is astragalus saponin III and astragalus saponin IV.
The medicine composition for treating premature ovarian failure is a mixture of monomer astragaloside III and monomer astragaloside IV.
The above-mentioned astragaloside III is the astragaloside III in the composition, its molecular formula is C 41 H 68 O 14 The molecular weight is 784.97, and the pure product is colorless needle crystal.
The astragaloside IV isThe astragaloside IV, also called astragaloside IV, is a lanolate alcohol-shaped tetracyclic triterpene saponin with molecular formula of C 41 H 68 O 14 The molecular weight is 784.97.
The pharmacological actions reported above for astragaloside IV mainly include the following:
1. enhancing immunity and disease resistance; 2. antiviral activity; 3. an anti-stress effect; 4. a growth promoter; 5. improving heart and lung function, enhancing heart contractility, protecting cardiac muscle, and resisting heart failure. However, there is no report on the pharmacological activity of astragaloside IV in the treatment or prevention of premature ovarian failure.
Furthermore, in the pharmaceutical composition for treating premature ovarian failure, the mass fraction of the astragaloside III and the astragaloside IV is 1-3.
In the above-mentioned pharmaceutical composition for treating premature ovarian failure, the mass ratio of the two components of the astragalus saponin III and the astragalus saponin IV may be different combinations, for example, the two compounds may be arbitrarily combined according to the following mass fractions on the premise that the mass fractions are 1-3, so as to form 5 pharmaceutical compositions with different proportions:
table 1, examples of different proportions of Astragaloside III and IV in the compositions
The mass ratio aims at the pure product ratio between two monomer compounds and does not comprise auxiliary materials and additives.
Further, the mass fraction ratio of the astragalus saponin III to the astragalus saponin IV is 1:1. experiments are carried out by using a plurality of groups of pharmaceutical compositions for treating premature ovarian failure, which are composed of astragalus saponin III and astragalus saponin IV with different mass fractions, wherein the two compositions 1:1 in vitro experiments can give better therapeutic effect against premature ovarian failure than the other ratios in table 1.
Further, the pharmaceutical composition for treating premature ovarian failure is configured as a unit dose.
Further, the pharmaceutical composition for treating premature ovarian failure is configured to be an oral preparation or an injection.
The above-mentioned injection prepared from the composition of the present application includes, but is not limited to, liquid injection and powder injection.
The above oral formulations include, but are not limited to, solid formulations, liquid formulations.
The pharmaceutical composition for treating premature ovarian failure can also be configured into other dosage forms capable of achieving the effect and purpose of treating premature ovarian failure, and for example, can include but is not limited to: inhalation aerosols, skin-administrable external preparations, mucous membrane-administrable preparations, and the like, are not limited herein.
Further, the application concentration of the drug composition for treating premature ovarian failure is 10-200 mug/mL.
Further, the pharmaceutical composition for treating premature ovarian failure is formulated and applied at a concentration of 10 μg/mL.
In addition, the application also provides application of the pharmaceutical composition for treating premature ovarian failure in preparing medicines for treating ovarian injury caused by chemotherapeutic medicines.
Further, the chemotherapeutic drug is doxorubicin.
With the increase of the younger patients with tumor and the postoperative survival rate, the iatrogenic damage caused by the chemotherapeutics is increasingly emphasized, and especially the younger patients with breast cancer and lymphoma no longer consider survival as the only index of tumor treatment. The quality of life and fertility requirements of post-operative patients are also a concern for their needs, and the status of ovarian function of post-chemotherapy patients is a key indicator for evaluating the quality of life of young female tumor patients. At present, according to literature reports, chemotherapeutic drugs are one of causes of premature ovarian failure.
Currently, it is known from clinical treatment case statistics and literature reports that the functions caused by conventional premature ovarian failure (such as premature ovarian failure symptoms caused by genetic factors, environmental toxins such as tobacco, enzyme deficiency and the like) are damaged, are generally irreversible, and are accompanied by other symptoms, which are caused by long-term life behaviors and genetic causes aiming at ovarian damage caused by low-level hormone environments, such as long-term low-level estrogen, resulting in uterine atrophy. Compared with other types of premature ovarian failure, the premature ovarian failure caused by the chemotherapeutic medicine is characterized in that acute estrogen is reduced and ovarian injury is caused by short-term administration of the chemotherapeutic medicine, so that premature ovarian failure even progresses to ovarian cancer, the premature ovarian failure caused by the reason has light symptoms, the symptoms of patients can be timely relieved under the timely treatment condition, the cure probability is high, and even the fertility problem of the patients can be reserved and solved, so that premature ovarian failure caused by the chemotherapeutic medicine is an important subject in the current premature ovarian failure treatment.
