Selaginella tin A and its preparation method and application

Abstract

The present invention relates to a selaginella viniferin A and a preparation method and application thereof. The molecular formula of selaginella viniferin A is C 33 H 22 O4; A method for preparing Selaginella serrata A, comprising: extracting Selaginella serrata with alcohol 3 to 8 times; filtering, concentrating the filtrate under reduced pressure to obtain an extract for later use; dry-loading the sample onto a 100-200 mesh silica gel column for separation by chromatography, eluting with a chloroform:methanol gradient, and collecting different fractions; then dry-loading the eluted portion onto a 200-300 mesh silica gel column for separation by chromatography with a chloroform:methanol gradient in a volume ratio of 95:5, eluting with a chloroform:methanol gradient, and collecting different fractions; tracking the target component by thin-layer chromatography, combining the fractions containing the target component, and then repeatedly purifying the fractions by Sephadex LH-20 column chromatography using aqueous methanol as the elution solvent. The invention also relates to the use of Selaginella serrata A in the preparation of a drug for treating endometriosis, which can be easily prepared into a variety of pharmaceutical dosage forms for convenient clinical use.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine preparations, and in particular to selaginella tin A and a preparation method thereof, and application thereof in preparing a medicine for treating endometriosis. Background Art

[0002] Endometriosis is a chronic gynecological disease in which endometrial tissue (glands and stroma) migrates backward with menstrual blood and implants and grows in the lining of the uterine cavity and other areas outside the uterus. This leads to recurrent bleeding, dysmenorrhea, and infertility, which severely impact the health and quality of life of young and middle-aged women. Approximately 10% to 15% of women of childbearing age suffer from endometriosis, a condition estimated to affect over 176 million women.

[0003] Endometriosis is characterized by extensive lesions, diverse morphologies, and extremely invasive and recurrent characteristics. The etiology of endometriosis remains unclear, and current clinical treatments primarily rely on surgery and medication. Given that endometriosis is difficult to cure with surgery and prone to recurrence, medication remains crucial. Treatments for endometriosis primarily include five categories: nonsteroidal anti-inflammatory drugs (NSAIDs), progestins, combined oral contraceptives (COCs), gonadotropin-releasing hormone agonists (GnRH-a), and traditional Chinese medicine. Drugs for the treatment of endometriosis primarily act on the hypothalamic-pituitary-ovarian axis, creating a low estrogen and progesterone environment and inhibiting the growth of ectopic endometrium. However, liver damage and symptoms of low estrogen levels reduce patient tolerance, and these drugs are ineffective in eradicating the lesions, resulting in a high recurrence rate.

[0004] Traditional Chinese Medicine considers endometriosis to fall under the categories of "hypertension and mass," "dysmenorrhea," "infertility," and "irregular menstruation." Its primary pathogenesis is blood stasis, a basic syndrome type of "blood stasis syndrome," and therefore the primary treatment principle is to "activate blood circulation and remove stasis." The Chinese medicinal herb Selaginella, derived from the dried whole herb of Selaginella tarmariscina (Beauv.) Spring and Selaginella pumvinata (Hook. et Grev.) Maxim., both members of the Selaginellaceae family, is known for its ability to "activate blood circulation and unblock menstruation." Selaginella was first mentioned in the Shennong's Herbal Classic, stating, "Selaginella, with a pungent and warm flavor,...can treat pathogenic qi in the five internal organs, cold, heat, and pain in the female genitals, hypertension and mass, blood stasis, and infertility." Similar references to endometriosis can be found in ancient herbal compendiums, and it has been used for a variety of gynecological conditions. "Hypertension and mass" refers to conditions characterized by nodules or masses in the lower abdomen, including uterine fibroids, ovarian tumors, and endometriosis.

[0005] Patent publication number CN1695643A discloses a traditional Chinese medicine preparation and preparation method for treating endometriosis. The preparation uses the Chinese medicinal herb Selaginella as a raw material to extract the effective biflavonoid components for treating endometriosis. Modern pharmacological research has shown that Selaginella not only has traditional anti-inflammatory, detoxifying, and blood circulation and hemostasis effects, but also has anti-tumor, antiviral, blood sugar-lowering, and immune-enhancing effects. Selaginella contains various compounds, including biflavonoids, acetylphenols, and phenylpropanoids. The inventors discovered during their research that Selaginella A, a compound found in the herb, is more effective in treating endometriosis. Summary of the Invention

[0006] The purpose of the present invention is to provide a medicine for treating endometriosis and a preparation method thereof, wherein the medicine has a good effect in treating endometriosis.

[0007] To solve the above technical problems, the present invention adopts the following technical solutions:

[0008] Selaginella A, whose molecular formula is C 33 H 22 O4, the structural formula is:

[0009]

[0010] The present invention also provides a method for preparing the above-mentioned Selaginella tin A, comprising the following steps:

[0011] S1. Take Chinese herbal medicine Selaginella by adding 1:8~12 mass volume ratio of ≥70% alcohol and extract 3 to 8 times; filter, and concentrate the filtrate under reduced pressure to obtain an extract, set aside;

[0012] S2. Dry sample was loaded on a 100-200 mesh silica gel column for separation by chromatography, and eluted with a gradient of chloroform: methanol in a volume ratio of 100:0 to 90:10, and different fractions were collected;

[0013] S3. Collect the chloroform: methanol fractions in a volume ratio of 95:5 and dry-load them onto a 200-300 mesh silica gel column for separation by gradient elution with chloroform: methanol in a volume ratio of 98:2 and 95:5, respectively, to collect different fractions;

[0014] S4. Trace the target component by thin-layer chromatography, combine the fractions containing the target component, and then purify it by repeated Sephadex LH-20 column chromatography using aqueous methanol as the elution solvent.

[0015] Among them, the Chinese medicinal herb Selaginella can be the dried whole herb of Selaginella of the Selaginellaceae family or the pufferfish Selaginella. When the dried whole herb of Selaginella is used, after obtaining the extract in step S1, it needs to be washed three times with twice the weight of petroleum ether, and then filtered, and the petroleum ether is evaporated from the filter residue for later use; when it is the pufferfish, this operation is not required; the volume of alcohol used in step S1 is 10 times the mass of the Chinese medicinal herb Selaginella, and the alcohol concentration is 75%; the concentration of methanol in step S4 is 70%.

[0016] Finally, the present invention also relates to the use of the above-mentioned Selaginellarin A in medicines for treating endometriosis.

[0017] The selaginella viniferin A and a pharmaceutically acceptable carrier are prepared into a drug in the form of tablets, capsules, injections, powder injections, granules, fat emulsions, microcapsules, dripping pills, ointments or transdermal controlled-release patches.

