CN102579673A - Application of fresh rehmannia root aqueous extract in preparation of estrogen medicines - Google Patents
Application of fresh rehmannia root aqueous extract in preparation of estrogen medicines Download PDFInfo
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Abstract
The invention relates to an application of a fresh rehmannia root aqueous extract in preparation of estrogen medicines, which can effectively solve the problem of preparing a medicament for treating the related diseases caused by in-vivo female hormone secretion insufficiency, and further can effectively solve the problem of treating the related diseases caused by in-vivo female hormone secretion insufficiency. The preparation of the fresh rehmannia root aqueous extract comprises the following steps: decocting fresh rehmannia root with water accounting for 10 times the amount of the fresh rehmannia root for 2 times and 2 hours each time, merging the water decoctions of 2 times, and then carrying out reduced pressure concentration and drying to obtain the fresh rehmannia root aqueous extract. The fresh rehmannia root aqueous extract can be effectively used for preparing the medicament for treating the related diseases caused by in-vivo female hormone secretion insufficiency, and realize the application of the fresh rehmannia root aqueous extract in the preparation of the medicament for treating the related diseases caused by in-vivo female hormone secretion insufficiency. The preparation method of the aqueous extract is simple, stable, reliable, convenient and low in cost, and the aqueous extract is effectively used for preparing estrogen medicines and opens up a new medicinal application of fresh rehmannia root.
Description
Technical field
The present invention relates to medicine; The application of particularly a kind of Radix Rehmanniae water extract in preparation estrogens medicine; The Radix Rehmanniae water extract has estrogen-like effects, is effective to the medicine for treatment problem owing to relevant disease that estrogen hyposecretion in the body causes (like climacteric syndrome etc.).
Background technology
Radix Rehmanniae is the fresh tuber of scrophulariaceae rehmannia glutinosa plant (Rehmannia glutinosa Libosch.), and excavate autumn, removes removing LU, fibrous root and silt, and using fresh herb is Radix Rehmanniae.The successive dynasties famous expert all has argumentation and research to it, thinks that its property is sweet, bitter, cold, GUIXIN, liver, kidney channel, and clearing away heat and promoting production of body fluid, removing heat from blood, hemostasis is mainly used to consumption of YIN caused by febrile disease, and the crimson tongue excessive thirst is sent out the speckle dermexanthesis, haematemesis, epistaxis, laryngopharynx swelling and pain.Modern pharmacology and clinical research show that Radix Rehmanniae can be used for the treatment of inflammatory diseases, like otitis media, pharyngolaryngitis, arthritis, hepatitis, lupus erythematosus, nephritis, diabetes etc.But do not report relevant for the research of Radix Rehmanniae estrogenic activity aspect yet so far.
Summary of the invention
To above-mentioned situation; For overcoming the defective of prior art; The present invention's purpose just provides the application of a kind of Radix Rehmanniae water extract in preparation estrogens medicine; Can effectively solve preparation because the relevant disease that causes of estrogen hyposecretion in the body medicine of (like climacteric syndrome etc.), thus medicine for treatment problem effectively solved owing to relevant disease that the interior estrogen hyposecretion of body causes (like climacteric syndrome etc.).
The technical scheme that the present invention solves is; The application of Radix Rehmanniae water extract in the preparation estrogenic; This Radix Rehmanniae water extract is Radix Rehmanniae to be boiled 2 times at every turn each 2h with the decocting of its 10 times of weight; Merge 2 times decocting liquid; The concentrating under reduced pressure drying obtains the Radix Rehmanniae water extract, and this Radix Rehmanniae water extract can be effective to prepare treatment because the medicine of the relevant disease that the estrogen hyposecretion causes in the body, realizes the application in the Radix Rehmanniae water extract relevant disease that the estrogen hyposecretion causes in preparation is treated owing to body (like the climacteric syndrome etc.) medicine.
Water extract method for preparing of the present invention is simple, reliable and stable, and through test of many times, has all obtained identical or akin result, and convenient, cost is low, and its water extract is effective to prepare the estrogens medicine, has opened up the medicinal new purposes of Radix Rehmanniae, and clinical meaning is huge.
Description of drawings
Fig. 1 is influence
sketch map of Radix Rehmanniae water extract of the present invention to the MCF-7 cell proliferation.
