CN102091115A - Epimedium extract for treating climacteric syndrome - Google Patents
Epimedium extract for treating climacteric syndrome Download PDFInfo
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Abstract
The invention relates to an epimedium extract for treating climacteric syndrome, which can be used for effectively solving the treatment of the climacteric syndrome with medicines. The technical scheme in the invention is as follows: the epimedium extract is prepared by the steps of: crushing epimedium into medicine powder, and extracting for twice with alcohol through refluxing, wherein each time is for 1.5h; mixing extract solutions, filtering, decompressing filtrate and recycling the alcohol until no alcohol taste exists, concentrating, refrigerating and standing for 12h and then filtering, regulating the pH value with hydrochloric acid and then passing through a adsorbent resin column; and firstly eluting with water, and eluting with alcohol, removing eluent, recycling the eluent, concentrating and drying to obtain the epimedium extract containing general flavones, wherein the epimedium extract can be used for effectively preparing medicines for treating the climacteric syndrome. The preparation method is simple, and is easy to operate. The content of the general flavones in the epimedium extract is high; and the epimedium extract has good effect and can be applied to the preparation of the medicines for treating the climacteric syndrome.
Description
One, technical field
The present invention relates to medicine, particularly a kind of Herba Epimedii extract for the treatment of climacteric syndrome.
Two, background technology
(Climacteric Syndrome CS), is one of commonly encountered disease and frequently-occurring disease to climacteric syndrome (levying).Mainly be because the ovarian function decline causes that organism endocrine is lacked of proper care, immunity is underground and the syndrome of autonomic nervous dysfunction, causes a plurality of systems of whole body function and pathology pathological changes to occur.The ovarian function decline is its core pathogenesis and pathologic basis.At present the climacteric syndrome prevalence is about 70% in the middle aged and aged women, and wherein about 40% need treatment, has become one of main illness that has a strong impact on middle aged and aged women quality of life.
Modern medicine thinks that menopausal syndrome is because factors such as age growth, hardworking, delivery and feed infant, weak and sickly, oophorectomize or radiotherapies, and excessively reducing generation such as the neurotransmitter that causes due to hypothalamic-pituitary-ovarian axis or the dysadrenalism, hormone, cytokine because of estrogen level unbalance is the main cause of falling ill climacteric.So the neuroendocrine immunity is the closest with the relation of climacteric syndrome.Doctor trained in Western medicine often adopts Hormone Replacement Therapy (Hormon Replacement Therapy, HRT), the two dimension of oryzanol commonly used clinically B sheet, oryzanol injection, nilestriol, compound recipe ethinylestradiol medroxyprogesterone capsule, compound hydrochloric acid procaine (capsule), chlorotrianisene pills, estradiol valerate etc., though HRT has better curative effect, but clinical practice is found, life-time service estrogen can increase the sickness rate of carcinoma of endometrium and breast carcinoma, in July, 2002, NIH's heart, lung and Blood Research Institute are announced, WomanHealth initial action (WHI) uses estrogen to add the clinical trial of progestogen in healthy postmenopausal women, because breast carcinoma morbidity relative risk increases by 26%, and there is no overall benefit and give premature termination.Reproductive system proliferative lesion not only, the clinical practice of this method of particularly having found generation significant limitation that it can increase serious side effects such as cardiovascular and cerebrovascular vessel danger in the recent period.
Climacteric syndrome is commonly encountered diseases, frequently-occurring disease, also is a kind of disease for the treatment of and easily recurring than refractory, and it not only shows gynecological's scope, also relates to fields such as internal medicine, psychiatric department simultaneously.Because it is the focus of colleague's concern both at home and abroad that the risk of HRT is benefited always, seem very necessary and urgent so seek a kind of safer effective Therapeutic Method, under above-mentioned background to the new knowledge of HRT drawback, Chinese medicine is faced with the unprecedented new opportunity that goes to the world.Chinese medicine is to the history in existing thousands of years of understanding of primary disease." element ask ancient times innocent opinion " cloud: " woman seventy-seven, conception vessel void, and taichong channel declines few, exhaustion of kidney-essence with promoting reproductive function, menopause is so senile appearance and loss of fecundity are also." women enters into the climacteric period a series of physiology, pathological change can take place.Kidney qi is lost empty, and menses will exhaust, and appoints chong channel to decline, and menstruation gets muddled, so that menopause.Simultaneously, the syndrome of deficiency-fire produced in the interior a series of deficiency of kidney-YIN that cause because of deficiency of kidney-essence appears.As hectic fever, sweating, dysphoria with feverish sensation in the chest palms and soles, insomnia and dreamful sleep, soreness of the waist and knees etc.From clinically, climacteric syndrome mostly shows as interior-heat caused by deficiency of YIN, its treatment with nourishing YIN and clearing away heat for wanting, wherein should be based on nourishing kidney yin.Viewpoint from the traditional Chinese medical science, " kidney qi declines " is its main endogenous cause of ill, according to scientific research in recent years, the imbalance of YIN and YANG and the autonomic nervous dysfunction of kidney are closely related, and the kidney invigorating medicine has also proved this point to the clinical and experimentation of reproductive endocrine system effect.Therefore, clinically, must catch " suffering from a deficiency of the kidney " this main pathogenesis, in treatment, must emphasize based on the kidney invigorating, and study carefully it, the regulation of mental activities of holding concurrently, liver, spleen and Gu Qibiao are so cause the heart, liver, spleen, all cards of imbalance of YIN and YANG to be got to suffering from a deficiency of the kidney studying carefully better.
The climacteric syndrome clinical manifestation often has menoxenia, warms sweating, hypomnesis, vertigo and tinnitus, palpitation and insomnia, back are aching and tired, the sense of skin ant row, irritable emotion etc.
Herba Epimedii is the dry aerial parts that Berberidaceae plant Herba Epimedii Epimedium brevicornum Maxim. etc. belongs to various plants together.Warm in nature, sweet in the mouth, suffering are returned liver, kidney channel, have warming the kidney to invigorate YANG, bone and muscle strengthening, the effect of wind-damp dispelling.But Herba Epimedii extract does not see so far that as the medicine that is used for the treatment of climacteric syndrome open report is arranged.
Three, summary of the invention
At above-mentioned situation, for overcoming the defective of prior art, the present invention's purpose just provides a kind of Herba Epimedii extract for the treatment of climacteric syndrome, can effectively solve the problem of climacteric syndrome medication treatment.
The technical scheme that the present invention solves is epimedium herb to be ground into medicated powder, usefulness alcohol reflux 2 times, each 1.5h, merge extractive liquid, filters, decompression filtrate recycling ethanol concentrates to there not being the alcohol flavor, and 12 hours after-filtration are left standstill in cold preservation, after the hydrochloric acid adjust pH, by adsorption resin column, first water eluting, the reuse ethanol elution discards eluent, reclaims eluent, concentrate drying must contain the Herba Epimedii extract of total flavones, can be effective to prepare the medicine for the treatment of climacteric syndrome.
The present invention is also applicable to arrow leaf Herba Epimedii [Epimedium sagittatum (Sieb.et Zucc.) Maxim] or pubescence Herba Epimedii [Epiimedium pubescens Maxim] or lobus cardiacus Herba Epimedii [Epimedium breviicornum Maxim], also be the Herba Epimedii certified products of pharmacopeia regulation, same experiment proves that also the extract that contains total flavones has the effect of good treatment climacteric syndrome.
Preparation method of the present invention is simple, and is easy to operate, and the general flavone content height is effective in the extract, effectively is implemented in the application in the preparation treatment climacteric syndrome medicine.
