CN106176782B

CN106176782B - Application of chemical components of eclipta as phytoestrogen

Info

Publication number: CN106176782B
Application number: CN201610524066.XA
Authority: CN
Inventors: 高秀梅; 王彧; 韩立峰; 苗琳; 王雪妮; 刘二伟; 王虹
Original assignee: Tianjin University of Traditional Chinese Medicine
Current assignee: Tianjin University of Traditional Chinese Medicine
Priority date: 2016-07-06
Filing date: 2016-07-06
Publication date: 2021-04-20
Anticipated expiration: 2036-07-06
Also published as: CN106176782A; CN112915096A; CN112915096B; CN112972461A; CN112972461B; CN112843045A

Abstract

The embodiment of the invention discloses application of chemical components of eclipta as phytoestrogen, in particular to application of an eclipta extract in preparing a medicament for treating and/or preventing diseases related to estrogen secretion abnormity. The invention also relates to a pharmaceutical composition for treating and/or preventing diseases related to abnormal estrogen secretion, wherein the pharmaceutical composition comprises the eclipta alba extract and a pharmaceutically acceptable carrier or excipient. The compound obtained from the eclipta has the effects of phytoestrogens in different degrees, and the compound obtained by the invention or the eclipta extract and the pharmaceutical composition containing the compound can be expected to be used for treating and/or preventing diseases related to estrogen secretion abnormity, and further can be used for preparing medicaments for treating and/or preventing the diseases related to estrogen secretion abnormity.

Description

Application of chemical components of eclipta as phytoestrogen

Technical Field

The invention relates to the field of new application of chemical components of eclipta, in particular to application of the chemical components of the eclipta as phytoestrogen.

Background

After menopause, estrogen secretion is reduced in women, and causes problems with memory loss, hot flashes, fatigue, irritability, and decreased bone density. Conventional therapies such as estrogen replacement therapy have been once popular, but their side effects are large, and long-term use increases the incidence of uterine cancer and breast cancer, thus being limited in use. Scientists then found that a class of heterocyclic polyphenols active ingredients similar in structure to endogenous estrogens existed in plants. It is capable of binding to the Estrogen Receptor (ER) of mammals or humans, producing estrogen-like or anti-estrogen-like activity effects, and is called Phytoestrogen (PE). On one hand, when the estrogen level in the organism is lower than the normal level, the phytoestrogen has the effect of simulating estrogen, and the effect can prevent and treat female climacteric syndrome, prostatic cancer, osteoporosis, cardiovascular diseases and the like; on the other hand, when the estrogen level in the body is higher than the normal level (such as hyperplasia of mammary glands, hysteromyoma and other diseases), the estrogen antagonism can be generated, and the response of target cells to the estrogen can be effectively weakened. It would be desirable to those skilled in the art to find phytoestrogens that have estrogenic activity without estrogen side effects, as dietary supplements, or to provide new therapeutic options for the clinic.

Eclipta is aerial part of Eclipta prostrate L of Eclipta prostrata of Compositae, is called Eclipta alba, and has the functions of enriching blood, cooling blood to stop bleeding, clearing heat and detoxicating, nourishing yin and tonifying liver and kidney. Modern pharmacological research shows that the yerbadetajo herb has pharmacological effects of resisting tumor, regulating immunity, resisting inflammation and the like, and is clinically used for treating coronary heart disease, hemorrhagic diseases and the like.

Disclosure of Invention

The inventors have conducted intensive studies on the basis of the pharmacodynamic substances of eclipta, prepared an extract of eclipta and further extracted and isolated 17 compounds therefrom, and have unexpectedly found that these compounds have phytoestrogen activity for the most part and can be used as dietary supplements for supplementing estrogen or for the preparation of medicaments for the treatment and/or prevention of diseases associated with abnormal estrogen secretion. The present invention has been completed based on the above finding.

The invention provides in a first aspect the use of an extract of eclipta in the manufacture of a medicament for the treatment and/or prevention of a disease associated with abnormal estrogen secretion.

Use according to any one of the first aspect of the invention wherein the extract of eclipta contains at least one compound selected from the group consisting of: ecliptin IV, 3' -hydroxybiochanin A, 5-but-1 ' -alkyne-3 ' -hydroxy-4 ' -chloro- (2-pent-1 ', 3' -diyne) -thiophene, luteolin 7-O-beta-D glucoside, ecliptin I, luteolin, 5-aldehyde-2, 2',5', 2' -trithiol, echinocystic acid-28-O-beta-D glucoside, protocatechuic acid ethyl ester, stigmasterol, 5-aldehyde-5 ' - (3-butene-1-alkynyl) -2,2' -bithiol, wedelolactone and ecliptin A.

Use according to any one of the first aspect of the invention wherein each gram of eclipta contains 10-4000 microgram of luteolin 7-O-beta-D-glucoside, 7-450 microgram of luteolin, 300-1400 microgram of ecliptin IV, 900-3000 microgram of ecliptin A and 10-350 microgram of echinocystic acid-28-O-beta-D-glucoside.

Use according to any one of the first aspect of the invention wherein the extract of eclipta is prepared by the following method:

1) extracting Ecliptae herba with ethanol water solution; concentrating until no alcohol exists to obtain a crude extract; wherein, the ethanol water solution is preferably 50-90% by volume, more preferably 60-80% by volume, and most preferably 70% ethanol water solution; the amount of the ethanol aqueous solution is preferably 2 to 10 times, more preferably 4 to 7 times, and most preferably 5 times that of eclipta. The extraction time is preferably 1 to 4 hours, more preferably 2 to 3 hours; the extraction times are preferably 1-6 times, more preferably 2-4 times, and most preferably 3 times;

2) dissolving the obtained crude extract in water (3-20 times of water, preferably 6-15 times of water, and most preferably 10 times of water. ) Then petroleum ether and ethyl acetate are used for extraction, and after the solvent is removed, petroleum ether extraction part extract, ethyl acetate extraction part extract and water layer extract are respectively obtained; wherein, petroleum ether and ethyl acetate are preferably used for extraction for 1 to 6 times respectively, more preferably 2 to 4 times, and most preferably 3 times; the solvent removal can be carried out by a method commonly used in the art such as distillation under reduced pressure, and the present invention is not limited thereto.

3) Combining the extracts obtained in the step 2) and drying to obtain the eclipta extract. Drying may be carried out in a manner commonly used in the art, and the present invention is not limited thereto.

The use according to any one of the first aspect of the invention, wherein the disease associated with abnormal estrogen secretion is a disease caused by estrogen levels higher than normal or a disease caused by estrogen levels lower than normal, including but not limited to: climacteric syndrome, prostatic cancer, osteoporosis, cardiovascular diseases, hyperplasia of mammary glands, breast cancer and hysteromyoma.

In a second aspect, the present invention provides a pharmaceutical composition for the treatment and/or prevention of a disease associated with abnormal estrogen secretion, wherein the pharmaceutical composition comprises an extract of eclipta alba and a pharmaceutically acceptable carrier or excipient.