In order to prevent or alleviate ovarian damage caused by chemotherapy, there are several main methods in clinic: 1. supplementary sex hormone treatment: including estrogens and progestogens. However, the use of such drugs does not solve the fertility problem and may increase the risk of recurrence of hormone sensitive tumors such as ovarian cancer and breast cancer. 2. Use of gonadotrophin releasing hormone analogues: including GnRH agonists, gnRH antagonists. The family of drugs still cannot meet the fertility needs of patients. 3. Fertilized egg and embryo cryopreservation techniques: although pregnancy rates reach 18%, they are not suitable for use in non-married women and there is a high likelihood of delay in treatment time. 4. Oocyte cryopreservation technology and ovarian cortex cryopreservation and transplantation technology: the pregnancy rate of the operation is lower than 3 percent, and the technology is still immature. Therefore, the existing treatment method cannot fundamentally relieve the ovarian injury caused by the chemotherapeutic drugs, and a new way is urgently required to be opened up, and the problem of fertility of patients is solved.
The prevention and treatment effects of total saponins of astragalus and related saponins of astragalus on ovarian injury caused by chemotherapeutic drugs are not reported in China.
Experiments prove that the astragalus total saponins and related saponins can reduce the release of FSH hormone and promote the synthesis of E2 hormone, and can protect the release of various levels of follicles and granulosa cells of an Doxorubicin (DOXO) -induced premature ovarian failure mouse model.
In addition, the application also provides a preparation method of the pharmaceutical composition for treating premature ovarian failure, which comprises the following steps:
extracting radix astragali total saponins, radix astragali saponins I, radix astragali saponins II, radix astragali saponins III and radix astragali saponins IV respectively from the pharmaceutical composition for treating premature ovarian failure according to any one of the above methods; one or more of the above materials are combined to obtain the pharmaceutical composition for treating premature ovarian failure;
the natural product extraction method can be one or more of decoction, reflux extraction, ultrasonic extraction, microwave extraction, enzyme extraction, supercritical extraction, flash extraction, high-speed countercurrent chromatography and high-performance liquid chromatography.
The radix astragali can be root of Astragalus mongholicus bge of Leguminosae.
The above natural product extraction method includes various methods including, but not limited to, decoction, reflux extraction, ultrasonic extraction, microwave extraction, enzyme extraction, supercritical extraction, flash extraction, high-speed countercurrent chromatography, and high-performance liquid chromatography.
The decoction method can be that water 16 times is used to decoct radix astragali for 60min for 4 times, and extract with content of 16.88mg/g can be obtained. Wherein the extract is radix astragali total saponin extract.
The reflux extraction method is a method of extracting raw material components by using volatile solvents such as ethanol, water or a mixed solvent thereof, heating and distilling the leaching solution, cooling the solvent after distilling off, and repeatedly flowing back to a leaching container to extract the raw material, so that the process is repeated until the reflux extraction of the effective components is complete. In the application, water for astragalus total saponins at normal pressure can be used as a solvent for reflux extraction, because the water solubility of the astragalus total saponins is higher than the ethanol solubility.
The ultrasonic extraction method adopts ultrasonic auxiliary solvent for extraction, and the sound wave generates high-speed and strong cavitation effect and stirring effect to destroy cells of the plant medicinal materials, so that the solvent permeates into the medicinal material cells, the extraction time is shortened, and the extraction rate is improved. In the application, the extract of the total saponins of astragalus can be obtained by carrying out ultrasonic extraction for 40 minutes by taking 15 times of 70% ethanol as a solvent.
The above-mentioned microwave extraction method, i.e., microwave-assisted extraction method, is a newly developed technology for extracting substances by using microwave energy, and is a technology and a method for extracting various chemical components from natural plants, minerals or animal tissues in a microwave reactor by using a suitable solvent. The radix astragali can be extracted by microwave-assisted extraction method.
The enzyme extraction method has certain catalysis effect on improving the extraction rate of the total saponins of the astragalus, the catalysis condition of the extraction can be that the pH=4.38, the enzymolysis time is 132 minutes, the temperature is 51 ℃, the amount of the cellulase is 8.8mg, and the radix scutellariae is extracted under the condition.