Fig. 2 is influence
sketch map of Radix Rehmanniae water extract of the present invention to sex immature Mouse Uterus coefficient.
Fig. 3 is reporter gene transient expression result
sketch map of Radix Rehmanniae water extract of the present invention to the ERE regulation and control.
Fig. 4 is reporter gene transient expression result
sketch map of Radix Rehmanniae water extract of the present invention to the ERE regulation and control.
The specific embodiment
Concrete condition of the present invention is elaborated with relevant testing data below in conjunction with embodiment.
The present invention in practical implementation, the application of described Radix Rehmanniae water extract in preparation estrogens medicine, this Radix Rehmanniae water extract is to be provided by following examples:
The present invention is in practical implementation, and described Radix Rehmanniae water extract is that Radix Rehmanniae (Rehmannia glutinosa Libosch.) 100g is decocted 2 times with the water 1000g (being 1000ml) of its 10 times of weight; Each 2h merges decocting liquid twice, and the concentrating under reduced pressure drying obtains the Radix Rehmanniae water extract; Yield is 12.03%; This water extract can prepare the estrogens medicine, is used to treat the disease that causes owing to estrogen hyposecretion in the body, like the climacteric syndrome medicine.
Embodiment 2
The present invention is in practical implementation, and described Radix Rehmanniae water extract is that Radix Rehmanniae (Rehmannia glutinosa Libosch.) 200g is decocted 2 times with the water 2000g (being 2000ml) of its 10 times of weight; Each 2h merges decocting liquid twice, and the concentrating under reduced pressure drying obtains the Radix Rehmanniae water extract; Yield is 15.84%; This water extract can prepare the estrogens medicine, is used to treat the disease that causes owing to estrogen hyposecretion in the body, like the climacteric syndrome medicine.
Embodiment 3
The present invention is in practical implementation, and described Radix Rehmanniae water extract is that Radix Rehmanniae (Rehmannia glutinosa Libosch.) 300g is decocted 2 times with the water 3000g (being 3000ml) of its 10 times of weight; Each 2h merges decocting liquid twice, and the concentrating under reduced pressure drying obtains the Radix Rehmanniae water extract; Yield is 14.99%; This water extract can prepare the estrogens medicine, is used to treat the disease that causes owing to estrogen hyposecretion in the body, like the climacteric syndrome medicine.
Can make the Radix Rehmanniae water extract of any amount through suitability for industrialized production in the ratio of Radix Rehmanniae and water according to said method; And, all having obtained identical and akin result through experiment repeatedly, method is reliable and stable; Gained Radix Rehmanniae water extract is used to prepare the estrogens medicine; Effectively solve because the disease that the estrogen hyposecretion causes in the body medication treatment problem of (like climacteric syndrome etc.), and obtained fully proving through test, about testing data following:
Adopt cell proliferation experiment, zoopery and reporter gene technology that it has been carried out fully proving, its relevant testing data is following:
At first through MCF-7 cell proliferation experiment (E-SCREEN), find that the Radix Rehmanniae water extract can promote the propagation of MCF-7 cell, explain that it has an estrogen-like effects external.
Secondly through Mouse Uterus weightening finish experiment, prove that the Radix Rehmanniae water extract can significantly increase the uterus coefficient of sex immature female mice, explain that it has estrogen-like effects in vivo.
Reporter gene transient expression through ERE regulation and control detects at last, further verify the Radix Rehmanniae water extract be through with estrogen receptor ER β combine to bring into play estrogen-like effects.
The inventor has proved the new purposes of medicine of the present invention in preparation estrogens medicine through experiment.Its main embodiment is following:
One, experiment material and method
1. experiment medicine
The fresh tuber of scrophulariaceae rehmannia glutinosa plant (Rehmannia glutinosa Libosch.).Excavate autumn, removes removing LU, fibrous root and silt, is Radix Rehmanniae.
Radix Rehmanniae boils 2 times with the decocting of 10 times of weight, each 2h, and decocting liquid drying under reduced pressure concentrate drying obtains the Radix Rehmanniae water extract.