The pharmaceutical composition according to any one of the second aspect of the present invention, wherein the disease associated with abnormal estrogen secretion is a disease caused by estrogen levels higher than normal or a disease caused by estrogen levels lower than normal, including but not limited to: climacteric syndrome, prostatic cancer, osteoporosis, cardiovascular diseases, hyperplasia of mammary glands, breast cancer and hysteromyoma.

In a third aspect, the present invention provides a pharmaceutical composition for treating and/or preventing a disease associated with abnormal estrogen secretion, wherein the pharmaceutical composition comprises at least one pharmaceutically active ingredient selected from the group consisting of: ecliptin IV, 3' -hydroxybiochanin A, 5-but-1 ' -alkyne-3 ' -hydroxy-4 ' -chloro- (2-pent-1 ', 3' -diyne) -thiophene, luteolin 7-O-beta-D glucoside, ecliptin I, luteolin, 5-aldehyde-2, 2',5', 2' -trithiol, echinocystic acid-28-O-beta-D glucoside, protocatechuic acid ethyl ester, stigmasterol, 5-aldehyde-5 ' - (3-butene-1-alkynyl) -2,2' -bithiol, wedelolactone and ecliptin A.

The pharmaceutical composition according to any one of the third aspect of the present invention, wherein the pharmaceutically active ingredient contained in the pharmaceutical composition is extracted from eclipta.

The method for extracting the active ingredients from the eclipta is as follows:

obtaining a petroleum ether extraction part extract, an ethyl acetate extraction part extract and a water layer extract according to the step 2) in the preparation method of the eclipta alba extract;

separating the water layer extract by macroporous resin adsorption, performing gradient elution, subjecting the eluate to silica gel column chromatography, and preparing compound 6 (luteolin) and compound 7 (apigenin) by preparative liquid chromatography;

subjecting part of the extract to silica gel column chromatography, gradient eluting with dichloromethane-methanol to obtain 15 fractions, and subjecting to Sephadex LH-20 gel column chromatography (methanol elution) and Flash medium pressure chromatography (ODS-C)₁₈) And/or preparing a liquid chromatography separation to obtain a compound 14 (wedelolactone), a compound 10 (echinocystic acid-28-O-beta-D-glucoside), a compound 17 (ecliptin A), a compound 1 (ecliptin IV), a compound 4 (luteolin 7-O-beta-D-glucoside), a compound 5 (ecliptin I), a compound 2(3 '-hydroxybiochanin A, an English name 3' -hydroxybiochanin A), a compound 11 (protocatechuic acid ethyl ester) and a compound 16 (echinocystic acid);

subjecting part of the extract to silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate to obtain 17 fractions, and subjecting to Sephadex LH-20 gel column chromatography (methanol elution) and Flash medium pressure chromatography (ODS-C)₁₈) And/or preparing a liquid chromatographic separation to obtain a compound 9 (5-aldehyde-2, 2'; 5',2 "-trithiol), compound 13 (5-carboxaldehyde-5 ' - (3-buten-1-ynyl) -2,2' -bithiol), compound 3 (5-but-1 ' -yn-3 ' -hydroxy-4 ' -chloro- (2-pent-1", 3 "-diyne) -thiophenol), compound 15 (5-hydroxymethyl-2, 2 '; 5',2 "-terphiol), compound 12 (stigmasterol) and compound 8(2, 2'; 5',2 "-terphiol).

The pharmaceutical composition according to any one of the third aspect of the present invention, wherein the disease associated with abnormal estrogen secretion is a disease caused by estrogen levels higher than normal or a disease caused by estrogen levels lower than normal, including but not limited to: climacteric syndrome, prostatic cancer, osteoporosis, cardiovascular diseases, hyperplasia of mammary glands, breast cancer and hysteromyoma.

The term "treatment" as used herein has its ordinary meaning and refers herein in particular to the treatment of a mammalian subject (preferably a human) already suffering from a disorder associated with abnormal estrogen secretion as described herein with a medicament of the invention in order to effect a treatment, cure, alleviation or the like of said disorder. Similarly, the term "prevention" as used herein has its ordinary meaning and herein refers in particular to the treatment of a mammalian subject, who may suffer from or is at risk of suffering from a disorder associated with abnormal estrogen secretion as described herein, with a medicament of the present invention in order to prevent, arrest, abrogate, etc. said disorder.

As used herein, "pharmaceutically acceptable" means having no substantial toxic effect when used in the usual dosage amounts, and thus being approved by the government or equivalent international organization or approved for use in animals, more particularly in humans, or registered in the pharmacopoeia.

The "pharmaceutically acceptable carrier or excipient" useful in the pharmaceutical compositions of the invention may be any conventional carrier in the art of pharmaceutical formulation, and the selection of a particular carrier will depend on the mode of administration or the type and state of the disease used to treat a particular patient. The preparation of suitable pharmaceutical compositions for a particular mode of administration is well within the knowledge of those skilled in the pharmaceutical art. For example, solvents, diluents, dispersing agents, suspending agents, surfactants, isotonic agents, thickening agents, emulsifiers, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavors, antioxidants, and the like, which are conventional in the pharmaceutical field, may be included as the pharmaceutically acceptable carrier or excipient. If necessary, a flavor, a preservative, a sweetener and the like may be further added to the pharmaceutical composition.

As used herein, the term "pharmaceutical composition" has its ordinary meaning. In addition, the 'pharmaceutical composition' of the invention can also be present or provided in the form of health products, functional foods, food additives and the like. The pharmaceutical composition of the present invention can be prepared by obtaining the active ingredients of the raw materials of the pharmaceutical composition of the present invention by extraction, separation and purification means commonly used in pharmaceutical manufacturing, optionally mixing with one or more pharmaceutically acceptable carriers or excipients, and then forming a desired dosage form, using conventional techniques in the pharmaceutical field, particularly in the field of formulation. The pharmaceutical composition according to the present invention is a pharmaceutical formulation which may be suitable for oral, parenteral or topical, topical administration. The pharmaceutical composition can be prepared into various forms such as tablets, powder, granules, capsules, oral liquid and the like. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field. Specifically, according to the pharmaceutical compositions of the present invention, the pharmaceutical dosage forms include, but are not limited to: tablet, capsule, granule, powder, injection, powder for injection, transdermal patch, ointment, gel, suppository, oral solution, oral suspension, emulsion for injection, oral emulsion, etc., sustained release tablet, and controlled release tablet. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

Dosage forms for oral administration may include, for example, tablets, pills, hard or soft capsules, solutions, suspensions, emulsions, syrups, powders, fine granules, pellets, elixirs and the like, without limitation. In addition to the active ingredient, these preparations may contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), lubricants (e.g., silica, talc, stearic acid or its magnesium salt, calcium salt, and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone. If necessary, it may further contain pharmaceutically acceptable additives such as disintegrating agents (e.g., starch, agar, alginic acid or sodium salt thereof), absorbents, coloring agents, flavoring agents, sweetening agents, and the like. Tablets may be prepared according to conventional mixing, granulating or coating methods.

