CN102579673B - Application of fresh rehmannia root aqueous extract in preparation of estrogen medicines - Google Patents
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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 particularly application of a kind of Radix Rehmanniae water extract in preparing estrogens medicine, Radix Rehmanniae water extract has estrogen-like effects, is effective to the medicine for treatment problem due to the relevant disease (as climacteric syndrome etc.) that in body, estrogen hyposecretion causes.
Background technology
Radix Rehmanniae is the fresh tuber of scrophulariaceae rehmannia glutinosa plant (Rehmannia glutinosa Libosch.), and excavate autumn, and except removing LU, fibrous root and silt, using fresh herb, is Radix Rehmanniae.Successive dynasties famous expert all has and discusses and research 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 crimson tongue excessive thirst is sent out speckle dermexanthesis, spits blood, epistaxis, laryngopharynx swelling and pain.Modern pharmacology and clinical research show that Radix Rehmanniae can be used for the treatment of inflammatory diseases, as otitis media, pharyngolaryngitis, arthritis, hepatitis, lupus erythematosus, nephritis, diabetes etc.But relevant for the research of Radix Rehmanniae estrogenic activity aspect, do not report yet so far.
Summary of the invention
For above-mentioned situation, for overcoming the defect of prior art, the present invention's object is just to provide the application of a kind of Radix Rehmanniae water extract in preparing estrogens medicine, can effectively solve preparation due to the medicine of relevant disease that in body, estrogen hyposecretion causes (as climacteric syndrome etc.), thereby effectively solve the medicine for treatment problem due to relevant disease that in body, estrogen hyposecretion causes (as climacteric syndrome etc.).
The technical scheme that the present invention solves is, the application of Radix Rehmanniae water extract in preparing estrogenic, this Radix Rehmanniae water extract is, Radix Rehmanniae is boiled 2 times with the decocting of its 10 times of weight at every turn, each 2h, merge 2 times decocting liquid, the dry Radix Rehmanniae water extract that obtains of concentrating under reduced pressure, this Radix Rehmanniae water extract can be effective to preparation treatment due to the medicine of the relevant disease that in body, estrogen hyposecretion causes, realize Radix Rehmanniae water extract preparation treatment due to body in application in the relevant disease that causes of estrogen hyposecretion (as climacteric syndrome etc.) medicine.
Water extract preparation method 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 estrogens medicine, has opened up the medicinal new purposes of Radix Rehmanniae, and clinical meaning is huge.
Accompanying drawing explanation
Fig. 1 is the impact of Radix Rehmanniae water extract of the present invention on MCF-7 cell proliferation
schematic diagram.
Fig. 2 is the impact of Radix Rehmanniae water extract of the present invention on sex immature Mouse Uterus coefficient
schematic diagram.
Fig. 3 is the reporter gene transient expression result of Radix Rehmanniae water extract of the present invention to ERE regulation and control
schematic diagram.
Fig. 4 is the reporter gene transient expression result of Radix Rehmanniae water extract of the present invention to ERE regulation and control
schematic diagram.
The specific embodiment
Below in conjunction with embodiment and related tests data, concrete condition of the present invention is elaborated.
The present invention in concrete enforcement, the application of described Radix Rehmanniae water extract in preparing estrogens medicine, this Radix Rehmanniae water extract is to be provided by following examples:
The present invention is in concrete enforcement, described Radix Rehmanniae water extract is, 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, the dry Radix Rehmanniae water extract that obtains of concentrating under reduced pressure, yield is 12.03%, this water extract can be prepared estrogens medicine, is used for the treatment of due to the disease that in body, estrogen hyposecretion causes, as climacteric syndrome medicine.
Embodiment 2
The present invention is in concrete enforcement, described Radix Rehmanniae water extract is, 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, the dry Radix Rehmanniae water extract that obtains of concentrating under reduced pressure, yield is 15.84%, this water extract can be prepared estrogens medicine, is used for the treatment of due to the disease that in body, estrogen hyposecretion causes, as climacteric syndrome medicine.
