CN107540643A - Ganoderma lucidum composition GL 1 and as estrogen replacement in terms of application - Google Patents

Abstract

This application discloses ganoderma lucidum composition GL 1 estrogen action and Primary Study its mechanism of action.GL 1 and ERs binding ability are calculated by computer aided animation, mtt assay detects the influence that GL 1 breeds to the cells of MCF 7 and the cells of MDA MB 231, GL 1 induces estrogen influence and the estrogen-receptor inhibitor ICI182 of the cells of MCF 7 propagation, the influence for the cells of MCF 7 propagation that 780 couples of GL 1 are induced；With protein immunization method（Western blot）Detect ER α, ER β monoclonal antibody expressions.As a result show, ganoderma lucidum composition GL 1 is good with the binding ability of erss, has estrogen-like action, may by being combined with ERs, and influence estrogen secretion and ER β expression play a role.

Description

Ganoderma lucidum composition GL-1 and as estrogen replacement in terms of application

Technical field

The present invention provides a kind of application of traditional Chinese medicine ingredients, specially ganoderma lucidum composition GL-1 and as answering on estrogen-like With.

Background technology

Estrogen is a kind of female sex hormone as caused by ovary and placenta, and adrenal cortex can also secrete a little estrogen. After adolescing, the ovary of Female Children also can secrete estrogen in this period, at the same time, endometrial hyperplasia, In addition, this estrogen can promote the development of vagina, fallopian tubal, uterus and ovary in itself, so as to cause menstrual onset.Women Internal estrogen, ovary is responsible for secretion when before menopause.After blood is entered, these are existed by the estrogen of ovarian secretion Circulation is formed in vivo.Therefore, estrogen is at some positions, such as uterus, bone, blood vessel, heart and brain formation distal end target spot, so as to Play coordinative role.Circulation estradiol level in blood has fluctuation in determining female physiological periodicity, and it can ovulate Phase reaches maximum, and still, inside rodent, oestrus is the peak place of estradiol.Estrogen can keep calcium It is deposited in bone joint, can promotes and promote sodium and water in vivo.In addition, subcutaneous fat enriches, zaftig, the proliferation of mammary gland with And promoting sexual desire etc. is influenceed by the estrogen in human body.Estrogen is largely in follicular cell and ovarian follicle Theca cell.In Ovarian Follicles, first testosterone under the stimulation of luteinising hormone by ovarian follicle endocrine, then in ovum Granular cell under bubble stimulin stimulates is changed into estradiol, and this is referred to as " the double promoting sexual gland hormone binding modes of double cells ".Remove Outside this, adrenal cortex, the position such as placenta and male testis can also secrete a small amount of estrogen.Estrogen can also be defined For estradiol, estradiol can be reduced rapidly after the deterioration of ovary, similar with climacteric metancholia so as to cause The generation of disease, caused by these diseases are substantially and lacked by estradiol.When ovarian follicle is in early development, only a little estrogen It can be secreted, the time of about physiological period or so, the amount of estrogen of ovarian follicle secretion can steeply rise, and can be Preovulatory formation peak, still, the secretory volume of estrogen can decrease in post-ovulatory phase.About one to two probably after ovulation My god, because corpus luteum starts to secrete estrogen, therefore estrogen level will rise in blood circulation of human body.Also, arranging About seven days or so after ovum, now corpus luteum is substantially ripe, and in blood circulation, estrogen forms the second peak, but with ovulation Preceding peak is compared to relatively low.Regardless of origin, all materials with heat function are referred to as estrogen, estrin by we And a kind of estrogen, the part metabolin of the estrogen and its serial C18 steroids of substitute, some are to come from animal, More also from other places unrelated with animal.