Dosage forms for parenteral administration may include, for example, injections, drops for medical use, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, patches and the like, without being limited thereto.

The pharmaceutical compositions according to the present disclosure may be administered orally or parenterally, for example rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, or subcutaneously.

As used herein, the term "about" generally refers to a range of error permitted in the art, such as ± 10%, e.g., ± 5%, e.g., ± 2%.

The compound obtained from the eclipta has the effects of phytoestrogens in different degrees, and the compound obtained by the invention or the eclipta extract and the pharmaceutical composition containing the compound can be expected to be used for treating and/or preventing diseases related to estrogen secretion abnormity, and further can be used for preparing medicaments for treating and/or preventing the diseases related to estrogen secretion abnormity.

Detailed Description

The technical solutions of the present invention will be described below with reference to specific embodiments, and the described embodiments are only a part of embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation of active ingredients in Ecliptae herba

20kg of Ecliptae herba (purchased from Anguo san shan Yao Co., Ltd., lot number: mhl0001, origin: Hebei) was extracted with 100L of 70% ethanol under reflux for 2 hours for 3 times. Distilling at 45 deg.C under reduced pressure, concentrating until no alcohol exists, and estimating the mass of the extract to be about 1 kg.

Dissolving the crude extract in 10L water, and extracting with petroleum ether and ethyl acetate for 3 times to obtain 255g (named as EPA-P) of petroleum ether extract, 150g (named as EPA-E) of ethyl acetate extract and 600g (named as EPA-W) of water layer extract.

600g of water layer part (EPA-W) is adsorbed and separated by macroporous resin D101, and is eluted by pure water, 30% ethanol, 70% ethanol and pure ethanol, and 30% ethanol eluate is EPA-W-D3.

The EPA-W-D3 fraction (25 g) is mixed with silica gel (50 g), and the mixture is loaded on a silica gel column (250 g) and subjected to silica gel column chromatography (600 ml). Performing gradient elution with dichloromethane-methanol to obtain 16 fractions; TLC (thin layer chromatography) detection was followed by pooling, and pooled fractions 1-4 were recorded as: EPA-W-D3-1; the combined fractions 5-6 were recorded as: EPA-W-D3-5; the combined fractions 7-8 were recorded as: EPA-W-D3-7; the combined fractions 9-10 were recorded as: EPA-W-D3-9; the combined fractions 11-13 were recorded as: EPA-W-D3-11; fraction 14 was recorded as: EPA-W-D3-14. Wherein EPA-W-D3-11 was prepared by preparative liquid chromatography to give compounds 6(348mg) and 7(83 mg).

150g of ethyl acetate fraction (EPA-E) was stirred with 200g of silica gel and loaded on a column of 1.6Kg of silica gel, and silica gel column chromatography was carried out in a column volume of 4L. The elution was performed with a gradient of dichloromethane-methanol (dichloromethane-methanol volume ratio) of 1:0, 100:1, 50:1, 25:1, 12.5:1, 7:1, 4:1, 1:1, 0:1, two column volumes were collected for each gradient. The fractions were checked by TLC analysis for a total of 15 fractions.

The 8 th and 9 th fractions were combined and designated EPA-E8, and separated by Sephadex LH-20 gel column chromatography (methanol elution) and preparative liquid chromatography to give compound 14(32 mg). The 11 th fraction is named EPA-E11, and is eluted by Sephadex LH-20 gel and methanol, and subjected to Flash medium pressure chromatography (ODS-C)₁₈) Gradient elution with 40% methanol → 100% methanol, preparative liquid chromatography, elution with methanol water of serial ratios, TLC and HPLC detection of each fraction eluted, and combination of the same fractions gave compound 10(130mg) and compound 17(1290 mg). The 13 th, 14 th and 15 th fractions were combined and designated EPA-E13, and separated by Sephadex LH-20 gel preparative liquid chromatography to give Compound 1(95mg), Compound 4(25mg) and Compound 5(210 mg). Combining the 5 th and 6 th fractions, named EPA-E5, and performing silica gel column chromatography, Sephadex LH-20 gel, and Flash medium pressure chromatography (ODS-C)₁₈) And preparative liquid chromatography gave compound 2(49mg), compound 11(114mg), and compound 16(43 mg).

255g of petroleum ether fraction (EPA-P) are stirred with 500g of silica gel, and the mixture is subjected to silica gel column chromatography and gradient elution by using petroleum ether-ethyl acetate (volume ratio is 1:0 → 0: 1) to obtain 17 fractions. Analyzing and detecting each component by TLC, combining to obtain 8 components, and naming the fraction 1 as EPA-P-1; fraction 2-4 is EPA-P-2; fraction 5-7 is EPA-P-5; fraction 8-10 is EPA-P-8; fraction 11-14 is EPA-P-11; fraction 15 is EPA-P-15; fraction 16 was EPA-P-16; fraction 17 was EPA-P-17.

The component EPA-P-1 was prepared by preparative liquid chromatography to give compound 8(88 mg). The EPA-P-5 component is processed by Flash medium pressure chromatography (ODS-C)₁₈) EPA-P-5-F6 was isolated and prepared by preparative liquid chromatography to give compounds 9(98mg) and 13(45 mg). The EPA-P-8 component is subjected to Flash medium pressure chromatography (ODS-C)₁₈) EPA-P-8-F3 and EPA-P-8-F7 were isolated and prepared by preparative liquid chromatography to give Compound 3(43 mg). The EPA-P-11 fraction was subjected to repeated Sephadex LH-20 gel separation, preparative liquid chromatography and recrystallization to give compounds 15(61mg) and 12(149 mg). The percentages in this example are volume percentages; the fraction designations are all named in the order of elution.

Example 2: determination of structures of Compounds 1-17

By passing¹H、¹³C-NMR is carried out, and the structures of the compounds 1-17 are determined; the structures of identified compounds 1-17 are shown in table 1.

