Sargentodoxa cuneata-dahurian patrinia herb extract as well as pharmaceutical composition and application thereof

Abstract

The invention discloses a sargentgloryvine stem-dahurian patrinia herb extract as well as a pharmaceutical composition and application thereof. The extract is extracted by a specific method, has specific active components, can significantly improve inflammatory reaction through various mechanisms, and has an application prospect as a drug for treating pelvic inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to an extract of Sargentodoxa cuneata - Patrinia scabiosaefolia and its pharmaceutical composition and application, and particularly to an ethyl acetate extract of Sargentodoxa cuneata - Patrinia scabiosaefolia and its pharmaceutical composition and application. Background Art

[0002] Pelvic inflammatory disease refers to infectious diseases of the female upper reproductive tract, including inflammation of the internal genital organs and their surrounding connective tissues and pelvic peritoneum. If it lingers without healing, it can lead to diseases such as infertility, ectopic pregnancy, and chronic pelvic pain, seriously affecting women's reproductive health. According to different clinical symptoms, signs, and pathogenesis, pelvic inflammatory disease can be classified into the categories of "menstrual disorders", "leucorrhea disorders", "abdominal pain in women", and "infertility" and other disease syndromes. Traditional Chinese medicine believes that pelvic inflammatory disease is mainly caused by damp - heat pouring downward, so the treatment principle is to clear heat and remove dampness, and promote blood circulation to remove stasis. Western medicine believes that pelvic inflammatory disease is mainly caused by infection, and antibiotics are often used for clinical treatment mainly for anti - infection, resulting in side effects such as drug resistance and repeated attacks. Therefore, the anti - inflammatory research of traditional Chinese medicine has certain value.

[0003] Both International Immunopharmacology 120(2023)110337 and Journal of Ethnopharmacology 318(2024)116855 have disclosed that the alcohol extract of Sargentodoxa cuneata - Patrinia scabiosaefolia has the activity of treating pelvic inflammatory disease. However, the components of the alcohol extract are complex and the curative effect is limited, and it still cannot meet the clinical treatment needs. Summary of the Invention

[0004] Object of the Invention: The present invention aims to provide an ethyl acetate extract of Sargentodoxa cuneata - Patrinia scabiosaefolia with excellent activity, its pharmaceutical composition and application.

[0005] Technical Solution: The preparation method of the extract of Sargentodoxa cuneata - Patrinia scabiosaefolia according to the present invention includes the following steps:

[0006] (1) Mix Sargentodoxa cuneata and Patrinia scabiosaefolia in a mass ratio of 1:1, extract twice with 50% V / V ethanol, combine the filtrates, remove the solvent, and dry to obtain a dry extract.

[0007] (2) Dissolve the extract prepared in step (1) in water, then extract three times with ethyl acetate, combine the ethyl acetate phases, remove the solvent, and dry to obtain the extract of Sargentodoxa cuneata - Patrinia scabiosaefolia.

[0008] The present invention further separates different extracts from the alcohol extract of Sargentodoxa cuneata - Patrinia scabiosaefolia. Among them, the ethyl acetate extract is rich in flavonoid and phenolic acid active ingredients. Compared with the alcohol extract and other extracts, its curative effect on pelvic inflammatory disease is significantly improved.

[0009] Preferably, in the Sargentodoxa cuneata - Patrinia scabiosaefolia extract, the abundance values of vanillic acid, chrysophanol, salidroside, emodin, luteolin, kaempferol, apigenin, acacetin, procyanidin B1, epicatechin, luteoloside, isoquercitrin, madecassic acid, sinapic acid, caffeic acid, asiatic acid, acteoside, naringenin, cirsimaritin, cynarin, iristectorigenin, isorhamnetin, genkwanin, genistein, physcion, quercetin, genistin, curcumol, diosgenin, scutellarein, formononetin, tyrosol, quercitrin are greater than 0.5.

[0010] More preferably, in the Sargentodoxa cuneata - Patrinia scabiosaefolia extract, the abundance values of vanillic acid, chrysophanol, salidroside, emodin, luteolin, kaempferol, apigenin, acacetin, procyanidin B1, epicatechin, luteoloside, isoquercitrin, madecassic acid, sinapic acid, caffeic acid, asiatic acid, acteoside, naringenin, cirsimaritin, cynarin, iristectorigenin, isorhamnetin, genkwanin, genistein, physcion, quercetin, genistin, curcumol, diosgenin, scutellarein, formononetin, tyrosol, quercitrin are greater than 1.