ERs is to form hormone-receptor compound after a kind of and hormone is specifically bound, so as to play life Most of the protein molecule of thing effect, this protein molecule is in the intracellular of target organ.ERs is to include two Kind：A kind of typical nuclear receptor, this nuclear receptor are to play its effect by adjusting estrogen genotype, that is, Say, by adjusting the transcription of specific objective gene, so as to show the effect of " genotype " regulation, this nuclear receptor, which includes, to be present In ER α and the ER β of nucleus.Second is film acceptor, and they are the effects for intervening quick non genotype, passes through the second letter Make system, so as to play indirect transcripting regulating function.But some of them, which seem only local in the brain, to work.This Two kinds of acceptor has study, remembers, cognition, reproduction and other regulatory functions, and this is with their distributions in body Cell-specific be closely related.ERs (estrogen receptor, ER) may reside in cell membrane, carefully In kytoplasm or nucleus.The protein of classical nuclear receptor can stay for a while in endochylema upon translation, and this receptor is located at cell In core, because can be stopped in endochylema, we can detect it in cytoplasm.The gene of downstream gene transcription Regulatory mechanism, it is due to that the female hormone for being diffused into nucleus is combined with its nuclear receptor so as to triggering.ER α and ER β are being tied It is nearly all much like on structure, but but have very big difference in Tissue distribution, encoding gene and their expression in each tissue It is different, especially they with after ligand binding, therefore and caused biological effect also has obvious difference.In addition, ER β are less than ER α to the affinity of estradiol.But in activated gene transcription, research finds that ER α are more notable than ER β effects, can See that both acceptors have apparent difference in mediate transcription activity part, significantly turn in addition, ER β act on than ER α in suppressor In terms of record.Therefore, ER_αIt is probably the activator mainly as genetic transcription, and ER β are probably mainly as genetic transcription Inhibitor.Some researchers are shown by experimental study：If lack ER α into the cell, then, low concentration estrogen is basic Responsive transcription cannot be produced；If there is ER α presence into the cell, then estrogen will produce significant transcription and live Property.But now ER β contents increased in the cell, then this transcription risen by ER α will decrease, This is also with regard to explanation：ER β can turn into a kind of ER α active regulator.Research finds that apiolin is to breast cancer cell line mcf-7 Cell propagation have facilitation, this with apiolin activation ER α acceptors be it is closely related, still, when apiolin is fewer, It is that inhibitory action is played to Cells Proliferation of Human Breast Cancer by ER β.In recent years, research is found, estrogen and is present in cytoplasm Or after the ER in cell membrane combines, second system of couriers is started.Estrogen is used as medium in human body, turns Many biological effects have been led, but in the early stages, people think that only nuclear receptor can be adjusted to the function of estrogen Section control, that is to say, that estrogen is expressed by being combined regulatory gene with the estrogen response element on target gene, and it is with certainly By the mode spread, it is intimately associated by cytoplasma membrane and with nuclear receptor, or, estrogen interacts with other nucleoprotein, Therefore the transcriptional activity of gene is changed.Research in recent years also found that some can be as the film estrogen of medium Acceptor, they can transduce quick non-essential edge, and these film ERs include G-protein coupling ERs 1 again Referred to as g protein coupled receptor 30.

Estrogen is a kind of regulation hormone, and it plays an important role in female body, and it is both to female bone The normal function of the physiological systems such as bone system, central nervous system and cardiovascular system plays an important role, also to women's The growth and development of reproductive system have important regulating and controlling effect.Estrogen is a kind of steroid hormone, and it is in normal mammary gland development With playing key effect in the forming process of breast cancer, its show in vivo extensive biological activity be by with difference The estrogenic receptor subtype of gene code combines.Women is after climacteric is entered, it may appear that climacteric syndrome, its is internal Estrogen level can also drastically reduce.Menopausal syndrome will occurs in women, and can cause ovarian function decay degenerate with And the decline of estrogen level in vivo, in addition, also resulting in the hair of the diseases such as osteoporosis, senile dementia, angiocardiopathy Sick rate increase.With the development in epoch, people gradually expect can obviously relieve the female of these above-mentioned menopause symptoms with a kind of HRT, but this method can not uninterruptedly use in a period of time, because if women long-term use estrogen, The incidence rate of the gynecological diseases such as carcinoma of endometrium and breast cancer can not only be increased, and can cause to trigger high blood clotting shape The poison such as state, oedema, hypertension, dementia is secondary to be influenceed.For a long time, it is clinically to be used to alleviate woman using artificial synthesized estrogen Female's climacteric syndrome symptom replaces the various diseases that treatment is triggered by estrogen deficiency.However, this treatment method institute The bad side reaction such as the generation of the disease promoted such as genital system and breast cancer and endometrial hyperplasia influences its application Prospect.Therefore, people take up to find：Both above-mentioned bad side reaction had been avoided that, and can enough retains estrogen to angiocarpy A kind of substitute materials of estrogen of the key effects such as system.Then, this kind of material is adjusted also referred to as selective estrogen receptor Agent is saved, they have dual regulation, that is to say, that when estrogen level is relatively low in vivo, there is similar estrogen to make With when estrogen level is higher in vivo, with antiestrogenic effect.For a long time, contained by many Chinese herbal medicines Similar estrogen compound is played an important role in health care of women and the treatment of gynecological diseases, and these compounds are played the part of wherein The key player drilled can not be ignored.