Four, the specific embodiment
Below in conjunction with concrete condition and experimental data, the specific embodiment of the present invention is elaborated.
The present invention in force, get epimedium herb, be ground into medicated powder, the mass concentration that at every turn adds 12 times of medicated powder weight is 70% alcohol reflux 2 times, each 1.5h, merge extractive liquid, filters, decompression filtrate recycling ethanol is to there not being the alcohol flavor, and the concentrated solution that to be concentrated into 50 ℃ of relative densities be 1.12-1.15, cold preservation was left standstill 12 hours, filtered, hydrochloric acid adjust pH with 1mol/L is 5.0-5.5, by AB-8 type macroporous adsorptive resins, with the distilled water eluting of 4 times of column volumes, the mass concentration of 4 times of column volumes of reuse is 10% ethanol elution earlier, discard eluent, the mass concentration with 8 times of column volumes of continuing is 70% ethanol elution, collects 70% ethanol elution, and decompression recycling ethanol also concentrates, dry, general flavone content reaches Herba Epimedii extract more than 60%, have the effect of good treatment climacteric syndrome, be effective to prepare the medicine for the treatment of climacteric syndrome, and obtained sufficient proof through experiment, relevant experimental data is as follows:
Test the influence of 1 Herba Epimedii extract (Herba Epimedii total flavones) to mice climacteric model
1 experiment material
1.1 laboratory animal
Cleaning level mice, the Kunming kind, female, 22～25g is provided by Henan Province's Experimental Animal Center.Quality certification numbering: SCXK (Henan) 2005-0001.
1.2 experiment medicine
Herba Epimedii extract (Herba Epimedii total flavones), garden, sky, Xi'an biological preparation factory, lot number: TY200080116; The GENGNIANAN capsule, Changchun Ying Ping pharmaceutcal corporation, Ltd, lot number: 20070521;
Times promise health soybean isoflavone sheet, Nanning Fresh-life Technology Co., Ltd., Nan Wei food card word (2006) 450101-000389.
1.3 reagent and instrument
Pentobarbital sodium, Shanghai chemical reagents corporation of Chinese Medicine group, lot number: F20030816; Formaldehyde, Chinese Laiyang City is the chemical industry company limited in pairs, lot number: 20040519; Sodium chloride injection, Yonghe County, Zhengzhou Pharmacy stock Co., Ltd, lot number: 20060302; Benzylpenicillin sodium for injection, Huabei Pharmaceutic Co., Ltd, lot number: X0603113; The estradiol radioimmunoassay kit, Kemei Dongya Biological Technology Co., Ltd., Beijing, lot number: the accurate word S20063125 of traditional Chinese medicines; The lutropin radioimmunoassay kit, Kemei Dongya Biological Technology Co., Ltd., Beijing, lot number: the accurate word S20063073 of traditional Chinese medicines; The follotropin radioimmunoassay kit, Kemei Dongya Biological Technology Co., Ltd., Beijing, lot number: the accurate word S20063072 of traditional Chinese medicines.
FA (N)/JA (N) series electronic balance, Shanghai Min Qiao precision instrument company limited; SN-695B type intelligence is put and is exempted from the γ measuring instrument, day ring instrument one factory of Shanghai nuclear research institute.
2 experimental techniques
2.1 modeling and administration
Modeling method: get 80 of Kunming mouses, female, 22～25g is divided into 7 groups at random, is respectively the blank group, model group, GENGNIANAN Capsules group, soybean isoflavone group, the high, medium and low dosage group of Herba Epimedii total flavones.Except that the blank group, other are respectively organized mouse peritoneal injection pentobarbital sodium 50mg/kg anesthesia venter posterior position and fix, then under the most last rib of mouse back, at midaxillary line with apart from the about 1cm infall cropping of the spinal column outside, skin and the about 0.5～1cm of dorsal muscles are cut in the sterilization back, the shinny liparitosis of a visible milky in the otch visual field, ovary be embedding wherein.Clamp liparitosis gently with pincet and pull out outside the otch, fractionation of fatty group can see a thin-line-shaped irregular ovary that is yellowish red color.Earlier fallopian tube under the ovary (comprising fat) is used the fine rule ligation during clip, extract ovary again, postoperative is taken advantage of a situation cornua uteri is put back in the abdominal cavity, and suture muscles and skin are extractd bilateral ovaries with method.Postoperative is raised meticulously, intramuscular injection penicillin prevention infection, 3d continuously.Begin behind the operation 5d by only carrying out the rat vagina plate coating checking, every day 1 time, whether 5d excises fully to determine ovary continuously.The animal that smear presents emotionally reaction discard need not, selects 60 castrations completely animal evenly be divided into 6 groups at random for experiment usefulness.
Medication: CMC-Na is dissolved in the distilled water that boils, makes 0.5% solution as solvent, wherein with following each medicine suspendible.Get the high, medium and low dosage of Herba Epimedii total flavones: 20mgmL
-1, 10mgmL
-1, 5mgmL
-1, press 400mgkg
-1, 200mgkg
-1, 100mgkg
-1Irritate stomach (be equivalent to 30 times of the clinical consumption of crude drug, 15 times, 7.5 times); GENGNIANAN capsule: 33.75mgmL
-1, press 675mgkg
-1Irritate stomach (be equivalent to clinical consumption 15 times); Soybean isoflavone: 6.25mgmL
-1, press 125mgkg
-1Irritate stomach; Model group and blank group give the CMC-Na Ji liquid with volume, and the administration volume is 0.2mL/10g, every day 1 time, continuous 3 weeks.
2.2 observation item and detection method
Each is organized the mice last and irritates 2h (fasting 15 hours) behind the stomach, and the socket of the eye vein is got blood, and separation of serum adopts E in the serum measured by radioimmunoassay
2, LH, FSH content; Dissect mice, extract thymus, spleen, uterine cancer cell, its weight in wet base of weighing is also calculated organ index (organ index=internal organs weight in wet base mg/ rat body weight g); Thymus, spleen, uterus, cerebral tissue are fixed in 10% formalin solution, paraffin embedding, section, HE dyeing, the tectology that light microscopic is observed each group down changes.
2.2.1 the assay method of estradiol
Measuring principle: radioimmunoassay method is adopted in this experiment.Add calibration object or sample, label and antibody follow procedure regulation in the test tube successively earlier, labelling and non-labeled antigen are competed mutually with finite quantity antibody to be combined, after the question response balance, add separating medium, centrifugation, free antigen is separated with antigen antibody complex, measure sedimentary radioactive intensity.Increase the then corresponding reduction of its radioactive intensity with E2 content.
Calibration reagent:
125I-E
2, 1 bottle (red liquid);
Antibody, 1 bottle (blue liquid);
Calibration object, 7 bottles (liquid), concentration is 0,10,50,100,250,750,2000pg/mL;
Separating medium, 1 bottle (suspension) is with before shaking up;
Quality controlled serum, 2 bottles (liquid) contains low, high two dosage.
Operation sequence: with round bottom polystyrene test tube numbering (by multiple pipe operation), according to the form below procedure operation
Application of sample is measured flow sheet unit: μ L
Date processing: the program of working out in advance with the γ calculating instrument has directly provided related parameter, calibration curve and sample concentration.
