Metabolites of eucommia ulmoides in vivo, and preparation method and use thereof

Abstract

This invention discloses the in vivo metabolites of Eucommia ulmoides, their preparation methods, and uses. Guided by ultra-high performance liquid chromatography-time-of-flight mass spectrometry, urine and feces were collected from rats after gavage administration of Eucommia ulmoides aqueous extract to prepare six metabolites with phytoestrogenic effects: 4-hydroxy-3,4-bis(3-hydroxyphenyl)dihydrofuran-2(3H)-alkane, (-)-2,3-di(3-hydroxybenzyl)butane-1,2,4-triol, (-)-3,3',5'-trihydroxy-9-o-8-8',9-O-9'-lignan, (R)-3-(3-hydroxy-5-(1-hydroxy-3-(3-hydroxyphenyl)propane-2-substituted)phenyl)propionic acid, 3-(3-hydroxy-5-(3-hydroxyphenylethyl)phenyl)propionic acid, and tomentosanan B. These metabolites can be used as pharmaceuticals for the treatment and / or prevention of diseases related to insufficient estrogen secretion in mammals.

Description

Technical Field

[0001] This invention belongs to the field of medical applications and discloses the preparation and use of metabolites of Eucommia ulmoides in rats. In particular, the metabolites were prepared from urine and feces collected after oral administration of Eucommia ulmoides aqueous extract to rats using ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-TOF-MS) for a novel pharmaceutical use as phytoestrogens. Background Technology

[0002] Traditional Chinese medicine (TCM), a cultural treasure with thousands of years of history, has played an irreplaceable role in the prosperity and continuation of Chinese civilization. While TCM is mostly administered orally, its relatively low bioavailability makes it difficult to explain its efficacy. TCM has complex chemical components; for it to exert its effects in the body, absorption into the bloodstream is a prerequisite (except for gastrointestinal diseases). After oral administration of traditional Chinese medicine, some chemical components are directly absorbed into the bloodstream, primarily exerting their effects in their original form. Most components undergo biotransformation in the digestive tract, intestinal flora, and / or liver metabolic enzymes, being absorbed into the body as metabolites. These metabolites then circulate through the bloodstream to target organs and tissues throughout the body, binding to specific enzymes or receptors on target cell membranes or within cells. This binding influences intracellular second messenger molecules, producing biological effects and therapeutic efficacy. [1-3] Therefore, identifying the active ingredients in traditional Chinese medicine based on in vivo metabolic products is the most direct and rapid approach.

[0003] After menopause, as women experience ovarian decline and estrogen secretion decreases, various estrogen-mediated physiological processes are affected, leading to physiological and psychological symptoms such as palpitations, chest tightness, anxiety, depression, irritability, insomnia, and memory loss. These symptoms are collectively known as menopausal syndrome. One beneficial method for treating and / or preventing these symptoms is estrogen supplementation. [4-5] Estrogen exerts different biological effects in different diseases or in different tissues and cells within the same disease. This is due to the complexity of the molecular mechanisms of estrogen and its receptor effects, which determines the specificity of estrogen responses in different tissues / cells. The effects of estrogen are mainly mediated by two receptors—ERα and ERβ. [6-7] However, years of clinical practice have shown that long-term estrogen use significantly increases the risk of breast cancer and endometrial cancer. [8] Therefore, it is necessary to find phytoestrogens with estrogen-like activity but without the side effects of estrogen. Phytoestrogens have a similar structure to estrogen and bind to two estrogen receptors (ERα and ERβ) in vivo, exhibiting estrogen-like activity; simultaneously, they also competitively bind to estradiol (ER) in vivo, producing a competitive antagonistic effect, thus exerting a dual regulatory role. [9-10]Modern pharmacological studies have shown that phytoestrogens play an important role in improving perimenopausal symptoms, improving osteoporosis, preventing cardiovascular diseases, and treating neurodegenerative diseases.

[11] .