Ganoderma lucidum [Canoderma lucidum] is a kind of large-scale medicinal fungi, belongs to basidiomycetes hilllock Polyporaceae Ganoderma Fungi, be most initially to be recorded in《Sheng Nong's herbal classic》In, ganoderma lucidum is classified as top grade by successive dynasties medical book, its have tonifying middle-Jiao and Qi, Strengthen the body resistance to consolidate the constitution, the multiple efficacies such as enriching yin is strong, promotes longevity.In the medium-height grass Drug Storage of our countries, ganoderma lucidum have these are only Special effect enjoys a widespread reputation.The big physician of this Ming Dynasty of Li Shizhen (1518-1593 A.D.),《Compendium of Materia Medica》In just ganoderma lucidum is write it is upper and other items, Also, he show ganoderma lucidum be it is a kind of be difficult can be as treasure of the medicine and can as food.Ganoderma lucidum is by as medicinal The earliest fungi of fungi.The biology that this species of fungi is more and colony is huge is extensive in distributed in nature, and they are different from general Logical green plants, chloroplaset is not contained, be so as to obtaining nutrition and energy by decomposing organism that other contain organic matter.People Macro fungi is had gained some understanding a long time ago.Up to now, fungi has kind more than 60000 to have been observed that, fungal resources by Gradually turn into the important living resources that a country realizes the strategy of sustainable development.Fungus circle species probably have more than 150 ten thousand kinds, from Fungus species found in right boundary almost has 400,000 kinds.The fungal resources in China are very abundant, estimate 20 with species More than ten thousand, wherein about more than 90% is not appreciated that also.The traditional medicine in China is used and has the fungus of medicinal function The kind more than 800, it can be seen that, fungi has great application prospect.China is the earliest country of the application of fungi, and it is applied Can trace back to before more than 2000 years, people in order to treat the gastrointestinal disease of common indigestion, having indigestion etc., using " Divine Comedy " come This kind of disease is cured, the treatment effect of this medicine is fine, is then also conceivable that use when people run into similar disease now It is cured.And " Divine Comedy " as a member in China's traditional Chinese medicine, its another identity --- fungi, as originated from fungus The representative of medicine, while expanding medicament sources for traditional Chinese medicine, also provide new thinking side to Natural Medicine Chemistry worker To.In the traditional Chinese medicine in China, some higher funguses such as ganoderma lucidum, cordyceps sinensis, stone-like omphalia, Poria cocos etc. are opened from the 1950s Beginning is just used always.These higher funguses have carried out every research by researcher, and existing a collection of medicinal application is faced On bed.Because of the continuous development of the field of medicaments subject such as clinical medicine, pharmacology, people little by little recognize medicinal fungi in food With the effect in life as important composition, development etc. is innovated on the mechanism of action of medicinal fungi, technique, application and new drug The research of aspect has marked improvement.In the course of much time using higher fungus, people have recognized many macro fungis There are many medicinal functions：Such as improve memory, immunological regulation, antitumor, anti-aging, hypoglycemic etc., and ganoderma lucidum is to be made For the earliest fungi of medicinal fungi.

According to Analysis of Nutritive Composition result it is known that the nutritional ingredient very abundant that ganoderma lucidum contains, such as amino acid, constant And trace element, aliphatic acid, vitamin e etc..As can be seen here, its nutritive value is also especially high.In addition, ganoderma lucidum contain it is needed by human Carrotene and bioactive polysaccharide, be dietary fiber necessary to health extraordinary source.Although some grind The person's of studying carefully isolated plurality of active ingredients from ganoderma lucidum (most of is artificial cultivation thing), such as polysaccharide, triterpenes, sterol Class, alkaloids etc., still, the much research on estrogen-like action is not carried out to its bioactivity, but mainly Study antitumor, anti-aging, antiviral and raising immune function of human body etc..《Chinese Pharmacopoeia》Middle ganoderma lucidum is included as one Rare traditional Chinese medicine is planted, effective component such as polysaccharide, triterpene, alkaloid, amino acid polypeptide, the trace element etc. included in ganoderma lucidum makes it As the dual-purpose treasure of a kind of medicine and food.Modern pharmacological research shows, one of active component of ganoderma lucidum ganoderma lucidum triterpene Compound, this kind of compound have liver protection, antitumor, anti-HIV-1 and HIV-1 eggs from enzymatic activity, antihistaminicum release, suppress blood vessel Angiotensin Converting Enzyme, anti-oxidant etc. act on.

So far, the successful separation and Extraction from ganoderma lucidum goes out 200 multiple compounds to domestic and international researcher, and to these changes Compound has carried out molecular structure identification.However, some research work are just with laboratory, this small platform is extracted Mask work, cause the compound amounts separated insufficient, therefore, it is difficult to carry out the depth of pharmacology and internal drug metabolism People studies, and is only merely that some researchs have been carried out in terms of chromatography and molecular structure identification, greatly affected ganoderma lucidum Continual exploitation and utilization.

As can be seen here, ganoderma lucidum is a kind of fungi for being worth being studied, therefrom extract obtained all to have some pharmacology mostly Activity.Isolate and purify to obtain extract GL-1 estrogen-like action and its mechanism of action in main research ganoderma lucidum herein.Pass through It is a large amount of to consult pertinent literature, according to the continuous discovery in pertinent literature and experimentation with summarizing, with cells in vitro testing sieve Select compound GL-1 estrogen-like action, recycle western blot detection methods, detect its whether by estrogen by Body surface reaches, and so as to promote the continual exploitation of ganoderma lucidum and utilization, and the research for health products, medicine provides scientific basis.