[0018] When the selaginella viniferin A provided by the present invention is made into tablets, the selaginella viniferin A and lactose or corn starch are mixed with lubricant magnesium stearate when necessary, mixed evenly, granulated, and then compressed into tablets.

[0019] When the Selaginellarin A provided by the present invention is prepared into capsules, the Selaginellarin A and carrier lactose or corn starch are evenly mixed, granulated, and then filled into capsules to prepare the capsules.

[0020] When the selaginella viniferin A provided by the present invention is prepared into granules, the selaginella viniferin A and a diluent, lactose or corn starch, are uniformly mixed, granulated, and dried to prepare the granules.

[0021] The selaginella tin A provided by the present invention is prepared by adding a carrier according to a conventional pharmaceutical method when being made into a powder injection or an injection.

[0022] The selaginella viniferin A provided by the present invention is prepared by adding a carrier according to a conventional pharmaceutical method when being prepared into dosage forms such as fat emulsion, ointment or transdermal controlled release patch.

[0023] Although endometriosis is a benign lesion, it has biological behaviors similar to malignant tumors, such as adhesion, invasion, and angiogenesis, and is associated with autoimmunity. Oral administration of 1mg / kg of selaginella selaginellae can significantly inhibit the growth of ectopic lesions in mice, angiogenesis and inflammatory infiltration of ectopic tissues; it can also regulate the expression of helper T cells in the peripheral blood of mice. 4+ , killer T cell CD 8+ , and CD 4+ / CD 8+ The ratio of CD in mouse peritoneal fluid was 4+ 、CD S+ T cells, and CD 4+ / CD 8+The ratio of selaginella to endometriosis also showed a downward trend, suggesting that selaginella A may play a therapeutic role in endometriosis by alleviating immunosuppression and inflammatory responses in the peritoneal microenvironment. HE staining did not show liver or kidney toxicity, and had no effect on the uterine and ovarian coefficients.

[0024] Endometriosis is a hormone-dependent disease characterized by chronic, invasive growth of ectopic tissue. The apoptotic activity of ectopic endometrial cells is significantly lower than that of normal and in situ endometrium. Studies have shown that β-estradiol promotes the proliferation and migration of endometrial cancer Ishikawa cells, induces the expression of the anti-apoptotic protein Bcl-2, and reduces the expression of the apoptotic proteins cleaved caspase-3 and Bax in Ishikawa cells, thereby increasing cell viability and resistance to apoptosis. Selaginellin A significantly inhibits the in vitro growth of Ishikawa cells and induces apoptosis by downregulating Bcl-2 expression and upregulating cleaved caspase-3 and Bax. β-estradiol upregulates the expression of VEGF and MMP9 in Ishikawa cells, promoting angiogenesis and epithelial-mesenchymal transition, and increasing cell migration and invasion. Selaginellin A at 40 μM significantly inhibits the expression of MMP9 and VEGF, thereby inhibiting cell migration.

[0025] The present invention provides the use of a monomeric compound, Selaginella vinifera A, from the traditional Chinese medicine Selaginella vinifera in a drug for treating endometriosis. The dosage is much lower than the dosage of the Selaginella vinifera extract in invention patent CN 1695643 A. Selaginella vinifera A can inhibit the growth of ectopic tissues, induce apoptosis, inhibit invasion and angiogenesis, and exert its effects by regulating the autoimmunity of the body and the peritoneal microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The growth of ectopic lesions and organ coefficients in mice (compared with the model group, ***P<0.001);

[0027] Figure 2 This is the HE pathological section of mouse liver tissue;

[0028] Figure 3 This is the HE pathological section of mouse kidney tissue;

[0029] Figure 4 This is the HE pathological section of mouse ectopic tissue;

[0030] Figure 5 CD4 T lymphocytes in mouse blood 4+ / CD 8+ Proportional flow diagram;

[0031] Figure 6 CD4 T lymphocytes in mouse peritoneal fluid 4+ / CD 8+ Proportional flow diagram;

[0032] Figure 7 The bar graph shows the proportion of T lymphocytes in mouse blood and peritoneal fluid;

[0033] Figure 8 This figure shows the effect of Selaginellin A on the level of TNF-α in the peritoneal fluid of mice;

[0034] Figure 9 This is a diagram showing the effect of Selaginellin A on the expression of related proteins in mouse ectopic endometrium;

[0035] Figure 10 This figure shows the effect of Selaginellin A on the proliferation of Ihsikawa cells;

[0036] Figure 11 This figure shows the effect of Selaginellin A on the migration ability of Ihsikawa cells;

[0037] Figure 12 Statistical analysis of Selaginellin A-induced Ishikawa cells;

[0038] Figure 13 This figure shows the effect of Selaginellin A on protein expression in Ishikawa cells. DETAILED DESCRIPTION

[0039] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following examples. It should be understood that the following text is only used to describe one or more specific embodiments of the present invention and does not strictly limit the scope of protection of the present invention.

[0040] Example 1

[0041] Selaginella A, whose molecular formula is C 33 H 22 O4, the structural formula is:

[0042]

[0043] Example 2

[0044] This embodiment is a method for preparing Selaginella selaginella A:

[0045] First, take 500g of Selaginella pulvinata and add 5000mL of 75% alcohol to extract five times; filter, and concentrate the filtrate under reduced pressure to obtain an extract, which is set aside;

[0046] (1) Crude fraction: 100-200 mesh silica gel, dry loading, gradient elution with chloroform: methanol (volume ratio 100:0), chloroform: methanol (volume ratio 98:2), chloroform: methanol (volume ratio 95:5), and chloroform: methanol (volume ratio 90:10), and different fractions were collected;

[0047] (2) Fine segmentation: 200-300 mesh silica gel, dry-load the fraction in (1) with a volume ratio of 95:5 chloroform:methanol, and sequentially elute with a gradient of 98:2 and 95:5 chloroform:methanol, collecting different fractions;

[0048] (3) Purification of target component: The target component is tracked by thin layer chromatography, and the fractions containing the target component are combined and then purified by repeated Sephadex LH-20 column chromatography using aqueous methanol as the elution solvent.

[0049] The obtained Selaginella A is a red powder with the molecular formula C 33 H 22 O4.