2. laboratory animal and cell strain and plasmid
Kunming mouse, female, be born 21 days (ablactation just), body weight 9～12g purchases the Experimental Animal Center in Henan Province.Human breast cancer cell (MCF-7) is provided by Chinese BIO ENGINEERING INST MILITARY.The HEK293 cell strain is purchased in China typical culture collection center.(β-galactosidase, β-gal) control plasmid p β gal-Control, reorganization reporter gene pERE-TAL-luc are so kind as to give by the dense doctor of leaf chess of BIO ENGINEERING INST MILITARY beta galactosidase.Recombined human ER α (humanER α, hER α) expression vector pCXN2-hER α and recombined human ER β (humanER β, hER β) expression vector pCXN2-hER β are so kind as to give by Satoshi doctor Inoue of Tokyo University department of medial science.
3. main agents
RPMI1640 culture medium, calf serum (Newborn Calf Serum, NCS) with liposome LipofectamineTM2000Reagent available from Gibco Invitrogen company; Hyclone, no phenol red RPMI1640 culture medium, 17 beta estradiols (17 β-estrogen, E
2), ampicillin (Amp), Tris alkali and active carbon (Charcoal) be all available from Sigma company; Glucosan T-70 (Dextran-70) is available from Shanghai chemical reagents corporation; Synestrin tablets (Hefei joins pharmacy for a long time); Neighbour-Nitrobenzol-β-D-gala pyranoside (O-Nitrophenyl-β-D-galactopy ranoside, ONPG), EDTA, MTT and DMSO be the Amresco Company products; Steady-
Stablize luciferase detection system test kit (Steady-
Luciferase Assay Systerm), glittering lysis buffer (Glo lysis Buffer) is available from Promega company; All the other reagent are homemade analytical pure.
4. used key instrument
The general surgery apparatus; CO2 gas incubator (REVCO); Inverted microscope (NIKON ECLIPSE TS100); KDC-160HR high-speed low temperature refrigerated centrifuger (Keda Innovation Co., Ltd); ELIASA (BIO-RAD 680); Pure water appearance (Sartorius 611VF); 90-3 magnetic stirring apparatus (Shanghai shake prompt experimental facilities company limited); The full-automatic novel air dry oven of ZRD-7080 (Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.); Micro sample adding appliance (Nichipet EX PLUS); AB204-N electronical reading analytical balance (Mettler-Toledo Instrument's product); 10cm culture dish, 96 well culture plates, frozen pipe are Corning company and produce; Superclean bench (Jiangsu Su Jing group); UV-7504PC type ultraviolet-visible spectrophotometer (foreign scientific and technological Instr Ltd. is good in Foochow); VeritasTM microplate photometer (Turner BioSystems company); SK6200H type ultrasonic cleaner (Shanghai High Kudos Science Instrument Co., Ltd.); The biochemical incubator of SHP-150 type (going up the grand experimental facilities company limited of Nereid).
5. experimental technique
5.1MCF-7 cell proliferation experiment
The MCF-7 cell through do not have phenol red contain 10% remove hormone serum RPMI1640 culture medium culturing after 2 weeks; Choose the exponential phase cell, PBS washes twice, behind 0.25% trypsinization; Add no phenol red contain 2% remove hormone serum RPMI1640 culture medium piping and druming evenly, with 1 * 10
4The concentration of individual/ml is inoculated in 96 orifice plates, and it is 200 μ l that cumulative volume is cultivated in every hole.After cultivation 24h treats cell attachment, be changed to the pastille culture fluid and continue to cultivate.37 ℃, 5%CO
2After cultivating 72h, every hole adds MTT solution (5mg/ml) 20 μ l, and 37 ℃ are continued to cultivate 4h, the culture fluid that carefully exhausts, and every hole adds 150 μ lDMSO, and concussion 5～10min dissolves crystal fully.With the DMSO zeroing, under 490nm, measure each hole absorbance (A) with ELIASA, the calculating average A value and the rate of increase (Proliferation Rate, RP).Test parallel triplicate.
RP=experimental group A value/blank control group A value)
-1* 100%
5.2 Mouse Uterus weightening finish experiment
Mice is balanced by body weight and principle is at random divided into groups.Administration continues 7 days.Blank group distilled water every day 0.2ml/10g irritates stomach; Positive controls diethylstilbestrol every day suspension (0.35mg/kg/d) is irritated stomach; The Radix Rehmanniae water extract calculates by 20 times of amount * extraction ratios of clinical application, is made into the suspension of respective concentration, presses 0.2ml/10g every day and irritates stomach.Behind the last administration 24h, take off neck and put to death mice, win the uterus immediately and weigh, calculate uterus coefficient (uterus weight in wet base * (body weight)
-1* 100%).Test parallel triplicate.The result compares with blank control group, and calculating the investigation of P value has not statistically significant.