[0004] Eucommia ulmoides Oliv., the dried bark of the Eucommia ulmoides plant (Eucommia family), is a precious and unique tonic medicinal herb in my country. It has a sweet taste and warm properties, entering the liver and kidney meridians. It is used to tonify the liver and kidneys, strengthen tendons and bones, and calm the fetus. It is primarily used to treat liver and kidney deficiency, lower back and knee pain, weakness of tendons and bones, dizziness, threatened miscarriage, and threatened abortion. Its main chemical components include lignans, iridoids, flavonoids, and polysaccharides. [12-13] Eucommia ulmoides contains the most lignans, among which pinoresinol diglucoside is an indicator component, with the 2020 edition of the Chinese Pharmacopoeia stipulating a content of no less than 0.10%. Modern pharmacological studies have shown that Eucommia ulmoides has multiple effects, including bidirectional regulation of estrogen, regulation of blood pressure, lowering lipids and blood sugar, antibacterial and anti-inflammatory effects, anti-tumor effects, and liver protection.

[14] According to literature reports, traditional Chinese medicinal herbs with kidney-tonifying and bone-strengthening effects have broad biological activities in bone metabolism, cardiovascular system, and immune regulation.

[15] This may be related to its estrogen-like effects. Although lignans, iridoids, and flavonoids in Eucommia ulmoides have been reported to have strong estrogen-like activity. [16-17] However, the bioavailability of these components in vivo is very low, and the true pharmacologically active forms in vivo are not very clear. There is very little research on the metabolites of Eucommia ulmoides in vivo. Only in 2003 did Japanese scholars prepare 15 metabolites by anaerobic co-culturing human enterobacteria with pinoresinol diglucoside, but further activity evaluation was not carried out.

[0005] Currently, there are no reports on the metabolites, preparation methods, and uses of Eucommia ulmoides in vivo. Based on the above, this invention aims to isolate and prepare the metabolites of Eucommia ulmoides extract in rats by gavage, starting with easily accumulated excreta (urine and feces), using modern chromatographic separation techniques guided by ultra-high performance liquid chromatography-time-of-flight mass spectrometry. Furthermore, the activity of the prepared metabolites will be evaluated using MCF-7 cell proliferation assays and reporter gene transcription activation assays as research models to identify estrogen-like active substances in vivo. Summary of the Invention

[0006] Based on the shortcomings and needs of existing technologies, we propose the in vivo metabolites of Eucommia ulmoides, their preparation methods, and applications;

[0007] The technical solution of the present invention is as follows:

[0008] Method for preparing eucommia metabolites in vivo: After rats were gavaged with eucommia water extract, their excreted urine and feces were enriched. Under the guidance of ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q / TOF-MS), a combination of D101 macroporous adsorption resin column chromatography, reversed-phase C18 silica gel column chromatography (ODS), dextran gel (Sephadex LH-20) column chromatography, and preparative high performance liquid chromatography (HPLC) was used to prepare phytoestrogens-like active metabolites.

[0009] The preparation method of Eucommia ulmoides metabolites in vivo is described in detail, including the following steps:

[0010] Male rats were administered Eucommia ulmoides aqueous extract by gavage for several consecutive days. Urine and feces were collected daily and stored at -80°C. The preparation of metabolites from the urine included the following steps:

[0011] ① The obtained urine was separated by D101 macroporous adsorption resin column chromatography and eluted with an ethanol-water mixed solvent gradient with increasing ethanol content. UPLC-Q / TOF-MS analysis showed that the metabolites were mainly present in the 50% ethanol-water elution fraction.

[0012] ② The 50% ethanol-water elution fraction in step ① was separated by D101 macroporous adsorption resin column chromatography, with gradient elution of ethanol-water mixed solvents with increasing ethanol content, and UPLC-Q / TOF-MS analysis to find the 30% ethanol-water elution fraction of the metabolite fraction.

[0013] ③ The 30% ethanol-water elution fraction in step ② was separated by ODS column chromatography and eluted with a methanol-water solvent system with increasing methanol content to obtain 10%, 20%, 30%, 50%, and 90% methanol-water elution fractions;

[0014] ④ The 20% methanol-water elution fraction in step ③ was separated by Sephadex LH-20 column chromatography and isocratically eluted with methanol-water solvent at a volume ratio of 1:1 to obtain the metabolite (-)-2,3-di(3-hydroxybenzyl)butane-1,2,4-triol (labeled as M2);

[0015] ⑤ The 50% methanol-water elution fraction in step ③ was purified by preparative HPLC chromatography using methanol-water with a volume ratio of 3:7 as the mobile phase to obtain the metabolite tomentosanan B (labeled as M6).