As the supercritical extraction method, supercritical CO can be used 2 The method for extracting the total saponins of astragalus is used for experiments. Aiming at the crushed coarse particles of the traditional Chinese medicine astragalus with the granularity of 60 meshes, the pressure is 35MPa at the temperature of 45 ℃, the ethanol solution with the content of 95 percent is used as entrainer, the extraction time is controlled to be 2.5 hours, and the CO is controlled to be the same as the ethanol solution 2 The flow rate was 3.7L/h.
The flash extraction method is also called a tissue disruption extraction method, and can break materials into tiny particles in a few seconds by means of high-speed mechanical shearing force, and meanwhile, chemical components in plant tissues can reach internal and external balance rapidly by high-speed stirring and vibration, so that the extraction efficiency of effective components is improved.
The high-speed countercurrent chromatography, HSCCC for short, is a liquid-liquid chromatographic separation technology, and has the advantages of no loss of samples, no pollution, high efficiency, high speed, large preparation amount separation and the like, and the stationary phase and the mobile phase of the high-speed countercurrent chromatography are both liquids without irreversible adsorption. HSCCC separation and purification can be carried out by adopting a two-phase solvent system of n-hexane-ethyl acetate-methanol-water (1:10:2:10), ethyl acetate-ethanol-n-butanol-water (30:10:6:50), wherein the lower phase is a mobile phase, the flow rate is 2.5m L/min,2.0mL/min and the rotating speed is 900r/min, and the detection wavelength is 254nm.
The high performance liquid chromatography, abbreviated as HPLC, uses liquid as mobile phase, adopts a high pressure transfusion system to pump single solvent with different polarities or mixed solvent with different proportions, buffer solution and other mobile phases into chromatographic column filled with stationary phase, after each component in the column is separated, the column enters into detector for detection, thus realizing analysis of sample. In the separation of total saponins and saponins of astragalus, the separation and purification are mainly carried out by adopting a mode of combining a preparation type high-efficiency liquid phase and an analytical high-efficiency liquid phase, for example, a forward column can be used for coarse separation, and a reverse column can be used for further subdivision.
For example, the extraction method of the main active ingredients in astragalus may be as follows:
step 1, crushing astragalus mongholicus by using a crusher, dissolving the crushed astragalus mongholicus in 5-15 times of ethanol water solution which can be 50% -100% (pure ethanol), carrying out heating reflux extraction, and carrying out reduced pressure drying and concentration to obtain crude extract;
step 2, dissolving the crude extract in 10 times of water to obtain a water-soluble crude extract;
step 3, extracting the water-soluble crude extract in petroleum ether, dichloromethane, ethyl acetate and n-butanol in sequence to obtain a water layer, a petroleum ether layer, a dichloromethane layer, an ethyl acetate layer and an n-butanol layer;
step 4, taking an n-butanol layer, utilizing macroporous adsorption resin (D101 can be adopted), then performing gradient elution by using methylene dichloride-methanol solution (the gradient elution solution can be ethanol water solution, for example, 50% ethanol can be used for eluting to 100% ethanol), obtaining more than 50% of the n-butanol layer, tracking by TLC to obtain a target mixture, and separating and further purifying the target mixture by sephadex LH-20; separating and purifying with normal phase silica gel, and tracking by TLC to obtain radix astragali total saponin extract;
and 5, separating and purifying by preparing a liquid phase (reverse silica gel column), and tracking by TLC to obtain the astragaloside I, the astragaloside II, the astragaloside III and the astragaloside IV respectively.
The application is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the application in any way.
Example 1: component confirmation of monomer and extract
The components are confirmed after separation and purification of the total saponins of astragalus, the saponins of astragalus I, the saponins of astragalus II, the saponins of astragalus III and the saponins of astragalus IV.
The specific method and the result are as follows:
in this embodiment, the total saponins of astragalus extract, as well as the saponins of astragalus I, the saponins of astragalus II, the saponins of astragalus III and the saponins of astragalus IV, are tested by a high resolution liquid chromatography-mass spectrometer, so as to confirm the components thereof.