The content of the invention

The present invention provides a kind of ganoderma lucidum composition GL-1, and structural formula is as follows：

Application of the present invention by described ganoderma lucidum composition GL-1 on estrogen-like action.Specially ganoderma lucidum composition GL-1 It can be combined with ERs ER β.And ganoderma lucidum composition GL-1 can promote MCF-7 cells to breed, ganoderma lucidum composition GL- during being somebody's turn to do 1 concentration is 10^-5~10^-6mol/L。

The present invention utilizes the binding ability of computer aided calculation GL-1 and ERs

Molecular docking can carry out rapidly the stable conformation of certain space to ligand molecular, within a certain period of time similar medicine The compound of thing is searched for from the substantial amounts of data of database and obtained.Using area of computer aided, medicine is designed and carrys out analog drug In the interaction of thing and bind receptor molecule, or molecular structure and Active substance by analyzing known drug connection System, rationally design out the lead compound of new structure.

Mtt assay detection cell propagation of the present invention

Up to the present, the method that we have many kind detection cytoactives can be used, but some of methods All there is a little shortcoming, such as:Isotope (³HTdR,¹²⁵Iud R) and incorporation methods, although both approaches have stability good, clever The advantages that sensitivity is high, but operate during with both approaches very complicated, step is various, in addition, it is necessary to special equipment is used, Most important problem is that the method has radioactivity, and testing staff can be damaged.However, mtt assay is not only easy to operate, quick The advantages that, also with high sensitivity, stability is good the characteristics of.The principle of mtt assay：Mainly can be with amber in mitochondria using MTT Amber acidohydrogenase reacts, and generates a kind of royal purple crystal, it is difficult dissolubility that the crystal, which is, and one kind precipitation can sink to cell In, but MTT does not react with dead cell.We not only should be noted thin in the hole in each orifice plate before testing The situation of intracellular growth, observation cell whether grow uniformly, and needed to be grasped the cell oneself cultivated adherent rate and Growth curve under the conditions of different vaccination cell number.In addition, we need to be grasped the number of cells that each hole is cultivated in experiment, And the cell culture time is grasped, so it is prevented that because cell growth excessively causes culture to terminate, otherwise will make experiment As a result directly affected.We should not be applied directly to hole when the culture medium or addition more renewed needs the medicine studied In cell on, and should be slowly added into along the edge of 96 orifice plates, therefore can avoids some from causing cell growth irregular The situation of title.Only in this way, we just can ensure that MTT crystallizes the amount to be formed with cell number in necessarily linear.In addition, exist We are also needed to after cell first first is cultivated into certain time with the nutrient solution for not containing serum before experiment, add thiazole basket, from And avoid the interference of serum.Because during with containing certain hyclone nutrient solution culture cell, after thiazole basket is added, because of hair Raw reaction is it is possible that precipitate, and this precipitation is difficult dissolving and also therefore absorbance can be had an impact, so that can shadow Ring experimental result.In addition, it would be desirable to a zeroing hole is set in experimentation, to be contrasted with other holes, so as to Reducing the error brought by nutrient solution or other operating process influences the accuracy of result.