[0016] The preparation of metabolic products from feces includes the following steps:

[0017] ① After the feces were crushed, they were extracted with methanol by ultrasound and concentrated to obtain a methanol extract;

[0018] ②The methanol extract was suspended in pure water and then extracted with petroleum ether to obtain the petroleum ether extract, the aqueous extract and the precipitate.

[0019] ③ The precipitate was dissolved in methanol and then separated by Sephadex LH-20 column chromatography with methanol as the eluent. UPLC-Q / TOF-MS analysis was performed to find the fraction containing metabolites (molecular weight between 250-500 Da).

[0020] ④ The fractions in step ③ were separated by ODS column chromatography and eluted with a methanol-water solvent system with increasing methanol content to obtain 10%, 20%, 30%, 50%, 70%, and 90% methanol-water eluted fractions.

[0021] ⑤ The 70% methanol-water elution fraction in step ④ was purified by preparative HPLC chromatography using acetonitrile-water at a volume ratio of 3.5:6.5 as the mobile phase to prepare the metabolites 4-hydroxy-3,4-bis(3-hydroxyphenyl)dihydrofuran-2(3H)-alkane (labeled as M1), (-)-3,3',5'-trihydroxy-9-o-oxy-8-8',9-O-9'-lignan (labeled as M3) and 3-(3-hydroxy-5-(3-hydroxyphenylethyl)phenyl)propionic acid (labeled as M5);

[0022] ⑥ The 20% methanol-water elution fraction in step ④ was purified by preparative HPLC chromatography using acetonitrile-water as the mobile phase at a volume ratio of 3:7 to obtain the metabolite (R)-3-(3-hydroxy-5-(1-hydroxy-3-(3-hydroxyphenyl)propane-2-substituted)phenyl)propionic acid (labeled as M4).

[0023] The in vivo metabolites of Eucommia ulmoides prepared by the method of this invention can be used as medicines for diseases related to insufficient estrogen secretion.

[0024] The in vivo metabolites of Eucommia ulmoides prepared by the method of this invention can be used as drugs for the treatment of menopausal syndrome, osteoporosis, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, and breast cancer.

[0025] The beneficial effects of this invention are:

[0026] Traditional Chinese medicine is mostly administered orally. After administration, the bioavailability of most chemical components is relatively low. They undergo metabolic transformation in the digestive tract, intestinal flora, and liver metabolic enzymes, ultimately exerting their medicinal effects as metabolites. Eucommia ulmoides has been used clinically for thousands of years to treat estrogen-related diseases such as osteoporosis, menopausal syndrome, and cardiovascular diseases, with proven efficacy. Therefore, identifying estrogen-like substances from the in vivo metabolites of Eucommia ulmoides is the most direct and effective approach. Based on this concept, this invention, using ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-TOF-MS) guided by the urine and feces collected from rats after gavage administration of Eucommia ulmoides extract, used modern chromatographic separation techniques to track, separate, and prepare six in vivo metabolites of Eucommia ulmoides. The estrogen-like activity of these metabolites was evaluated using MCF-7 cell proliferation assays and reporter gene transcription activation assays. The results showed that these in vivo metabolites all exhibit good phytoestrogenic effects and can bind to estrogen receptors to varying degrees to exert their medicinal effects. They can be used in pharmaceuticals to treat and / or prevent diseases related to insufficient estrogen secretion in mammals (especially humans). Attached Figure Description

[0027] Figure 1 The effects of the positive control drug (E2) and its six metabolites on the proliferation of MCF-7 cells were investigated. Compared with the normal group, *P<0.05, **P<0.01, ***P<0.001 indicated statistical significance.

[0028] Figure 2 The effects of the positive control drug (E2) and its six metabolites on ERα and ERβ were compared with those of the normal group. *P<0.05 # P < 0.05, **P < 0.01 ## P < 0.01, ***P < 0.001, ### P < 0.001 indicates that the difference is statistically significant.

