Extraction method of philippine flemingia root extract and application of philippine flemingia root extract in preparation of anti-inflammatory or gynecological disease treatment medicine

Abstract

The invention discloses a preparation method of a philippine flemingia root extract and application of the philippine flemingia root extract in preparation of anti-inflammatory or gynecological disease treatment medicines. The extraction method comprises the following steps: mixing philippine flemingia root and an alcohol solvent, carrying out alcohol extraction, and concentrating an alcohol extract to remove the alcohol solvent so as to obtain an alcohol-removed solution; then filtering by adopting a nano ceramic membrane and an ultrafiltration membrane to obtain clear liquid; loading, leaching and eluting the clarified liquid by adopting a high-performance preparative liquid chromatography system, and collecting ethanol eluted liquid to obtain eluent; a preparative column of the high-performance preparative liquid chromatography system is PS-NVP; and concentrating and drying the eluent to obtain the philippine flemingia root extract. According to the method disclosed by the invention, alcohol extraction is combined with filtration of an ultrafiltration membrane and a nano-membrane to remove impurities, and then enrichment adsorption is carried out by adopting a specific preparation column, so that the anti-inflammatory effect of the philippine flemingia root extract can be better improved, and the extracted philippine flemingia root extract has a better effect than gynaecological Qianjin capsules in the aspect of resisting inflammation or treating gynecological diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of extracting Herba Lycopodiellae, and more specifically, relates to an extraction method of Herba Lycopodiellae extract and application of the extract in preparing anti-inflammatory or gynecological disease treatment drugs. Background Art

[0002] Moghania philippinensis Merr.et Rolfe is a perennial herbaceous plant of the genus Moghania in the family Fabaceae. It was first recorded in the Illustrated Records of Plant Names and Realities. Moghania philippinensis is rich in flavonoids, phenols, glycosides, sterols, coumarins, amino acids and other chemical components, but the content of each type of compound is not high. Among them, the content of flavonoids is only about 3%, which is relatively low. Modern pharmacological studies have shown that flavonoids in Moghania philippinensis have multiple pharmacological activities such as anti-inflammatory, anti-tumor and antibacterial.

[0003] Among the common gynecological diseases, premenstrual syndrome, vaginal inflammation and breast hyperplasia are the most obvious. Vaginal inflammation is a common and frequent gynecological disease, which seriously affects the quality of life of patients. The Gynecological Qianjin Capsule, which has good therapeutic effects and sales on the market, has a good therapeutic effect on vaginal inflammation and is the first choice for female gynecological inflammation. It has the effects of nourishing blood, replenishing qi, anti-inflammatory, removing dampness, strengthening waist and unblocking meridians. It is used for women's acute and chronic pelvic inflammatory disease, endometritis, cervicitis, leucorrhea and other diseases. Qianjinba in its components is one of the key ingredients.

[0004] At present, in the preparation method of Gynecological Qianjin Capsule, the extraction process of the Herba Lycopodii, one of its main raw materials, is water extraction, and the Herba Lycopodii extraction method is relatively extensive. Patent CN 102940667 A discloses that the Herba Lycopodii total flavonoids are extracted by alcohol extraction and ultrasonic extraction, but the Herba Lycopodii extraction method is mainly aimed at extracting the lipid-lowering and thrombotic components in the Herba Lycopodii, and the method is also limited in the extraction of anti-inflammatory related active substances in the raw Herba Lycopodii and the removal of impurities. Therefore, how to optimize the extraction process of the raw Herba Lycopodii, improve the anti-inflammatory effect of the Herba Lycopodii extract, and maintain the stability of the preparation process has become a technical problem that needs to be solved urgently. Summary of the invention

[0005] In view of the above existing technical problems, the primary purpose of the present invention is to provide an extraction method of the extract of the Herba Lycopodii. The extraction method of the present invention can improve the anti-inflammatory effect of the Herba Lycopodii extract, has an excellent therapeutic effect on gynecological diseases, and has better efficacy than the Herba Lycopodii capsule in inhibiting inflammation.

[0006] The second object of the present invention is to provide a method for extracting the above-mentioned Herba Lycopodii extract and obtain the Herba Lycopodii extract.

[0007] The third object of the present invention is to provide the use of the above-mentioned Eleutherodactyla truncatula extract in the preparation of drugs for anti-inflammatory or treatment of gynecological diseases.