[0050] The UV spectrum characteristics of Selaginella A are as follows: UV(MeOH)λ max (logε): 297(3.15), 430(3.27);

[0051] Infrared IR (KBr), cm -1 : 3150, 2799, 2669, 1595, 1511, 1456, 1377, 1336, 1241, 1162, 902, 836; ESI-MS m / z 505[M+Na] + ;

[0052] The H NMR spectrum data are: 1 H NMR (acetone-d6, 500 MHz) δ H: 6.34 (1H, d, J = 10.0Hz, H-2), 7.52 (1H, d, J = 10.0Hz, H-3), 7.32 (1H, d, J = 10.0Hz, H-5), 6.32 (1H, d, J = 10.0Hz, H-6), 6.80 (2H, d, J = 8.5Hz, H-8, 12), 6.68 (2H, d, J = 8.5Hz, H-9, 11), 7.64 (1H, d, J = 8.0Hz, H-15), 7.54 (1H, t, J =8.0Hz, H-16), 7.34 (1H, d, J = 8.0Hz, H-17), 6.86 (2H, d, J = 8.5Hz, H-20 / 24), 6.63 (2H, d, J = 8.5Hz, H-21 / 23), 7 .06 (2H, d, J = 8.5Hz, H-28 / 32), 6.72 (2H, d, J = 8.5Hz, H-29 / 31), 8.90 (1H, brs), 8.82 (1H, brs), 8.44 (1H, brs); 13 C NMR (acetone-d6, 125 MHz) δ C : 185.0(C-1), 116.1(C-2), 138.5(C-3), 129.8(C-4), 138.5(C-5), 116.1(C-6), 149.0(C-7), 136.7(C-8 ), 115.9(C-9), 159.4(C-10), 115.9(C-11), 136.7(C-12), 133.2(C-13), 125.5(C-14), 130.9(C-15), 129 .9(C-16), 130.7(C-17), 143.9(C-18), 141.8(C-19), 130.5(C-20 / 24), 115.6(C-21 / 23), 157.9(C-22), 132.0(C-25), 87.2(C-26), 94.5(C-27), 133.8(C-28 / 32), 116.3(C-29 / 31), 159.3(C-30), 113.8(C-33).

[0053] The purity calculated by HPLC-UV area normalization method was >95%.

[0054] Example 3

[0055] This example is a study on the inhibitory effect of Selaginellacin A on ectopic lesions in mice with endometriosis.

[0056] 1. Materials

[0057] 1.1 Animals 75 C57BL / 6J female mice, 6-8 weeks old, weighing 18-20 g, were housed under SPF conditions, maintained at 22 ± 2°C and humidity between 55% and 65%. A 12-hour light and 12-hour dark cycle was maintained, with bedding changed weekly. Sterile nutrient pellets and sterile water were available 24 hours a day.

[0058] 2. Methods

[0059] 2.1 Reagent Configuration

[0060] Preparation of Shutai combined with xylazine hydrochloride solution: accurately weigh 60.0 mg of Shutai powder, add 300 μL of xylazine hydrochloride injection and mix thoroughly, then add 9.7 mL of normal saline and mix well;

[0061] Preparation of ampicillin sodium solution: accurately weigh 60.0 mg of ampicillin sodium and add 10 mL of normal saline and mix well;

[0062] Preparation of β-estradiol solution: Accurately weigh 3.0 mg of β-estradiol, add 100 mL of corn oil and mix well;

[0063] 2.2 Establishment of mouse endometriosis model

[0064] After 7 days of adaptive feeding of C57BL / 6J mice, 15 mice were randomly selected as donor mice (recipient mice: donor mice = 4:1). 0.1 mL of β-estradiol solution was subcutaneously injected at 8-10 am every day for 7 consecutive days. Endometrial transplantation was performed 1 hour after the injection on the 7th day. The donor mice in estrus were euthanized by CO2 asphyxiation, and the uterus of the mice was removed, opened longitudinally, and biopsied into 2×2 mm 2 The uterine fragments were divided into small pieces and placed in cold saline for later use.

[0065] After fasting for 24 hours, 60 recipient mice were anesthetized with an intraperitoneal injection of 0.1 mL of a combination of 30 mg / kg of valproate and 10 mg / kg of xylazine hydrochloride. The mice's abdomens were depilated with a depilatory cream, cleaned with cotton balls, and disinfected with povidone-iodine. A 1 cm midline incision was made to expose the pelvis. The inner side of the uterine fragment was pressed against the abdominal wall, and the fragment was sutured to the peritoneal wall with 6-0 black sutures, one on each side. The abdominal muscles and skin were sutured with 4-0 black sutures, and the skin was cleaned with povidone-iodine cotton. The sham-operated group was constructed as described above, except that the endometrial fragments were sutured with fat of the same size to the abdominal wall instead of sutured. Mice were kept on a heating pad throughout the surgery. Ampicillin 0.1 mL at 30 mg / kg was administered intramuscularly for three consecutive days postoperatively to prevent infection. β-estradiol 0.1 mg / kg was administered subcutaneously starting the day after surgery and administered every four days for a total of four times to promote ectopic endometrial growth. The mice were weighed on days 4, 7, 11, and 14.

[0066] 2.3 Grouping and Dosing

[0067] On the 14th day after modeling, the mice were randomly divided into 5 groups: sham group (Sham), model group (Model), and low-, medium-, and high-dose groups of Selaginella A (1 mg / kg, 7.5 mg / kg, and 15 mg / kg); the sham group and model group were given equal volumes of normal saline containing 1% DMSO, and the mice were killed after 28 consecutive days of administration.

[0068] 2.4 Specimen collection

[0069] After anesthesia, blood was collected from the mouse's orbital cavity. Some samples were stored in EP tubes at -80°C, while others were collected in anticoagulant tubes and allowed to stand for 40-60 minutes. The samples were then centrifuged at 1500 rpm for 10 minutes, and the supernatant was collected and stored at 4°C for flow cytometry analysis. After blood collection, the mouse was euthanized by CO2 asphyxiation. After confirming asphyxiation, the mouse was sacrificed by cervical dislocation and immersed in 75% alcohol for 2-3 minutes. 2 mL of PBS solution was drawn using a 5 ml syringe and injected into the mouse's peritoneal cavity. The abdomen was gently massaged for 1 minute. A small incision was made in the mouse's abdomen, and the mouse peritoneal fluid was aspirated using a pipette into a centrifuge tube. The fluid was stored at -80°C, and some samples were centrifuged at 1500 rpm for 10 minutes at 4°C. The supernatant was collected and stored at 4°C for flow cytometry analysis. The mouse abdominal cavity was opened, and the lesions on both sides of the abdominal wall were removed (normal uterine tissue was taken from the sham operation group). The length, width, and height of the lesions were measured with a vernier caliper, and the mass of the lesions was measured with a 1 / 10,000 analytical balance. The left lesion was fixed in 4% formaldehyde fixative, and the right lesion was placed in a cryopreservation tube and stored in liquid nitrogen. The liver and kidney tissues of the mice were placed in a cryopreservation tube and stored in liquid nitrogen. After the experiment, the samples in the cryopreservation tube were transferred to a -80°C freezer for future use. The formula for calculating the volume of mouse ectopic lesions is as follows:

[0070] V=0.52×length×width×height

[0071] 2.5 Hematoxylin-eosin (HE) staining and histomorphological observation

[0072] Mouse tissues were excised and preserved in 4% paraformaldehyde, embedded in paraffin, cut into 4 μM sections, and stained with HE. The sections were observed and photographed under an upright white-field microscope to examine morphological changes.