The model number of the NMR instrument is: BRUKER AVANCE iii 500 superconducting nuclear magnetic resonance spectrometer (TMS as internal standard, BRUKER, switzerland);

specifically, the method comprises the following steps:

compound 1 was characterized as follows:

¹H-NMR(500MHz,pyridine-d₅) The spectral low field region has 2 hydrogens: Δ 5.65(1H, br s, H-12) and 5.25(1H, br s, H-16). The low and medium field regions can observe that: delta.3.31 (1H, m) is the H-3 hydrogen signal; δ 4.92(1H, d, J ═ 7.0Hz, H-1') and δ 5.37(1H, d, J ═ 7.5Hz, H-1") suggest the presence of two glucose groups. 7 characteristic methyl peak signals can also be observed in the high field region: δ 1.85(3H, s, H-27),1.28(3H, s, H-23),1.19(3H, s, H-30),1.10(3H, s, H-24),1.07(3H, s, H-29),1.03(3H, s, H-26), and 0.87(3H, s, H-25).¹³C-NMR(125MHz，pyridine-d₅) Spectral data are ascribed as follows: 38.8(C-1), 26.7(C-2), 89.0(C-3), 39.6(C-4), 56.0(C-5), 18.6(C-6), 33.4(C-7), 39.9(C-8), 47.2(C-9), 37.0(C-10), 23.9(C-11), 122.4(C-12), 145.2(C-13), 42.2(C-14), 36.2(C-15), 74.7(C-16), 48.9(C-17), 41.5(C-18), 47.4(C-19), 31.1(C-20), 36.3(C-21), 32.9(C-22), 28.3(C-23), 16.9(C-24), 15.6(C-25), 17.5(C-26), 27.3(C-27), 180.0 (C-29), 33.6(C-29), 24.8(C-30), 105.1(C-1'), 83.4(C-2'), 78.3(C-3'), 71.6(C-4'), 77.9(C-5'), 62.7(C-6'), 106.1(C-1"), 77.1(C-2"), 78.0(C-3"), 71.6(C-4"), 77.9(C-5"), 62.8 (C-6").

Compound 2 was characterized as follows:

¹H-NMR(500MHz,DMSO-d₆) The spectrum is in a low field region, and delta 12.98(1H, s) can be seen as an association signal of a hydroxyl at the 5-position and a carbonyl; 6.23(1H, d, J ═ 1.5Hz, H-6),6.39(1H, d, J ═ 1.5Hz, H-8). The signal delta of the low field region of the hydrogen spectrum is 8.36(1H, s, H-2), which indicates that the compound is isoflavone. The low field region can also see a set of ABX signals for the B-ring: 7.14(1H, d, J ═ 1.5Hz, H-2'),6.82(1H, d, J ═ 8.5Hz, H-5'),6.99(1H, dd, J ═ 1.5,8.5Hz, H-6'), suggesting that ring B3 ', 4' was taken twiceGeneration; a methoxy signal can also be observed in the hydrogen spectrum: delta.3.80 (3H, s,4' -OCH)₃). In that¹³C-NMR(125MHz,DMSO-d₆) The corresponding isoflavone carbon signal appears on the spectrum: Δ:93.6(C-8),98.9(C-6),104.3(C-10),113.2(C-2'),115.2(C-5'),121.5(C-3),121.6(C-6'),122.2(C-1'),146.6(C-4'),147.2(C-3'),154.1(C-2),157.4(C-9),161.9(C-5),164.2(C-7),180.1(C-4), and the methoxy signal Δ 56.0(4' -OCH)₃)。

Compound 3 was characterized as follows:

¹H-NMR(500MHz,CDCl₃) The spectral low field region has 2 hydrogens: δ 7.10(1H, d, J ═ 4.0Hz) and 7.06(1H, d, J ═ 4.0Hz) are the H-3 and 4 hydrogen signals, respectively, on the thiophene ring; the low and medium field regions can observe that: δ 4.82(2H, dd, J ═ 4.5, 6.5Hz) is the H-3' hydrogen signal; δ 3.79(1H, dd, J ═ 4.5, 11.5Hz) and 3.73(1H, dd, J ═ 6.5, 11.5Hz) are the hydrogen signals at the H-4' position, respectively; there is a clear methyl peak signal in the high field region: delta.2.04 (3H, s, H-5 ").¹³C-NMR(125MHz，CDCl₃) Spectral data are ascribed as follows: Δ 4.8(C-5"), 48.7(C-4'), 63.1(C-3'), 64.1(C-3"), 66.3(C-2"), 79.1(C-1'), 79.7(C-1"), 83.6(C-4"), 90.7(C-2'), 123.4(C-5), 124.5(C-2), 132.7(C-4), 133.6 (C-3).

Compound 4 was characterized as follows:

¹H-NMR(500MHz,DMSO-d₆) The spectrum is in a low field region, and delta 12.99(1H, s) can be seen as an association signal of a hydroxyl at the 5-position and a carbonyl; δ 6.45(1H, d, J ═ 2.0Hz) and δ 6.79(1H, d, J ═ 2.0Hz) are the 6-and 8-position hydrogen signals, respectively; 6.75(1H, s) is the 3-position hydrogen proton signal, and the low field region can also see a group of ABX signals of the B ring: δ 6.91(1H, d, J ═ 8.5Hz, H-5'), 7.42(1H, d, J ═ 2.0Hz, H-2') and 7.45(1H, dd, J ═ 2.0,8.5Hz, H-6'), suggesting a B ring 3', 4' disubstitution; in addition, a set of β -D glucose signals was observed: δ 5.08(1H, d, J ═ 7.5Hz, H-1"),3.28(1H, m, H-2"),3.30(1H, m, H-3"),3.19(1H, m, H-4"),3.45(1H, m, H-5"),3.71(1H, dd, J ═ 4.0,11.5Hz, H-6" b),3.49(1H, m, H-6 "a). In that¹³C-NMR(125MHz,DMSO-d₆) The corresponding 15 carbon signals of the flavonoid aglycone appear on the spectrum: delta 94.6(C-8),99.4(C-6),103.1(C-3),105.2(C-10),113.5(C-2'),115.9(C-5'),119.1 (C-10)C-6'),121.3(C-1'),145.7(C-3'),149.8(C-4'),156.8(C-9),161.0(C-5),162.8(C-7),164.4(C-2) and 181.8 (C-4); a corresponding set of β -D glucose signals was also observed in the carbon spectra: delta 99.8(C-1"),73.0(C-2"),76.3(C-3"),69.4(C-4"),77.2(C-5"),60.5 (C-6").

Compound 5 was characterized as follows:

¹H-NMR(500MHz,pyridine-d₅) The spectral low field region has 2 hydrogens: Δ 5.63(1H, br s, H-12) and 5.32(1H, br s, H-16). The low and medium field regions can observe that: delta.3.43 (1H, m) is the H-3 hydrogen signal; δ 4.96(1H, d, J ═ 7.5Hz, H-1') and δ 6.34(1H, d, J ═ 8.0Hz, H-1") suggest the presence of two glucose groups. 7 characteristic methyl peak signals can also be observed in the high field region: δ 1.86(3H, s, H-27),1.30(3H, s, H-23),1.15(3H, s, H-26),1.05(3H, s, H-30),1.01(3H, s, H-29),1.01(3H, s, H-24) and 0.90(3H, s, H-25).¹³C-NMR(125MHz，pyridine-d₅) Spectral data are ascribed as follows: 38.9(C-1), 26.6(C-2), 88.9(C-3), 39.5(C-4), 55.9(C-5), 18.6(C-6), 33.5(C-7), 40.1(C-8), 47.2(C-9), 37.1(C-10), 23.9(C-11), 122.7(C-12), 144.5(C-13), 42.1(C-14), 36.2(C-15), 74.2(C-16), 49.1(C-17), 41.3(C-18), 47.2(C-19), 30.9(C-20), 36.0(C-21), 32.2(C-22), 28.3(C-23), 17.1(C-24), 15.6(C-25), 17.6(C-26), 27.3(C-27), 176.0 (C-27), 28.29.2 (C-29), 24.6(C-30), 106.9(C-1'), 75.8(C-2'), 79.4(C-3'), 71.9(C-4'), 78.3(C-5'), 63.1(C-6'), 96.0(C-1"), 74.2(C-2"), 79.4(C-3"), 71.1(C-4"), 78.8(C-5"), 62.2 (C-6").