Test conditions:
astragalus saponins I, II, III, IV and Astragalus total saponins are respectively dissolved in 50% acetonitrile, and the final concentration is 5ppm. A UPLC BEH C18 chromatographic column (100 mm×2.1mm,1.7 μm) was used, with 0.1% aqueous formic acid and acetonitrile as mobile phase A and mobile phase B, respectively, and the elution was carried out in a gradient (0-2.5 min,95% A.fwdarw.70% A;2.5-4.5min,70% A.fwdarw.65% A;4.5-6min,65% A;6-7min,65% A.fwdarw.60% A;7-9min,60% A.fwdarw.40% A;9-14min,40% A.fwdarw.3% A;14-15min,3% A.fwdarw.95% A) at a flow rate of 350. Mu.L/min, a column temperature of 35℃and a sample injection amount of 5. Mu.L. The mass spectrum detection adopts ESI source and selects positive ion mode to carry out selective reaction monitoring Scanning (SRM). Spray Voltage (Spray Voltage) 3600V, sheath Gas flow rate (Sheath Gas) 40arb, auxiliary Gas flow rate (auxgas) 10arb, atomizer temperature (atomizer Temp) 325 ℃, ion transport tube temperature (ion transfer tube Temp) 350 ℃.
Test results:
1. total saponins of astragalus: referring to FIG. 1, it can be seen that the molecular weights of the main components of Astragaloside III and Astragaloside IV contained in the components have been detected, and that the mixture is confirmed to be Astragalus total saponin extract;
2. (1) Astragalus saponin I is shown in figure 2, and the detection results are shown in the following table;
(2) Astragalus saponin II is shown in figure 3, and the detection results are shown in the following table;
(3) Astragalus saponin III is shown in figure 4, and the detection results are shown in the following table;
(4) Astragaloside IV is shown in figure 5, and the detection results are shown in the following table.
Summarizing:
through the test of a high resolution liquid chromatography-mass spectrometry instrument, the components of astragalus total saponins, astragalus saponins I, astragalus saponins II, astragalus saponins III and astragalus saponins IV are confirmed.
Table 2 Mass Spectrometry parameters for Astragalus saponins I, II, III and IV
| Name of the name | Relative molecular mass | Ionization mode | Parent ion m/z | Ion m/z | Collision energy eV |
| Astragalus saponin I | 869.04 | ESI + | 869.76 | 437.32 | 18 |
| Astragalus saponin II | 827.01 | ESI + | 827.72 | 437.37 | 18 |
| Astragalus saponin III | 784.97 | ESI + | 785.56 | 437.32 | 20 |
| Astragalus saponin IV | 784.97 | ESI + | 785.56 | 143.15 | 20 |
Pharmacological Activity assays in vivo and in vitro (examples 2-4)
Experimental materials:
1. sample to be measured: total saponins of Astragalus membranaceus, astragalus saponins I, astragalus saponins II, astragalus saponins III and Astragalus saponins IV.
2. Reagents and samples: pregnant mare serum gonadotrophin PMSG, prospechot-272; DMEM-F12 phenol red free medium, thermo; fetal bovine serum, gibco; accumax TM solution, sigma; hanks buffer, thermo; cell titer-Glo luminous cell viability assay, promega; doxorubicin, sigma; mouse Follicle Stimulating Hormone (FSH) ELISA kit, bio-ramp; mouse estradiol (E2) ELISA kit, bio-wash; 4% paraformaldehyde tissue fixative, bio-wash; hematoxylin-eosin dye solution, baso;1% hydrochloric acid alcohol, baso.
3. Instrument apparatus: a cell incubator, thermo; inverted microscope, ZEISS; inverted dissecting microscope, ZEISS; a small-sized low-temperature centrifuge, eppendorf; a cytometer, countess II; multifunctional microplate detector, bioTek.
Example 2: proliferation effect of sample to be tested on mouse ovary granular cells isolated and cultured in vitro
1. The experimental method comprises the following steps:
(1) Taking SPF female mice of 5-6 weeks old, and injecting pregnant horse serum gonadotropin PMSG (8-10U) and Human Chorionic Gonadotrophin (HCG) into the abdominal cavity; after 48 hours, the mice are sacrificed by cervical dislocation, the ovaries of the mice are separated, PBS is used for cleaning, and the surrounding fat and the envelope are removed under a stereoscopic microscope;
(2) Puncturing follicle with 25-gauge needle in mixed basal medium to release granulosa cells and oocytes, adding enzyme for digestion, filtering with 40 mesh screen, centrifuging filtrate, and discarding supernatant; the mixed complete culture medium is used for resuspension of cell sediment and inoculated in a culture dish, and CO 2 Culturing in incubator for 48 hr, changing liquid, culturing in incubator for 5-7 days, changing culture medium, and adding medicine 24 hr after synchronization.