5.3ERE the reporter gene transient expression of regulation and control detects
5.3.1 cell inoculation
The HEK293 cell inoculation that 72h will be in exponential phase before transfection is in 24 orifice plates, and density is 16.0 * 10
4Individual/the 0.5ml/ hole, and culture fluid be contain 10% remove estrogen serum no phenol red RPMI1640 culture fluid, blank hole, estradiol hole and medicine to be measured hole are set respectively.
5.3.2 the transfection of plasmid
(1) begin transfection behind the 72h, the careful former culture medium of sucking-off, add 400 μ l contain 10% remove estrogen serum no phenol red RPMI1640 culture medium.
(2) get 3 EP pipes, adding 500 μ l in the ER alphatrons does not have phenol red RPMI1640 culture medium, 8 μ lpCXN2-hER α plasmids, 8 μ lpERE-TAL-luc plasmids, 8 μ lp β gal-Control plasmids; Add 500 μ l in the ER β pipe and do not have phenol red RPMI1640 culture medium, 8 μ lpCXN2-hER β plasmids, 8 μ lpERE-TAL-Iuc plasmids, 8 μ l β gal-Control plasmids; Add 1000 μ l in the liposome pipe and do not have phenol red RPMI1640 culture medium and 24 μ l liposomees.
(3) reversing mixing liposome pipe, room temperature is placed 5min, gets 500 μ l and adds ER α and ER β pipe respectively, and room temperature is placed 20min;
(4) reversing mixing ER α and ER β pipe gets that liposome/DNA complex adds 24 orifice plate ER α experimental ports respectively in the 80 μ lER alphatrons, get 80 μ lER β manage in liposome/DNA complex add 24 orifice plate ER β experimental ports respectively.Front and back shake up, and put into incubator.
(5) add control drug and detection of drugs behind the 6h.
5.3.3ERE the reporter gene transient expression of regulation and control detects
The cell of 24h after the dosing is inhaled and is removed culture fluid, washes once with 1mlPBS, and removes PBS.Stablize luciferase detection system test kit (
Luciferase Assay Systerm) and glittering lysis buffer (Glo lysis Buffer) evolutionary operation procedure according to
; Collecting cell cracking supernatant; Add luciferase reaction substrate (fluorescein); Place the dark place, with VeritasTM microplate photometer fluorescence intensity.
Detect 5.3.4 β-gal is active
In order to investigate transfection efficiency, internal reference β-gal activity is detected.
(1) 54 μ l beta-mercaptoethanol adds among the 40ml Z-buffer, and mixing is got the EP pipe, and every pipe adds 1800 μ l;
(2) every pipe adds 20 μ l cracking supernatant;
(3) every pipe adds 400 μ l ONPG, the reversing mixing;
(4) place 37 ℃ of incubators, to displaing yellow;
(5) add Na
2CO
3The 1ml cessation reaction;
(6) survey OD
420Value;
Two, statistical disposition
Experimental data representes that with
SPSS13.0 carries out one factor analysis of variance (One-Way ANOVA) statistical disposition.
Three, experimental result
1. the Radix Rehmanniae water extract is to the experimental result of MCF-7 cell proliferation influence
The result shows, the dosage of Radix Rehmanniae water extract compares the MCF-7 cell with blank control group when 0.01～1mg/ml all have and significantly urge proliferation function (P＜0.01).Explain that the Radix Rehmanniae water extract has an estrogen-like effects external.See table 1, Fig. 1.
Table 1 Radix Rehmanniae water extract is to the influence
of MCF-7 cell proliferation
Annotate: compare with the blank group, ☆ representes P＜0.05, and ★ representes P＜0.01; Compare with positive group, △ representes P＜0.05, ▲ expression P＜0.01.
2. the Radix Rehmanniae water extract is to the experimental result of Mouse Uterus coefficient influence
The result shows that Radix Rehmanniae water extract group and the increase (P＜0.01) of the remarkable inductivity immaturity of specific energy Mouse Uterus coefficient mutually of blank group explain that the Radix Rehmanniae water extract has estrogen-like effects in vivo.See table 2, Fig. 2.