[0029] Figure 3 For metabolite M1 1 H NMR spectrum;

[0030] Figure 4 For metabolite M1 13 C NMR spectrum;

[0031] Figure 5 For metabolite M2 1 H NMR spectrum;

[0032] Figure 6 For metabolite M2 13 C NMR spectrum;

[0033] Figure 7 For metabolite M31 H NMR spectrum;

[0034] Figure 8 For metabolite M3 13 C NMR spectrum;

[0035] Figure 9 For metabolite M4 1 H NMR spectrum;

[0036] Figure 10 For metabolite M4 13 C NMR spectrum;

[0037] Figure 11 For metabolite M5 1 H NMR spectrum;

[0038] Figure 12 For metabolite M5 13 C NMR spectrum;

[0039] Figure 13 For metabolite M6 1 H NMR spectrum;

[0040] Figure 14 For metabolite M6 13 C NMR spectrum; Detailed Implementation

[0041] The technical solution of the present invention will now be described with reference to specific embodiments. These embodiments are merely some, not all, of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0042] Example 1

[0043] The method for preparing in vivo metabolites of Eucommia ulmoides includes the following steps:

[0044] (1) Male rats were given Eucommia ulmoides water extract by gavage. The administration was carried out continuously for 5 days and then rested for 2 days. The administration was carried out continuously for 1 month. Urine and feces were collected every day and stored in a -80℃ refrigerator. A total of about 22L of urine and 900g of feces were collected.

[0045] (2) Preparation of metabolites in urine, including the following steps:

[0046] ① The obtained urine was separated by D101 macroporous adsorption resin column chromatography and eluted with water, 10%, 50%, and 95% ethanol-water gradients. UPLC-Q / TOF-MS analysis showed that the metabolites were mainly present in the 50% ethanol-water elution fraction.

[0047] ② The 50% ethanol-water elution fraction in step ① was separated by D101 macroporous adsorption resin column chromatography, with gradient elution of ethanol-water mixed solvents with increasing ethanol content, and UPLC-Q / TOF-MS analysis to find the 30% ethanol-water elution fraction of the metabolite fraction.

[0048] ③ The 30% ethanol-water elution fraction in step ② was separated by ODS column chromatography and eluted with a methanol-water solvent system with increasing methanol content to obtain 10%, 20%, 30%, 50%, and 90% methanol-water elution fractions;

[0049] ④ The 20% methanol-water elution fraction in step ③ was separated by Sephadex LH-20 column chromatography and isocratically eluted with methanol-water solvent at a volume ratio of 1:1 to obtain the metabolite (-)-2,3-di(3-hydroxybenzyl)butane-1,2,4-triol (M2, 11.0 mg).

[0050] ⑤ The 50% methanol-water elution fraction in step ③ was purified by preparative HPLC chromatography with a methanol-water mobile phase of 3:7 (v / v) and a detection wavelength of 280 nm to obtain the metabolite tomentosanan B (retention time 20.5 min, M6, 2.4 mg).

[0051] (3) Preparation of metabolites from feces, including the following steps:

[0052] ① After the feces were crushed, they were ultrasonically extracted three times with 10 times the amount of methanol solvent, 3L / time, 3h each time, and then concentrated to obtain the methanol extract;

[0053] ②The methanol extract was suspended in 3 times its weight of pure water and then extracted 3 times with an equal volume of petroleum ether to obtain the petroleum ether layer extract, the aqueous layer extract and the precipitate.

[0054] ③ The precipitate was dissolved in methanol and then separated by Sephadex LH-20 column chromatography with methanol as the eluent. UPLC-Q / TOF-MS analysis was performed to find the fraction containing metabolites (molecular weight between 250-500 Da).

[0055] ④ The fractions in step ③ were separated by ODS column chromatography and eluted with a methanol-water solvent system with increasing methanol content to obtain 10%, 20%, 30%, 50%, 70%, and 90% methanol-water eluted fractions.