Compound 6 was characterized as follows:

¹H-NMR(500MHz,DMSO-d₆) The spectrum is in a low field region, and delta 12.98(1H, s) can be seen as an association signal of a hydroxyl at the 5-position and a carbonyl; δ 6.19(1H, d, J ═ 2.0Hz) and δ 6.45(1H, d, J ═ 2.0Hz) are the 6-and 8-position hydrogen signals, respectively; 6.67(1H, s) is the 3-position hydrogen proton signal. In addition, the low field region can also see a set of ABX signals of the B-ring: δ 6.89(1H, d, J ═ 8.0Hz, H-5'), 7.40(1H, d, J ═ 2.0Hz, H-2'), and 7.41(1H, dd, J ═ 2.0,8.0Hz, H-6'), suggesting a B ring 3', 4' disubstitution. In that¹³C-NMR(125MHz,DMSO-d₆) There are 15 corresponding carbon signals present on the spectrum: delta 93.7(C-8),98.7(C-6),102.8(C-3)103.6(C-10),113.3(C-2'),115.9(C-5'),118.9(C-6'),121.4(C-1'),145.6(C-3'),149.6(C-4'),157.2(C-9),161.4(C-5),163.8(C-2),164.0(C-7) and 181.6 (C-4).

Compound 7 was characterized as follows:

¹H-NMR(500MHz,DMSO-d₆) The spectrum is in a low field region, and delta 12.96(1H, s) can be seen as an association signal of a hydroxyl at the 5-position and a carbonyl; δ 6.19(1H, d, J ═ 2.0Hz) and δ 6.48(1H, d, J ═ 2.0Hz) are the 6-and 8-position hydrogen signals, respectively; 6.78(1H, s) is the signal for the hydrogen proton at the 3-position. In addition, the low field region can also see a set of AA 'BB' signals of the B-ring: δ 6.93(2H, dd, J ═ 2.0,8.5Hz, H-3',5') and 7.93(2H, dd, J ═ 2.0,8.5Hz, H-2',6'), suggest para substitution of the B ring. In that¹³C-NMR(125MHz,DMSO-d₆) There are 13 corresponding carbon signals (including two symmetric carbon signals) present on the spectrum: Δ 93.9(C-8),98.7(C-6),102.8(C-3),103.6(C-10),115.9(C-3',5'),121.1(C-1'),128.4(C-2',6'),157.2(C-9),161.1(C-4'),161.4(C-5),163.7(C-2),164.0(C-7) and 181.7 (C-4).

Compound 8 was characterized as follows:

¹H-NMR(500MHz,CDCl₃) The spectrum is in a low field region, and delta 7.08(br s,2H) can be seen as proton signals at 3 'and 4'; 7.02(2H, dd, J ═ 3.5,5.0Hz, H-4, 4"), 7.17(2H, dd, J ═ 1.0,3.5Hz, H-3, 3"), and 7.22(2H, dd, J ═ 1.0,5.0Hz, H-5, 5"), suggest the presence of symmetric structures in the structure. In that¹³C-NMR(125MHz,CDCl₃)6 aromatic carbons, 123.7(C-3, 3"), 124.3(C-3', 4'), 124.5(C-5, 5"), 127.9(C-4, 4"), 136.2(C-2', 5'), 137.1(C-2, 2') appeared in the spectrum.

Compound 9 was characterized as follows:

¹H-NMR(500MHz,CDCl₃) A spectrum low field region, wherein delta 9.86 is a 5-position aldehyde proton signal; δ 7.67(1H, d, J ═ 4.0Hz) and 7.24(1H, overlapped) are the hydrogen signals at positions 4 and 3 on the a ring; 7.28(1H, overlapped) and 7.27(1H, overlapped) are the signals for H-4 'and 3' on the B-ring, respectively; 7.23(1H, overlapped), 7.13(1H, d, J ═ 4.0Hz) and 7.05(1H, dd, J ═ 4.0,5.0Hz) are the signals for H-3",5" and 4", respectively, on the C ring. Specific carbon Spectroscopy data (125MHz, CDCl)₃) The following were used: delta 124.0(C-3), 124.5(C-3"), 124.7(C-5")、125.4(C-4')、126.9(C-3')、128.1(C-4")、134.5(C-2')、136.4(C-5')、137.3(C-4)、139.2(C-2")、141.6(C-5)、146.8(C-2)、182.4(5-CHO)。

Compound 10 was characterized as follows:

¹H-NMR(500MHz,pyridine-d₅) The spectral low field region has 2 hydrogens: Δ 5.63(1H, br s, H-12) and 5.29(1H, br s, H-16). The low and medium field regions can observe that: δ 3.44(1H, dd, J ═ 5.0,11.0Hz) is the H-3 hydrogen signal; δ 6.31(1H, d, J ═ 8.5Hz, H-1') suggests the presence of a glucose group. 7 characteristic methyl peak signals can also be observed in the high field region: δ 1.82(3H, s, H-27),1.21(3H, s, H-23),1.16(3H, s, H-26),1.05(3H, s, H-30),1.03(3H, s, H-24),1.00(3H, s, H-29) and 0.97(3H, s, H-25).¹³C-NMR(125MHz，pyridine-d₅) Spectral data are ascribed as follows: 39.1(C-1), 28.1(C-2), 78.1(C-3), 39.4(C-4), 56.0(C-5), 18.9(C-6), 33.2(C-7), 40.2(C-8), 47.3(C-9), 37.5(C-10), 23.9(C-11), 122.7(C-12), 144.5(C-13), 42.1(C-14), 36.0(C-15), 74.1(C-16), 48.9(C-17), 41.4(C-18), 47.3(C-19), 30.9(C-20), 36.1(C-21), 32.3(C-22), 28.8(C-23), 16.6(C-24), 15.8(C-25), 17.6(C-26), 27.3(C-27), 25.28.6 (C-29), 33.6(C-29), 24.6(C-30), 95.9(C-1'), 74.1(C-2'), 79.4(C-3'), 71.0(C-4'), 78.8(C-5'), 62.2 (C-6').