Western blot detect estrogen receptor expression situation

Immunoblotting is Western Blot.It is a kind of experiment detection method, and this method is in biochemistry, molecule Biology and immunogenetics are often applied to.Its general principle is that sample is carried out by specific antibody Coloring, the biological tissue and cell that these samples treat from gel electrophoresis, we are by analyzing the bar by coloring Bring the expression for judging the protein in biological tissue and cell.The detection method and other hybridizing methods compare class Seemingly, for example Southern hybrid methods, difference are that Western Blot carry out electrophoresis with polyacrylamide gel, The sample of method detection is protein, is using antibody as probe, using secondary antibody as being developed the color after label.By poly- The sample of acrylamide gel electrophoresis separation, we are transferred them to above solid phase carrier, and protein is by solid phase carrier with non- Covalent bond adsorbs, and the polypeptide forms of the protein and the bioactivity of itself all do not change.We will pass through Sample after PAGE experiments, transfers it to and antigen samples is used as on solid phase carrier, and antigen occurs anti-with corresponding antibody Should, reacted followed by enzyme or the secondary antibody of isotope marks, the expression of GAP-associated protein GAP then can be detected by development.The inspection Survey method is for the first time written in Neil Bai Naite (Neal Burnette)《Analytical biochemistry》In be referred to as Western Blot's.Southern be do earliest trace research researcher, DNA chains be he study to picture, he is his This detection technique is referred to as Southern blot.Below, people are gradually found that a research object is RNA, and another grinds Study carefully the similar technology of two kinds of experimentations that object is protein, be referred to as Northern and Western.Blot experiment is general Including three steps：First, protein is separated by electrophoresis；2nd, by the Protein transfer of gel separation on solid phase carrier, and Unadsorbed protein domain on solid film is closed with nonreactive activity and non-specific molecules；3rd, immunology detection. First stage carries out electrophoresis with SDS-polyacrylamide gel.Protein sample etc. is as antigen and band after SDS reactions Negative charge, protein sample swimming from cathode to anode in polyacrylamide acyl gel in electrophoresis process, the smaller albumen of molecular weight Sample, its speed will be faster.Second stage is electrotransfer process.The band being successfully separated in gel will be transferred to nitre On acid cellulose film, now from high current and low-voltage, shift after about 45min and just complete.Phase III is to use Enzyme is immunized to position.Successfully transfer is had to the nitrocellulose filter of protein band, it is anti-with specific antibody and second respectively Body effect after, then with enzyme reaction substrate effect can dye band.The band of positive reaction is clearly, can substantially to divide Distinguish, and the molecular weight standard that adds when we can be according to electrophoresis estimates the molecular weight of each component.Protein immunoblot is A kind of protein detection techniques, detection method of protein have many, such as：Enzyme-linked immunosorbent assay.Detect albumen Matter is to determine a kind of mode of gene expression, it is possible to which this inspection similar to detection RNA is immunized with western in selection Survey method of protein.This detection technique of detected by Western blot is widely used to gene and ground in the expression of protein level Study carefully, antibody activity detection and disease early diagnosis etc. many aspects.The detection technique overcomes the drawbacks of prior immunization credit analysis, Such as：Immunoassay is directly carried out on the gel after electrophoresis.It combines the high of SDS-polyacrylamide gel electrophoresis The sensitiveness and high specific of resolution capability and enzyme-linked immunosorbent assay, drastically increase its resolution ratio, utilization rate And sensitivity.The detection technique is an effective analysis method, be not only widely used in the various antigen components of analysis and The related activity of these antigens, and can diagnose the illness, such as：The detection technique is in AIDS viral infection by as making a definite diagnosis Experiment.This problem is studied Ganodenna Lucidum P.E GL-1 by the detection method, detects whether it is sent out by ERs The effect of waving.

The external concentration 10 of GL-1^-4Mol/L and the above are inhibited to MCF-7, and 10^-6Mol/L and and upper right MDA-MB-231 is inhibited, shows that GL-1 has antitumor action, selects suitable dosage such as 10^-5Mol/L, substitute Estrogen-like uses, it is ensured that has estrogen action, can overcome the risk of the possible induced tumor of estrogen again.

Outer experiment proves that GL-1 has estrogen-like action inside the present invention, and alternative estrogen encloses menopause for therapy The oestrogen deficiencies symptom such as phase syndrome.Major advantage is the suitable dosage of selection, substitutes estrogen and uses, it is ensured that has female Hormone-like effect, the risk of the possible induced tumor of estrogen can be overcome again.

Brief description of the drawings

Fig. 1 is GL-1 and ER β combination figure.

Fig. 2 is the expression influence that Western-blot detects albumen.

Embodiment

Embodiment 1

Medicine and cell

(mycology National Key Laboratory of institute of microbiology of the Chinese Academy of Sciences provides GL-1, isolated from ganoderma lucidum.Purity 98%)；17 beta estradiols (are purchased from Sigma)；MCF-7 cell strainHJ2mm, MDA-MB-231 are (purchased from BeiJing, China typical case Culture collection).

Main agents

DMEM dry powder (being purchased from Gibco companies), L-15 dehydrated mediums (being purchased from Invitrogen Corporation)；Tire Cow's serum, calf serum (being purchased from Zhejiang Tian Hang bio tech ltd)；Tetrazolium bromide (MTT) is (public purchased from U.S. Amresco Department)；Trypsase (is purchased from Sigma companies)；ER α, ER β monoclonal antibodies (being purchased from Abcam companies of the U.S.)；β-actin Dan Ke Grand antibody (being purchased from Cell Signaling companies of the U.S.)；Secondary antibody (is purchased from Shanghai You Ningwei bio tech ltd)；Without ammonia Base yeast nitrogen (Difco) (is purchased from Angel Yeast joint-stock company)；Dimethyl sulfoxide (DMSO) DMSO (is purchased from close europeanized of Tianjin section Reagent Co., Ltd) etc..

Key instrument

The a ten thousandth electronic balance of ME215S types ten (German Sartorios companies)；CO₂Incubator (SHELLJB)； 5415R small desks high speed freezing centrifuge (German Eppendorf AG)；The turbula shakers of Vortex-Genie 2 are (beautiful State Scientific Industries)；TDZ4-WS table-type low-speed centrifuges (Changsha Xiang Yi centrifuges Instrument Ltd.)；Enzyme Join immune detector Stat Fax-2100 (AWARENESS TECHNOLOGY INC)；24DN electrophoresis apparatuses (the biology of Beijing 61 Science and Technology Ltd.)；Decolorization swinging table STS-1 types (its woods Bel's instrument manufacturing Co., Ltd of Haimen City).