2.2.2 the assay method of interstitialcellstimulating hormone (ICSH)
Measuring principle: non-equilibrium radioimmunoassay method is adopted in this experiment, makes the LH in calibration object or the test serum carry out immunoreation with antibody earlier, adds a certain amount of when reaction does not reach balance
125I-LH makes itself and cold LH to unconjugated antibody still competitive combination take place.LH content in the sample is high more, and still unconjugated antibody is just few more, and institute can be bonded
125I-LH is also just few more.Add separating medium, centrifugation makes free antigen separate with antigen antibody complex, measures sedimentary radioactive intensity.Increase the then corresponding minimizing of its radioactive intensity with LH content.
Calibration reagent:
125I-LH, 1 bottle (red liquid);
Antibody, 1 bottle (blue liquid);
Calibration object, 7 bottles (liquid), concentration is 0,5,10,20,40,100,200mIU/mL;
Separating medium, 1 bottle (suspension) is with before shaking up;
Quality controlled serum, 2 bottles (liquid) contains low, high two dosage.
Operation sequence: with round bottom polystyrene test tube numbering (by multiple pipe operation), according to the form below procedure operation
Application of sample is measured flow sheet unit: μ L
Date processing: the program of working out in advance with automatic γ calculating instrument has directly provided related parameter, calibration curve and sample concentration.
2.2.3 short fourth of the twelve Earthly Branches bubble generates plain assay method
Measuring principle: non-equilibrium radioimmunoassay method is adopted in this experiment, makes the FSH in calibration object or the test serum carry out immunoreation with antibody earlier, adds a certain amount of when reaction does not reach balance
125I-FSH makes itself and cold FSH to unconjugated antibody still competitive combination take place.FSH content in the sample is high more, and still unconjugated antibody is just few more, and institute can be bonded
125I-FSH is also just few more.Add separating medium, centrifugation makes free antigen separate with antigen antibody complex, measures sedimentary radioactive intensity.Increase the then corresponding minimizing of its radioactive intensity with FSH content.
Calibration reagent:
125I-FSH, 1 bottle (red liquid);
Antibody, 1 bottle (blue liquid);
Calibration object, 7 bottles (liquid), concentration is 0,2.5,5,10,20,40,100mIU/mL;
Separating medium, 1 bottle (suspension) is with before shaking up;
Quality controlled serum, 2 bottles (liquid) contains low, high two dosage.
Operation sequence: with round bottom polystyrene test tube numbering (by multiple pipe operation), according to the form below procedure operation
Application of sample is measured flow sheet unit: μ L
Date processing: the program of working out in advance with the γ calculating instrument has directly provided related parameter, calibration curve and sample concentration.
2.3 statistical procedures
Data analysis carries out the statistical procedures of data information with SPSS 13.0 for windows statistical packages, and measurement data is with average ± standard deviation
Variance analysis (LSD method) is relatively used in expression between group.
The result
1 medicine is to the influence of climacteric model mice organ index
See Table 1.
Table 1 medicine is to climacteric mouse thymus, spleen, the exponential influence in uterus
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As can be seen from the above table, compare with the blank group, the uterus index of model group significantly reduces (P＜0.01), illustrates that the climacteric model of extracing ovary causes metratrophia.Compare with model group, the high dose Herba Epimedii total flavones can obviously improve thymus index (P＜0.05); Low, middle dosage Herba Epimedii total flavones can obviously improve uterus index (P＜0.05); High dose Herba Epimedii total flavones and soybean isoflavone can significantly improve uterus index (P＜0.01),
2 medicines are to the influence of climacteric model mice serum gonadal hormone content
See Table 2.
Table 2 medicine is to E in the climacteric model mice serum
2, FSH, LH content influence
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As can be seen from the above table, compare E in the model group serum with the blank group
2Level significantly descends (P＜0.01), and LH, FSH level all significantly raise (P＜0.01), and the sex hormone level disorder in the model serum of climacteric due to the excision ovary be described.Compare all can significantly the raise serum E of climacteric animal pattern of each administration group with model group
2Level (P＜0.01); GENGNIANAN capsule and the LH level (P＜0.05) low, that the high dose Herba Epimedii total flavones can obviously reduce rising, middle dosage Herba Epimedii total flavones can significantly reduce LH level (P＜0.01); The GENGNIANAN capsule can obviously reduce the FSH level (P＜0.05) of rising, and each dosage group Herba Epimedii total flavones and soybean isoflavone all can significantly reduce FSH level (P＜0.01).
3 medicines are to the influence of climacteric model mice organ-tissue form
3.1 medicine is to the influence of climacteric model mice uterine cancer cell form
Adopt micrometer to measure the widest part and the narrowest place of endometrium and flesh layer thickness, ask mean, use method of counting, calculate body of gland quantity in the inner membrance, ask mean, the results are shown in Table 3.
Table 3 medicine is to the influence of climacteric model mice uterus pathological change
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As can be known from the above table, compare with the blank group, model group endometrium of mouse thickness, flesh layer thickness significantly reduce (P＜0.01), and the body of gland number significantly reduces (P＜0.01), illustrate that mice makes metratrophia behind the climacteric model, and body of gland reduces.Compare with model group, each medicine all can significantly increase endometrium thickness (P＜0.01); High dose Herba Epimedii total flavones (P＜0.01) can remarkable and obviously increase the flesh layer thickness respectively with GENGNIANAN capsule (P＜0.05); High, middle dosage Herba Epimedii total flavones and GENGNIANAN capsule all can significantly improve body of gland quantity in the inner membrance (P＜0.01).
3.2 medicine is to the influence of climacteric model mice thymic tissue form
Adopt micrometer to measure the widest part and the narrowest place of thymic cortex, ask mean, drop on lymphocyte number on the baseline, the results are shown in Table 4 with the baseline calculating of micrometer.
Table 4 medicine is to the influence of climacteric model mice thymus pathological change
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As can be known from the above table, compare with the blank group, model group mouse thymus cortical thickness and cortilymph number of cells significantly reduce (P＜0.01), illustrate that mice makes the long-pending atrophy of climacteric model metathorax body of gland.Compare with model group, high, middle dosage Herba Epimedii total flavones can significantly thicken thymic cortex (P＜0.01), and the GENGNIANAN capsule can obviously thicken thymic cortex (P＜0.05); High, middle dosage Herba Epimedii total flavones and GENGNIANAN capsule all can significantly increase cortilymph number of cells (P＜0.01).
3.3 medicine is to the influence of climacteric model mice spleen tissue form
Adopting the baseline of micrometer to drop on the splenic nodule, is the splenic nodule thickness size that both sides are measured at the center respectively with the central artery, asks mean; Calculate the number that drops on the lymphocyte number of both sides on the baseline simultaneously, ask mean, the results are shown in Table 5.
Table 5 medicine is to the influence of climacteric model mice spleen pathological change
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As can be known from the above table, compare with the blank group, model group mice spleen brief summary volume and lymphocyte number significantly reduce (P＜0.01), illustrate that mice makes spleen atrophy behind the climacteric mould.Compare with model group, each medicine all can enlarge markedly the splenic nodule volume, and improves lymphocyte quantity (P＜0.01).
Test of the influence of 2 Herba Epimedii total flavones to rat climacteric model
1 experiment material
1.1 laboratory animal
The Wistar rat, female, 160～200g is provided by Hebei province's medical experiment animal center, the quality certification number: 701022.
1.2 experiment medicine
Herba Epimedii extract (Herba Epimedii total flavones), garden, sky, Xi'an biological preparation factory, lot number: TY200080116;
The GENGNIANAN capsule, Changchun Ying Ping pharmaceutcal corporation, Ltd, lot number: 20070521;
Times promise health soybean isoflavone sheet, Nanning Fresh-life Technology Co., Ltd., Nan Wei food card word (2006) 450101-000389.