Compound 11 was characterized as follows:

¹H-NMR(500MHz,CD₃OD) spectrum low field region, a set of ABX aromatic hydrogen proton signals can be seen: δ 7.43(1H, d, J ═ 2.0Hz, H-2),7.42(1H, dd, J ═ 2.0,8.0Hz, H-6),6.80(1H, d, J ═ 8.0Hz, H-5), a set of ethyl hydrogen proton signals: δ 4.29(2H, q, J ═ 7.0Hz, H-8); δ 1.35(3H, t, J ═ 7.0Hz, H-9). In that¹³C-NMR(125MHz,CD₃OD) spectrum with six corresponding aromatic carbon signals: δ 122.9(C-1),117.4(C-2),146.2(C-3),145.7(C-4),115.9(C-5) and 123.6(C-6), a carboxyl carbon signal: delta 168.5(C-7), a set of ethyl carbon signals delta 61.7(C-8),14.7 (C-9).

Compound 12 was characterized as follows:

¹H-NMR(500MHz,CDCl₃) The spectral low field region has 3 hydrogens: delta 5.34(1H, d, J ═ 5.0Hz, H-6), 5.15(1H, dd, J ═ 9.0, 15.0Hz, H-22) and δ 5.02(1H, dd, J ═ 9.0, 15.0Hz, H-23). The middle and low field zone delta 3.52(1H, m, H-3) is a continuous oxygen hydrogen signal at the 3-position. The high field region presents 6 methyl signals: δ 1.02(3H, d, J ═ 7.0Hz, H-21), 1.01(3H, s, H-19), 0.85(3H, d, J ═ 7.0Hz, H-26), 0.81(3H, t, J ═ 7.0Hz, H-29), 0.80(3H, d, J ═ 7.0Hz, H-27) and 0.70(3H, s, H-18).¹³C-NMR(125MHz，CDCl₃) Spectral specific data are ascribed δ: 12.1(C-18), 12.3(C-29), 19.0(C-27), 19.4(C-19), 21.1(C-11,16), 21.2(C-21), 24.4(C-15), 25.4(C-28), 28.9(C-16), 31.7(C-25), 31.9(C-2,7,8), 36.5(C-10), 37.3(C-1), 39.7(C-12), 40.5(C-20), 42.2(C-13), 42.3(C-4), 50.2(C-9), 51.3(C-24), 56.0(C-17), 56.9(C-14), 71.8(C-3), 121.7(C-6), 129.3(C-23), 138.3(C-22), 140.8 (C-5).

Compound 13 was characterized as follows:

¹H-NMR(500MHz,CDCl₃) The delta 9.86 in the spectrum is a 5-position aldehyde proton signal; δ 7.67(1H, d, J ═ 4.0Hz) and 7.24(1H, d, J ═ 4.0Hz) are the hydrogen signals at the 4 and 3 positions on the a ring; 7.22(1H, d, J ═ 4.0Hz) and 7.14(1H, d, J ═ 4.0Hz) are the signals for H-3 'and 4' on the B ring, respectively; 6.02(1H, dd, J ═ 11.5,17.5Hz, H-4 "); 5.76(1H, dd, J ═ 1.0,17.5Hz) and 5.60(1H, dd, J ═ 1.0,11.5Hz) are the alkene hydrogen signals at the 5 "position.¹³C-NMR(125MHz，CDCl₃) Spectral data are ascribed as follows: δ 82.7(C-2"), 94.3(C-3"), 116.6(C-4"), 124.6(C-3), 124.8(C-5'), 126.0(C-3'), 127.8(C-5"), 133.1(C-4'), 137.2(C-2'), 137.2(C-4), 142.1(C-5), 146.1(C-2), 182.5 (5-CHO).

Compound 14 was characterized as follows:

in that¹H-NMR(500MHz,DMSO-d₆) In the low field region of the spectrum, 4 aromatic hydrogen proton signals can be seen: δ 6.47(1H, d, J ═ 2.0Hz, H-8),6.63(1H, d, J ═ 2.0Hz, H-6),7.18(1H, s, H-13),7.25(1H, s, H-10). In that¹³C-NMR(125MHz,DMSO-d₆) The spectrum shows 12 aromatic carbons, 2 olefinic carbons, one carbonyl carbon and one methoxy carbon signals: delta 158.8(C-1),101.6(C-2),155.2(C-3),96.6(C-4),154.7(C-5),93.1(C-6),162.1(C-7),98.1(C-8),157.7(C-9),104.5(C-10),145.3(C-11),144.2(C-12),98.8(C-13),113.6(C-14),148.8(C-15),55.6(C-16)。

compound 15 was characterized as follows:

¹H-NMR(500MHz,CDCl₃) The low field of the spectrum has 7 hydrogens, δ 7.22(1H, d, J ═ 5.0Hz), 7.17(1H, d, J ═ 3.0Hz) and 7.02(1H, overlapped) are the H-5", 3" and 4 "hydrogen signals on the C ring, respectively; 7.06(1H, d, J ═ 3.5Hz) and 7.05(1H, d, J ═ 3.5Hz) are the signals for H-4 'and 3' on the B ring, respectively; 7.03(1H, overlapped) and 6.92(1H, d, J ═ 3.0Hz) are signals for H-3 and 4 on the a ring, respectively. There is a distinct broad single peak in the mid-low field region: delta.4.81 (2H, br s, 5-CH)₂OH). Carbon spectrum specific data (125MHz, CDCl)₃) The following were used: delta 60.2 (5-CH)₂OH)、123.3(C-3)、123.8(C-3")、124.3(C-3'、4')、124.5(C-5")、126.3(C-4)、127.9(C-4")、136.1(C-5')、136.4(C-2')、137.1(C-2")、137.4(C-2)、143.1(C-5)。

Compound 16 was characterized as follows:

¹³C-NMR(125MHz，pyridine-d₅) Spectral data are ascribed as follows: 39.1(C-1), 28.2(C-2), 78.1(C-3), 39.4(C-4), 55.9(C-5), 18.9(C-6), 33.4(C-7), 40.0(C-8), 47.3(C-9), 37.5(C-10), 23.9(C-11), 122.5(C-12), 145.2(C-13), 42.2(C-14), 36.2(C-15), 74.7(C-16), 48.9(C-17), 41.5(C-18), 47.3(C-19), 31.1(C-20), 36.2(C-21), 32.9(C-22), 28.8(C-23), 16.6(C-24), 15.7(C-25), 17.6(C-26), 27.3(C-27), 180.0 (C-29), 33.6(C-29), 24.8 (C-30).

Compound 17 was characterized as follows:

¹H-NMR(500MHz,CD₃OD) spectrum low field has 2 hydrogens: Δ 5.29(1H, br s, H-12) and 4.46(1H, br s, H-16). The low and medium field regions can observe that: delta.3.18 (1H, m) is the H-3 hydrogen signal; δ 4.32(1H, d, J ═ 7.5Hz, H-1') suggests the presence of a glucose group. 7 characteristic methyl peak signals can also be observed in the high field region: δ 1.37(3H, s, H-27),1.05(3H, s, H-23),0.97(3H, s, H-30),0.96(3H, s, H-25),0.88(3H, s, H-29),0.85(3H, s, H-24) and 0.79(3H, s, H-26).¹³C-NMR(125MHz，CD₃OD) number of spectraThe attribution is as follows: 38.0(C-1), 27.1(C-2), 90.9(C-3), 36.3(C-4), 57.2(C-5), 19.4(C-6), 34.4(C-7), 40.2(C-8), 47.8(C-9), 36.6(C-10), 24.5(C-11), 123.5(C-12), 145.1(C-13), 40.7(C-14), 39.9(C-15), 75.3(C-16), 47.8(C-17), 42.1(C-18), 42.7(C-19), 31.5(C-20), 36.3(C-21), 32.8(C-22), 28.6(C-23), 17.1(C-24), 16.1(C-25), 17.8(C-26), 27.3(C-27), 181.2 (C-27), 28.5 (C-23), 33.25-29), 25.0(C-30), 106.8(C-1'), 75.8(C-2'), 78.4(C-3'), 71.7(C-4'), 77.7(C-5'), 62.9 (C-6').