[0073] 2.6 Data Processing and Analysis

[0074] All experimental data are expressed as "mean ± standard error" All data were statistically analyzed using GraphPad Prism 9. Multiple group comparisons were performed using one-way analysis of variance, and P < 0.05 was considered statistically significant.

[0075] 3. Results

[0076] 3.1 Selaginellin A inhibits ectopic endometrium in EMs mice

[0077] Continuous administration of Selaginellin A at different doses for 28 days significantly inhibited the growth of ectopic endometrium. Compared with the model group, the volume of ectopic lesions in the treatment group was significantly reduced (P < 0.01) and the weight was significantly reduced (P < 0.01). There were no significant differences in the uterine coefficient and ovarian coefficient (P > 0.05). The body weight of mice in the high-dose group decreased significantly compared with the sham-operated group starting from the 21st day (P < 0.05).

[0078] Table 1 Effects of drugs on body weight of endometriosis model mice

[0079]

[0080] Note: Compared with Sham, * P<0.05, ** P<0.01, *** P<0.001

[0081] Table 2 Effects of drugs on mouse uterus and ovary

[0082]

[0083] Note: Uterine (ovary) coefficient = uterine (ovary) weight mg / mouse body weight g×100%

[0084] Table 3 Effects of drugs on the area and volume of ectopic membranes and their inhibition rates

[0085]

[0086] Note: Compared with the model group, ***P<0.001

[0087] 3.2 HE pathological section results

[0088] Liver pathological section results showed that ( Figure 2 ), the liver cells in each group radiated from the central vein to the periphery, with normal cell morphology, close arrangement between cells, and no inflammatory cell infiltration. Figure 3 ), the renal tubules and glomeruli were intact, with no inflammation or edema. These results indicate that Selaginellin A does not cause liver or kidney damage.

[0089] The pathological section of the ectopic lesion showed ( Figure 4 , Figure 4 The dotted arrows in the figure represent angiogenesis, the long solid arrows represent glands, and the short solid arrows represent inflammatory cells. Compared with the sham-operated group, the tissue cells in the model group were loose, with a large number of new blood vessels and inflammatory cell infiltration, and increased glandular tissue. After intervention with Selaginellin A, the ectopic endometrial tissue in the low-, medium-, and high-dose groups showed varying degrees of atrophy, the number of glands was significantly reduced, and angiogenesis and inflammatory cell infiltration were effectively inhibited in a dose-dependent manner.

[0090] Example 4

[0091] This example is a study on the in vivo mechanism of Selaginella tin A in intervening in endometriosis in mice.

[0092] 1. Methods

[0093] 1.1 Detection of T cell levels in mouse blood and peritoneal fluid by flow cytometry

[0094] To 50 μL of fresh blood, add 5 μL of FcR Blocking, vortex for 2 seconds, let it stand at room temperature for 2 minutes, add flow cytometry antibodies, and incubate at 4°C in the dark for 15 minutes; add 1 mL of red blood cell lysis buffer, incubate at room temperature for 10 minutes, centrifuge at 300g for 5 minutes, discard the supernatant, add 1 mL of staining buffer, vortex for 1 second, centrifuge at 300g for 5 minutes, discard the supernatant, resuspend in 150 μL of staining buffer, transfer to a flow cytometry tube, and detect on the instrument;

[0095] Fresh peritoneal fluid was centrifuged at 300g for 10 minutes, part of the supernatant was discarded, and about 100 μL of the supernatant was kept. 5 μL of FcR Blocking was added, vortexed for 2 seconds, and placed at room temperature for 2 minutes. Flow cytometry antibodies were added, and the cells were incubated at 4°C in the dark for 15 minutes. The cells were centrifuged at 300g for 5 minutes, and the supernatant was discarded. 1 ml of staining buffer was added, vortexed for 1 second, and centrifuged at 300g for 5 minutes. The supernatant was discarded, and the cells were resuspended with 150 μL of staining buffer. The cells were transferred to a new flow cytometry tube and then tested on the flow cytometer.

[0096] 1.2 Elisa kit to detect the levels of inflammatory factors in mouse peritoneal fluid

[0097] (1) Add 300 μl of 1× wash solution to the ELISA plate, let it soak for 30 seconds, discard the wash solution, and pat the microwells dry on absorbent paper;

[0098] (2) Add 100 μL of diluted standard solution to the standard wells and 100 μL of standard diluent to the blank wells; add 50 μL of peritoneal fluid sample and 50 μL of detection buffer to the sample wells;

[0099] (3) Seal the plate with sealing film, shake at 200 rpm / min, and incubate at room temperature for 2 h;

[0100] (4) Discard the liquid and add 300 μL of washing solution to each well to wash the plate six times, patting the plate dry on absorbent paper after each wash;

[0101] (5) Add 100 μL of detection antibody to each well, seal the plate with a new sealing film, shake at 200 rpm / min, and incubate at room temperature for 45 min;

[0102] (6) Repeat step (3);

[0103] (7) Add 100 μL of chromogenic substrate TMB to each well and incubate at room temperature in the dark for 5-30 min. When the S5 well marked with the curve is light blue and the Blank well is not obviously blue, add 100 μL of stop solution to each well to terminate the reaction.

[0104] (8) The OD value was measured at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm. The OD value after calibration was OD 450nm -OD 630nm .

[0105] 1.3 Western blotting assay to detect related protein expression in ectopic lesions

[0106] 1.3.1 Total protein extraction and BCA protein quantification

[0107] Remove the endometrial tissue sample from a -80°C refrigerator, weigh 20 mg using a 1 / 10,000 micrometer balance, transfer to a 1.5 mL centrifuge tube, add 200 μL of Ripa lysis buffer, 20 μL of PMSF, 20 μL of phosphatase inhibitor A, and 20 μL of phosphatase inhibitor B, add two steel beads, and immediately homogenize for 180 seconds. Lyse the homogenate on ice for 20 minutes, then sonicate for 30 seconds using a sonicator. Centrifuge at 4°C, 12,000 rpm, for 15 minutes. Aspirate the supernatant, transfer it to a centrifuge tube, and store on ice.