1.3 reagent and instrument
Pentobarbital sodium, Shanghai chemical reagents corporation of Chinese Medicine group, lot number: F20030816; Formaldehyde, Chinese Laiyang City is the chemical industry company limited in pairs, lot number: 20040519; Sodium chloride injection, Yonghe County, Zhengzhou Pharmacy stock Co., Ltd, lot number: 20060302; Benzylpenicillin sodium for injection, Huabei Pharmaceutic Co., Ltd, lot number: X0603113; The estradiol radioimmunoassay kit, Kemei Dongya Biological Technology Co., Ltd., Beijing, lot number: the accurate word S20063125 of traditional Chinese medicines; The lutropin radioimmunoassay kit, Kemei Dongya Biological Technology Co., Ltd., Beijing, lot number: the accurate word S20063073 of traditional Chinese medicines; The follotropin radioimmunoassay kit, Kemei Dongya Biological Technology Co., Ltd., Beijing, lot number: the accurate word S20063072 of traditional Chinese medicines; The interleukin II radioimmunoassay kit, Beijing pul great achievement bio tech ltd; The Bone Gla protein radioimmunoassay kit, Beijing pul great achievement bio tech ltd.
FA (N)/JA (N) series electronic balance, Shanghai Min Qiao precision instrument company limited; SN-695 B type intelligence is put and is exempted from the γ measuring instrument, day ring instrument one factory of Shanghai nuclear research institute.
2 experimental techniques
2.1 modeling and administration
Modeling method: get 80 of Wistar rats, female, 160～200g is divided into 7 groups at random, is respectively the blank group, model group, GENGNIANAN Capsules group, soybean isoflavone group, the high, medium and low dosage group of Herba Epimedii total flavones.Except that the blank group, each is organized the equal lumbar injection pentobarbital sodium of rat 50mg/kg anesthesia venter posterior position and fixes, then under the most last rib of rat back, at midaxillary line with apart from the about 2cm infall cropping of the spinal column outside, skin and the about 1.5～2cm of dorsal muscles are cut in the sterilization back, the shinny liparitosis of a visible milky in the otch visual field, ovary be embedding wherein.Clamp liparitosis gently with pincet and pull out outside the otch, fractionation of fatty group can see a thin-line-shaped irregular ovary that is yellowish red color.Earlier fallopian tube under the ovary (comprising fat) is used the fine rule ligation during clip, extract left ovary fully, the right side extracts 80%.Postoperative is taken advantage of a situation cornua uteri is put back in the abdominal cavity, suture muscles and skin, and postoperative is raised meticulously, and the injection penicillin is in case infect continuous 3d.Begin behind the operation 5d by only carrying out the rat vagina plate coating checking, every day 1 time, whether 5d excises fully to determine ovary continuously.The animal that smear presents emotionally reaction discard need not, selects 60 castrations completely animal evenly be divided into 6 groups at random for experiment usefulness.
Medication: CMC-Na is dissolved in the distilled water that boils, makes 0.5% solution as solvent, wherein with following each medicine suspendible.The high, medium and low dosage of Herba Epimedii total flavones: 10.5mgmL
-1, 7mgmL
-1, 3.5mgmL
-1, press 210mgkg
-1, 140mgkg
-1, 70mgkg
-1Irritate stomach (be equivalent to 15 times of the clinical consumption of crude drug, 10 times, 5 times); GENGNIANAN capsule: 22.5mgmL
-1, press 450mgkg
-1Irritate stomach (be equivalent to clinical consumption 10 times); Soybean isoflavone: 4.25mgmL
-1, press 85mgkg
-1Irritate stomach (be equivalent to clinical consumption 15 times); Model group and blank group give the CMC-Na solution with volume, and the administration volume is 2mL/100g, every day 1 time, continuous 5 weeks.
2.2 observation item and detection method
Each is organized the rat last and irritates 2h (fasting 15 hours) behind the stomach, and the socket of the eye vein is got blood, and separation of serum adopts E in the serum measured by radioimmunoassay
2, LH, FSH (method with experiment 2) and IL-2, BGP content; Dissect rat, extract thymus, spleen, uterine cancer cell, its weight in wet base of weighing is also calculated organ index (organ index=internal organs wet difficult mg/ rat body weight g); Thymus, spleen, uterus, cerebral tissue are fixed in 10% formalin solution, paraffin embedding, section, HE dyeing, the tectology that light microscopic is observed each group down changes.
2.2.1 the assay method of interleukin-2
Measuring principle: utilize the liquid phase competition to suppress principle, adopt counterbalanced procedure that sample is measured.Testing sample or standard add being at war with property association reaction together with antiserum of limiting the quantity of and labelled antigen, after reacting completely, add immune release agent, isolate antigen-antibody complex, measure the CMP number on gamma counter.
Reagent preparation: buffer (PBS) liquid: 1 bottle (liquid), 30mL shakes up the back and directly uses;
IL-2 standard substance: 5 bottles (white, lyophilized powder): with buffer 0.4mL dissolving, concentration is 1,3,9,27,81ng/mL;
IL-2 antiserum: 1 bottle (white, lyophilized powder): with buffer 12mL dissolving;
125I-IL-2:1 bottle (white, lyophilized powder): with before dissolving with buffer 10.5mL;
The PR separating medium: 1 bottle (liquid), 60mL fully shakes up before the use, directly uses.
Operation sequence: counterbalanced procedure is adopted in this experiment, gets polystyrene test tube numbering, the according to the form below operation.
IL-2RIA liquid feeding program unit: μ L
Date processing: the program of working out in advance with the γ calculating instrument has directly provided related parameter, calibration curve and sample concentration.
2.2.2 the assay method of Bone Gla protein
Measuring principle: utilize the liquid phase competition to suppress principle, adopt counterbalanced procedure that sample is measured.Testing sample or standard add being at war with property association reaction together with antiserum of limiting the quantity of and labelled antigen, after reacting completely, add immune release agent, isolate antigen-antibody complex, measure the CMP number on gamma counter.
Reagent preparation: buffer (PBS) liquid: 1 bottle (liquid), 10mL is diluted to 50mL with preceding adding distil water;
BGP standard substance: 5 bottles (white, lyophilized powder): with preceding every bottle with buffer 1mL dissolving, concentration is 1,2,4,8,16ng/mL;
BGP antiserum: 1 bottle (white, lyophilized powder): with buffer 10.5mL dissolving;
125I-BGP:1 bottle (white, lyophilized powder): with before dissolving with buffer 10.5mL;
The PR separating medium: 1 bottle (liquid), 60mL fully shakes up before the use, directly uses.
Operation sequence: counterbalanced procedure is adopted in this experiment, gets polystyrene test tube numbering, the according to the form below operation.
BGP RIA liquid feeding program unit: μ L
Date processing: the program of working out in advance with the γ calculating instrument has directly provided related parameter, calibration curve and sample concentration.
2.3 statistical procedures
Data analysis carries out the statistical procedures of data information with SPSS 13.0 for windows statistical packages, and measurement data is with average ± standard deviation
Variance analysis (LSD method) is relatively used in expression between group.
The result
1 medicine is to the influence of climacteric rat model organ index
See Table 6.
Table 6 medicine is to climacteric rat model thymus, spleen, the exponential influence in uterus
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As seen from the above table, compare with the blank group, the index and spleen index of model group, uterus index all significantly descend (P＜0.01), illustrates that not exclusively spleen and metratrophia appear in the hot flushes in rats model due to the excision ovary.Compare the index and spleen index of each administration group be significantly increased (P＜0.01) with model group; The dosage group can obviously improve its uterus index (P＜0.05) in the Herba Epimedii total flavones, and the uterus index of Herba Epimedii total flavones high dose group significantly improves (P＜0.01).
