CN102579673B - Application of fresh rehmannia root aqueous extract in preparation of estrogen medicines - Google Patents

Abstract

The invention relates to the application of fresh rehmannia glutinosa water extract in the preparation of estrogen drugs, which can effectively solve the preparation of related diseases caused by insufficient secretion of estrogen in the body, thereby effectively solving the related diseases caused by insufficient secretion of estrogen in the body For the treatment of medication problems, the fresh rehmannia water extract is to decoct the fresh rehmannia glutinosa twice with water 10 times its weight each time, for 2 hours each time, combine the decoctions twice, concentrate and dry under reduced pressure to obtain the fresh rehmannia water extract The water extract of fresh rehmannia glutinosa can be effectively used in the preparation of medicines for treating related diseases caused by insufficient secretion of estrogen in the body, and realize that the water extract of fresh rehmannia glutinosa can be used in the preparation of medicines for related diseases caused by insufficient secretion of estrogen in the body. application, the preparation method of the water extract of the present invention is simple, stable, reliable, convenient, low in cost, effectively used for preparing estrogen drugs, and opens up a new medicinal use of fresh rehmannia glutinosa.

Description

Application of fresh rehmannia glutinosa water extract in the preparation of estrogen drugs

technical field

The invention relates to medicine, in particular to the application of fresh rehmannia glutinosa water extract in the preparation of estrogen drugs. The fresh rehmannia glutinosa water extract has estrogen-like effects and is effectively used for related diseases caused by insufficient secretion of estrogen in the body ( Such as the treatment of women's menopause syndrome, etc.).

Background technique

Fresh Rehmannia glutinosa is the fresh root tuber of Rehmannia glutinosa Libosch., which is excavated in autumn, removed the reed heads, fibrous roots and sediment, and freshly used, which is Fresh Rehmannia glutinosa. Famous experts of all dynasties have discussed and studied it, thinking that it is sweet, bitter, and cold in nature, returns to the heart, liver, and kidney meridian, clears away heat and promotes body fluid, cools blood, and stops bleeding. Rash, vomiting blood, epistaxis, sore throat. Modern pharmacological and clinical studies have shown that fresh rehmannia can be used in the treatment of inflammatory diseases, such as otitis media, pharyngitis, arthritis, hepatitis, lupus erythematosus, nephritis, diabetes, etc. But there is still no research report on the estrogen-like activity of fresh rehmannia glutinosa.

Contents of the invention

In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide an application of fresh rehmannia glutinosa water extract in the preparation of estrogen drugs, which can effectively solve the related diseases caused by insufficient secretion of estrogen in the body. (such as women's climacteric syndrome, etc.), thereby effectively solving the problem of the treatment of related diseases (such as women's climacteric syndrome, etc.) caused by insufficient secretion of estrogen in the body.

The technical scheme solved by the present invention is the application of the fresh rehmannia water extract in the preparation of estrogen drugs. The fresh rehmannia root water extract is decocting fresh rehmannia rehmannia twice with water 10 times its weight each time for 2 hours , combined two decoctions, concentrated and dried under reduced pressure to obtain fresh rehmannia water extract, the fresh rehmannia water extract can be effectively used to prepare medicines for treating related diseases caused by insufficient secretion of estrogen in the body, and realize the fresh rehmannia water extract Application of the drug in the preparation of drugs for treating related diseases (such as women's climacteric syndrome, etc.) caused by insufficient secretion of estrogen in the body.

The preparation method of the water extract of the present invention is simple, stable and reliable, and after many tests, the same or similar results have been obtained, which is convenient and low in cost. New medicinal use, great clinical significance.

Description of drawings

示意图。Fig. 1 is the effect of fresh rehmannia glutinosa water extract of the present invention on MCF-7 cell proliferation

schematic diagram.

示意图。Fig. 2 is the influence of fresh rehmannia glutinosa water extract of the present invention on the uterine coefficient of immature mice

schematic diagram.

示意图。Fig. 3 is the transient expression result of the reporter gene regulated by fresh rehmannia glutinosa water extract of the present invention to ERE

schematic diagram.