[0056] ⑤ The 70% methanol-water elution fraction from step ④ was subjected to preparative HPLC chromatography with acetonitrile-water at a volume ratio of 3.5:6.5 as the mobile phase and a detection wavelength of 280 nm to prepare the metabolites 4-hydroxy-3,4-bis(3-hydroxyphenyl)dihydrofuran-2(3H)-alkane (retention time 17.0 min, M1, 4.9 mg), (-)-3,3',5'-trihydroxy-9-o-oxy-8-8',9-O-9'-lignan (retention time 21.5 min, M3, 6.2 mg) and 3-(3-hydroxy-5-(3-hydroxyphenylethyl)phenyl)propionic acid (retention time 37.0 min, M5, 103.2 mg);

[0057] ⑥ The 20% methanol-water elution fraction from step ④ was purified by preparative HPLC using acetonitrile-water (3:7 v / v) as the mobile phase and 280 nm as the detection wavelength to obtain the metabolite (R)-3-(3-hydroxy-5-(1-hydroxy-3-(3-hydroxyphenyl)propane-2-substituted)phenyl)propionic acid (retention time 25 min, M4, 21.0 mg).

[0058] The physicochemical constants of the six metabolites are as follows:

[0059] Metabolite M1 (4-hydroxy-3,4-bis(3-hydroxybenzyl)dihydrofuran-2(3H)-one): white amorphous powder, m / z 313.1079 [MH] as determined by HRESI-MS. - (Calculated value 313.1076), the molecular weight of this compound is estimated to be 314, and the molecular formula is determined to be C based on NMR data. 18 H 18 O5, calculated unsaturation degree is 10. [α]25D-35.0 (c 0.04, MeOH); ECD: 220nm (Δε+3.4), 242nm (Δε-9.9); IR (KBr)v max :3062,2937,1607,1366,775cm -1 ; 1 H-NMR (600MHz, CD3OD), see Table 1 and Figure 3 ; 13 C-NMR (150MHz, CD3OD), see Table 1 and Figure 4 .

[0060] Metabolite M2 ((-)-2,3-bis(3-hydroxybenzyl)butane-1,2,4-triol): white amorphous powder, HRESI-MS measured m / z 317.1417 (calculated value 317.1389), suggesting a molecular weight of 318. Combined with NMR data, the molecular formula was determined to be C2. 18 H 22 O5, calculated unsaturation degree is 8. [α]25D-10.0(c 0.06,MeOH); ECD: 230nm(Δε-9.5), 280nm(Δε-2.1); IR(KBr)v max :1652,1565,1372,1359,1149cm -1 ; 1 H-NMR (600MHz, CD3OD), see Table 1 and Figure 5 ; 13 C-NMR (150MHz, CD3OD), see Table 1 and Figure 6 .

[0061] Metabolite M3 ((-)-3,3',5'-trihydroxy-9-o-8-8',9-O-9'-lignan): white amorphous powder, m / z measured by HRESI-MS 313.1086 (calculated value 313.1076), suggesting a molecular weight of 314. Combined with NMR data, the molecular formula was determined to be C1. 18 H 18 O5, calculated unsaturation degree is 10. [α]25D-16.7 (c 0.05, MeOH). 1 H-NMR (600MHz, CD3OD), see Table 1 and Figure 7 ; 13 C-NMR (150MHz, CD3OD), see Table 1 and Figure 8 .

[0062] Metabolite M4 ((R)-3-(3-hydroxy-5-(1-hydroxy-3-(3-hydroxyphenyl)propane-2-substituted)phenyl)propionic acid): white amorphous powder, m / z 315.1234 (calculated value 315.1232) measured by HRESI-MS, suggesting a molecular weight of 316. Combined with NMR data, the molecular formula is determined to be C2. 18 H 20 O5, calculated degree of unsaturation is 8. [α]25D-18.2(c 0.05,MeOH). 1 H-NMR (600MHz, CD3OD), see Table 2 and Figure 9 ; 13 C-NMR (150MHz, CD3OD), see Table 2 and Figure 10 .

[0063] Metabolite M5 (3-(3-hydroxy-5-(3-hydroxyphenylethyl)phenyl)propionic acid): white amorphous powder, m / z measured by HRESI-MS 285.1137 (calculated value 285.1127), suggesting a molecular weight of 286. Combined with NMR data, the molecular formula was determined to be C2. 17 H 18 O4, the calculated degree of unsaturation is 9. 1 H-NMR (600MHz, CD3OD), see Table 2 and Figure 11 ; 13 C-NMR (150MHz, CD3OD), see Table 2 and Figure 12 .