2 medicines are to the influence of climacteric rat model serum gonadal hormone content
See Table 7.
Table 7 medicine is to E in the climacteric rat model serum
2, FSH, LH content influence
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As seen from the above table, compare the serum E of model group with the blank group
2Level significantly descends (P＜0.01), and LH, FSH level all significantly raise (P＜0.01), illustrates that not exclusively extracing ovary causes the sexual hormone disturbance of hot flushes in rats model.Compare with model group, each dosage group of Herba Epimedii total flavones and soybean isoflavone group all can significantly improve serum E
2Level (P＜0.01), Herba Epimedii total flavones high dose group and soybean isoflavone group can significantly reduce the serum Lh level, and the Herba Epimedii total flavones high dose group also can significantly reduce FSH level (P＜0.01).
3 medicines are to the influence of IL-2, BGP content in the climacteric rat model serum
See Table 8.
Table 8 medicine is to the influence of IL-2, BGP content in the climacteric rat model serum
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As seen from the above table, compare with the blank group, serum il of model group-2 and BGP level all significantly reduce (P＜0.01), hot flushes in rats model immunity of organisms be describeds not exclusively due to the excision ovary and bone is calcareous decreases.Compare with model group, GENGNIANAN capsule (P＜0.01) and high dose Herba Epimedii total flavones (P＜0.05) respectively can be significantly and obvious raising serum il-2 level; Soybean isoflavone significantly improves serum BGP level (P＜0.05), and other each administration groups all can obviously improve serum BGP level (P＜0.05).
4 medicines are to the influence of climacteric rat model organ-tissue form
4.1 medicine is to the influence of climacteric rat model uterine cancer cell form
Body of gland under the endometrial mucosa thickness of testing each treated animal, mucosal epithelium cell, the mucosa is carried out three-dimensional metrological mensuration, the results are shown in Table 9.
Table 9 medicine is to the influence of climacteric rat model uterine cancer cell pathological change
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As can be known from the above table, compare with the blank group, model group rat endometrium thickness, flesh layer thickness significantly reduce (P＜0.01), illustrate that rat makes metratrophia behind the climacteric model.Compare with model group, high and low dose Herba Epimedii total flavones and GENGNIANAN capsule can significantly increase endometrium thickness (P＜0.01), and middle dosage Herba Epimedii total flavones can obviously increase endometrium thickness (P＜0.05); Each medicine all can significantly increase body of gland quantity in mucomembranous epithelial cell and the inner membrance (P＜0.01).
4.2 medicine is to the influence of climacteric rat model thymus form
Adopt micrometer to measure the widest part and the narrowest place of thymic cortex, ask mean, drop on lymphocyte number on the baseline with the baseline calculating of micrometer.Measurement result sees Table 10.
Table 10 each treated animal thymic cortex thickness of experiment and the Cytometric measurement result of cortilymph
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As can be known from the above table, compare with the blank group, model group rat chest gland cortical thickness and cortilymph number of cells significantly reduce (P＜0.01), illustrate that rat makes the long-pending atrophy of climacteric model metathorax body of gland.Compare with model group, high, medium and low dosage Herba Epimedii total flavones and GENGNIANAN capsule all can significantly thicken thymic cortex (P＜0.01); High, middle dosage Herba Epimedii total flavones, GENGNIANAN capsule and soybean isoflavone all can significantly increase cortilymph number of cells (P＜0.01).
4.3 medicine is to the influence of climacteric rat model spleen tissue form
Adopting the baseline of micrometer to drop on the splenic nodule, is the splenic nodule thickness size that both sides are measured at the center respectively with the central artery, asks mean; Calculate the number that drops on the lymphocyte number of both sides on the baseline simultaneously, ask mean.Record and the results are shown in Table 11.
Table 11 medicine is to the influence of hot flushes in rats model spleen pathological change
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As can be known from the above table, compare with the blank group, model group rat splenic nodule volume significantly reduces (P＜0.01), and the lymphocyte number obviously reduces (P＜0.01), illustrates that rat makes spleen atrophy behind the climacteric model.Compare with model group, middle dosage Herba Epimedii total flavones, GENGNIANAN capsule, soybean isoflavone all can enlarge markedly splenic nodule volume (P＜0.01), and soybean isoflavone can significantly improve lymphocyte quantity (P＜0.01).
4.4 medicine is to the influence of climacteric rat model hypothalamus form
See Table 12.
The influence that table 12 medicine changes hot flushes in rats model hypothalamus pathology
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As can be known from the above table, compare with the blank group, the remarkable atrophy of model group rat hypothalamus neurocyte (P＜0.01), the neurocyte endochylema obviously reduces (P＜0.05), illustrates that rat makes hypothalamus generation pathological changes behind the climacteric model.Compare with model group, low dosage Herba Epimedii total flavones, GENGNIANAN capsule, soybean isoflavone all can significantly increase hypothalamus neurocyte quantity (P＜0.01); The GENGNIANAN capsule can significantly increase hypothalamus neurocyte endochylema content (P＜0.01).
Test of the influence of 3 Herba Epimedii total flavones to rat involutional depression model
1 experiment material
1.1 laboratory animal
The Wistar rat, female, 160～200g is provided by Hebei province's medical experiment animal center, the quality certification number: 701022.
1.2 experiment medicine
Herba Epimedii extract (Herba Epimedii total flavones), garden, sky, Xi'an biological preparation factory, lot number: TY200080116;
The GENGNIANAN capsule, Changchun Ying Ping pharmaceutcal corporation, Ltd, lot number: 20070521;
Times promise health soybean isoflavone sheet, Nanning Fresh-life Technology Co., Ltd., Nan Wei food card word (2006) 450101-000389.
1.3 reagent and instrument
Pentobarbital sodium, Shanghai chemical reagents corporation of Chinese Medicine group, lot number: F20030816; Formaldehyde, Chinese Laiyang City is the chemical industry company limited in pairs, lot number: 20040519; Sodium chloride injection, Yonghe County, Zhengzhou Pharmacy stock Co., Ltd, lot number: 20060302; Benzylpenicillin sodium for injection, Huabei Pharmaceutic Co., Ltd, lot number: X0603113; O-phthalaldehyde(OPA), chemical reagent purchase and supply 5-linked chemical plant, Shanghai produces, lot number 20060101; N-butyl alcohol, the production of reagent company limited, lot number 050105 are learned in triumphant Tonghua, Tianjin; Hydrochloric acid, Laiyang City chemical industry company limited are in pairs produced lot number 20060120; Normal heptane, four-way chemical plant, Tianjin produces, lot number 20051105; Dopamine (DA), Sigma company; 5-hydroxy tryptamine (5-HT), Sigma company; Sodium metaperiodate, Tianjin good fortune chemical reagent in morning factory produces, lot number 20021105; EDTA-2Na, the production of precious letter bio tech ltd, lot number 20051021; Sodium hydrogen phosphate, the production of reagent company limited, lot number 20051212 are learned in triumphant Tonghua, Tianjin; Sodium dihydrogen phosphate, Tianjin good fortune chemical reagent in morning factory produces, lot number 20040105; Sodium acetate, chemical reagent three factories in Tianjin produce, lot number 20030808; Sodium sulfite, the production of reagent company limited, lot number 20051108 are learned in triumphant Tonghua, Tianjin; Dehydrated alcohol, Laiyang City chemical industry company limited are in pairs produced lot number 20061203; Iodine, Tianjin Kermel Chemical Reagent Co., Ltd. produces, lot number 20050405; The estradiol radioimmunoassay kit, Kemei Dongya Biological Technology Co., Ltd., Beijing, lot number: the accurate word S20063125 of traditional Chinese medicines; The lutropin radioimmunoassay kit, Kemei Dongya Biological Technology Co., Ltd., Beijing, lot number: the accurate word S20063073 of traditional Chinese medicines; The follotropin radioimmunoassay kit, Kemei Dongya Biological Technology Co., Ltd., Beijing, lot number: the accurate word S20063072 of traditional Chinese medicines; The interleukin II radioimmunoassay kit, Beijing pul great achievement bio tech ltd; The Bone Gla protein radioimmunoassay kit, Beijing pul great achievement bio tech ltd.