Computer Simulation calculation binding ability

The binding ability of methyl maclurin A and ERs is calculated by Computer-aided Molecular rendezvous method, prediction is female Hormone dependent interaction and its Interactions Mode between ERs.The three-dimensional of estrogen receptor alpha, erss Structure coordinate, downloaded from Protein Data Bank.Connection module Glide is divided using semi-flexible in Schrodinger's software Son docking, judge that the effect of methyl maclurin A and ERs is strong and weak with the negative value size of Gscore values, Gscore negative values It is smaller, represent stronger with compound effects, Gscore negative values are bigger, illustrate weaker with compound effects.

Cell culture

Using open monolayer adherence culture human breast carcinoma MCF-7 (ER+) cell, nutrient solution is phenol red DMEM (tire ox bloods Clear 10%, penicillin 100U/mL, the μ g/mL of streptomysin 100), condition of culture is 37 DEG C of constant temperature, 5%CO₂, relative saturation humidity, Sterile culture.General two to three days passes a generation, during passage, suctions out culture medium, adds 3 milliliters of pancreatin, gently rock blake bottle, make Pancreatin digestive juice flows through bottle wall, after standing a few minutes, under the microscope after cell shrinks and is rounded, discards pancreatin digestive juice, adds Enter culture medium and terminate digestion.Cell is blown and beaten into after single cell suspension, moved in centrifuge tube.After the completion of centrifugation, supernatant is abandoned in suction, Add after culture medium mixes and be drawn in blake bottle, be placed in incubator culture of uncapping.

Human breast carcinoma born of the same parents MDA-MB-231 uses closed monolayer adherence culture, and the culture medium of MDA-MB-231 cells is L- 15 culture mediums (calf serum 10%, penicillin 100U/mL, 100 μ g/mL), 37 DEG C of constant temperature, relative saturation humidity, sterile culture. It is basically identical to pass on step, but needs in incubator to cover tightly lid culture.GL-1 breeds to MCF-7 and MDA-MB-231 cells Influence

Take the logarithm growth period MCF-7 cell, with DMEM culture mediums (containing 10% hyclone, 0.5% penicillin, streptomysin) Culture, after cell is adherent in 96 orifice plates, pancreatin digestive juice is added in blake bottle, so as to vitellophag, and in microscope Lower observation cell detachment situation, until attached cell completely falls off, add equivalent nutrient solution and terminate digestion, be made for cell and hang After liquid, cover lid and centrifuged with certain rotating speed, inhaled after centrifugation and abandon supernatant, cell concentration is diluted to 1 × 10⁴Individual/mL, inoculation In 96 orifice plates, blank group, positive group of (E are set respectively₂, 1.00 ×^-9Mol/L), 10^-4Mol/L, 10^-5Mol/L, 10^-6Mol/L, 10^-7Mol/L, 10^-8Mol/L medicines, totally 7 groups, every group sets 6 multiple holes.Culture 24h makes its adherent, and medicine is added after adherent, 37 DEG C, 5%CO₂After cultivating 96h in incubator, bred with mtt assay measure cell, 20 μ L MTT (5 mg/mL) added per hole, 150 μ L DMSO are added after culture 4h, OD values are determined under 492nm wavelength conditions with ELIASA.

Take the logarithm growth period MDA-MB-231 cell, 1640 culture mediums are changed to L-15 nutrient solutions, other processing methods are same On.

GL-1 induces estrogen the influence of MCF-7 cells propagation

The processing method of MCF-7 cells adds 10 into system simultaneously with " 2.2 " when adding test medicine^-8mol/L 17 beta estradiols, cultivate under the same conditions.

Estrogen-receptor inhibitor ICI182, the influence of the MCF-7 cells propagation of 780 pairs of GL-1 inductions

The processing method of MCF-7 cells is with " mtt assay detection cell propagation ", when adding test medicine simultaneously respectively to body 10 are added in system^-7Mol/L estrogen-receptor inhibitor (ICI182,780), is cultivated under the same conditions.