Embodiment 3
The present invention is in concrete enforcement, described Radix Rehmanniae water extract is, 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, the dry Radix Rehmanniae water extract that obtains of concentrating under reduced pressure, yield is 14.99%, this water extract can be prepared estrogens medicine, is used for the treatment of due to the disease that in body, estrogen hyposecretion causes, as climacteric syndrome medicine.
According to said method, in the ratio of Radix Rehmanniae and water, can make by suitability for industrialized production the Radix Rehmanniae water extract of any amount, and through experiment repeatedly, all obtained identical and akin result, method is reliable and stable, gained Radix Rehmanniae water extract is for the preparation of estrogens medicine, effectively solve the medication treatment problem due to disease that in body, estrogen hyposecretion causes (as climacteric syndrome etc.), and obtained fully proving through test, related tests data is as follows:
Adopt cell proliferation experiment, zoopery and reporter gene technology to carry out fully proving to it, its related tests data is as follows:
First by MCF-7 cell proliferation experiment (E-SCREEN), find that Radix Rehmanniae water extract can promote the propagation of MCF-7 cell, illustrates that it has estrogen-like effects in vitro.
Secondly by the Mouse Uterus experiment of increasing weight, prove that Radix Rehmanniae water extract can significantly increase the uterus coefficient of sex immature female mice, illustrate that it has estrogen-like effects in vivo.
The reporter gene transient expression finally regulating and controlling by ERE detects, and further verifies that Radix Rehmanniae water extract is by bringing into play estrogen-like effects with the combination of estrogen receptor ER β.
Inventor has proved the new purposes of medicine of the present invention in preparing estrogens medicine by experiment.Its main scheme for implementing said method is as follows:
One, experiment material and method
1. Experimental agents
The fresh tuber of scrophulariaceae rehmannia glutinosa plant (Rehmannia glutinosa Libosch.).Excavate autumn, except 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 Radix Rehmanniae water extract.
2. laboratory animal and cell strain and plasmid
Kunming mouse, female, be born 21 days (just weaning), body weight 9～12g, is purchased from Henan Province's Experimental Animal Center.Human breast cancer cell (MCF-7) is provided by Bioengineering Research Institute of Chinese military medicine academy of science.HEK293 cell strain is purchased from Chinese Typical Representative culture collection center.Beta galactosidase (β-galactosidase, β-gal) control plasmid p β gal-Control, restructuring reporter gene pERE-TAL-luc are so kind as to give by the dense doctor of leaf chess of BIO ENGINEERING INST MILITARY.Recombinant human ER α (humanER α, hER α) expression vector pCXN2-hER α and recombinant human ER β (humanER β, hER β) Satoshi doctor Inoue of expression vector pCXN2-hER βYou Tokyo University department of medial science are so kind as to give.
3. main agents
RPMI1640 culture medium, calf serum (Newborn Calf Serum, NCS) and liposome LipofectamineTM2000Reagent are purchased from Gibco Invitrogen company; Hyclone, without phenol red RPMIl640 culture medium, 17 beta estradiols (17 β-estrogen, E
2), ampicillin (Amp), Tris alkali and active carbon (Charcoal) be all purchased from Sigma company; Glucosan T-70 (Dextran-70) is purchased from Solution on Chemical Reagents in Shanghai company; Synestrin tablets (Hefei joins pharmacy for a long time); O-Nitrobenzol-β-D-synthesis (O-Nitrophenyl-β-D-galactopy ranoside, ONPG), EDTA, MTT and DMSO are Amresco company product;
stablize luciferase detection system test kit
luciferase Assay Systerm), glittering lysis buffer (Glo lysis Buffer) is purchased from Promega company; All the other reagent are domestic analytical pure.