示意图。Fig. 4 is the transient expression result of the reporter gene regulated by fresh rehmannia glutinosa water extract of the present invention to ERE

schematic diagram.

Detailed ways

Below in conjunction with embodiment and relevant test data the concrete situation of the present invention is described in detail.

In the specific implementation of the present invention, the application of the described fresh rehmannia glutinosa water extract in the preparation of estrogen drugs, the fresh rehmannia glutinosa water extract is given by the following examples:

Example 1

In the specific implementation of the present invention, the fresh rehmannia water extract is that fresh rehmannia glutinosa (Rehmannia glutinosa Libosch.) 100g is decocted twice with 10 times the weight of water 1000g (i.e. 1000ml), each time for 2h, and combined twice The water decoction was concentrated and dried under reduced pressure to obtain the water extract of fresh rehmannia glutinosa with a yield of 12.03%. The water extract can be used to prepare estrogen drugs for the treatment of diseases caused by insufficient secretion of estrogen in the body, such as women's climacteric syndrome disease medicine.

Example 2

In the specific implementation of the present invention, the fresh rehmannia water extract is that fresh rehmannia (Rehmannia glutinosa Libosch.) 200g is decocted twice with 2000g (i.e. 2000ml) of water 10 times its weight, each time for 2h, and combined twice The water decoction was concentrated and dried under reduced pressure to obtain the water extract of fresh rehmannia glutinosa with a yield of 15.84%. The water extract can be used to prepare estrogen drugs for the treatment of diseases caused by insufficient secretion of estrogen in the body, such as women's climacteric syndrome disease medicine.

Example 3

In the specific implementation of the present invention, the fresh rehmannia water extract is that fresh rehmannia (Rehmannia glutinosa Libosch.) 300g is decocted twice with 3000g (i.e. 3000ml) of water 10 times its weight, each time for 2h, and merged twice The water decoction was concentrated and dried under reduced pressure to obtain the water extract of fresh rehmannia glutinosa with a yield of 14.99%. The water extract can be used to prepare estrogen drugs for the treatment of diseases caused by insufficient secretion of estrogen in the body, such as women's climacteric syndrome disease medicine.

According to the above method, any amount of fresh rehmannia water extract can be produced by industrial production according to the ratio of fresh rehmannia glutinosa and water, and through repeated experiments, the same and similar results have been obtained. The method is stable and reliable, and the obtained fresh rehmannia glutinosa water extract It is used for the preparation of estrogen drugs, which can effectively solve the problem of drug treatment of diseases (such as women's climacteric syndrome) caused by insufficient secretion of estrogen in the body, and has been fully proved by tests. The relevant test data are as follows:

It has been fully proved by cell proliferation experiments, animal experiments and reporter gene technology, and the relevant test data are as follows:

First, through the MCF-7 cell proliferation experiment (E-SCREEN), it was found that fresh rehmannia glutinosa water extract can promote the proliferation of MCF-7 cells, indicating that it has estrogen-like effects in vitro.

Secondly, through the mouse uterine weight gain experiment, it is proved that the water extract of fresh rehmannia glutinosa can significantly increase the uterine coefficient of immature female mice, indicating that it has estrogen-like effects in vivo.

Finally, through the transient expression detection of the reporter gene regulated by ERE, it was further verified that the water extract of fresh rehmannia glutinosa exerted estrogen-like effects by binding to the estrogen receptor ERβ.

The inventor has proved the new application of the drug of the present invention in the preparation of estrogen drugs through experiments. Its main implementation scheme is as follows:

1. Experimental materials and methods

1. Experimental drug

The fresh tuber root of Rehmannia glutinosa Libosch. Excavated in autumn, remove reed heads, fibrous roots and sediment, that is fresh rehmannia glutinosa.

Fresh Rehmannia glutinosa was decocted twice with 10 times the weight of water, each time for 2 hours, and the decoction was dried under reduced pressure, concentrated and dried to obtain fresh rehmannia glutinosa water extract.