Western-blot detects the expression of albumen

After cell is digested with pancreatin digestive juice, it is made 1 × 10 after single cell suspension⁵/ mL is inoculated in 6 orifice plates, culture After 24h is adherent, GL-1 10 is separately added into^-5mol/L、10^-6mol/L、10^-7mol/L、10^-8Mol/L, 17 β estradiol (E₂ 10^{- 9}mol/L)；After processing 24, after blotting nutrient solution, 1mL, the PBS of 4 DEG C of precoolings are added per hole, keeps flat and gently shakes washing cell, Then cleaning solution is discarded, repeat more than operation twice, wash three times cell so as to washing away nutrient solution.After PBS is exhausted, culture Bottle is placed on ice, is added cell pyrolysis liquid and is cracked 30min on ice.After having cracked, with clean scraper rapidly by cell from 6 holes Plate bottom is swept, and is then moved in the disposable centrifuge tubes of 1.5mL cell fragment and lysate with liquid-transfering gun, is used centrifuge 12000rpm, 4 DEG C of centrifugation 10min (opening centrifuge precooling in advance), after BCA method protein quantifications, 50-100 μ g albumen is through 10% It is transferred to after the separation of SDS-PAGE glue on pvdf membrane, the TBST solution closing 1.5h containing 5% milk.Various primary antibodies 4 are incubated respectively DEG C overnight, clean film 5 times with the TBST now prepared, every time with shaking table clean 5 minutes, incubation secondary antibody room temperature 1h, then use TBST cleanings film 5 times, 5 minutes every time, then carry out exposure imaging with ECL luminous substrates.With ImageJ analysis purpose bands OD value.

Statistical method

Data are handled with SPSS11.5 statistical softwares.Examined with the normal distribution of the advanced row data of the software Test and homogeneity test of variance, using variance analysis between group compare.Continuous data is to use mean ± standard deviation (X ± S) table Show, group difference uses one-way analysis of variance, between multiple sample averages the comparison of each two mean examined using Q.P< 0.05 explanation significant difference, P<0.01 is that difference is extremely notable.

Simulate result of calculation

As shown in figure 1, GL-1 and estrogen beta (PDB code names：Molecular docking, docking point 1U3R) are carried out using Glide methods Value Gscore is -8.37kcal/mol, and GL-1 long-chains are in hydrophobic pocket, π-π phase interactions between its phenyl ring and Phe356 be present With.

The influence that GL-1 breeds to MCF-7 cells

Shown in table 1, when estrogen deficiency in cell culture system, GL-1 can be obviously promoted MCF-7 cells propagation (P< 0.01), as the increase of drug concentration gradient, cel l proliferation are remarkably reinforced.When GL-1 concentration reaches 10^-5During mol/L, Maximum to the proliferation function of MCF-7 cells, difference has conspicuousness (P compared with blank group<0.01), when concentration proceeds to 10^{- 4}During more than mol/L, inhibitory action is shown, concentration continues to increase, inhibitory action enhancing.It is anti-swollen to show that high concentration GL-1 has Knurl acts on.

Influence that the GL-1 of table 1 breeds to MCF-7 cells (N=6)

Note：Compared with blank group, * P ＜ 0.05, * * P ＜ 0.01

The influence that GL-1 breeds to MDA-MB-231 cells

As shown in table 2, GL-1 can suppress the propagation of MDA-MB-231 cells, 10^-5It is poor compared with blank group during mol/L It is different to have conspicuousness (P<0.05).As a result show, GL-1 has inhibitory action to tumour cell.Further demonstrate that high concentration GL-1 has Antitumor action.

Influence that the GL-1 of table 2 breeds to MDA-MB-231 cells (N=6)

Note：Compared with blank group, * P ＜ 0.05, * * P ＜ 0.01

GL-1 induces estrogen the influence of MCF-7 cells propagation

As shown in table 3, when in system simultaneously add E₂During with the GL-1 of various concentrations, GL-1 can suppress E₂Increasing to MCF-7 Grow, with E₂Group compares, and cell survival rate declines, and difference has conspicuousness (P<0.01).

Table 3GL-1 estrogen is induced MCF-7 cells propagation influence (N=6)

Note：Compared with blank group, * P ＜ 0.05, * * P ＜ 0.01

Estrogen-receptor inhibitor ICI182, the influence of the MCF-7 cells propagation of 780 pairs of GL-1 inductions

As shown in table 4, estrogen-receptor inhibitor ICI182.780 being added in cell culture system can significantly inhibit Propagation (the P of MCF-7 cells<0.01).GL-1 and estrogen-receptor inhibitor ICI182.780 co-cultured cells, ERs Inhibitor ICI182.780 can block proliferation function (Ps of the tested concentration GL-1 to MCF-7 cells<0.01).

The ICI182 of table 4, the influence of the MCF-7 cells propagation of 780 pairs of GL-1 inductions

Note：Compared with blank group, * P ＜ 0.05, * * P ＜ 0.01, #P ＜ 0.05

The influence that GL-1 is expressed estrogen receptor

As shown in Fig. 2 GL-1 is 10^-5During mol/L, ER α protein expressions substantially reduce (P compared with blank group<0.01), GL-1 10^-8mol/L-10^-6During mol/L, compared with blank group, ER α protein expression differences are without conspicuousness；GL-1 is 10^{- 5}During mol/L, ER β protein expressions are with blank group comparing difference without conspicuousness, and GL-1 is 10^-7mol/L-10^-6During mol/L, ER β eggs White expression relatively dramatically increases (P with blank group<0.01).