4. key instrument used
General surgery apparatus; CO2 gas incubator (REVCO); Inverted microscope (NIKON ECLIPSE TS100); KDC-160HR high-speed low temperature refrigerated centrifuger (University of Science and Technology innovation Ban Fen company limited); Microplate reader (BIO-RAD 680); Pure water instrument (Sartorius 611VF); 90-3 magnetic stirring apparatus (Shanghai Zhen Jie experimental facilities company limited); ZRD-7080 full-automatic novel air dry oven (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, cryopreservation tube are Corning company and produce; Superclean bench (Jiangsu Su Jing group); UV-7504PC type ultraviolet-visible spectrophotometer (foreign scientific and technological Instrument Ltd. is good in Foochow); VeritasTM microplate photometer (Turner BioSystems company); SK6200H type ultrasonic cleaner (Shanghai High Kudos Science Instrument Co., Ltd.); SHP-150 type biochemical cultivation case (the above grand experimental facilities of Nereid company limited).
5. experimental technique
5.1MCF-7 cell proliferation experiment
MCF-7 cell is through containing 10% and go the RPMI1640 culture medium culturing of hormone serum after 2 weeks without phenol red, choose exponential phase cell, PBS washes twice, with after 0.25% trypsinization, add without phenol red and contain the 2% RPMI1640 culture medium piping and druming of removing hormone serum 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.Cultivate 24h after cell attachment, be changed to pastille culture fluid and continue to cultivate.37 ℃, 5%CO
2cultivate after 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 completely.With DMSO zeroing, by microplate reader, under 490nm, measure each hole absorbance (A), calculate average A value and the rate of increase (Proliferation Rate, RP).Test in triplicate parallel.
RP=experimental group A value/blank group A value)
-1* 100%
5.2 Mouse Uterus weightening finish experiments
Mice is divided into groups by the balanced and random principle of body weight.Administration continues 7 days.Blank group distilled water every day 0.2ml/10g gavage; Positive controls diethylstilbestrol every day suspension (0.35mg/kg/d) gavage; 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 gavage every day.After last administration 24h, de-neck is put to death mice, wins immediately uterus and weighs, and calculates uterus coefficient (uterus weight in wet base * (body weight)
-1* 100%).Test in triplicate parallel.Result is compared with blank group, and calculating the investigation of P value has not statistically significant.
The reporter gene transient expression of 5.3ERE regulation and control detects
5.3.1 cell inoculation
Before transfection, 72h is inoculated in 24 orifice plates by the HEK293 cell in exponential phase, and density is 16.0 * 10
4individual/0.5ml/ hole, and culture fluid for containing 10% go estrogen serum without 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) after 72h, start transfection, the careful former culture medium of sucking-off, add 400 μ l containing 10% go estrogen serum without phenol red RPMI1640 culture medium.
(2) get 3 EP pipes, in ER alphatrons, add 500 μ l without phenol red RPMI1640 culture medium, 8 μ lpCXN2-hER α plasmids, 8 μ lpERE-TAL-luc plasmids, 8 μ lp β gal-Control plasmids; In ER β pipe, add 500 μ l without phenol red RPMI1640 culture medium, 8 μ lpCXN2-hER β plasmids, 8 μ lpERE-TAL-Iuc plasmids, 8 μ l β gal-Control plasmids; In liposome pipe, add 1000 μ l without phenol red RPMI1640 culture medium and 24 μ l liposomees.
(3) reversing mixes liposome pipe, and room temperature is placed 5min, gets 500 μ l and adds respectively ER α and ER β pipe, and room temperature is placed 20min;
(4) reversing mixes ER α and ER β pipe, gets liposome/DNA complex in 80 μ lER alphatrons and adds respectively 24 orifice plate ER α experimental ports, gets liposome/DNA complex in 80 μ lER β pipes and adds respectively 24 orifice plate ER β experimental ports.Front and back shake up, and put into incubator.
(5) after 6h, add control drug and detection of drugs.
5.3.3ERE the cell of 24h after the reporter gene transient expression of regulation and control detection dosing, sucks culture fluid, washes once, and remove PBS with 1mlPBS.According to
stablize luciferase detection system test kit (Steady-
luciferase Assay Systerm) and glittering lysis buffer (Glo lysis Buffer) evolutionary operation step operation, collecting cell cracking supernatant, add luciferase reaction substrate (fluorescein), be placed in dark place, by VeritasTM microplate photometer fluorescence intensity.