2. Experimental animals, cell lines and plasmids

Kunming mice, female, 21 days old (just weaned), weighing 9-12 g, were purchased from Experimental Animal Center of Henan Province. Human breast cancer cells (MCF-7) were provided by the Institute of Bioengineering, Chinese Academy of Military Medical Sciences. HEK293 cell lines were purchased from China Center for Type Culture Collection. The β-galactosidase (β-galactosidase, β-gal) control plasmid pβgal-Control and the recombinant reporter gene pERE-TAL-luc were donated by Dr. Ye Qinong, Institute of Bioengineering, Academy of Military Medical Sciences. The recombinant human ERα (humanERα, hERα) expression vector pCXN2-hERα and recombinant human ERβ (humanERβ, hERβ) expression vector pCXN2-hERβ were donated by Dr. Satoshi Inoue, Faculty of Medicine, University of Tokyo.

3. Main reagents

稳定荧光素酶检测系统试剂盒

Luciferase AssaySysterm)、闪亮裂解缓冲液(Glo lysis Buffer)购自Promega公司；其余试剂均为国产分析纯。RPMI1640 medium, calf serum (Newborn Calf Serum, NCS) and liposome LipofectamineTM2000Reagent were purchased from Gibco Invitrogen; fetal bovine serum, phenol red-free RPMI1640 medium, 17β-estradiol (17β-estrogen, E ₂ ), Ampicillin (Amp), Tris base and activated carbon (Charcoal) were purchased from Sigma; dextran T-70 (Dextran-70) was purchased from Shanghai Chemical Reagent Company; diethylstilbestrol tablets (Hefei Jiulian Pharmaceutical); Benzene-β-D-galactopyranoside (O-Nitrophenyl-β-D-galactopy ranoside, ONPG), EDTA, MTT and DMSO are products of Amresco;

Stable Luciferase Assay System Kit

Luciferase Assay System), Shiny lysis buffer (Glo lysis Buffer) were purchased from Promega Company; the rest of the reagents were domestic analytical grade.

4. Main instruments used

General surgical instruments; carbon dioxide incubator (REVCO); inverted microscope (NIKON ECLIPSE TS100); KDC-160HR high-speed low-temperature refrigerated centrifuge (KUST Innovations Co., Ltd.); microplate reader (BIO-RAD 680); pure water instrument ( Sartorius 611VF); 90-3 magnetic stirrer (Shanghai Zhenjie Experimental Equipment Co., Ltd.); ZRD-7080 fully automatic new blast drying oven (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.); micro sampler (Nichipet EX PLUS) ; AB204-N electronic reading analysis balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd. product); 10cm culture dish, 96-well culture plate, cryopreservation tube are all produced by Corning Company; Group); UV-7504PC UV-Vis Spectrophotometer (Fuzhou Jianyang Technology Instrument Co., Ltd.); VeritasTM Microplate Photometer (TurnerBioSystems); SK6200H Ultrasonic Cleaner (Shanghai Kedao Ultrasonic Instrument Co., Ltd.); SHP- 150-type biochemical incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.).

5. Experimental method

5.1 MCF-7 cell proliferation assay

After MCF-7 cells were cultured in RPMI1640 medium containing 10% hormone-free serum without phenol red for 2 weeks, the cells in the logarithmic growth phase were selected, washed twice with PBS, digested with 0.25% trypsin, and added with 2% serum without phenol red The RPMI1640 medium with hormone-free serum was pipetted evenly, and inoculated in a 96-well plate at a concentration of 1×10 ⁴ cells/ml, and the total culture volume of each well was 200 μl. After culturing for 24 hours, after the cells adhered to the wall, they were replaced with drug-containing medium to continue culturing. After culturing at 37°C and 5% _CO2 for 72 hours, add 20 μl of MTT solution (5 mg/ml) to each well, continue to cultivate at 37°C for 4 hours, carefully aspirate the culture solution, add 150 μl DMSO to each well, and shake for 5-10 minutes to completely dissolve the crystals . Adjust to zero with DMSO, measure the absorbance value (A) of each well at 490 nm with a microplate reader, and calculate the average A value and the proliferation rate (Proliferation Rate, RP). The experiment was repeated three times in parallel.