[0108] The total protein concentration in each sample was calculated using the BCA assay. The following protocol was used: 0, 1, 2, 4, 8, 12, 16, and 20 μL of a 25 mg / mL protein standard was added to a 96-well plate. Subsequently, 20, 19, 18, 16, 12, 8, 4, and 0 μL of PBS buffer were added. Samples were then added to the 96-well plate at a rate of 2 μL / well, and 18 μL of PBS buffer was added. 200 μL of BCA working solution (BCA reagent A:BCA reagent B = 50:1) was added to each well. After incubation at 37°C for 30 min, the OD value of each well was measured at 570 nm using a microplate reader. The total protein concentration in each sample was calculated based on the standard curve. One-quarter volume of protein loading buffer was added, mixed thoroughly, and the protein was denatured in a 100°C thermostatted metal bath for 10 min. After the protein cooled, the sample was aliquoted and stored at -80°C until further use.

[0109] 1.3.2 Target protein detection

[0110] (1) SDS-PAGE gel configuration

[0111] Align the bottoms of the two glass plates, placing them in a glue clamp and securing them securely. Add sufficient double-distilled water to test for leaks. Observe whether the water level in the glass plate has dropped. Discard the double-distilled water and blot with absorbent paper. To a 50mL EP tube, add 6.4mL of double-distilled water, 12.8mL of 30% Acr-Bis (29:1), 12.16mL of 1M Tris-HCl (pH 8.8), 0.32mL of 10% SDS, 0.32mL of 10% APS, and 0.013mL of TEMED. Mix thoroughly and immediately inject into the glass plates, adding 7.5mL to each. Fill the remaining space with anhydrous ethanol. After the lower gel has solidified, discard the ethanol and add the upper gel solution. The upper gel formulation is: 5.44 mL of double-distilled water, 1.36 mL of 30% Acr-Bis (29:1), 1.04 mL of 1M Tris-HCl (pH 6.8), 0.08 mL of 10% SDS, 0.08 mL of 10% APS, and 0.008 mL of TEMED. Immediately insert a 15-well comb and let it sit for 30 minutes until the upper gel solidifies before loading the sample and performing electrophoresis.

[0112] (2) Configuration of electrophoresis fluid

[0113] Weigh 3.03 g of Tris, 14.4 g of Glycine, and 1 g of SDS, add 1 L of double-distilled water, and stir to dissolve.

[0114] (3) Sample loading and gel electrophoresis

[0115] Secure the prepared SDS-PAGE gel glass plate to the electrophoresis holder. Add the prepared electrophoresis buffer to the top of the plate. Pull the comb vertically out of the plate and load the sample into the loading well formed by the comb and gel. Load the protein marker, Sham, Model, Low, Middle, and High samples one at a time, starting from left to right. Connect the power supply and run the gel at 80V for 20 minutes, followed by 120V for 60 minutes.

[0116] (4) Preparation of transfer solution

[0117] Weigh 3.03 g of Tris and 14.4 g of Glycine, add 800 mL of double-distilled water, stir the solvent, and then add 200 mL of methanol to mix evenly.

[0118] (5) Transfer

[0119] According to the size of the colloid, cut the PVDF membrane to the appropriate size. Soak the PVDF membrane in methanol for 1-2 minutes, and then soak it in the transfer solution for later use. Take out the gel from the glass plate, and place a porous pad, three filter papers, gel, membrane, three filter papers, and a porous pad ("sandwich" structure) on the plywood in sequence. Place the transferred membrane in the transfer tank, add the transfer solution, and add an ice box to the transfer tank. Transfer the transfer tank to a foam box filled with ice cubes and turn on the power. Transfer the membrane at a constant current of 250mA for 90 minutes. After the transfer is completed, take out the transferred PVDF membrane and soak it in TBST for later use.

[0120] (6) Closed

[0121] Weigh 2g of skim milk powder and add 40mL of TBST to mix until a 5% skim milk solution is obtained. Place the transferred PVDF membrane in the 5% skim milk powder and block on a shaker at room temperature for 60 minutes. Remove the PVDF membrane and add sufficient TBST to elute on a shaker three times for 10 minutes each.

[0122] (7) Primary antibody incubation

[0123] Cut the target protein strips according to the protein marker indication and incubate them in the prepared primary antibody solution at 4°C overnight. Remove the strips the next day. Add sufficient TBST and elute on a shaker three times for 10 minutes each.

[0124] (8) Secondary antibody incubation

[0125] Place the eluted bands in the secondary antibody and incubate on a shaker at room temperature for 60 minutes. Remove the bands and add sufficient TBST to elute on a shaker three times, each time for 10 minutes.

[0126] (9) ELC development

[0127] After the ELC developer is prepared at a ratio of 1:1, it is poured onto the strips and developed on a Tanon chemiluminescence system.

[0128] 1.4 Semi-quantitative detection of 308 indicators in mouse serum based on solid-phase protein chip technology

[0129] 1.4.1 Sample purification

[0130] Place the spin column in a 500 μL conical collection tube and centrifuge at 1000 g for 3 minutes to remove the storage buffer. Discard the flow-through. Clear the column three times with 300 μL of labeling buffer and centrifuge at 1000 g for 3 minutes to remove the flow-through. Aspirate the bottom of the column to remove excess liquid and transfer to a new collection tube. Over the next few minutes, apply the sample to the resin. Centrifuge at 1000 g for 3 minutes and collect the flow-through, which contains the sample.

[0131] 1.4.2 Biotin-labeled samples

[0132] Before using the labeling reagent, quickly centrifuge the labeling reagent tube. Add 100 μL of 1X PBS to dissolve the powder and mix by pipetting up and down to prepare a 1X labeling reagent solution. Add the appropriate amount of sample and labeling reagent to a new centrifuge tube. Mix quickly and incubate on a shaker at room temperature for 30 minutes. Gently flick the centrifuge tube every 5 minutes to mix the reaction reagents. Add 3 μL of stop solution. Unscrew the bottom cap of the spin column and loosen the lid (do not remove it). Place the spin column in a 500 μL conical collection tube and centrifuge at 1000 g for 3 minutes to remove the storage buffer. Discard the flow-through. Clear the column three times with 300 μL of labeling buffer and centrifuge at 1000 g for 3 minutes to remove the flow-through. Aspirate the bottom of the column to remove excess liquid and transfer to a new collection tube. Over the next few minutes, apply the sample to the resin. Centrifuge at 1000 g for 3 minutes and collect the flow-through containing the sample.