[0011] Preferably, in the 2 μg / mL Sargentodoxa cuneata - Patrinia scabiosaefolia extract solution, it contains 100 - 150 ng / mL of salidroside, 30 - 60 ng / mL of kaempferol, 50 - 100 ng / mL of quercitrin, 50 - 100 ng / mL of epicatechin and 40 - 80 ng / mL of luteoloside.

[0012] More preferably, in the 2 μg / mL Sargentodoxa cuneata - Patrinia scabiosaefolia extract solution, it contains 120 - 140 ng / mL of salidroside, 40 - 50 ng / mL of kaempferol, 60 - 90 ng / mL of quercitrin, 80 - 90 ng / mL of epicatechin and 50 - 60 ng / mL of luteoloside.

[0013] Even more preferably, in the 2 μg / mL Sargentodoxa cuneata - Patrinia scabiosaefolia extract solution, it contains 130 - 135 ng / mL of salidroside, 40 - 45 ng / mL of kaempferol, 70 - 75 ng / mL of quercitrin, 85 - 90 ng / mL of epicatechin and 55 - 60 ng / mL of luteoloside.

[0014] Still more preferably, in the 2 μg / mL Sargentodoxa cuneata - Patrinia scabiosaefolia extract solution, it contains 133.79 ng / mL of salidroside, 42.22 ng / mL of kaempferol, 73.46 ng / mL of quercitrin, 88.18 ng / mL of epicatechin and 57.22 ng / mL of luteoloside.

[0015] Preferably, in step (2) of the preparation method of the Sargentodoxa cuneata - Patrinia villosa extract, the volume ratio of the amount of ethyl acetate to the amount of water each time is successively (1 - 1.5):1, (1 - 1.2):1, and (0.8 - 1):1.

[0016] More preferably, in step (2) of the preparation method of the Sargentodoxa cuneata - Patrinia villosa extract, the volume ratio of the amount of ethyl acetate each time is 1.2:1:0.8.

[0017] Preferably, in step (1) of the preparation method of the Sargentodoxa cuneata - Patrinia villosa extract, the total material - to - liquid ratio for extraction is 1:5 m / V.

[0018] More preferably, in step (1) of the preparation method of the Sargentodoxa cuneata - Patrinia villosa extract, the volume ratio of the amount of 50% V / V ethanol each time is 4:1.

[0019] The pharmaceutical composition of the present invention comprises the Sargentodoxa cuneata - Patrinia villosa extract of the present invention and a pharmaceutically acceptable carrier.

[0020] Preferably, the pharmaceutically acceptable carrier can be an excipient widely used in the field of drug production. Excipients are mainly used to provide a safe, stable, and functional pharmaceutical composition, enabling the active ingredient to dissolve at a desired rate after the subject receives the administration, or promoting the effective absorption of the active ingredient after the subject receives the composition administration. The pharmaceutical excipient can be an inert filler or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipient can include one or more of the following excipients: binder, suspending agent, emulsifier, diluent, filler, granulating agent, adhesive, disintegrant, lubricant, anti - sticking agent, glidant, wetting agent, gelling agent, absorption retardant, dissolution inhibitor, enhancer, adsorbent, buffer, chelating agent, preservative, colorant, flavoring agent, sweetening agent.

[0021] The pharmaceutical composition of the present invention can be prepared by any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, freeze - drying.

[0022] The pharmaceutical composition of the present invention can be administered in any form, including mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical, parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra - arterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be in a controlled - release or sustained - release dosage form (such as liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, hard capsules, cachets, soft capsules, tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops. Examples of preparations for parenteral administration include, but are not limited to, injection solutions, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, injection suspensions, injection emulsions. Examples of other dosage forms include, but are not limited to, eye drops, other ophthalmic preparations; aerosols, such as nasal sprays, inhalants; suppositories, lozenges suitable for parenteral administration.

[0023] The Sargentodoxa cuneata - Patrinia villosa extract or its pharmaceutical composition of the present invention is used in the preparation of a drug for treating pelvic inflammatory disease.

[0024] Preferably, the drug is a drug for treating pelvic inflammatory disease with damp - heat stasis - binding syndrome.

[0025] Preferably, the drug is a drug for treating endometritis, salpingitis, tubo - ovarian inflammation, tubo - ovarian abscess or pelvic peritonitis.

[0026] Preferably, the drug can improve uterine tissue damage, reduce inflammatory cell infiltration, and promote the recovery of uterine tissue.

[0027] Preferably, the drug can inhibit the proliferation of macrophages in uterine tissue.

[0028] More preferably, the drug can inhibit the proliferation of macrophages in uterine tissue in a concentration - dependent manner.