TABLE 1 list of compounds

Example 3 determination of Compound content in Ecliptae herba extract

(a) Preparation of reference substance and test solution

Weighing a proper amount of each reference substance respectively, precisely weighing, placing in a 10mL volumetric flask, adding 50% methanol-water solution to scale to obtain a reference substance series solution, i.e., luteolin 7-O-beta-D-glucoside (A, 18.15mg for short), luteolin (B, 4.64mg for short), drynarioside IV (C, 11.85mg for short), apigenin (D, 1.56mg for short), drynarioside A (E, 21.50mg for short), echinocystic acid-28-O-beta-D-glucoside (F, 2.60mg for short) and echinocystic acid (G, 8.45mg for short). Taking 1mL of the comparison series solution (I) and respectively placing the comparison series solution (I) into a 10mL volumetric flask, and adding 50% methanol-water solution by volume fraction to scale to obtain a comparison series solution (II). Mixing the control solutions in equal volume, and adding 50% methanol-water solution for dilution to obtain a control mixed standard solution; the linear range of each compound obtained finally is as follows: 0.028-20.17 mu g/mL (A), 0.012-8.59 mu g/mL (B), 0.060-43.89 mu g/mL (C), 0.008-5.78 mu g/mL (D), 0.033-23.89 mu g/mL (E), 0.013-9.63 mu g/mL (F), 0.013-9.39 mu g/mL (G).

Taking about 1.00g of eclipta coarse powder sample, precisely weighing, placing the sample in a 50mL measuring flask, precisely adding 25mL of methanol-water solution with the volume fraction of 50%, weighing, carrying out ultrasonic treatment for 30 minutes at room temperature, placing the sample for 30 minutes, adding methanol to complement the weight, filtering, taking 1mL of subsequent filtrate, placing the subsequent filtrate in a 10mL volumetric flask, adding the methanol-water solution with the volume fraction of 50%, dissolving the subsequent filtrate in a fixed solution, taking 1mL of fixed solution in a centrifugal tube, centrifuging the solution for 10 minutes at 13171g, and taking supernatant to obtain the sample solution.

(b) HPLC-QQQ (high Performance liquid chromatography-triple quadrupole mass spectrometry) analysis of eclipta

HPLC mobile phase: acetonitrile (A) -water (containing 0.1% formic acid; B); adopting a gradient elution mode: 0-5 minutes, 30-100% A; 5-10 minutes, 100% A; 100-30% A for 10-11 min; 11-20 minutes, 30% a; flow rate 0.5 mL/min; the column temperature is 35 ℃; injection volume 1. mu.L.

QQQ: electrospray ion source (ESI); adopting an anion mode; the liquid nitrogen is atomized gas and dry gas, and the high-purity nitrogen is collision gas; drying airflow rate of 8.0L/min, temperature of 350 ℃; atomizing gas pressure 45 psig; capillary voltage 4000V; 7 compounds were monitored simultaneously in MRM (multiple reaction monitoring) mode (see table 2);

TABLE 2MRM quantitative parameters

(c) Investigation of linear relationships

Sucking a proper amount of a reference substance mixed standard solution, adding 50% methanol for dilution, preparing 7 parts of mixed standard solutions with different concentrations in total, determining according to the HPLC-QQQ condition in the step (b), determining each sample for 3 times, taking the peak area as a vertical coordinate Y and the sample concentration (mu g/mL) as a horizontal coordinate X, performing linear regression, drawing a standard curve, and obtaining a good linear relation of results, wherein R is²Are all above 0.9990, as detailed in Table 3.

TABLE 3 quantitative Standard Curve, quantitative Limit and detection Limit

(d) Detection limit and quantification limit

The control mixed standard solution was diluted with 50% methanol (volume percentage) and the lowest limit of detection (LOD) and lowest limit of quantitation (LOQ) were calculated as HPLC-QQQ conditions in (b) above with signal-to-noise ratios (S/N) of 3 and 10, respectively, and the results are shown in Table 3.

(e) Precision and repeatability

Intra-day Precision (Intra-day Precision): and (3) taking the same reference substance mixed standard solution at the middle point of the standard curve, continuously injecting samples for 6 times according to the HPLC-QQQ condition, recording an LC-MS spectrogram, integrating, calculating a peak area, calculating a Relative Standard Deviation (RSD), inspecting the precision of an instrument, and indicating that the RSD of each compound is less than 2.37 percent, wherein the precision in the day is good, and the results are shown in Table 4.

Daytime Precision (Inter-day Precision): and (3) mixing the standard solution with the same reference substance at the middle point of the standard curve, continuously measuring the mixed solution once every 12 hours according to the HPLC-QQQ condition, recording an LC-MS spectrogram, integrating, calculating peak areas, and calculating RSD, wherein the RSD of each compound is less than 2.49% according to the result, the precision in the daytime is good, and the result is shown in Table 4.

And (3) repeatability test: 6 parts of the same sample (S9) are parallelly taken, a test solution is prepared according to the method under item 3.1, peak areas are measured according to the HPLC-QQQ conditions, RSD is calculated, and the RSD of each compound is less than 2.99 percent, which indicates that the test preparation method has good repeatability. The results are shown in Table 4.

TABLE 4 precision, repeatability and recovery of samples

(f) Sample measurement and results

The content of 13 batches (S1-S13) of Ecliptae herba from different origins was measured by HPLC-QQQ in (b) above, and each sample was measured 3 times. The results are shown in Table 5.

The production area of each eclipta batch is as follows:

s1: hunan; s2: north of river; s3: henan; s4: henan; s5: north of river; s6: unknown; s7: jiangsu; s8: unknown; s9: north of river; s10: unknown; s11: unknown; s12: north of river; s13: an emblem.