5.3.4 β-gal is active detects
In order to investigate transfection efficiency, internal reference β-gal activity is detected.
(1) 54 μ l beta-mercaptoethanol adds in 40ml Z-buffer, mixes, and gets 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, and reversing mixes;
(4) place 37 ℃ of incubators, to displaing yellow;
(5) add Na
2cO
31ml cessation reaction;
(6) survey OD
420value;
Two, statistical disposition
Experimental data with
represent, SPSS13.0 carries out one factor analysis of variance (One-Way ANOVA) statistical disposition.
Three, experimental result
1. the experimental result that Radix Rehmanniae water extract affects MCF-7 cell proliferation
Result shows, the dosage of Radix Rehmanniae water extract when 0.01～1mg/ml and blank group compare MCF-7 cell and all have remarkable proliferation (P < 0.01).Illustrate that Radix Rehmanniae water extract has estrogen-like effects in vitro.In Table 1, Fig. 1.
The impact of table 1 Radix Rehmanniae water extract on MCF-7 cell proliferation
Note: with the comparison of blank group, ☆ represents P < 0.05, and ★ represents P < 0.01; With the comparison of positive group, △ represents P < 0.05, ▲ expression P < 0.01.
2. the experimental result that Radix Rehmanniae water extract affects Mouse Uterus coefficient
Result shows, the increase (P < 0.01) of Radix Rehmanniae water extract group and the remarkable inductivity immaturity of blank group phase specific energy Mouse Uterus coefficient, illustrates that Radix Rehmanniae water extract has estrogen-like effects in vivo.In Table 2, Fig. 2.
The impact of table 2 Radix Rehmanniae water extract on sex immature Mouse Uterus coefficient
Note: with the comparison of blank group, ☆ represents P < 0.05, and ★ represents P < 0.01; With positive controls comparison, △ represents P < 0.05, ▲ expression P < 0.01.
The reporter gene transient expression of 3.ERE regulation and control detects evaluates the estrogen-like effects whether Radix Rehmanniae water extract is brought into play by receptor pathway
Utilize the interactional impact of reporter gene technology for detection Radix Rehmanniae water extract and estrogen receptor (Estrogen Receptors, ERs), positive controls is 17-estradiol (E
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 demonstration, no matter be beta mediated by ER α or ER, E
2uciferase activity after standardization is all significantly higher than blank well (P < 0.01).When alpha mediated by ER, the uciferase activity after the standardization of tetra-dosage groups of Radix Rehmanniae water extract 0.001mg/ml～1mg/ml is compared all not statistically significants (P > 0.05) with blank well, in Table 3, Fig. 3.When beta mediated by ER, the uciferase activity after Radix Rehmanniae water extract 0.001mg/ml, 0.01mg/ml, 0.1mg/ml, the standardization of 1mg/ml group is all significantly higher than blank well (P < 0.01), and has dose dependent.In Table 4, Fig. 4.
The reporter gene transient expression testing result of table 3 Radix Rehmanniae water extract to ERE regulation and control
Note: with the comparison of blank group, ☆ represents P < 0.05, and ★ represents P < 0.01; With the comparison of positive group, △ represents P < 0.05, ▲ expression P < 0.01.
The reporter gene transient expression testing result of table 4 Radix Rehmanniae water extract to ERE regulation and control
Note: with the comparison of blank group, ☆ represents P < 0.05, and ★ represents P < 0.01; With the comparison of positive group, △ represents P < 0.05, ▲ expression P < 0.01.
MCF-7 cell proliferation experiment result shows, during the concentration 0.01mg/ml～1mg/ml of Radix Rehmanniae water extract, MCF-7 cell is had to significant proliferation function, and effect is the strongest when 0.1mg/ml.
Mouse Uterus weightening finish experimental result shows, Radix Rehmanniae water extract can significantly increase sex immature Mouse Uterus coefficient, illustrate Radix Rehmanniae water extract in vivo, externally all there is estrogenic activity.