RP=experimental group A value/blank group A value) ^-1 × 100%

5.2 Mouse uterus weight gain experiment

The mice were divided into groups according to the principle of weight balance and randomization. Dosing continued for 7 days. The blank control group was intragastrically administered with 0.2ml/10g of distilled water every day; the positive control group was intragastrically administered with diethylstilbestrol suspension (0.35mg/kg/d) every day; Make a suspension of corresponding concentration, and give it to the stomach at 0.2ml/10g every day. 24 hours after the last administration, the mice were killed by cervical dislocation, and the uterus was immediately removed and weighed, and the uterine coefficient was calculated (wet weight of uterus × (body weight) ^{- 1} × 100%). The experiment was repeated three times in parallel. Results Compared with the blank group, calculate the P value to see if there is any statistical significance.

5.3 Detection of transient expression of reporter genes regulated by ERE

5.3.1 Cell Seeding

72 hours before transfection, HEK293 cells in the logarithmic growth phase were inoculated on a 24-well plate at a density of 16.0× ¹⁰⁴ /0.5ml/well, and the culture medium was phenol red-free RPMI1640 culture containing 10% estrogen-depleted serum solution, respectively set blank control wells, estradiol wells and drug wells to be tested.

5.3.2 Transfection of plasmids

(1) Transfection was started after 72 hours, the original medium was carefully sucked out, and 400 μl of phenol red-free RPMI1640 medium containing 10% estrogen-depleted serum was added.

(2) Take 3 EP tubes, add 500 μl phenol red-free RPMI1640 medium, 8 μlpCXN2-hERα plasmid, 8μlpERE-TAL-luc plasmid, 8μlpβgal-Control plasmid to ERα tube; add 500μl phenol red-free RPMI1640 medium, 8 μl of CXN2-hERβ plasmid, 8 μl of ERE-TAL-Iuc plasmid, 8 μl of βgal-Control plasmid; 1000 μl of phenol red-free RPMI1640 medium and 24 μl of liposomes were added to the liposome tube.

(3) Invert and mix the liposome tube, place at room temperature for 5 minutes, take 500 μl into ERα and ERβ tubes respectively, and place at room temperature for 20 minutes;

(4) Invert and mix the ERα and ERβ tubes, add 80 μl of the liposome/DNA complexes in the ERα tubes to the ERα experimental wells of the 24-well plate, and add 80 μl of the liposome/DNA complexes in the ERβ tubes to the ERβ experimental wells of the 24-well plate respectively . Shake back and forth and place in the incubator.

(5) Add control drug and test drug after 6 hours.

稳定荧光素酶检测系统试剂盒(Steady-

Luciferase Assay Systerm)及闪亮裂解缓冲液(Glolysis Buffer)改良操作步骤操作，收集细胞裂解上清液，加入荧光素酶反应底物(荧光素)，置于暗处，用VeritasTM微板光度计检测荧光强度。5.3.3 Detection of transient expression of reporter gene regulated by ERE For the cells 24 hours after drug addition, the culture medium was aspirated, washed once with 1ml PBS, and the PBS was removed. according to

Stable luciferase detection system kit (Steady-

Luciferase Assay System) and shiny lysis buffer (Glolysis Buffer) to improve the operation steps, collect the cell lysate supernatant, add the luciferase reaction substrate (luciferin), put it in the dark, and detect it with a VeritasTM microplate photometer The fluorescence intensity.

5.3.4 β-gal activity detection

In order to examine the transfection efficiency, the internal reference β-gal activity was detected.

(1) Add 54μl β-mercaptoethanol to 40ml Z-buffer, mix well, take EP tubes, add 1800μl to each tube;

(2) Add 20 μl lysed supernatant to each tube;

(3) Add 400μl ONPG to each tube, invert and mix;

(4) Place in a 37°C incubator until it turns yellow;

(5) Add Na ₂ CO ₃ 1ml to terminate the reaction;

(6) Measure OD ₄₂₀ value;

2. Statistical processing

表示，SPSS13.0进行单因素方差分析(One-Way ANOVA)统计处理。Experimental data with

Said, SPSS13.0 for one-way analysis of variance (One-Way ANOVA) statistical processing.