[0064] Metabolite M6 (tomentosanan B): a white amorphous powder. HRESI-MS measured m / z 301.1072 (calculated value 301.1076), suggesting a molecular weight of 302. Combined with NMR data, the molecular formula was determined to be C2. 17 H 18 O5, the calculated degree of unsaturation is 9. 1 H-NMR (600MHz, CD3OD), see Table 2 and Figure 13 ; 13 C-NMR (150MHz, CD3OD), see Table 2 and Figure 14 .

[0065] Table 1. Carbon and hydrogen spectral data of metabolites M1-M3

[0066]

[0067]

[0068] Note: The solvent used for metabolites M1-M3 was CD3OD, and the 1H NMR spectrum was measured (600MHz) and the 1C NMR spectrum was measured (150MHz).

[0069] Table 2. Carbon and hydrogen spectral data of metabolites M4-M6

[0070]

[0071] Note: The solvent used for metabolites M4-M6 was CD3OD, and the 1H NMR spectrum was measured (600MHz) and the 1C NMR spectrum was measured (150MHz).

[0072] The structural formulas of the six metabolites are as follows:

[0073]

[0074] Example 2

[0075] The proliferation assay of the six metabolites of this invention in MCF-7 cells confirmed their estrogenic activity:

[0076] MCF-7 cells were cultured in a monolayer at 37°C and 5% CO2 in complete medium (DMEM) supplemented with FBS (10%) and penicillin (100 U / mL). -1 ) and streptomycin (100 U·mL) -1 (1%). MCF-7 cells were digested with trypsin. 1.5 × 10⁻⁶ 3 Cells were seeded in 96-well plates and incubated for 24 hours. Cells were then treated with 0.1% DMSO (control group) and 10... -8 10 -7 10 -6 10 -5 Cells were treated with mol / L Eucommia ulmoides metabolites, which were dissolved in the complete culture medium. Cell viability was determined using the MTT assay 24 h after metabolite treatment. Data were analyzed using one-way ANOVA.

[0077] Using β-estradiol (E2) as the positive control, compared with their respective control groups, E2 and its metabolites all promoted the proliferation of MCF-7 cells. Specific results are shown in Table 3 and... Figure 1 The positive control drug E2 significantly promoted the proliferation of MCF-7 cells (p<0.01), and its effect was significant at 10... -8 ~10 -6 It showed a significant promoting effect at all concentrations of mol / L, with the maximum being 10 mol / L. -6 At a concentration of mol / L, the promotion rate was 32.4%. Metabolites M1, M2, M4, and M6 showed a promoting effect at sufficiently high concentrations, as evidenced by a 10 mol / L increase. -5 At concentrations of mol / L, it can significantly promote the proliferation of MCF-7 cells, increasing proliferation by 16.8%, 35.8%, 29.7%, and 26.1%, respectively (p<0.05). Metabolite M3 is the most effective component in promoting MCF-7 cell proliferation, at 10 mol / L. -8 At a concentration of mol / L, M3 can significantly promote the proliferation of MCF-7 cells, and at 10 mol / L... -8 mol / L to 10 -5 Significant MCF-7 cell proliferation was observed across the entire concentration range, with the effect increasing with increasing concentration. The promoting effects at the four concentrations were 21.6%, 37.5%, 38.7%, and 44.1% (p<0.05 or p<0.001). The metabolite M5 was only present at concentrations of 10 mol / L. -8 mol / L can promote the proliferation of MCF-7 cells, while 10 mol / L can promote the proliferation of MCF-7 cells. -7 mol / L to 10 -5Within the mol / L range, it did not significantly promote the proliferation of MCF-7 cells, and the proliferation increased with increasing concentration to 10 mol / L. -5 At mol / L, cell proliferation tends to be inhibited.

[0078] Table 3. Effects of six metabolites M1-M6 on MCF-7 cell proliferation (MEAN±SEM)

[0079]

[0080]

[0081] Example 3

[0082] The six metabolites of this invention were confirmed to activate estrogen receptor levels in HEK293 cell assays.