[0133] 1.4.3 Complete drying of the slide

[0134] Take the glass slide chip out of the box, equilibrate it at room temperature for 1 hour, open the packaging bag, remove the sealing strip, and then place the chip in a vacuum desiccator to dry for 1 hour.

[0135] 1.4.4 Blocking and incubation

[0136] (1) Add 400 μL of 1× blocking solution to each chip well and incubate on a shaker at room temperature for 1 h to avoid bubbles.

[0137] (2) Remove the blocking solution and add 400 μL of sample to each well, one sample per array, and incubate at 4°C with shaking overnight.

[0138] (3) Dilute the sample 100-fold with blocking solution;

[0139] (4) Remove the sample and add approximately 1 mL of 1× Wash Solution I (20× Wash Solution diluted with deionized water) to each well. Shake at room temperature and wash the slides four times, 5 minutes each time.

[0140] (5) Remove 1× Wash Solution I and add 1× Wash Solution II, shaking at room temperature, and wash the slides 4 times, 5 min each time;

[0141] (6) Remove 1× Wash Buffer II, add 1 ml of Cy3 equivalent blocking buffer, and dilute 5-fold. Add 400 μL to each well and incubate at room temperature in the dark for 2 hours with shaking.

[0142] (7) Wash the slides according to steps (4) and (5).

[0143] 1.4.5 Fluorescence detection

[0144] Scanning was performed using an InnoScan 300 Microarray Scanner instrument, with the Cy3 or green channel selected and the excitation frequency set at 532 nm.

[0145] 1.4.6 Data Analysis

[0146] The raw data from the microarray scans were processed using Raybiotech software for chip background removal and inter-chip normalization. Normalization 2 without background data was selected for analysis. Differential proteins were screened using fold change analysis under the following conditions:

[0147] (1) fold Change = < 0.83 or fold Change > = 1.2.

[0148] (2) It is recommended to select an average (fluorescence) signal value of each group > 150.

[0149] The ClusterProfile package was used to perform enrichment analysis (GO enrichment and KEGG enrichment) of differentially expressed genes. The screening criteria were that the number of differentially expressed proteins on a certain GO / KEGG was >= 2 and p_value < 0.01.

[0150] 2. Results

[0151] 2.1 Effect of Selaginellin A on CD4 T lymphocytes in mouse blood and peritoneal fluid 4+ / CD 8+ The impact of level

[0152] Compared with the sham operation group (ref. Figure 5 ), helper T cell CD4+ T cells in the blood of the model group 4+ Increased cytotoxic T cell CD 8 + Lower, CD 4+ / CD 8+ The proportion of CD4+ in the blood increased significantly (P<0.01), while Selaginellin A reversed this trend. Low-dose Selaginellin A significantly reduced the level of CD4+ in the blood. 4+ / CD 8+ (P<0.01) ratio, and the callback trend of medium and high doses was not statistically significant.

[0153] Compared with the sham operation group (ref. Figure 6 ), CD in peritoneal fluid of model group 4+ Lower, CD 8+ Increase, CD 4+ / CD 8+ The ratio decreased, and there was a trend of callback after administration of different doses of Selaginellin B, but there was no statistical significance.

[0154] Figure 7 is the ratio of T lymphocytes in mouse blood and peritoneal fluid, compared with Sham, ## P < 0.01; **P < 0.01 compared with Mod.

[0155] 2.2 Effect of Selaginellin A on TNF-α Levels in Mouse Peritoneal Fluid

[0156] Compared with the sham operation group, the TNF-α level in the peritoneal fluid of the model group mice was significantly increased (P<0.05), and different doses of Selaginellin A significantly reduced the TNF-α level in the peritoneal fluid (P<0.05) in a dose-dependent manner.

[0157] Figure 8 The effect of Selaginellin A on the level of TNF-α in the peritoneal fluid of mice was compared with Sham. # P<0.05; compared with Mod, * P<0.05, ** P<0.01, *** P<0.01.

[0158] 2.3 Effect of Selaginellin A on the Expression of Ectopic Lesion-Related Proteins in Mice

[0159] Compared with the sham-operated group, the expressions of VEGF and MMP9 in the ectopic lesions of mice in the model group were significantly increased (P<0.05). Selaginellin A could significantly inhibit the expression of VEGF (P<0.01) in a dose-dependent manner. Low and medium doses of Selaginellin A had no significant inhibitory effect on the expression of MMP9 in ectopic lesions (P>0.05), while high doses of Selaginellin A could significantly inhibit the expression of MMP9 (P<0.01). Detection of apoptosis-related proteins revealed that the level of Bcl-2 in the ectopic lesions of mice in the model group was significantly higher than that in the sham-operated group (P<0.05), and different doses of Selaginellin A could inhibit the expression of Bcl-2 in the ectopic lesions (P<0.05), but there was no dose-dependency. The effect of Bax was opposite to that of Bcl-2 and it could promote cell apoptosis. The level of Bax in the lesions of the model group was significantly lower than that in the sham-operated group (P<0.05), and Selaginellin A significantly reversed this trend (P<0.01). Figure 9 The effect of Selaginellin A on the expression of related proteins in mouse ectopic endometrium is shown in Figure 2. Compared with Sham, # P<0.05, ## P<0.01, ### P < 0.001; compared with Mod, *P < 0.05, **P < 0.01, ***P < 0.01.

[0160] 2.4 Regulatory effect of selaginellin A on inflammatory immune-related proteins in mouse serum

[0161] Compared with the sham operation group, 29 proteins were significantly downregulated in the serum of endometriosis mice, of which 11 proteins, including rolactin, Soggy-1, CXCR2, VEGF-B, DKK-1, TGF beta R2, Ubiquitin+1, IFN-gammaR1, TIMP-4, SAA and CXCR6, were restored by low-dose Selaginellin A, while 25 of them were restored by high-dose Selaginellin A. 237 proteins were significantly upregulated in the serum of endometriosis mice, of which 182 proteins were restored by low-dose Selaginellin A, while 8 of them were restored by high-dose Selaginellin A. KEGG analysis of drug-induced callback proteins mainly involved chemokine signaling pathway (mmu04062: Chemokine signaling pathway), interleukin 17 signaling pathway (mmu04657: IL-17signaling pathway), PI3K-Akt signaling pathway (mmu04151: P13K-Akt signaling pathway), TNF signaling pathway (mmu04668: TNF signaling pathway), NF-κB signaling pathway (mmu04064: NF-kappa Bsignaling pathway) and Th17 cell differentiation (mmu04659: Th17 cell differentiation), which are related to immunity and inflammation.