The reporter gene transient expression testing result of ERE regulation and control shows, the estrogen-like effects of Radix Rehmanniae water extract is mainly beta mediated by 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 the genetic transcription alpha mediated to ER, Radix Rehmanniae water extract is compared with blank well all without impact when 0.001mg/ml, 0.01mg/ml, 0.1mg/ml, 1mg/ml.
In sum, animal and the demonstration of cell experiment result, Radix Rehmanniae water extract has certain estrogen-like effects, and it is mainly to bring into play estrogen-like effects by the beta mediated genetic transcription of ER.
Four, conclusion
MCF-7 cell is the human breast cancer cell strain of estrogen receptor positive, can be subject to specifically estrogen or estrogenic activity material regulate and breed, and 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 experiment MCF-7 cell proliferation being affected at Radix Rehmanniae water extract, Radix Rehmanniae water extract all has significant proliferation to MCF-7 cell when 0.01mg/ml～1mg/ml, has illustrated that Radix Rehmanniae water extract has estrogenic activity in vitro.
Uterus weight experiment is the classical way of the detection estrogen activity set up the earliest, when evaluating estrogen activity, it considered material absorption, metabolism, be combined with plasma protein and the many factors such as pharmacokinetics, directly reflect the total Biology effect of tested medicine after internal metabolism.Therefore, this experiment adopts sex immature female mice uterus weight in wet base and ratio (uterus coefficient) conduct of body weight to evaluate the index of estrogenic activity.Result demonstration, Radix Rehmanniae water extract can significantly increase sex immature Mouse Uterus coefficient, proves that 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, Radix Rehmanniae water extract in vivo, externally all there is certain estrogen-like effects.
Reporter gene (reporter gene) is the genoid that its expression product be very easy to detect, more easily distinguishes with endogenous background albumen.By genetic engineering means, response element is connected with reporter gene, by examining report activity of gene expression, can learn the active of signal transduction pathway and on the regulation and control of gene expression in cell and impact.Reporter gene sensitivity is high, selectivity is strong and simple to operate.In Radix Rehmanniae water extract is tested the inducing action of Reporter gene vector pERE-TAL-luc transient expression, transient transfection in order to ERE regulation and control reporter gene, ER α will be loaded with respectively, the restructuring Reporter gene vector plasmid transient cotransfection target cell (HEK 293) that ER β and the ERE sequence of take build as controlling element is medicaments sifting model, add after the tested medicine of variable concentrations, by the expression of visual report gene, be the height of fluorescence intensity can detect tested material whether can with estrogen receptor (ER) combination, thereby the power of understanding Radix Rehmanniae water extract estrogen-like effects with and with the size of ER α or ER β affinity.From experimental result, Radix Rehmanniae water extract can pass through the expression of estrogen receptor (ER) induction luc fluorescence, and Radix Rehmanniae water extract activated gene is transcribed mainly beta mediated by ER, this is consistent to the higher bibliographical information of ER β affinity with phytoestrogen.
Organism is a complicated system, and vivo and vitro result exists certain deviation.In this experimental applications In vitro cell experiment, body, Integral animal experiment and reporter gene technology combine and jointly verify the estrogenic activity of tested material, have guaranteed the accuracy of result.
By above-mentioned data, fully prove that 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 is mainly by the beta mediated estrogen-like effects of bringing into play of ER.That is to say, Radix Rehmanniae water extract has estrogen-like effects, can be effective to preparation treatment due to the medicine of the relevant disease that in body, estrogen hyposecretion causes, is the innovation of Chinese medicine Radix Rehmanniae in purposes.
Claims (1)
1. the application in preparing estrogens medicine as unique active component Radix Rehmanniae water extract, this Radix Rehmanniae water extract is Radix Rehmanniae to be boiled 2 times at every turn to each 2h with the decocting of its 10 times of weight, merge 2 times decocting liquid, the dry Radix Rehmanniae water extract that obtains of concentrating under reduced pressure.
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