FA (N)/JA (N) series electronic balance, Shanghai Min Qiao precision instrument company limited; SN-695 B type intelligence is put and is exempted from the γ measuring instrument, day ring instrument one factory of Shanghai nuclear research institute; F-4500 type spectrofluorophotometer, Hitachi, Ltd produces; Thermostat water bath, the bright instrument plant in Beijing produces; Centrifuge, Medical Instruments repair shop in Beijing produces; Adjustable pipette, Lei Bo Analytical Instrument Co., Ltd in Shanghai produces; Calorstat, the bright forever Medical Instruments in Beijing factory produces; The TGL-16G High speed refrigerated centrifuge, Anting Scientific Instrument Factory, Shanghai produces; Rat opens case, self-control.
2 experimental techniques
2.1 modeling and administration
Modeling method: get 80 of Wistar rats, female, 160～200g is divided into 7 groups at random, is respectively the blank group, model group, GENGNIANAN Capsules group, soybean isoflavone group, the high, medium and low dosage group of Herba Epimedii total flavones.Except that the blank group, each is organized the equal lumbar injection pentobarbital sodium of rat 50mg/kg anesthesia venter posterior position and fixes, then under the most last rib of rat back, at midaxillary line with apart from the about 2cm infall cropping of the spinal column outside, skin and the about 1.5～2cm of dorsal muscles are cut in the sterilization back, the shinny liparitosis of a visible milky in the otch visual field, ovary be embedding wherein.Clamp liparitosis gently with pincet and pull out outside the otch, fractionation of fatty group can see a thin-line-shaped irregular ovary that is yellowish red color.Earlier fallopian tube under the ovary (comprising fat) is used the fine rule ligation during clip, extract left ovary fully, the right side extracts 80%.Postoperative is taken advantage of a situation cornua uteri is put back in the abdominal cavity, suture muscles and skin, and postoperative is raised meticulously, injects penicillin with prevention infection, continuously 3d.Operation begins behind the 5d by only carrying out the rat vagina plate coating checking, every day 1 time, the animal that 5d continuously, smear present emotionally reaction discard need not, selects 60 castrations completely animal evenly be divided into 6 groups at random for experiment usefulness.After the two weeks, 5 raisings of the every cage of normal group, normal diet drinking-water is not given outside any stimulation, and the modeling type is every cage for 6 groups and raises 1 rat, gives different stimulated every day at random.7 kinds of stress factors are used in 20d by random method: 1. illumination (24h) all night; 2. fasting (24h); 3. prohibit water (24h); 4. 4 ℃ of cold water were swum 5 minutes; 5. pressed from both sides tail in 1 minute; 6. level vibration in 5 minutes; 7. 2h behavior restriction.Give a kind of stimulation every day at random, every kind of stimulation can not occur continuously, continuously 20d.
Medication: CMC-Na is dissolved in the distilled water that boils, makes 0.5% solution as solvent, wherein with following each medicine suspendible.The high, medium and low dosage of Herba Epimedii total flavones: 10.5mgmL
-1, 7mgmL
-1, 3.5mgmL
-1, press 210mgkg
-1, 140mgkg
-1, 70mgkg
-1Irritate stomach (be equivalent to 15 times of the clinical consumption of crude drug, 10 times, 5 times); GENGNIANAN capsule: 22.5mgmL
-1, press 450mgkg
-1Irritate stomach (be equivalent to clinical consumption 10 times); Soybean isoflavone: 4.25mgmL
-1, press 85mgkg
-1Irritate stomach (be equivalent to clinical consumption 15 times); Model group and blank group give the CMC-Na solution with volume, and the administration volume is 2mL/100g, every day 1 time, continuous 5 weeks.
2.2 observation item and detection method
2.2.1 sucrose solution intake test
Chronic stress stimulate to finish the back test and respectively organizes rat 1% sucrose water consumption, prohibits water 12 hours before the test, measure 24 hours in the sucrose water intake.
2.2.2Open field method test
Experimental provision is the spacious case of cube, high 40cm, and length and width 80cm, perisporium, bottom surface are black, 25 of being equated by area in bottom surface form, and divide with white line.During experiment rat is placed in the grid of spacious case center, observe rat was passed through bottom surface block number (grid that four paws all enters can count) in 30 minutes horizontal anomalous movement (crossing) score, and vertical activity (rearing) score of rear number of times (two fore paws soar or seek connections with wall).Feces thoroughly must be removed after each the experiment, every animal 2 hours mensuration 1 time after administration is respectively organized score difference.This experiment is quietly being carried out in the room.
2.2.3 influence to monoamine neurotransmitter in the brain
To put to death behind the rat extracting blood, get brain, preparation brain tissue homogenate is with the content of monoamine neurotransmitter 5-HT, DA in the fluorescence spectrometry brain tissue homogenate.
Weigh after cerebral tissue removed blood film, olfactory bulb and cerebellum, in ice-water bath, make homogenate with 5 times of amount acidify n-butyl alcohol (to add concentrated hydrochloric acid 0.85mL in the 1000mL n-butyl alcohol).Vortex vibration 5min, centrifugal 5min (3000 rev/mins).Get supernatant 2.5mL, put into another band plug centrifuge tube, add normal heptane 5mL and 0.1molL
-1Hydrochloric acid 1.2mL, vortex vibration 5min, centrifugal 5min, (water I contains 5-HT, DA) to obtain layering solution.
5-HT, DA standard substance are used 0.01mol respectively
-L
1Hydrochloric acid is mixed with 500 μ gmL
-1As the standard stock solution, deposit in 2 ℃ of refrigerators, face the time spent and respectively get above-mentioned stock solution 0.25mL, use 0.01molL
-1Hydrochloric acid is diluted to 100mL standard application liquid, each standard substance concentration 1.25 μ gmL
-1Water intaking phase I 0.5mL adds 0.5% cysteine (0.1molL
-1The fresh preparation of hydrochloric acid) 0.1mL, 0.005% o-phthalaldehyde(OPA) 3mL is (with 10molL
-1Hydrochloric acid is made into temporarily), 0.02% sodium iodate 0.1mL, boiling water bath 10 minutes is cooled to room temperature, measures.
Standard pipe is got standard application liquid 0.5mL, and blank pipe is got double distilled water 0.5mL, adds acidify n-butyl alcohol 4.5mL, vortex vibration 5min, and centrifugal 5min (3000 rev/mins) gets supernatant 2.5mL, puts into another band plug centrifuge tube, adds normal heptane 5mL and 0.1molL
-1Hydrochloric acid 1.0mL, vortex vibration 5min, centrifugal 5min obtains layering solution (water I contains 5-HT, DA).Water intaking phase I 0.5mL adds 0.5% cysteine 0.1mL, and 0.005% o-phthalaldehyde(OPA) 3mL is (with 10molL
-1Hydrochloric acid is made into), 0.02% sodium metaperiodate 0.1mL, boiling water bath 10 minutes is cooled to room temperature, measures.