[0162] Example 5

[0163] This example is a study on the mechanism by which Selaginella tin A inhibits the proliferation and migration of Ishikawa cells.

[0164] 1. Methods

[0165] 1.1 Cell culture

[0166] The cells were cultured in RPMI1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were cultured at 37°C in a 5% CO2 incubator. The medium was changed every two days by discarding the existing medium from the culture flask, washing the cells with PBS, and adding 10 mL of culture medium. The cells were passaged when they reached approximately 80% confluency.

[0167] 1.2 MTT assay to detect cell proliferation

[0168] 1.2.1 MTT assay to detect the effect of Selaginellin A on Ishikawa cell proliferation

[0169] Ishikawa cells in the logarithmic phase with good growth status were taken and cultured at 4×10 3 Cells were seeded into 96-well plates at 100 μL / well and cultured in a 37°C, 5% CO2 incubator for 24 hours, after which the culture medium was discarded. A control group (100 μL complete culture medium) and a selaginellin A group (100 μL culture medium containing 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, 75 μM, and 100 μM Selaginellin A, respectively) were set up. After 24 and 48 hours of culture, the culture medium was discarded, and 100 μL of complete culture medium containing 10% MTT was added to each well. The plates were incubated in an incubator for 4 hours, then removed and the culture medium discarded. 150 μL of DMSO solution was added to each well. After shaking in the dark for 10 minutes, the absorbance of each well was measured at 490 nm using a microplate reader.

[0170] 1.2.2 Effect of β-estradiol on Ishikawa cell proliferation

[0171] The cells were 4×10 3 Cells were seeded into 96-well plates and cultured in a 37°C, 5% CO2 incubator for 24 hours, after which the culture medium was discarded. A control group (100 μL complete culture medium) and a β-estradiol group (100 μL culture medium containing 10 nM, 50 nM, 100 nM, 500 nM, 1000 nM, 1500 nM, or 2000 nM β-estradiol) were set up. After 24 and 48 hours of culture, the culture medium was discarded, and 100 μL complete culture medium containing 10% MTT was added to each well. The plates were incubated in an incubator for 4 hours, then removed and the culture medium discarded. 150 μL of DMSO solution was added to each well. After shaking in the dark for 10 minutes, the absorbance OD value of each well was measured at 490 mm on a microplate reader.

[0172] 1.2.3 Effect of Selaginellin A on β-estradiol-induced Ishikawa cell proliferation

[0173] The cells were 4×10 3Cells were seeded into 96-well plates at 37°C, 5% CO₂ incubator for 24 hours, after which the culture medium was discarded. A control group (Control) was treated with 100 μL of complete culture medium, while a model group (Model) was treated with β-estradiol (50 nM) and a β-estradiol + selaginellin A group (50 nM β-estradiol at 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, 75 μM, and 100 μM selaginellin A, respectively) were treated. After 24 and 48 hours of incubation, the culture medium was discarded, and 100 μL of complete culture medium containing 10% MTT was added to each well. The plates were incubated for 4 hours, removed from the incubator, and the culture medium discarded. 150 μL of DMSO solution was added to each well. After shaking in the dark for 10 minutes, the OD value of each well was measured at 490 nm using a microplate reader.

[0174] 1.3 Cell scratch assay

[0175] Set up a control group, a β-estradiol group (100nM), and a β-estradiol + Selaginellin A group (10μM, 20μM, and 40μM Selaginellin A containing 100nM β-estradiol were added, respectively). Before plating, use a marker to draw a line every 0.5-1cm on the back of the 6-well plate, ensuring that there are 5 lines passing through each well. Inoculate 4×10 cells per well. 5 Cells were cultured in a 37°C, 5% CO2 incubator for 24 hours, the culture medium was discarded, and scratches were made perpendicular to the marked line using a 10 μL sterile pipette tip. The cells were then washed 2-3 times with PBS and immediately photographed under an inverted phase-contrast microscope (0 h). The scratch width at 0 h was used as the scratch width. Subsequently, photographs were taken at 24 and 48 h. The scratch width was calculated using Image-Pro Plus 6.0 software: cell migration distance = 0 h distance - 12 h distance / 24 h distance.

[0176] 1.4 Flow cytometry detection of cell apoptosis

[0177] 48 hours after administration, cells were collected together with cells that were not firmly attached (cells suspended in the culture medium), washed with 2 mL of PBS buffer, and digested at 37°C for 5 min with 1 mL of 0.25% trypsin without EDTA. The cells were suspended and centrifuged at 1000 g for 5 min, and the supernatant was discarded. The cells were resuspended in 2 mL of PBS, 200 μL of the cell suspension was transferred to a 1.5 mL centrifuge tube, centrifuged at 1000 g for 5 min, and the supernatant was discarded. The cells were resuspended in 195 μL of V-FITC conjugate solution, 5 μL of V-FITC and 10 μL of PI were added, and the cells were incubated at room temperature in the dark for 15 min and detected by the microscope.

[0178] 1.5 Western blotting experiment

[0179] 4×10 5 Seed cells at a density of 100 μL / well and culture at 37°C for 24 hours. Discard the culture medium and culture for 48 hours after dosing. Discard the culture medium and wash three times with 4°C pre-chilled PBS. Aspirate the PBS thoroughly and place the dish on ice. Add 100 μL / well of the prepared protein lysis buffer. Lyse on ice for 30 minutes. Scrape the cells with a scraper and transfer them to a 1.5 mL centrifuge tube. Centrifuge at 12,000 rpm at 4°C for 15 minutes. Collect the supernatant.

[0180] Subsequent protein quantification and other operations were the same as those in 1.3 of Example 4.

[0181] 1.6 Data processing and analysis

[0182] The same operation as 1.4.6 of Example 4 is performed.

[0183] 2. Results

[0184] 2.1Selaginellin A can significantly inhibit β-estradiol-induced Ishikawa cell proliferation

[0185] like Figure 10 As shown in (A), different concentrations of Selaginellin A can inhibit the proliferation of Ishikawa cells. 1μM Selaginellin A showed a significant inhibitory effect after 24 hours of administration, and the effect became more pronounced with increasing doses, and even more pronounced with prolonged exposure to 48 hours. EMs are estrogen-dependent diseases, and β-estradiol promotes the proliferation of Ishikawa cells. Figure 10 As shown in (B), at 24 h, low-dose β-estradiol had no obvious effect on the proliferation of Ishikawa cells, and only 1500 nM and 2000 nM β-estradiol could significantly promote the proliferation of Ishikawa cells; at 48 h, β-estradiol had a more obvious effect on the proliferation of Ishikawa cells, and was dose-dependent.