[0029] Preferably, the drug can inhibit the release of NO and the expression of iNOS in macrophages of uterine tissue.

[0030] More preferably, the drug can inhibit the release of NO and the expression of iNOS in macrophages of uterine tissue in a concentration - dependent manner.

[0031] Preferably, the drug can improve uterine tissue swelling and congestion.

[0032] Preferably, the drug can down - regulate uterine coefficient and spleen index.

[0033] Preferably, the drug can improve uterine tissue glandular hyperplasia, glandular fusion, vascular hyperplasia, and inflammatory cell infiltration.

[0034] Preferably, the drug can down-regulate the expression of F4 / 80 and CD86 in uterine tissue and promote the expression of CD206 in uterine tissue.

[0035] Preferably, the drug can inhibit the M1 polarization of macrophages in uterine tissue and promote the M2 polarization of macrophages in uterine tissue.

[0036] Preferably, the drug can inhibit the expression of glycolysis-related proteins in uterine tissue.

[0037] More preferably, the drug can inhibit the expression of hexokinase II, hexokinase I, pyruvate kinase M2, and pyruvate dehydrogenase in uterine tissue.

[0038] Even more preferably, the drug can inhibit the expression of hexokinase II in uterine tissue.

[0039] Preferably, the drug can inhibit the NF-κB signaling pathway and the phosphorylation of IκBα.

[0040] Preferably, the drug can have a synergistic effect with 2-deoxy-D-glucose.

[0041] More preferably, the dosage ratio of the drug to 2-deoxy-D-glucose is 1:2.

[0042] More preferably, the administration method of the drug and 2-deoxy-D-glucose is selected from simultaneous administration or sequential administration.

[0043] Even more preferably, when the drug and 2-deoxy-D-glucose are administered sequentially, there is no requirement for the administration order.

[0044] Preferably, based on the active ingredient, the administration dosage of the drug is 50-200 μg / mL, more preferably 50-100 μg / mL, and even more preferably 100 μg / mL.

[0045] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0046] In the present invention, a specific ethyl acetate extract of Sargentodoxa cuneata and Patrinia scabiosaefolia is obtained by extraction, and the active ingredients are retained to the greatest extent. The inflammatory response is significantly improved through various mechanisms such as improving uterine tissue damage, reducing inflammatory cell infiltration, and promoting tissue recovery, and the curative effect of the ethyl acetate extract on pelvic inflammatory disease is enhanced. Compared with the existing alcohol extracts, the in vitro and in vivo curative effects are better, and it has the application prospect as a drug for treating pelvic inflammatory disease. Description of the Drawings

[0047] Figure 1Component analysis results of different extracts; A. Negative ion mode diagram; B. Positive ion mode diagram; C. Heat map of differences in component abundance values in different extracts;

[0048] Figure 2 Effects of different parts of Sargentodoxa cuneata - Patrinia scabiosaefolia on the proliferation of macrophages and LPS - induced macrophages; A - C. Detection of the effects of different parts of Sargentodoxa cuneata - Patrinia scabiosaefolia on the proliferation of macrophage RAW264.7 by CCK8 method; D - F. Detection of the effects of different parts of Sargentodoxa cuneata - Patrinia scabiosaefolia on the proliferation of LPS - induced macrophage RAW264.7 by CCK8 method; # Compared with the Blank group, # P < 0.05, ## P < 0.01, ### P < 0.001; * Compared with the DMSO or LPS group, *P < 0.05, **P < 0.01, ***P < 0.001;

[0049] Figure 3 Effects of different parts of Sargentodoxa cuneata - Patrinia scabiosaefolia on LPS - induced NO release and iNOS expression in macrophages; A - C. Effects of different parts of Sargentodoxa cuneata - Patrinia scabiosaefolia on LPS - induced NO release in macrophage RAW264.7; # Compared with the Blank group, # P < 0.05, ## P < 0.01, ### P < 0.001; * Compared with the LPS group, *P < 0.05, **P < 0.01, ***P < 0.001; D. Detection of the effects of EtOAc and S&P on LPS - induced iNOS expression in macrophage RAW264.7 by Western Blot; # Compared with the S&P group, *P < 0.05, **P < 0.01, ***P < 0.001;