Table 2 Radix Rehmanniae water extract is to the influence
of sex immature Mouse Uterus coefficient
Annotate: compare with the blank group, ☆ representes P＜0.05, and ★ representes P＜0.01; Compare with positive controls, △ representes P＜0.05, ▲ expression P＜0.01.
3.ERE detecting, the reporter gene transient expression of regulation and control estimates the estrogen-like effects whether the Radix Rehmanniae water extract passes through the receptor pathway performance
(positive controls is 17-estradiol (E for Estrogen Receptors, ERs) interactional influence to utilize reporter gene technology for detection Radix Rehmanniae water extract and estrogen receptor
2) final concentration that adds is 10
-8Mol/L, Radix Rehmanniae water extract adding consistency is respectively 0.001mg/ml, 0.01mg/ml, 0.1mg/ml, 1mg/ml.Result's demonstration, no matter be beta mediated by ER α or ER, E
2Uciferase activity after the standardization all is significantly higher than blank well (P＜0.01).When alpha mediated, the uciferase activity after four dose groups standardization of Radix Rehmanniae water extract 0.001mg/ml～1mg/ml is compared all not statistically significants (P＞0.05) with blank well, see table 3, Fig. 3 by ER.When beta mediated, the uciferase activity after Radix Rehmanniae water extract 0.001mg/ml, 0.01mg/ml, 0.1mg/ml, the standardization of 1mg/ml group all is significantly higher than blank well (P＜0.01), and has dose dependent by ER.See table 4, Fig. 4.
Table 3 Radix Rehmanniae water extract is to the reporter gene transient expression testing result
of ERE regulation and control
Annotate: compare with the blank group, ☆ representes P＜0.05, and ★ representes P＜0.01; Compare with positive group, △ representes P＜0.05, ▲ expression P＜0.01.
Table 4 Radix Rehmanniae water extract is to the reporter gene transient expression testing result
of ERE regulation and control
Annotate: compare with the blank group, ☆ representes P＜0.05, and ★ representes P＜0.01; Compare with positive group, △ representes P＜0.05, ▲ expression P＜0.01.
MCF-7 cell proliferation experiment result shows, during the concentration 0.01mg/ml of Radix Rehmanniae water extract～1mg/ml the MCF-7 cell is had significant proliferation function, and effect is the strongest when 0.1mg/ml.
Mouse Uterus weightening finish experimental result shows that the Radix Rehmanniae water extract can significantly increase sex immature Mouse Uterus coefficient, explain the Radix Rehmanniae water extract in vivo, externally all have an estrogenic activity.
The reporter gene transient expression testing result of ERE regulation and control shows; The estrogen-like effects of Radix Rehmanniae water extract mainly is beta mediated through ER; Concentration all can activate the beta mediated genetic transcription of ER at 0.001mg/ml, 0.01mg/ml, 0.1mg/ml, 1mg/ml, and has dose dependent; But comparing with blank well, the genetic transcription alpha mediated to ER, Radix Rehmanniae water extract all do not have influence when 0.001mg/ml, 0.01mg/ml, 0.1mg/ml, 1mg/ml.
In sum, animal and cell experiment result show that the Radix Rehmanniae water extract has certain estrogen-like effects, and it mainly is to bring into play estrogen-like effects through the beta mediated genetic transcription of ER.
Four, conclusion
The MCF-7 cell is the human breast cancer cell strain of estrogen receptor positive, can receive estrogen or estrogenic activity material to regulate and breed specifically, is the cell strain of the most frequently used detection estrogenic activity.MCF-7 cell estrogen-like proliferation test is very sensitive, can distinguish estrogen agonist and antagonist, thereby is widely used in external a large amount of, rapid screening and evaluation environmental estrogens and phytoestrogen.In the experiment of Radix Rehmanniae water extract to MCF-7 cell proliferation influence, the Radix Rehmanniae water extract all has significant short proliferation function to the MCF-7 cell when 0.01mg/ml～1mg/ml, has explained that the Radix Rehmanniae water extract has an estrogenic activity external.