[0186] Ishikawa cells were treated with 50 nM β-estradiol and then treated with different concentrations of Selaginellin A for 24 h and 48 h. Figure 10 As shown in (C), different doses of Selaginellin A can inhibit cell proliferation induced by β-estradiol, and the intensity of the effect increases with increasing drug dose and action time.

[0187] Figure 10 The effect of Selaginellin A on the proliferation of Ihsikawa cells is compared with the control. ### P<0.001; compared with β-estradiol, *P<0.05, **P<0.01, ***P<0.01.

[0188] 2.2 Selaginellin A inhibits β-estradiol-induced Ishikawa cell migration

[0189] The cell scratch test was used to investigate whether Selaginellin A could inhibit β-estradiol-induced Ishikawa cell migration. The experimental results showed that ( Figure 11 ): Compared with the control, 100nMβ-estradiol could significantly promote the migration of Ishikawa cells at 24h (P<0.01), with a migration rate of 22.97%±2.80%. The migration promoting effect became more significant with the passage of time (P<0.001), and the migration rate reached 36.51%±5.04% at 48h. Compared with 100nMβ-estradiol, different concentrations of Selaginellin A could significantly inhibit the migration of Ishikawa cells. At 24h, the cell migration rates induced by β-estradiol of 10μM, 20μM and 40μM Se-A were 9.03%±0.5%, 5.25%±0.71% and 5.64%±2.14%, respectively; the migration rates at 48h were 13.21%±1.18%, 6.10%±1.03% and 8.04%±1.95%.

[0190] 2.3 Selaginellin A induces apoptosis in Ishikawa cells

[0191] Annexin V-FITC apoptosis detection kit was used to detect the effect of Selaginellin A on apoptosis of Ishikawa cells. The results showed that ( Figure 12 ): After 48 hours of β-estradiol treatment, the apoptosis rate of Ishikawa cells decreased to 12.45±1.07, which was significantly lower than that of the Control group (20.45±4.01). After administration of different doses of Selaginellin A, the cell apoptosis rate was significantly increased compared with the β-estradiol group (P<0.001), which were 26.57±3.3, 32.14±4.12 and 29.92±2.90, respectively.

[0192] 2.4 Effect of Selaginellin A on the Expression of Related Proteins in Ishikawa Cells

[0193] like Figure 13 As shown in the results, β-estradiol significantly increased the expression of VEGF and MMP9 in Ishikawa cells (P<0.001), and Selaginellin A significantly inhibited the expression of VEGF (P<0.05) in a dose-dependent manner; low-dose and medium-dose Selaginellin A had no significant inhibitory effect on the expression of MMP9 in ectopic lesions (P>0.05), while high-dose Selaginellin A significantly inhibited the expression of MMP9 (P<0.01). Apoptosis-related proteins were detected in Ishikawa cells treated with β-estradiol. The expression level of Bcl-2 was significantly increased in Ishikawa cells treated with β-estradiol compared with that in untreated cells (P<0.05). Different doses of Selaginellin A inhibited Bcl-2 expression in a dose-dependent manner (P<0.001). Cleaved caspase-3, which has the opposite effect of Bcl-2, can promote cell apoptosis. The level of cleaved caspase-3 in Mod lesions was significantly lower than that in Sham (P<0.01). Low-dose Selaginellin A significantly reversed this trend (P<0.05), and the reversal effect became more pronounced with increasing doses of Selaginellin A (P<0.001).

[0194] Example 6

[0195] Preparation of tablets: Take the Selaginella viniferin A prepared in Example 2 above, add appropriate amounts of pharmaceutical excipients such as starch and magnesium stearate, mix thoroughly, and press into tablets to prepare tablets for oral use.

[0196] Example 7

[0197] Preparation of capsules: Take the Selaginella vinifera A prepared in Example 2 above, add an appropriate amount of starch as a pharmaceutical excipient, mix thoroughly, and put into capsules to prepare capsules for oral use.

[0198] The above describes the implementation mode of the present invention in detail with reference to the embodiments. However, the present invention is not limited to the above implementation mode. After knowing the contents described in the present invention, ordinary technicians in this technical field can make several equivalent transformations and substitutions without departing from the principles of the present invention. These equivalent transformations and substitutions should also be regarded as falling within the scope of protection of the present invention.

Claims

1. Application of Selaginella A in the preparation of a drug for treating endometriosis, wherein the molecular formula of Selaginella A is C 33 H 22 O4, the structural formula is: .

2. The use of Selaginellarin A according to claim 1 in the preparation of a drug for treating endometriosis, wherein: The preparation method of Selaginella tin A comprises the following steps: S1. Extract the Chinese herbal medicine Selaginella by adding 70% or higher alcohol at a mass volume ratio of 1:8-12 for 3-8 times; filter, and concentrate the filtrate under reduced pressure to obtain an extract for later use; S2 dry sample was loaded on a 100 mesh ~ 200 mesh silica gel column chromatography, and eluted with a volume ratio of 100:0~90:10 chloroform: methanol gradient, and different fractions were collected; S3. The chloroform volume ratio of 95:5 was collected: methanol fractions were dry loaded on a 200-300 mesh silica gel column for separation, and then gradient eluted with chloroform volume ratios of 98:2 and 95:5: methanol to collect different fractions; S4. The target component is tracked by thin-layer chromatography, and the fractions containing the target component are combined and purified by repeated Sephadex LH-20 column chromatography using aqueous methanol as the elution solvent.

3. The use of Selaginella tin A according to claim 2 in the preparation of a drug for treating endometriosis, characterized in that: The Chinese medicinal herb Selaginella is the dried whole herb of Selaginella truncatula of the Selaginellaceae family. After obtaining the extract in step S1, it is washed three times with twice the weight of petroleum ether, and then filtered. The petroleum ether is evaporated from the filter residue for later use.

4. The use of Selaginellarin A according to claim 2 in the preparation of a drug for treating endometriosis, characterized in that: The Selaginella is the dried whole herb of Selaginella pulvinata of the Selaginellaceae family.

5. Use of Selaginellarin A according to claim 3 or 4 in the preparation of a drug for treating endometriosis, characterized in that: The volume of alcohol used in step S1 is 10 times the mass of the Chinese medicine Selaginella, and the alcohol concentration is 75%.

6. Use of Selaginellarin A according to claim 5 in the preparation of a medicament for treating endometriosis, characterized in that: The concentration of methanol in step S4 is 70%.

Images