[0050] Figure 4Effect of EtOAc on LPS-induced PID mice; A. Construction process of mouse PID model; B. Effect of EtOAc on body weight of LPS-induced PID mice; C. Effect of EtOAc on uterine tissue morphology of LPS-induced PID mice; D-E. Effect of EtOAc on uterine and spleen coefficients of LPS-induced PID mice; F. Detection of the effect of EtOAc on uterine histopathology of LPS-induced PID mice by HE staining; Red arrows represent plasma cells, yellow arrows represent neutrophils, and blue arrows represent lymphocytes; G-J. Detection of the effect of EtOAc on the expression of macrophage phenotype markers F4 / 80, CD86, and CD206 in uterine tissue of LPS-induced PID mice by immunohistochemistry; Compared with the S&P group, *P < 0.05, **P < 0.01, ***P < 0.001;

[0051] Figure 5 Effect of EtOAc on proteins related to glycolysis and inflammatory pathways in LPS-induced macrophages RAW264.7; A-E. Detection of the effect of EtOAc on glycolysis-related proteins in LPS-induced macrophages RAW264.7 by Western Blot; F-J. Detection of the effect of EtOAc and HK-II inhibitor 2-DG on glycolysis-related proteins in LPS-induced macrophages RAW264.7 by Western Blot; K-M. Detection of the effect of EtOAc and HK-II inhibitor 2-DG on glycolysis-related proteins in LPS-induced macrophages RAW264.7 by Western Blot; # Compared with the Blank group, # P < 0.05, ## P < 0.01, ### P < 0.001; *Compared with the LPS group, *P < 0.05, **P < 0.01, ***P < 0.001. Specific embodiments

[0052] The technical solutions of the present invention will be further described below in conjunction with the embodiments.

[0053] Example 1: Preparation of various extracts of Sargentodoxa cuneata and Patrinia scabiosaefolia

[0054] (1) Preparation of ethanol extract

[0055] After mixing 50 g of Sargentodoxa cuneata and 50 g of Patrinia scabiosaefolia, they were pulverized in a pulverizer to obtain traditional Chinese medicine powder. Then, 400 mL of ethanol with a concentration of 50% V / V was added and boiled, followed by heating with a gentle fire for 2 h and filtering with gauze. Then, 100 mL of 50% V / V ethanol was added to the medicinal residues, heated to boiling, and heated with a gentle fire for 1 h, followed by filtration. The filtrates from the two times were combined, and then concentrated on a rotary evaporator to form an extract, and finally dried in an oven (58 °C) to obtain a traditional Chinese medicine extract.

[0056] (2) Preparation of ethyl acetate extract

[0057] The traditional Chinese medicine extract prepared in (1) was dissolved in 50 mL of pure water, and then 60 mL of ethyl acetate was added. The mixture was placed in a separating funnel, inverted and mixed repeatedly, and left to stand. After significant stratification, the upper layer was poured out. Then, 50 mL of ethyl acetate was added, and the above steps were repeated. Finally, 40 mL of ethyl acetate was added, and the above steps were continued. The filtrates from the three times were mixed and dried in an oven (58 °C) to obtain the ethyl acetate extract of Sargentodoxa cuneata - Patrinia scabiosaefolia, which was stored in a 4 °C refrigerator.

[0058] (3) Preparation of other extracts

[0059] The ethyl acetate in (2) was replaced with petroleum ether and n - butanol respectively to obtain petroleum ether and n - butanol extracts. The separated aqueous phase was used as the aqueous phase extract.

[0060] Example 2: Component analysis of various extracts of Sargentodoxa cuneata - Patrinia scabiosaefolia

[0061] (1) Experimental method

[0062] 1) Sample treatment

[0063] The weighed sample was dissolved in chromatographic methanol and diluted to 2 μg / mL. Centrifugation was carried out at 4 °C and 12000 rpm for 10 min. The supernatant was transferred to a UHPLC vial for LC - MS / MS analysis.

[0064] 2) LC - MS / MS analysis

[0065] i) Chromatographic conditions

[0066] The sample was analyzed by SCIEX ExionLC ultra - high performance liquid chromatography (UHPLC) system, Phenomenex with a SecurityGuard UHPLC C18 column Chromatographic separation was carried out on a C18 column (100×2.1 mm, 2.6 μm), and the column temperature was maintained at 40 °C. A mobile phase system composed of 0.1% formic acid in water (V / V, solvent A) and acetonitrile (solvent B) was used for gradient elution, and the gradient elution procedure was shown in Table 1. The flow rate was 0.3 mL / min, and the injection volume was 2 μL. During the whole analysis process, the temperature of the autosampler was maintained at 4 °C.