3. Experimental results

1. Experimental results of the effect of fresh rehmannia glutinosa water extract on the proliferation of MCF-7 cells

The results showed that compared with the blank group, the fresh rehmannia glutinosa water extract had a significant proliferation-promoting effect on MCF-7 cells (P<0.01). It shows that the water extract of fresh rehmannia glutinosa has estrogen-like effects in vitro. See Table 1, Figure 1.

Table 1 The effect of fresh rehmannia glutinosa water extract on the proliferation of MCF-7 cells

Note: Compared with the blank control group, ☆ means P<0.05, ★ means P<0.01; compared with the positive group, △ means P<0.05, ▲ means P<0.01.

2. Experimental results of the effect of fresh rehmannia glutinosa water extract on the uterine coefficient of mice

The results showed that the fresh rehmannia water extract group could significantly induce the increase of the uterine coefficient of immature mice compared with the blank control group (P<0.01), indicating that the fresh rehmannia water extract had estrogen-like effects in vivo. See Table 2, Figure 2.

Table 2 The effect of fresh rehmannia glutinosa water extract on the uterine coefficient of immature mice

Note: Compared with the blank control group, ☆ means P<0.05, ★ means P<0.01; compared with the positive control group, △ means P<0.05, ▲ means P<0.01.

3. ERE-regulated reporter gene transient expression detection to evaluate whether fresh rehmannia glutinosa water extract exerts estrogen-like effects through receptor pathway

The effect of the interaction between fresh rehmannia glutinosa water extract and estrogen receptors (Estrogen Receptors, ERs) was detected by reporter gene technology, and the final concentration of 17-estradiol (E ₂ ) in the positive control group was 10 ^-8 mol/L , fresh rehmannia glutinosa water extract dosing concentrations were 0.001mg/ml, 0.01mg/ml, 0.1mg/ml, 1mg/ml. The results showed that, whether mediated by ERα or ERβ, the normalized luciferase activity of _E2 was significantly higher than that of blank wells (P<0.01). When mediated by ERα, the normalized luciferase activity of four dosage groups of fresh rehmannia glutinosa water extract 0.001mg/ml～1mg/ml was not statistically significant compared with the blank well (P>0.05), see Table 3, image 3. When mediated by ERβ, the luciferase activity after normalization of fresh rehmannia glutinosa water extract 0.001mg/ml, 0.01mg/ml, 0.1mg/ml, 1mg/ml group was significantly higher than that of the blank well (P<0.01), and It is dose-dependent. See Table 4 and Figure 4.

Table 3 Detection results of transient expression of reporter gene regulated by fresh rehmannia glutinosa water extract on ERE

Note: Compared with the blank control group, ☆ means P<0.05, ★ means P<0.01; compared with the positive group, △ means P<0.05, ▲ means P<0.01.

Table 4 Detection results of transient expression of reporter gene regulated by fresh rehmannia glutinosa water extract on ERE

Note: Compared with the blank control group, ☆ means P<0.05, ★ means P<0.01; compared with the positive group, △ means P<0.05, ▲ means P<0.01.

The results of MCF-7 cell proliferation experiment showed that fresh rehmannia glutinosa water extract had a significant proliferation effect on MCF-7 cells when the concentration was 0.01mg/ml-1mg/ml, and the effect was the strongest at 0.1mg/ml.

The results of mouse uterine weight gain experiments showed that the water extract of fresh rehmannia glutinosa can significantly increase the uterine coefficient of immature mice, indicating that the water extract of fresh rehmannia glutinosa has estrogen-like activity both in vivo and in vitro.

The test results of the transient expression of the reporter gene regulated by ERE showed that the estrogen-like effect of fresh rehmannia glutinosa water extract was mainly mediated by ERβ, and the concentration of 0.001mg/ml, 0.01mg/ml, 0.1mg/ml, 1mg/ml could Activation of gene transcription mediated by ERβ was dose-dependent; but for gene transcription mediated by ERα, fresh rehmannia glutinosa water extract was comparable to blank at 0.001mg/ml, 0.01mg/ml, 0.1mg/ml, 1mg/ml Holes have no effect.