Mouse Uterus weightening experiment

Weight-increase experiment on womb, it is the classical way for the detection estrogen active established earliest, is still widely used at present.Son Abundant ERs is contained in palace, after foreign compound is combined with ERs, can increase estrogen inducible protein (IPs) content in uterus Add, stimulate uterine growth.Therefore, determine animal uterus weight or calculate its organ coefficient, the estrogen that tested material can be evaluated is lived Property and its intensity.

Real face animal

Kunming female mice, 9~12g, SanXia University's Experimental Animal Center provide, production licence：SCXK (Hubei Province) 2011-0012。

Mouse is grouped at random by body weight.Administration continues 7 days, and the daily aquae destillata of control group presses 0.1ml/10g body weight gavages； Positive controls estradiol suspension presses 0.1mg/kg body weight gavages.24h after last time is administered, take off neck and put to death mouse, immediately Cut uterus to weigh, calculate Uterine coefficient (uterine wet weight/body weight × 100)

The medicine of table 6. to young mouse Uterine coefficient influence (N=10)

Asterisk tabular form has conspicuousness p ﹤ 0.01 (* *), p ﹤ 0.05 (*) with blank group comparing difference

As a result show：GL-1 has estrogen-like action in vivo, Mouse Uterus can be promoted to breed.

It is a series of disorderly with autonomic nervous system function caused by the front and rear sex hormone fluctuation or reduction occurred of postmenopausal women Based on unrest, with one group of syndrome of neuropsychological symptom, referred to as perimenopausal syndrome.Controversies in hormone replacement in the elderly climacteric The conventional treatments of syndrome, but long-term use of estrogen can make the pathogenetic increased risk of the diseases such as phlebothrombosis, hypertension, Also the risk of breast cancer or endometrium carcinogenesis can be increased.Thus, it is found that safer estrogen substitute, overcome makes for a long time It is significant with the adverse reaction of estrogen.Estrogen-like effects mainly have been done with regard to ganoderma lucidum composition GL-1 herein, and Its possible mechanism of action of Primary Study.Shown by computer simulation experiment, ganoderma lucidum composition GL-1 and ERs ER β With stronger binding ability, and there is no obvious binding ability with ERs ER α, it may be possible to which ERs ER β are special Specific binding agent, it is worth further investigation.

MCF-7 cells are estrogen receptor expression positive cells, and MDA-MB-231 cells are that estrogen receptor expression is negative Cell.By test-compound to MCF-7, MDA-MB-231 proliferation function difference, its estrogen-like action can be tentatively judged.It is real Test and show, GL-1 low concentrations have to MCF-7 cells promotes proliferation function, and same concentrations do not increase to MDA-MB-231 cells The effect of growing, showing that GL-1 there may be estrogen-like action, GL-1 high concentrations are inhibited to MCF-7, MDA-MB-231, Show that GL-1 there may be antitumor action, select suitable dosage, replacement estrogen is used and ensures that drug safety has Significance.

GL-1 can suppress E₂To MCF-7 proliferation function, meanwhile, GL-1 can be swashed to the proliferation function of MCF-7 cells by female Plain acceptor inhibitor ICI182.780 is blocked, and GL-1 can adjust the expression of ERs, improves ER β protein expressions, reduces ER α Protein expression, showing GL-1 estrogen-like action may be produced by being combined with ERs, and it is to ERs The regulation of expression, may be relevant with antiproliferative, it is necessary to further study

Ganoderma lucidum enjoys domestic and international extensive concern always as China's rare medicinal herbs, and contained active component is also special in ganoderma lucidum It is more, it is worth us to go to probe into.Experiment shows that Ganodenna Lucidum P.E GL-1 has estrogen-like action, and its mechanism of action is probably logical ERs combination is crossed, and influences estrogen secretion and estrogen receptor expression, so as to play its estrogen-like action, but The specific mechanism of GL-1 mediation estrogen receptor paths also needs to further probe into.

Claims (5)

1. ganoderma lucidum composition GL-1, it is characterised in that structural formula is as follows：

2. applications of the ganoderma lucidum composition GL-1 on estrogen-like action described in claim 1, alternative estrogen is used to treat Medicine in the disease of the oestrogen deficiencies of perimenopausal syndrome.

3. the application described in claim 2, it is characterised in that ganoderma lucidum composition GL-1 is combined with ERs ER β, and GL-1 is in body It is interior that there is estrogen-like action, Mouse Uterus can be promoted to breed.

4. the application described in claim 2, it is characterised in that ganoderma lucidum composition GL-1 promotes estrogen receptor positive cell MCF-7 Propagation, but do not cause estrogen receptor negative cell MDA-MB-231 to breed, GL-1 can be female to the proliferation function of MCF-7 cells Hormone receptor inhibitor ICI182.780 is blocked.

5. the application described in claim 3, it is characterised in that ganoderma lucidum composition GL-1 external concentration is 10^-8~10^-5mol/L。