[0067] Table 1 Gradient elution procedure

[0068]

[0069] ii) Q-TOF mass spectrometry analysis

[0070] A hybrid quadrupole time-of-flight mass spectrometer equipped with a Duospray ion source was used to collect data in the ESI positive (+) / negative (-) ion modes respectively. The ESI source conditions were set as follows: nitrogen was used as the nebulizer and auxiliary gas, the nebulizer gas 1 (GS1) was 55 psi; the auxiliary gas (GS2) was 55 psi; the curtain gas was 35 psi; the ion source temperature was 550 °C; the spray voltage was 5500 V (+) / -4500 V (-). In the TOFMS-IDA-MS / MS acquisition, the TOFMS spectral mass scanning range was 100 - 1300 m / z, and the accumulation time was set to 0.1 s / spectrum; the product ion scanning mass range was 50 - 1300 m / z, and the accumulation time was set to 0.05 s / spectrum. The information-dependent acquisition (IDA) was used and the high-sensitivity mode was selected for product ion scanning. To obtain better spectral quality, the parameters of the IDA mode were set as follows: the declustering voltage was 60 V (+) / -60 V (-), the collision energy was 40 ± 20 V (+) / -40 ± 20 V (-), isotopes within 4 Da were excluded, the ion tolerance was 50 mDa, and 10 candidate ions were monitored in each cycle. The dynamic background subtraction (DBS) function was turned on to improve the sensitivity of low-abundance or trace analytes. An external calibration system (CDS) was used to perform automatic calibration of TOFMS and TOFMS / MS every 5 samples.

[0071] iii) Component identification

[0072] The original LC-MS data files were converted to the open mzXML format using MSCon-vert (ProteoWizard, version 3.0.23114). The converted files were subjected to peak detection, alignment, and deconvolution using MS-DIAL software (version 4.9.221218). MS-DIAL parameter settings included a minimum peak height of 1,500 counts for MS1 data; an MS2 tolerance of 0.05 Da; and a retention time tolerance of 0.3 minutes. A multi-faceted approach was used for compound identification: the built-in libraries and algorithms in MS-DIAL were used for preliminary compound annotation. MS1 and MS2 spectra were matched against multiple databases including MassBank, GNPS, and RikenMSP, as well as a self-built library. Preliminary identification required a spectral similarity score of at least 70%. In silico fragmentation tools including CFM-ID 4.0 were used to predict the MS2 spectra of compounds not found in the spectral library. The predicted spectra were compared with the experimental data to assist in structure elucidation. Compounds of particular interest that could not be reliably identified by the automated method were subjected to manual interpretation of the MS2 spectra.

[0073] (2) Experimental results

[0074] Table 2 Components of different extraction fractions and S&P

[0075]

[0076]

[0077] As shown in Table 2, a total of 58 components were identified. The ethyl acetate extract (EtOAc) had the largest peak area, followed by the ethanol extract (S&P) ( Figure 1 A-B). Most components in the ethyl acetate extract (EtOAc) had the highest abundance values, including genkwanin, formononetin, quercitrin, isoquercitrin, asiatic acid, acteoside, cynarin, naringenin, cirsimaritin, luteolin, luteoloside, genistein, emodin, acacetin, tectorigenin, isorhamnetin, hydroxyasiatic acid, sinapic acid, procyanidin B1, vanillic acid, caffeic acid, salidroside, kaempferol, tyrosol, curcumol, epicatechin, chrysophanol, apigenin, physcion, diosgenin, genistin, and scutellarin. It is speculated that these bioactive components contribute to the optimal anti-inflammatory effect of EtOAc. Followed by the ethanol extract (S&P), including shikimic acid, trigonelline, tormentic acid, wogonin, eleutheroside E, cryptochlorogenic acid, isoorientin, syringic acid, isokaempferide, protocatechuic acid, procyanidin B2, procyanidin C1, catechin, pinocembrin, syringaresinol, and quercetin. The components in the n-butanol extract (n-BuOH) and the aqueous extract (Water) had the lowest abundance values ( Figure 3 C).

[0078] Example 3: Evaluation of cell proliferation

[0079] (1) Experimental method

[0080] RAW264.7 cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 1×10 6 cells / mL, with 100 μL of cell suspension in each well. Three replicate wells were set up for each group. After the cells adhered, they were stimulated with LPS (1 μg / mL) and three different parts (EtOAc, n-BuOH, and Water) at different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL) for 24 h. Then, 10 μL of CCK8 solution was added to each well, and after incubation in an incubator for 1 h, the OD values of each well were measured using an enzyme-linked immunosorbent assay (ELISA) reader at a detection wavelength of 450 nm.