To sum up, the results of animal and cell experiments show that fresh rehmannia glutinosa water extract has certain estrogen-like effects, which are mainly through ERβ-mediated gene transcription to exert estrogen-like effects.

4. Conclusion

MCF-7 cells are estrogen receptor-positive human breast cancer cell lines, which can be specifically regulated by estrogen or estrogen-like active substances to proliferate, and are the most commonly used cell lines for detecting estrogen-like activity. The MCF-7 cell estrogen-like proliferation assay is very sensitive and can distinguish estrogen agonists and antagonists, so it is widely used in large quantities and rapid screening and evaluation of environmental estrogens and phytoestrogens in vitro. In the experiment of the effect of fresh rehmannia water extract on the proliferation of MCF-7 cells, the fresh rehmannia water extract has a significant effect on the proliferation of MCF-7 cells at 0.01mg/ml～1mg/ml, which shows that fresh rehmannia water The extract has estrogen-like activity in vitro.

Uterine weight gain test is the earliest established classic method to detect estrogen activity. When evaluating estrogen activity, it takes into account the absorption, metabolism, binding to plasma protein and pharmacokinetics of substances and other factors, directly reflecting the test results. The overall biological effect of a drug after it has been metabolized in the body. Therefore, the ratio of uterine wet weight to body weight (uterine coefficient) of sexually immature female mice was used as an index for evaluating estrogen-like activity in this experiment. The results showed that the water extract of fresh rehmannia can significantly increase the uterine coefficient of immature mice, which proves that the water extract of fresh rehmannia has estrogen-like effects in vivo.

The above experiments were repeated many times, and all obtained the same or similar results, suggesting that the water extract of fresh rehmannia glutinosa has certain estrogen-like effects both in vivo and in vitro.

Reporter gene is a kind of gene whose expression product is very easy to detect and easy to distinguish from endogenous background protein. By means of genetic engineering, the response element is connected to the reporter gene, and by detecting the expression activity of the reporter gene, the activity of the signal transduction pathway and its regulation and influence on intracellular gene expression can be known. Reporter gene technology has high sensitivity, strong selectivity and simple operation. In the induction experiment of fresh rehmannia glutinosa water extract on the transient expression of reporter gene vector pERE-TAL-luc, ERE was used to regulate the transient transfection of the reporter gene, and the vectors carrying ERα, ERβ and ERE sequences as regulatory elements were constructed respectively. Recombinant reporter gene carrier plasmid transiently co-transfects target cells (HEK 293) as a drug screening model. After adding different concentrations of test drugs, by observing the expression of the reporter gene, that is, the level of fluorescence intensity can detect whether the test substance can Combine with estrogen receptor (ER), so as to understand the strength of estrogen-like effect of fresh rehmannia glutinosa water extract and its affinity with ERα or ERβ. It can be seen from the experimental results that the water extract of fresh rehmannia glutinosa can induce the expression of luc fluorescence through the estrogen receptor (ER), and the activation of gene transcription by the water extract of fresh rehmannia glutinosa is mainly mediated by ERβ, which is similar to the effect of phytoestrogens on ERβ The literature reports with higher affinity are consistent.

Organisms are complex systems, and there are certain deviations in in vivo and in vitro results. In this experiment, the combination of in vitro cell experiment, in vivo whole animal experiment and reporter gene technology is used to verify the estrogen-like activity of the test substance, which ensures the accuracy of the results.

The above data fully prove that the fresh rehmannia water extract can promote the proliferation of MCF-7 cells and increase the wet weight of the uterus of immature mice. Further ERE-regulated reporter gene transient expression detection technology suggests that the fresh rehmannia water extract is mainly through ERβ-mediated estrogen-like effects. That is to say, the water extract of fresh rehmannia glutinosa has estrogen-like effects, and can be effectively used to prepare medicines for treating related diseases caused by insufficient secretion of estrogen in the body, which is an innovation in the use of the traditional Chinese medicine fresh rehmannia glutinosa.

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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