TABLE 5 assay results (mean. + -. standard deviation,. mu.g/g, n ═ 3)

ND represents not detected;

EXAMPLE 4 Compound 1-17 Estrogen Activity screening assay

Reagent: DMEM/F-12, HEPES, no phenol red is

Manufactured by corporation; the specification of the product is 11039-sodium acetate 021 is 500 ml/bottle;

the activated carbon adsorption fetal bovine serum FBS is produced by BioInd company, has the product number of 04-201-1A and the specification of 500 ml/bottle;

the penicillin streptomycin solution is produced by hyclone company, and the product number is SV30010, and the specification is 100 ml/bottle;

FBS is Hyclone company production product number: SH30084.03E specification 500 ml;

ER α Antibody (F-10) is a product number of Santa Cruz: the specification of sc-8002 is 200 mu g/ml;

Donkey Anti-Mouse IgG H&L(Alexa

488) production order number for abcam corporation: ab 150105500 ug;

hochest33342 is SIGMA corporation product number: 14533, respectively;

tamoxifen is manufactured by Sigma-Aldrich company under the product number: T5648-1G

PBS was prepared from KH2PO4, Na2HPO4, NaCl, KCl powder all produced by Sigma using ultrapure water;

DMSO, produced by solibao corporation, cell culture grade, cat #: d8371 specification 50 ml/bottle;

4% paraformaldehyde manufactured by Solebao corporation, cat #: p1110 specification 500 ml/bottle;

TritonX-100 is 100 ml/bottle of the product number T8200 specification of Solebao company;

tween-20 is a product number of Solebao corporation: 100ml of T8220 specification;

instruments High Screening Assay (Operetta) purchased from Perkin Elmer;

CO2 incubator IL-161HI is manufactured by STIK corporation;

adjustable speed and timing vortex mixer (SI-T256)

The electric heating type constant temperature water bath kettle is produced by Tianjin Euro instruments company;

the liquid-transfering gun is produced by Eppendorf company;

nikon ECLIPSE Ti-U inverted biological microscope

1.6R desk-top multi-purpose centrifuge is manufactured by Thermo corporation;

the water generator is an American Milli-Q Century ultra-pure water system with model number of Milli-QACademic, Milli-Q Grandie, Milli-Q Biocel, Milli-Q Synthesis

Refrigerator with a temperature of 4 DEG C

-20 ℃ Heier refrigerator

Compound 1-17 sample preparation: dividing the weighed mass M of the sample by the relative molecular mass M of the sample to obtain the amount of material n of the sample, and then adding 10 x n volumes of DMSO to obtain 10^-1Initial concentration of mol/L. 10ul of volume is taken^-1The mol/L medicine is diluted to 100ul by DMSO to obtain the concentration of 10^-2The medicine with mol/L concentration is diluted to 10 by analogy^-4mol/L, 10ul of each tube, and storing at-20 ℃.

Screening compounds capable of selectively activating estrogen receptors into the nucleus by using an indirect immunofluorescence method. Indeed, the entry of estrogen receptors from the cytoplasm into the nucleus is the initial step in which they exert genomic pathways to regulate gene transcription.

1. Cells and culture thereof

MCF-7 cells (

Number:HTB-22^TM) Purchased from ATCC and subcultured from this chamber; MCF-7 cells were cultured in a cell culture incubator at 37 ℃ and 5% CO2, with 10% FBS/DMEM (4500mg/L D-glucose, L-glutamyl and 110mg/L sodium pyruvate, 100U/ml penicillin, 100. mu.g/ml streptomycin).

2. Experimental procedure for Nuclear migration

At 37 ℃ 5% CO₂MCF-7 cells were cultured in a cell culture incubator using phenol red-free DMEM/F12 medium containing 10% charcoal-adsorbed fetal bovine serum and 1% diabody. After 72h, the cell density is about 80%, the cells are paved in a 96-hole blackboard according to the density of 7000/hole, the middle 60 holes are experimental holes, serum containing 1% of activated carbon adsorption and 100ul of phenol red-free DMEM/F12 culture solution containing 1% of double antibodies are added into each hole, the peripheral holes are filled with 100ml of PBS, and the cells are cultured overnight until the cells are completely attached to the wall. Diluting the drug with phenol red-free DMEM/F12 medium (the drug is diluted to 10 degrees with DMSO)^-4M, subpackaging and storing in a refrigerator at-20 ℃) to 10^-7And M. The concentration of each experimental hole in the 96-hole plate is 100ul^-7M, positive drug is tamoxifen, a blank control is added into a phenol red-free DMEM/F12 culture medium containing 1 thousandth of DMSO, and each drug is provided with three multiple wells. After completion of the administration, 5% CO at 37 deg.C₂Incubated for half an hour and cells were fixed with 4% paraformaldehyde for half an hour (room temperature). Cells were washed three times with PBS, 200ul per well, one minute each time. 0.2% TritonX-100 was added and the wells were punched for half an hour (room temperature), and the cells were washed three times with 200ul of PBS per well for one minute each time. 5% fetal bovine serum (Hyclone calf serum, diluted with PBS) was added for 2 hours (room temperature), primary antibody (0.01% Tween-20+ 0.5% Hyclone fetal bovine serum + 1% primary antibody + PBS, primary antibody ER α) was added, incubation was overnight at 4 deg.C, cells were washed three times with 200ul per well for 5 minutes in PBST (PBS + 0.05% Tween-20), and then washed three times with PBS with 200ul per well for one minute. Adding a secondary antibody (0.01% Tween-20+ 0.5% Hyclone fetal bovine serum + one ten thousand Hochests + one two percent secondary antibody + PBS) in a dark place, and incubating for two hours at room temperature. PBST (PBS + 0.05% Tween-20) washed the cells three times, 200ul per well, 5 minutes per time, then washed three times with PBS, 200ul per well, one minute per time, and finally added 100ul PBS into the wells with high content. After the high content photographing is completed, the data is automatically processed by the high content, namely the fluorescence value in the nucleus is compared with the fluorescence value in the cytoplasm, and the average value of three empty values is taken. Graphpad mapping, SPSS processing of the data, and whether the drug group was statistically significant compared to the blank group. The results of the experiment are shown in table 6:

TABLE 6 results of activation of Estrogen receptor alpha by Compounds 1-17

P <0.05, p <0.01, p <0.001, ns represents null

From the activity experiments, it can be seen that the compounds 1-17 isolated from eclipta, except the compound 7, the compound 8, the compound 15 and the compound 16, can be combined with an estrogen receptor ER alpha to improve the transcriptional activity, and the compounds are proved to have estrogen-like effects. Therefore, it can be used for preventing and treating climacteric syndrome, postmenopausal osteoporosis, breast cancer, uterine cancer, prostate cancer and colon cancer, and its pharmacological action is expanded in the fields of cardiovascular system, reproductive system and immunoregulation.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. Use of a pharmaceutical composition for the manufacture of a medicament for the treatment and/or prevention of a disease associated with abnormal estrogen secretion, wherein the pharmaceutical composition comprises at least one pharmaceutically active ingredient selected from the group consisting of: ecliptin IV, ecliptin I and ecliptin A;

wherein the disorder associated with estrogen secretion abnormality is selected from the group consisting of: climacteric syndrome, prostatic cancer, osteoporosis, hyperplasia of mammary glands and hysteromyoma.

2. The use as claimed in claim 1, wherein the pharmaceutically active ingredient contained in the pharmaceutical composition is extracted from eclipta.