[0081] (2) Experimental results

[0082] Considering that the amount of the petroleum ether fraction was extremely small, this fraction was excluded from subsequent experiments. The CCK8 assay was used to detect the effect of different parts of Sargentodoxa cuneata - Patrinia scabiosaefolia on the proliferation of macrophages RAW264.7 and LPS-induced macrophages RAW264.7 after 24 h. Compared with the DMSO group, the low-concentration groups of EtOAc (50 μg / mL and 100 μg / mL) and n-BuOH (50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL) promoted the proliferation of RAW264.7 cells. The high-concentration groups of EtOAc (200 μg / mL, 400 μg / mL, and 800 μg / mL) and n-BuOH (800 μg / mL) significantly inhibited the proliferation of RAW.264.7 cells. Water had no significant effect on cell proliferation ( Figure 2 A-C). Compared with the LPS group, the high-concentration groups of EtOAc (400 μg / mL and 800 μg / mL) significantly inhibited the proliferation of LPS-induced RAW264.7 cells. The low-concentration groups of EtOAc (50 μg / mL, 100 μg / mL, and 200 μg / mL) had no significant effect on cell proliferation. n-BuOH and water had no obvious effect on cell proliferation ( Figure 2 D-F). Considering the cytotoxic effect, three concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL were used in subsequent experiments.

[0083] Example 4: Evaluation of nitrite and iNOS

[0084] (1) Experimental method

[0085] RAW264.7 cells were seeded at a density of 1×10 6Cells were seeded at a density of 1×10 cells / mL in a 96-well plate, with 100 μL of cell suspension in each well. Three replicate wells were set up for each group. After the cells adhered, they were stimulated with LPS (1 μg / mL) and three drugs (EtOAc, n-BuOH, Water) at different concentrations (50 μg / mL, 100 μg / mL, and 200 μg / mL) for 24 h. The cell supernatants were collected, and the standard NaNO was diluted according to the manufacturer's protocol. 2 50 μL of the cell supernatant and the diluted standard were added to a 96-well plate, and then 50 μL of Griess Reagent I and Griess Reagent II were added to each well. Three replicate wells were set up for each group. The plate was incubated at room temperature for 10 min, and the OD values of each well were measured at a detection wavelength of 540 nm using a microplate reader. The NO values of each group were calculated according to the standard curve.

[0086] RAW264.7 cells were seeded at a density of 1×10 6 cells / mL in a 100-mm culture dish and stimulated with different treatments for 24 h. The cells were scraped off with a cell scraper, and 100 μL of ice-cold cell lysis buffer was added to each sample. The cells were lysed on ice for 30 min to extract proteins, and then BCA protein quantification was performed. Proteins (30 μg) were separated by 10% SDS-PAGE at 100 V for 90 min, and then transferred to a PVDF membrane. The proteins were incubated with primary antibodies (1:1000) and secondary antibodies (1:2000) at appropriate concentrations, and the bands were visualized using a gel imaging system. The optical density of the bands was analyzed using Image J software.

[0087] (2) Experimental results

[0088] To detect the anti-inflammatory activities of different parts of Sargentodoxa cuneata and Patrinia scabiosaefolia, and to screen out the optimal anti-inflammatory part, the effects of different parts on the release of NO from LPS-induced macrophages RAW264.7 after 24 h were detected using a nitric oxide detection kit. It was found that both EtOAc and n-BuOH could inhibit the LPS-induced NO release in a concentration-dependent manner, and the inhibitory effect of EtOAc was more obvious, while the inhibitory effect of Water was not significant ( Figure 3 A-C). To further verify the anti-inflammatory effect of EtOAc, the effect of EtOAc on the expression of iNOS in LPS-induced macrophages RAW264.7 after 24 h was detected by Western Blot. The results showed that LPS significantly promoted the expression of iNOS, and EtOAc inhibited the LPS-induced expression of iNOS in a concentration-dependent manner, with a better effect than S&P ( Figure 3 D). Since 200 μg / mL of EtOAc also had an inhibitory effect on the cells, two concentrations of 50 μg / mL and 100 μg / mL of EtOAc were selected for subsequent experiments.

[0089] Example 5: Evaluation of the effect of the ethyl acetate extract of Sargentodoxa cuneata - Patrinia scabiosaefolia on LPS - induced pelvic inflammatory disease in mice

[0090] (1) Experimental method

[0091] Thirty mice (n = 30) were randomly divided into 5 groups, namely the control group (Control), the model group (Model), the EtOAc group, the S&P group, and the metronidazole group (MTZ). The mice were anesthetized by intraperitoneal injection of avertin (0.2 mL / 10 g). In the control group, 20 μL of normal saline was injected through the uterine horn, and in the other 4 groups, 20 μL of LPS (5 mg / mL) was injected through the uterine horn. The body weight of each group of mice was recorded daily. One day after modeling, gavage was started, once a day, 200 μL each time. The control group and the model group were gavaged with ultrapure water, and gavage was continued for 14 consecutive days.