Experimental grouping:
different concentration groups of samples to be tested: astragalus total saponins, astragalus saponins I, astragalus saponins II, astragalus saponins III and Astragalus saponins IV (10 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL);
composition group in the first step experiment: concentration: 10 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL; fixed ratio: 1:1, a step of; combination of compounds: I+II, I+III, I+IV, II+III, II+IV, III+IV;
composition group in the second step experiment: fixed concentration: 10 μg/mL; the component is III+IV, wherein III: the ratio of IV is: 1:1, 1:2, 1:3, 2:1, 2:3, 3:1, and 3:2;
the granulosa cells are grown on the wall after 5 to 7 days of separated granulosa cells, the culture medium is replaced, and the new culture medium is replaced 24 hours after the synchronous drug addition treatment;
(3) Cell viability was measured using CellTiter-Glo luminescent cell viability assay system (Promega).
Specifically, the method comprises two steps of experiments:
(1) Firstly, carrying out experiments aiming at different concentration groups of the sample to be tested and composition groups in the first step of experiments to obtain results respectively.
(2) And screening out the preferred concentration according to the experimental result, and carrying out experiments on the preferred composition combination of the sample to be tested, so as to find out the combination of the specific compounds or extracts in the specific proportion under the specific concentration.
The results are as follows.
2. Experimental results:
TABLE 3 proliferation effect of different groupings on mouse ovarian granulosa cells isolated cultured in vitro at a concentration of 10-200. Mu.g/mL
TABLE 4 proliferation effect of different proportions of III+IV on mouse ovarian granulosa cells isolated in vitro at a concentration of 10. Mu.g/mL
As can be seen from the results of Table 3, in the first step of experiment, by setting 4 concentration administration doses (10. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL), the ovarian granulosa cell activity after 24 hours of the action had different degrees of effect of promoting proliferation of granulosa cells in the low concentration administration groups (10. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL), as compared with the control group; and, wherein, at the concentration of 10 mug/mL, the astragalus total saponins, the astragalus saponins I, the astragalus saponins II, the astragalus saponins III and the astragalus saponins IV can obviously promote the proliferation of granulosa cells, have obvious differences, have statistical significance (P < 0.05), can intuitively draw the conclusion by referring to the figure 6, and the astragalus total saponins which are tested at the same time in comparison in the figure have obvious differences compared with a blank group at the concentration of mug/mL, namely have the effect of obviously promoting the proliferation of granulosa cells.
Furthermore, in the composition group in the first step experiment, at 1:1, and the combination effect of the III+IV is obvious, wherein the I+II, the I+III, the I+IV and the III+IV can promote the proliferation effect of granulosa cells at the concentration of 10 mug/mL.
In the second step of the experiment, further experiments were performed on the drug combinations of III+IV. Specifically, under the concentration of 10 mug/mL screened in the first step of experiment, aiming at III+IV combination, detection of different proportions of components is carried out, and the detection is respectively as follows: 1:1, 1:2, 1:3, 2:1, 2:3, 3:1 and 3:2, it can be seen from experimental results that the combination of the drugs III+IV can obviously promote proliferation of granulosa cells in the two groups of ratios of 1:1 and 1:2, and 1:1 the effect of promoting granulosa cells was most pronounced, with a significant difference compared to the blank.
The first step and the second step of experiments in the embodiment can prove that the combination of III+IV has better proliferation effect on the mouse ovary granular cells isolated and cultured in vitro, and the total saponins extract of astragalus can detect the main component of III+IV by a liquid chromatography-mass spectrometry, so that the total saponins of astragalus can be further identified to have certain proliferation effect on the mouse ovary granular cells isolated and cultured in vitro based on the main saponins of astragalus. In conclusion, the following experiments on total saponins of astragalus were further carried out according to the above experimental results.
Example 3: influence of Astragalus total saponins on granulosa cells after doxorubicin-induced ovarian injury
1. The experimental method comprises the following steps:
(1) After 5-7 days of isolated granulosa cells were grown on the wall, the medium was changed, the total saponins of Astragalus membranaceus extract was added simultaneously, the new medium was changed 24 hours after treatment, and 1. Mu. Mol of doxorubicin solution was added for 12 hours of incubation.
(2) Cell viability was measured using CellTiter-Glo luminescent cell viability assay system (Promega).