[0083] HEK-293 cells were cultured in a monolayer at 37°C and in 5% CO2 complete medium. The complete medium was DMEM supplemented with 10% CS-FBS and 100 U / mL penicillin. -1 ) and streptomycin (100 U·mL) -1 (1%). HEK-293 cells were digested with trypsin. 1×10 4 Cells were seeded in 96-well plates and incubated for 24 hours. Two EP tubes were labeled. DMEM, ESR1 plasmid (50 ng / well), ERE plasmid (50 ng / well), and Renilla plasmid (25 ng / well) were added to ESR1 tube; DMEM, ESR2 plasmid (50 ng / well), ERE plasmid (50 ng / well), and Renilla plasmid (25 ng / well) were added to ESR2 tube. The mixtures were gently mixed and allowed to stand at room temperature for a few minutes. Then, TransactionReagent (0.6 μL / well) was added to each well, the plates were inverted and shaken well, and allowed to stand for 15 minutes. Finally, the transfection compound was added to each well of the 96-well plate (100 μL / well). After culturing for 12 hours, the transfection complex was discarded, and the cells were cultured in complete culture medium for 24 hours. The cells were then cultured with complete culture medium (control group) and 10... -2Cells were treated with μmol / L β-estrogens and 10 μmol / L Eucommia ulmoides metabolites, dissolving them in complete culture medium. After 24 h of treatment, cell viability was determined using a dual-luciferase reporter gene assay. Cells were lysed using cell lysis buffer, and after thorough lysis, the supernatant was collected by centrifugation and placed in 96-well plates. Firefly luciferase assay reagent was added, and the chemiluminescence value was measured using a microplate reader. Renila luciferase assay reagent was then added, and the chemiluminescence value was measured again. The fluorescence values ​​of both firefly luciferase and Renila luciferase were recorded. The activation levels of the target reporter gene among different samples were compared using the firefly luciferase RLU / Renila luciferase RLU values. Data were analyzed using one-way ANOVA.

[0084] β-estradiol (E2) was a positive control, and compared with the control group, it significantly activated estrogen receptors in HEK293 cells, resulting in a 4.72-fold and 6.66-fold increase in the transcriptional expression levels of downstream genes of ERα and ERβ, respectively. Six metabolites, M1-M6, could also activate estrogen receptors ERα and ERβ, leading to increased transcription and expression levels of their downstream genes. Among them, M1 activated estrogen receptors, increasing the transcriptional expression levels of downstream genes of ERα and ERβ by 3.39 and 2.93 times, respectively, with a statistically significant effect on ERα (p<0.001); M2 increased the transcriptional expression levels of downstream genes of ERα and ERβ by 2.17 and 1.90 times, respectively; M3 increased the transcriptional expression levels of downstream genes of ERα and ERβ by 2.47 and 2.03 times, respectively, with a statistically significant effect on ERα (p<0.05); M4 showed significant agonistic effects on both ERα and ERβ, increasing the transcriptional expression levels of downstream genes of ERα and ERβ by 2.97 and 3.25 times, respectively (p<0.001 and p<0.05); M5 increased the transcriptional expression levels of downstream genes of ERα and ERβ by 2.83 and 2.34 times, respectively; and M6 increased the transcriptional expression levels of downstream genes of ERα and ERβ by 3.77 and 2.41 times, respectively, with a statistically significant effect on ERα (p<0.001). Specific results are shown in Table 4 and Figure 2 .

[0085] Table 4. Activation effects of six metabolites M1-M6 on estrogen receptors (MEAN±SEM)

[0086]

[0087] Estrogens can bind to estrogen receptors (ER), which are found in osteoblasts, osteoclasts, the cardiovascular system, and the mammary glands. Phytoestrogens are widely used as natural estrogen receptor modulators for the prevention and / or treatment of osteoporosis. Our research has confirmed that Eucommia ulmoides metabolites can bind to ER and enhance transcriptional activity, demonstrating that these metabolites exert estrogen-like effects in the clinical and pharmacological studies of Eucommia ulmoides. They could be used to treat osteoporosis, cardiovascular and cerebrovascular diseases, neurodegenerative diseases, menopausal syndrome, and breast cancer caused by insufficient estrogen secretion.