5-HT maximum emission wavelength (em) 475nm, maximum excitation wavelength (ex) 355nm.
Water intaking phase I 0.5mL adds 1/15molL
-1Phosphate buffered saline(PBS) (pH=7.2) 1.7mL, 0.1molL
-1EDTA2Na is (with 1molL
-1The sodium acetate solution preparation is with 10molL
-1Sodium hydroxide is transferred pH to 7.0) 0.4mL, add iodine reagent and (be configured to 5% IodineSodium Solution with 70% ethanol, with 0.02molL
-1Iodine liquid faces with preceding and mixes with 1: 1 volume ratio) 0.1mL, left standstill 2 minutes, add alkaline sodium sulfite solution 0.5mL (25% sodium sulfite 1.0mL, the interim mixing of 5mol/L sodium hydroxide 9mL), left standstill 2 minutes, add 6molL
-1Acetic acid solution 0.5mL, boiling water bath 20 minutes is cooled to room temperature, measures DA.DA maximum emission wavelength (em) 370nm, maximum excitation wavelength (ex) 322nm.
Computing formula: sample monoamine transmitters content (ugg
-1Cerebral tissue)=(Ri-Rib) (mL) * 1.25 (ugmL/(Rr-Rrb) * 0.5
-1) the ÷ real EEG heavily
Ri: be test sample fluorescence reading; Rib: be the blank fluorescence reading of test sample;
Rr: be reference substance fluorescence reading; Rrb: be the blank reading of reference substance.
2.2.4 the mensuration of serological index
Each is organized the rat last and irritates 2h (fasting 15 hours) behind the stomach, measures the socket of the eye vein and gets blood, and separation of serum adopts E in the serum measured by radioimmunoassay
2, LH, FSH, IL-2, BGP content (method is with experiment 2,3).
2.2.5 organ index and pathological section
Dissect rat, extract thymus, spleen, uterine cancer cell, its weight in wet base of weighing is also calculated organ index (organ index=internal organs weight in wet base mg/ rat body weight g); Thymus, spleen, uterus, cerebral tissue are fixed in 10% formalin solution, paraffin Bao Li, section, HE dyeing, the tectology that light microscopic is observed each group down changes.
2.3 statistical procedures
Data analysis carries out the statistical procedures of data information with SPSS 13.0 for windows statistical packages, and measurement data is with average ± standard deviation
Variance analysis (LSD method) is relatively used in expression between group.
The result
1 medicine is to the influence of involutional depression rat model sucrose solution consumption
See Table 13.
Table 13 medicine is to the influence of 24 hours sucrose solution intakes of involutional depression rat model
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As seen from the above table, compare with the blank group, 24 hours sucrose solution intakes of model group significantly reduce (P＜0.01), have reflected the extent of reaction reduction of rat to award.Compare with model group, GENGNIANAN Capsules group and low dosage Herba Epimedii total flavones all can obviously improve 24 hours sucrose solution intake of depression rat (P＜0.05), and middle and high dosage Herba Epimedii total flavones and soybean isoflavone all can significantly improve 24 hours sucrose solution intake of depression rat (P＜0.01).
2 medicines merge the influence that the depression model rat behavior is learned to climacteric
See Table 14.
Table 14 medicine is to the influence of involutional depression rat model level-vertical integration
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As seen from the above table, compare with the blank group, the level of model group, the integration that moves both vertically all significantly reduce (P＜0.01), have reflected the curious degree reduction of rat to fresh environment.Compare with model group, the GENGNIANAN capsule, middle and high dosage Herba Epimedii total flavones group all can significantly improve the level-integration that moves both vertically (P＜0.01) of rat, low dosage Herba Epimedii total flavones and soybean isoflavone can significantly improve its horizontal movement integration (P＜0.01), and obviously improve its integration that moves both vertically (P＜0.05).
3 medicines merge levels of monoamine neurotransmitters in the depression model rat brain to climacteric
See Table 15.
Table 15 medicine is to the influence of 5-HT, DA level in the involutional depression rat model brain
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As seen from the above table, compare with the blank group, the 5-HT in the model group brain homogenate, DA level all significantly reduce (P＜0.01), illustrate and make the success of rat chronic stress depression model.Compare with model group, GENGNIANAN capsule, middle dosage Herba Epimedii total flavones can obviously improve the 5-HT level (P＜0.05) in the brain homogenate, and soybean isoflavone, high dose Herba Epimedii total flavones can significantly improve its level (P＜0.01); GENGNIANAN capsule, soybean isoflavone can obviously improve the DA level (P＜0.05) in the brain homogenate, and middle and high dosage Herba Epimedii total flavones is its level of upward adverse flow of QI (P＜0.01) significantly.
4 medicines merge the exponential influence of depression model Rats Organs and Tissues to climacteric
See Table 16.
Table 16 medicine is to involutional depression rat model thymus, spleen, the exponential influence in uterus
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As seen from the above table, compare with the blank group, the model group index and spleen index obviously reduces (P＜0.05), and thymus index, uterus index significantly reduce (P＜0.01), illustrates that the atrophy of thymus, spleen and uterine cancer cell appears in the involutional depression rat model.Compare with model group, soybean isoflavone can significantly improve the thymus index (P＜0.01) of animal pattern, and the high dose Herba Epimedii total flavones can obviously improve its uterus index (P＜0.05).
5 medicines merge the influence of depression model rat blood serum gonadal hormone to climacteric
See Table 17.
Table 17 medicine is to E in the involutional depression rat model serum
2, FSH, LH content influence
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As seen from the above table, compare the serum E of model group with the blank group
2Level significantly descends (P＜0.01), and LH, FSH level all significantly raise (P＜0.01), and the sexual hormone disturbance of involutional depression rat model is described.Compare with model group, soybean isoflavone, high dose Herba Epimedii total flavones can significantly improve serum E
2Level (P＜0.01); Soybean isoflavone (P＜0.01), high dose Herba Epimedii total flavones (P＜0.05) can remarkable and obviously reduce the serum Lh level respectively; GENGNIANAN capsule, soybean isoflavone and middle dosage Herba Epimedii total flavones can obviously reduce serum FSH level (P＜0.05), and the high dose Herba Epimedii total flavones can significantly reduce FSH level (P＜0.01).
6 medicines are to the influence of IL-2, BGP content in the involutional depression rat model serum
See Table 18.
Table 18 medicine is to the influence of IL-2, BGP content in the involutional depression rat model serum
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As seen from the above table, compare with the blank group, model group serum il-2, BGP level significantly reduce (P＜0.01), and the immunity that the involutional depression rat is described decreases and osteoporosis occurs.Compare GENGNIANAN capsule, high dose Herba Epimedii total flavones serum il-2 level (P＜0.01) that can significantly raise, the middle dosage Herba Epimedii total flavones IL-2 level (P＜0.05) that can obviously raise with model group; The high dose Herba Epimedii total flavones can obviously improve serum BGP content (P＜0.05).
7 medicines are to the influence of involutional depression rat model organ-tissue form
7.1 medicine is to the influence of involutional depression rat model uterine cancer cell form
Body of gland under the endometrial mucosa thickness of testing each treated animal, mucosal epithelium cell, the mucosa is carried out three-dimensional metrological mensuration, the results are shown in Table 19.