Uterus weight experiment is the classical way of the detection estrogen activity set up the earliest; When estimating estrogen activity; It has considered the absorption, metabolism of material, combine with plasma protein and many-sided factor such as pharmacokinetics, and directly reflection receives the overall biological effect of reagent thing behind internal metabolism.Therefore, this experiment adopts the sex immature female mice uterus weight in wet base and ratio (uterus coefficient) conduct of body weight to estimate the index of estrogenic activity.The result shows that the Radix Rehmanniae water extract can significantly increase sex immature Mouse Uterus coefficient, proves that the Radix Rehmanniae water extract has estrogen-like effects in vivo.
Above-mentioned test is carried out repeatedly repeatedly, has all obtained identical or close result, prompting, the Radix Rehmanniae water extract in vivo, externally all have certain estrogen-like effects.
Reporter gene (reporter gene) is that its expression product be very easy to detect, is prone to a genoid of difference with endogenous background albumen.Through genetic engineering means, response element is linked to each other with reporter gene, through the examining report activity of gene expression, can learn the activity of signal transduction pathway and the regulation and control and the influence of the interior gene expression of pair cell thereof.The reporter gene sensitivity is high, selectivity is strong and simple to operate.In of the inducing action experiment of Radix Rehmanniae water extract to reporter gene carrier pERE-TAL-luc transient expression; Transient transfection in order to ERE regulation and control reporter gene; With being loaded with ER α, ER β respectively and being medicaments sifting model as the reorganization reporter gene vector plasmid transient cotransfection target cell (HEK 293) that controlling element makes up with the ERE sequence; Add variable concentrations receive the reagent thing after; Through visual report expression of gene situation, promptly whether the height of fluorescence intensity can detect and tried thing and can combine with estrogen receptor (ER), thus the power of understanding Radix Rehmanniae water extract estrogen-like effects with and with the size of ER α or ER β affinity.Visible from experimental result; The Radix Rehmanniae water extract can be induced the expression of luc fluorescence through estrogen receptor (ER); And Radix Rehmanniae water extract activated gene is transcribed mainly beta mediated through ER, and this is consistent to the higher bibliographical information of ER β affinity with phytoestrogen.
Organism is the system of a complicacy, and there is certain deviation in the vivo and vitro result.Whole animal experiment and reporter gene technology combine and verify the estrogenic activity that is tried thing jointly in the experiment of this experimental applications cell in vitro, the body, have guaranteed result's accuracy.
Prove fully that by above-mentioned data the Radix Rehmanniae water extract can promote the propagation of MCF-7 cell and the weight in wet base of increase sex immature Mouse Uterus, further the reporter gene transient expression detection technique prompting Radix Rehmanniae water extract of ERE regulation and control mainly is through the beta mediated estrogen-like effects of bringing into play of ER.That is to say that the Radix Rehmanniae water extract has estrogen-like effects, can be effective to prepare the medicine of treating the relevant disease that causes owing to estrogen hyposecretion in the body, is the innovation of Chinese medicine Radix Rehmanniae on purposes.
Claims (1)
1. the Radix Rehmanniae water extract application in preparation estrogens medicine, this Radix Rehmanniae water extract be, Radix Rehmanniae is boiled 2 times with the decocting of its 10 times of weight at every turn, and each 2h merges 2 times decocting liquid, and the concentrating under reduced pressure drying obtains the Radix Rehmanniae water extract.
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| CN103055021A (en) * | 2013-01-10 | 2013-04-24 | 济南康众医药科技开发有限公司 | Freeze-dried fresh rehmannia for treating systemic lupus erythematosus |
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| CN101269170A (en) * | 2008-03-05 | 2008-09-24 | 上海中医药大学 | Medicament for treating climacteric syndrome and preparation method thereof |
| CN101966254A (en) * | 2010-10-08 | 2011-02-09 | 河南中医学院 | Application of raw rehmannia aqueous extract in preparing medicines for treating woman climacteric syndrome caused by hyposecretion of in-vivo female hormones |
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| JP2004155779A (en) * | 2002-10-16 | 2004-06-03 | Taisho Pharmaceut Co Ltd | Peroral composition for preventing and treating climacteric symptom |
| CN1519016A (en) * | 2003-09-01 | 2004-08-11 | 江苏省中国科学院植物研究所 | Prepared Chinese medicine for improving function of female ovary as well as preparation method and application in pharmacy |
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