[0092] Dosage: According to the conversion of human and animal body surface areas, the equivalent dosage ratio of an adult (70 kg) to a mouse (20 g) is 0.0026. The dosage of raw Sargentodoxa cuneata - Patrinia scabiosaefolia for an adult is 60 g / day. After calculation, the dosage of raw Sargentodoxa cuneata - Patrinia scabiosaefolia for a mouse per day is 7.8 g / kg. According to the extraction rate, the dosage of S&P for a mouse per day is 420 mg / kg, and the dosage of EtOAc is 31.7 mg / kg. The dosage of metronidazole for an adult is 0.8 g / day. According to the calculation of human and animal body surface areas, the dosage of metronidazole for a mouse per day is 104 mg / kg.

[0093] Twenty - four hours after the last administration, samples were taken. Blood was collected from the eyeballs into 1.5 mL EP tubes, left to stand at room temperature for 4 h, centrifuged at 3500 rpm at 4 °C for 15 min, and the supernatant was collected for testing. The mice were sacrificed by cervical dislocation, and uterine tissue, spleen tissue, liver tissue, and kidney tissue were separated, photographed, and weighed.

[0094] H&E staining and immunohistochemical staining were used to evaluate the therapeutic effect of the ethyl acetate extract of Sargentodoxa cuneata - Patrinia scabiosaefolia on PID mice.

[0095] (2) Experimental results

[0096] A mouse PID model was constructed by directly injecting LPS through the uterine horn ( Figure 4 A). The day of modeling was the first day. It was observed that on the second day after modeling, except for the control group, the body weight of the mice decreased significantly. From the third day, the body weights of the mice in the model group and the drug - treated groups increased steadily, and the body weights of the EtOAc, S&P, and MTZ groups increased significantly compared with the model group ( Figure 4 B). After sampling, it was found that compared with the control group, the uterine tissue in the model group was significantly swollen and congested, and the degree of uterine tissue swelling in the EtOAc, S&P, and MTZ groups was significantly reduced ( Figure 4C). Next, the uterine coefficient and spleen coefficient of mice were calculated. Compared with the control group, the uterine coefficient and spleen coefficient in the model group increased; compared with the model group, the uterine coefficient and spleen coefficient in the EtOAc, S&P, and MTZ groups decreased, but there were no significant differences ( Figure 4 D-E). The results of H&E staining showed that the uterine tissue structure in the control group was intact, the glands were scattered, and there was no infiltration of inflammatory cells; compared with the control group, the glands in the model group showed hyperplasia and fusion, blood vessel hyperplasia, and a large number of inflammatory cell infiltrations; compared with the model group, the gland structure in the EtOAc, S&P, and MTZ groups returned to normal, the infiltration of inflammatory cells was significantly reduced, and the infiltration of inflammatory cells in the EtOAc group was less than that in the S&P group. Figure 4 F).

[0097] Through immunohistochemical staining, it was found that compared with the control group, the expression levels of F4 / 80 and CD86 in the uterine tissue of the model group were significantly up-regulated. After treatment with drugs, this trend could be significantly reversed, and the down-regulation effect in the EtOAc group was significantly better than that in the S&P group; the expression levels of CD206 in the uterine tissue of the control group and the model group were comparable. Compared with the model group, the expression of CD206 was significantly promoted after treatment with drugs, and the promoting effect of EtOAc was significantly stronger than that of S&P. Figure 4 G-J).

[0098] The above results indicate that EtOAc can inhibit the M1 polarization of macrophages in uterine tissue and promote M2 polarization, and its effect is better than that of S&P.

[0099] Example 6: Study on anti-inflammatory mechanism

[0100] (1) Experimental method

[0101] RAW264.7 cells were seeded in a 100 mm culture dish at a density of 1×10 6 cells / mL. The cells were stimulated with different treatments for 24 h. The cells were scraped off with a cell scraper. 100 μL of ice-cold cell lysis buffer was added to each sample, and the cells were lysed on ice for 30 min to extract proteins, and then BCA protein quantification was performed. Proteins (30 μg) were separated by 10% SDS-PAGE at a voltage of 100 V for 90 min, and the proteins were transferred to a PVDF membrane. The proteins were incubated with appropriate concentrations of primary antibody (1:1000) and secondary antibody (1:2000), and the gel imaging system was used to emit light, and the band optical density was analyzed by Image J software.