2. Experimental results:
as can be seen from fig. 8, in the ovarian granulosa cells under the action of doxorubicin, the cell viability of the astragalus total saponins with the effective administration concentrations (50 μg/mL and 100 μg/mL) is obviously higher than that of the same group of blank control, wherein AST-100 is most obvious and relatively stable, and the astragalus total saponins extract has a protective effect on the ovarian granulosa cells after the ovarian injury caused by doxorubicin.
Example 4: application of astragalus total saponins in doxorubicin-induced premature ovarian failure mouse model
1. The experimental method comprises the following steps:
(1) SPF female mice of 5-6 weeks of age were randomly assigned to 4 groups of 6 animals, wherein:
control mice (n=6) were perfused with physiological saline (20 ml/Kg) daily for 4 weeks.
The doxorubicin model group (n=6) was intragastrically (20 ml/Kg) daily with normal saline for 4 weeks, and received one intraperitoneal injection of doxorubicin (8 mg/Kg) in the fifth week.
Mice of the astragalus total saponin group (n=6) were intragastrically irrigated daily with astragalus total saponin extract (10 mg/kg) for 4 weeks.
Mice of the group of total saponins of astragalus + doxorubicin (n=6) were intragastrically given daily total saponins of astragalus extract (10 mg/kg) and received one intraperitoneal injection of doxorubicin (8 mg/kg) four weeks later.
(2) The changes in body weight, hair, behaviours, etc. of each group of mice were recorded continuously during the experiment. Mice were sacrificed 7 days after doxorubicin injection, serum was collected and serum hormone FSH, E2 levels were determined; the ovaries of the mice were harvested, weighed wet, paraffin sections were prepared for conventional H & E staining, and the morphological observation of the ovaries tissue and follicle count were observed.
2. Experimental results:
the results are shown in Table 5 and FIGS. 9 and 10. In the doxorubicin molding model mice, the ovary size and the ovary wet weight were significantly reduced; the astragalus total saponin extract of the application can obviously resist the above pathological changes of model mice.
From the results of fig. 11-14, it was shown that the number of primordial follicles, primary follicles, and mature follicles was significantly reduced in the model mice relative to the blank group, and that the number of follicles at each stage was significantly increased in the mice of the astragalus total saponin group, with the number of primordial follicles being most prominent. The use of astragalus total saponins can obviously restore the pathological changes of reduced number of original follicles and mature follicles in the model group.
The ELISA kit for FSH and E2 is used for detecting the serum of each group of mice, and the result is shown in figures 15-16, the FSH of the serum of the mice of the doxorubicin-treated model group is obviously increased, and the E2 is obviously reduced, so that the serum meets the biochemical index of the mice model of premature ovarian failure. The use of total saponins of Astragalus membranaceus can reduce FSH hormone release and promote E2 hormone synthesis.
TABLE 5 influence of test samples on ovarian size and wet weight after doxorubicin-induced ovarian injury
In the above table, AST is radix astragali total saponin extract; DOXO is doxorubicin.
While the preferred embodiments and examples of the present application have been described, it should be noted that those skilled in the art may make various modifications and improvements without departing from the inventive concept, including but not limited to, adjustments of proportions, procedures, and amounts, which fall within the scope of the present application. While the preferred embodiments and examples of the present application have been described, it should be noted that those skilled in the art may make various modifications and improvements without departing from the inventive concept, including but not limited to, adjustments of proportions, procedures, and amounts, which fall within the scope of the present application.
Claims (3)
1. The application of the pharmaceutical composition in preparing medicines for treating ovarian injury caused by doxorubicin is characterized in that the pharmaceutical composition is astragalus saponin III and astragalus saponin IV with the mass ratio of 1:1; the pharmaceutical composition is administered at a concentration of 10 μg/mL.
2. The use of claim 1, wherein the pharmaceutical composition is configured as a unit dose.
3. The use of claim 1, wherein the pharmaceutical composition is formulated as an oral formulation or injection.
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| 黄芪甲苷对多囊卵巢综合征大鼠性激素水平及氧化应激损伤的影响;李艳青等;中国病理生理杂志;第36卷(第12期);第2244-2250页 * |
| 黄芪甲苷通过抑制卵泡颗粒细胞凋亡缓解糖尿病雌鼠生殖损伤;任雪华等;动物医学进展;第42卷(第12期);"摘要"项下，第66页左边栏第1-2段，第2.1、2.2、2.3、3小节 * |
| 黄芪皂苷的提取及含量测定;王培培等;中国实验方剂学杂志;第16卷(第4期);第27-30页 * |
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