Table 19 medicine is to the influence of involutional depression rat model uterus pathological change
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As can be known from the above table, compare with the blank group, model group rat endometrium thickness, mucomembranous epithelial cell, submucosal gland body significantly reduce (P＜0.01), illustrate that rat makes metratrophia behind the involutional depression model.Compare with model group, high and low dose Herba Epimedii total flavones, soybean isoflavone all can significantly increase endometrium thickness (P＜0.01), and the GENGNIANAN capsule can obviously increase endometrium thickness; Each medicine all can significantly increase body of gland quantity (P＜0.01) in mucomembranous epithelial cell (P＜0.01), the inner membrance.
7.2 medicine is to the influence of involutional depression rat model thymus form
Adopt micrometer to measure the widest part and the narrowest place of thymic cortex, ask mean, drop on lymphocyte number on the baseline with the baseline calculating of micrometer.Measurement result sees Table 20.
Table 20 medicine is to the influence of involutional depression rat model thymus pathomorphism
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As can be known from the above table, model group is compared there was no significant difference with the blank group, and it is different little to illustrate that rat is made involutional depression model metathorax body of gland product moment.Compare with model group, high, medium and low dosage Herba Epimedii total flavones and GENGNIANAN capsule all can significantly thicken thymic cortex (P＜0.01); High, medium and low dosage Herba Epimedii total flavones, GENGNIANAN capsule all can significantly increase cortilymph number of cells (P＜0.01), and soybean isoflavone can obviously increase cortilymph number of cells (P＜0.05).
7.3 medicine is to the influence of involutional depression rat model spleen form
Adopting the baseline of micrometer to drop on the splenic nodule, is the splenic nodule thickness size that both sides are measured at the center respectively with the central artery, asks mean; Calculate the number that drops on the lymphocyte number of both sides on the baseline simultaneously, ask mean.Record and the results are shown in Table 21.
Table 21 medicine is to the influence of involutional depression rat model spleen pathological change
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
Table is compared with the blank group as can be known from the graph, and model group rat splenic nodule volume significantly reduces (P＜0.01), and the lymphocyte number obviously reduces (P＜0.01), illustrates that rat makes spleen atrophy behind the involutional depression model.Compare with model group, middle dosage Herba Epimedii total flavones, GENGNIANAN capsule, soybean isoflavone all can enlarge markedly splenic nodule volume (P＜0.01), and middle dosage Herba Epimedii total flavones, GENGNIANAN capsule can significantly improve lymphocyte quantity (P＜0.01).
7.4 medicine is to the influence of involutional depression rat model hypothalamus form
See Table 22.
The influence that table 22 medicine changes involutional depression rat model hypothalamus pathology
Annotate: compare with the blank group,
△P＜0.05,
△ △P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group
As can be known from the above table, compare with the blank group, the remarkable atrophy of model group rat hypothalamus neurocyte (P＜0.01), the neurocyte endochylema significantly reduces (P＜0.01), illustrates that rat makes hypothalamus generation pathological changes behind the involutional depression model.Compare with model group, high, medium and low dosage Herba Epimedii total flavones, GENGNIANAN capsule all can significantly increase hypothalamus neurocyte quantity (P＜0.01); High, middle dosage Herba Epimedii total flavones, GENGNIANAN capsule all can significantly increase the neural thin amount of hypothalamus (P＜0.01); High, middle dosage Herba Epimedii total flavones, GENGNIANAN capsule all can significantly increase hypothalamus neurocyte endochylema content (P＜0.01).
Above-mentioned experiment is through repeated multiple times (12 times), all obtained identical and similar result, the extraction and the experiment of said method also arrow leaf Herba Epimedii, pubescence Herba Epimedii, lobus cardiacus Herba Epimedii have been done simultaneously as stated above respectively, its extract has all obtained proof, have and the same or analogous result of the test of Herba Epimedii, therefore prove that fully Herba Epimedii has the effect of good treatment climacteric syndrome, its extract is effectively used in the medicine of preparation treatment climacteric syndrome, is that one on the Chinese medicine is innovated greatly.
Claims (2)
1. Herba Epimedii extract for the treatment of climacteric syndrome, it is characterized in that, this extract is, gets epimedium herb, is ground into medicated powder, the mass concentration that at every turn adds 12 times of medicated powder weight is 70% alcohol reflux 2 times, each 1.5h, merge extractive liquid, filters, decompression filtrate recycling ethanol is to there not being the alcohol flavor, and the concentrated solution that to be concentrated into 50 ℃ of relative densities be 1.12-1.15, cold preservation was left standstill 12 hours, filtered, hydrochloric acid adjust pH with 1mol/L is 5.0-5.5, by AB-8 type macroporous adsorptive resins, with the distilled water eluting of 4 times of column volumes, the mass concentration of 4 times of column volumes of reuse is 10% ethanol elution earlier, discard eluent, the mass concentration with 8 times of column volumes of continuing is 70% ethanol elution, collects 70% ethanol elution, and decompression recycling ethanol also concentrates, drying, general flavone content reaches Herba Epimedii extract more than 60%.
2. the described Herba Epimedii extract of claim 1 is the application in preparation treatment climacteric syndrome medicine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105943591A (en) * | 2016-06-08 | 2016-09-21 | 河南中医学院 | Application of fenugreek total flavones to preparation of medicine for treating perimenopausal syndromes |
| CN106138133A (en) * | 2016-09-21 | 2016-11-23 | 河南中医药大学 | The application in exhausted premenstrual syndrome medicine is treated in preparation of the Fructus Foeniculi total flavones |
| CN108094473A (en) * | 2018-02-01 | 2018-06-01 | 广州聚澜健康产业研究院有限公司 | A kind of green tea fried dough twist item and preparation method thereof |
| CN113332339A (en) * | 2021-05-21 | 2021-09-03 | 陆华 | Application of pharmaceutical composition in preparation of medicine for preventing and/or treating thin endometrium or infertility |
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| CN1977873A (en) * | 2005-12-09 | 2007-06-13 | 长春至宝药业科技开发有限责任公司 | Epimedium total flavone extracting process |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1977873A (en) * | 2005-12-09 | 2007-06-13 | 长春至宝药业科技开发有限责任公司 | Epimedium total flavone extracting process |
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| 《中国科学技术协会第十届年会21分会场论文汇编》 20081231 于燕等 淫羊藿及淫羊藿苷雌激素样作用研究 第181-185页 1-2 , * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105943591A (en) * | 2016-06-08 | 2016-09-21 | 河南中医学院 | Application of fenugreek total flavones to preparation of medicine for treating perimenopausal syndromes |
| CN106138133A (en) * | 2016-09-21 | 2016-11-23 | 河南中医药大学 | The application in exhausted premenstrual syndrome medicine is treated in preparation of the Fructus Foeniculi total flavones |
| CN106138133B (en) * | 2016-09-21 | 2019-10-29 | 河南中医药大学 | Fennel seeds general flavone treats the application in premenopausal syndrome drug in preparation |
| CN108094473A (en) * | 2018-02-01 | 2018-06-01 | 广州聚澜健康产业研究院有限公司 | A kind of green tea fried dough twist item and preparation method thereof |
| CN113332339A (en) * | 2021-05-21 | 2021-09-03 | 陆华 | Application of pharmaceutical composition in preparation of medicine for preventing and/or treating thin endometrium or infertility |
| CN113332339B (en) * | 2021-05-21 | 2023-01-31 | 陆华 | Application of pharmaceutical composition in preparation of medicine for preventing and/or treating thin endometrium or infertility |
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Application publication date: 20110615 |