[0102] (2) Experimental results

[0103] Since the M1 / M2 polarization of macrophages is affected by glucose metabolism, and the results of RNA sequencing in animal experiments show that EtOAc can inhibit the expression levels of genes related to glycolysis in uterine tissue, especially HK-II. To further explore this, the effect of EtOAc on the expression of proteins related to glycolysis in LPS-induced macrophages was further verified by cell experiments. It was observed that the expression of hexokinase II (HK-II), hexokinase I (HK-I), pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase (PD) in macrophages RAW264.7 was significantly promoted after LPS stimulation; the expression of these proteins was significantly inhibited after EtOAc treatment ( Figure 5 A-E). 2-Deoxy-D-glucose (2-DG) is a recognized HK-II inhibitor, and it was found that 2-DG can significantly inhibit the expression of HK-II, HK-I, PKM2 and PD induced by LPS. After combined treatment with EtOAc (100 μg / mL) and 2-DG, the inhibitory effect was more obvious ( Figure 5 F-J). As a protein kinase, HK-II can mediate the phosphorylation of IκBα T291 , promote its degradation and activate NF-κB. The NF-κB signaling pathway is a classical pro-inflammatory signaling pathway that can promote the secretion of various inflammatory cytokines. To further verify whether HK-II is a potential anti-inflammatory target of EtOAc, proteins related to the NF-κB pathway were detected. The results showed that LPS significantly promoted the phosphorylation of NF-κB and IκBα, while the phosphorylation could be inhibited by using 2-DG or EtOAc alone, and there was a synergistic inhibitory effect between the two ( Figure 5 K-M). The above results indicate that EtOAc can inhibit the inflammatory response by inhibiting the expression of proteins related to glycolysis, and HK-II may be a potential target.

Claims

1. A Caulis Sargentodoxae-Patrilinia affine extract, characterized in that: The preparation method comprises the following steps: (1) Mixing Caulis Sargentodoxae and Herba Patriniae in a mass ratio of 1:1, extracting twice with 50% V / V ethanol, combining the filtrates, removing the solvent, and drying to obtain a dry extract; (2) dissolving the extract obtained in step (1) in water, and then extracting with ethyl acetate three times, combining the ethyl acetate phases, removing the solvent, and drying to obtain the Sargentodoxae-Patrilinia chinensis extract.

2. The Caulis Sargentodoxae-Patrilinia affine extract according to claim 1, characterized in that Among them, the abundance values ​​of vanillic acid, chrysophanol, salidroside, rhamnol, luteolin, kaempferol, apigenin, acacetin, proanthocyanidin B1, epicatechin, luteolin, isoquercetin, madecassic acid, sinapinic acid, caffeic acid, asiatic acid, verbascoside, naringenin, cynarin, cynarin, irisin, isorhamnetin, genkwain, genistein, physacetin methyl ether, quercetin, genistein, curcumol, diosgenin, baicalein, formononetin, tyrosol, and quercetin were greater than 0.

5.

3. The Caulis Sargentodoxae-Patrilinia affine extract according to claim 1, characterized in that The extract solution at a concentration of 2 μg / mL contains 100 to 150 ng / mL of salidroside, 30 to 60 ng / mL of kaempferol, 50 to 100 ng / mL of quercetin, 50 to 100 ng / mL of epicatechin, and 40 to 80 ng / mL of olibanum.

4. The Caulis Sargentodoxae-Patrilinia affine extract according to claim 1, characterized in that In the step (2) of the preparation method, the volume ratio of the amount of ethyl acetate used each time to the amount of water used is (1-1.5):1, (1-1.2):1 and (0.8-1):1 respectively.

5. The Caulis Sargentodoxae-Patrilinia affine extract according to claim 4, characterized in that The volume ratio of ethyl acetate used each time in step (2) of the preparation method is 1.2:1:0.

8.

6. A pharmaceutical composition, characterized in that It comprises the red spatholobi-patriniae extract according to claim 1 and a pharmaceutically acceptable carrier.

7. Use of the Sargentodoxae-Patrilinia scabra extract according to claim 1 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating pelvic inflammatory disease.

8. The use according to claim 7, characterized in that: The medicine is a medicine for treating pelvic inflammatory disease with damp-heat and blood stasis syndrome.

9. The use according to claim 7, characterized in that: The medicine is a medicine for treating endometritis, salpingitis, salpingo-oophoritis, tubo-ovarian abscess or pelvic peritonitis.

10. The use according to claim 7, characterized in that: The medicine can improve uterine tissue damage, reduce inflammatory cell infiltration, and promote uterine tissue recovery.

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