[0088] The above embodiments are implementation methods of this patent. Without departing from the technical principles of this patent, appropriate improvements can be made, and such improvements should fall within the protection scope of this patent.

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[17] SUETSUGI M,Su L L,KARLSBERG K,et al.Flavone and IsoflavonePhytoestrogens Are Agonists ofEstrogen-Related Receptors.Molecular CancerResearch,2003,1(13):981-991。

Claims

1. A method for preparing in vivo metabolites of Eucommia ulmoides; characterized in that, After administering Eucommia ulmoides aqueous extract to rats via gavage, the excreted urine and feces were enriched. Guided by ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-TOF-MS), a combination of macroporous adsorption resin column chromatography, reversed-phase C18 silica gel column chromatography, dextran gel column chromatography, and preparative HPLC techniques were used to prepare phytoestrogens-like active metabolites. The steps included: Male rats were administered Eucommia ulmoides aqueous extract by gavage for several consecutive days. Urine and feces were collected daily and stored in a -80°C refrigerator. The preparation of metabolic products from urine includes the following steps: ① The obtained urine was separated by D101 macroporous adsorption resin column chromatography and eluted with a gradient of ethanol-water mixed solvent with increasing ethanol content. UPLC-Q / TOF-MS analysis showed that the metabolites were mainly present in the 50% ethanol-water elution fraction. ② The 50% ethanol-water elution fraction in step ① was separated by D101 macroporous adsorption resin column chromatography, with gradient elution of ethanol-water mixed solvents with increasing ethanol content, and UPLC-Q / TOF-MS analysis to find the 30% ethanol-water elution fraction of the metabolite fraction. ③ The 30% ethanol-water elution fraction in step ② was separated by ODS column chromatography and eluted with a methanol-water solvent system with increasing methanol content to obtain 10%, 20%, 30%, 50%, and 90% methanol-water elution fractions. ④ The 20% methanol-water elution fraction in step ③ was separated by Sephadex LH-20 column chromatography and isocratically eluted with methanol-water solvent at a volume ratio of 1:1 to obtain the metabolite (-)-2,3-di(3-hydroxybenzyl)butane-1,2,4-triol; ⑤ The 50% methanol-water elution fraction in step ③ was purified by preparative HPLC chromatography using methanol-water at a volume ratio of 3:7 as the mobile phase to obtain the metabolite tomentosanan B. The preparation of metabolic products from feces includes the following steps: ① After the feces were crushed, they were extracted with methanol by ultrasound and concentrated to obtain a methanol extract; ②The methanol extract was suspended in pure water and then extracted with petroleum ether to obtain the petroleum ether extract, the aqueous extract and the precipitate. ③ The precipitate was dissolved in methanol and then separated by Sephadex LH-20 column chromatography with methanol as the eluent. UPLC-Q / TOF-MS analysis was performed to find the fraction containing metabolites with a molecular weight between 250-500 Da. ④ The fractions in step ③ were separated by ODS column chromatography and eluted with a methanol-water solvent system with increasing methanol content to obtain 10%, 20%, 30%, 50%, 70%, and 90% methanol-water eluted fractions. ⑤ The 70% methanol-water washing fraction in step ④ was purified by preparative HPLC chromatography using acetonitrile-water at a volume ratio of 3.5:6.5 as the mobile phase to prepare the metabolites 4-hydroxy-3,4-bis(3-hydroxyphenyl)dihydrofuran-2(3H)-alkane, (-)-3,3',5'-trihydroxy-9-oxy-8-8',9-O-9'-lignan and 3-(3-hydroxy-5-(3-hydroxyphenylethyl)phenyl)propionic acid; ⑥ The 20% methanol-water washing fraction in step ④ was purified by preparative HPLC chromatography using acetonitrile-water at a volume ratio of 3:7 as the mobile phase to obtain the metabolite (R)-3-(3-hydroxy-5-(1-hydroxy-3-(3-hydroxyphenyl)propane-2-substituted)phenyl)propionic acid.

2. The use of the Eucommia ulmoides in vivo metabolites prepared by the method of claim 1 in the preparation of a medicament for a disease related to insufficient estrogen secretion; wherein the disease is menopausal syndrome.

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