[0008] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0009] The present invention claims a method for extracting the extract of the Herba Lycopodii, comprising the following steps:

[0010] S1. Mixing the 100-pound hyssop and an alcohol solvent, performing alcohol extraction, and concentrating the alcohol extract to remove the alcohol solvent to obtain an alcohol-free solution;

[0011] S2. The alcohol removal liquid is first filtered using a nano-ceramic membrane and then filtered using an ultrafiltration membrane to obtain a clarified liquid;

[0012] S3. The clarified liquid is loaded with a high-performance preparative liquid chromatography system, then rinsed with pure water, eluted with ethanol, and the liquid after elution with ethanol is collected to obtain an eluent; the preparative column of the high-performance preparative liquid chromatography system is PS-NVP;

[0013] S4. The eluate is concentrated and dried to obtain the Herba Lycopodii Fructus extract.

[0014] The present invention first uses an alcohol solvent to extract the components in the Herba Lycopersicum, so that the anti-inflammatory active ingredients such as flavonoids in the Herba Lycopersicum can be better extracted from the crude drug. After the alcohol extract removes the alcohol solvent, the nano-ceramic membrane treats the alcohol extract, which can remove macromolecular substances such as polysaccharides, pigments and insolubles, and can pre-remove some non-anti-inflammatory active substances with large molecular weights that will be enriched and adsorbed later, thereby reducing impurities in the final Herba Lycopersicum extract. Further, the present invention uses an ultrafiltration membrane for further filtration, which can effectively filter out the anti-inflammatory active ingredients such as flavonoids in the Herba Lycopersicum, and can further remove other non-anti-inflammatory active substances in the Herba Lycopersicum, such as polysaccharides, proteins and other substances. In addition, by using a combination of nano-ceramic membrane + ultrafiltration membrane, the alcohol extract can be subjected to two-stage screening and separation, which not only avoids the problem of macromolecular substances blocking the membrane pores and low extraction efficiency caused by directly using ultrafiltration membrane filtration, but also the two-stage extraction can better separate and remove macromolecular substances and non-anti-inflammatory active substances in the alcohol extract.

[0015] Furthermore, in the process of enrichment and capture, the selection of the preparative column of the high-performance preparative liquid chromatography system is crucial for the adsorption of anti-inflammatory active substances in the extract of the Herba Lycopodii. The inventors have found through research that, compared with other conventional preparative columns, the present invention uses a specific preparative column filler: PS-NVP, which has extremely strong adsorption for anti-inflammatory active ingredients such as flavonoids in the extract, thereby playing an excellent enrichment and capture role.

[0016] Through the optimization of the above process and in combination with other process conditions, the extract of the Herba Lycopodiellae extracted by the extraction method has excellent therapeutic effects on gynecological diseases. The Herba Lycopodiellae extract provided by the present invention can not only better reduce the damage to the spleen, thymus and uterus; it can also better reduce the levels of inflammatory factors TNF-α, IL-1β, IL-18, IL-6, as well as transforming growth factor TGF-β1 and vascular endothelial growth factor VEGF in serum. Therefore, it has a better effect in improving gynecological diseases such as chronic pelvic inflammatory disease.

[0017] Preferably, in step S2, the pore size of the nano-ceramic membrane is 30 to 500 nm. Further preferably, the pore size of the nano-ceramic membrane is 300 to 500 nm. Most preferably, the pore size of the nano-ceramic membrane is 500 nm. Nano-ceramic membranes with specific pore sizes can better remove macromolecular substances such as polysaccharides, pigments and insoluble substances, and can pre-remove some non-anti-inflammatory active substances with larger molecular weight that will be enriched and adsorbed later.

[0018] Preferably, in step S2, the pore size of the ultrafiltration membrane is 5 to 50 KD. Further preferably, the pore size of the ultrafiltration membrane is 40 to 50 KD. Most preferably, the pore size of the ultrafiltration membrane is 50 KD.

[0019] Ultrafiltration membranes with specific pore sizes can further remove other non-anti-inflammatory active substances in the herb, such as polysaccharides, proteins, etc. The combination of the above-mentioned nano-ceramic membranes with specific pore sizes and ultrafiltration membranes can achieve the selective separation of anti-inflammatory active ingredients such as total flavonoids in the herb.

[0020] Preferably, in step S2, during ultrafiltration membrane filtration, the temperature is controlled to be ≤50°C and the pressure is controlled to be 0.1-0.25MPa.

[0021] Preferably, in step S1, the alcohol solvent is ethanol with a volume concentration of 70-90%. More preferably, the volume concentration of ethanol is 70%.

[0022] Preferably, in step S1, the alcohol extraction is performed in a water bath, and the temperature of the water bath is 80-90° C. More preferably, the temperature of the water bath is 90° C.

[0023] Preferably, the solid-liquid ratio of the raw material mulberry to ethanol is 1:8 to 10. Further preferably, the solid-liquid ratio of the raw material mulberry to ethanol is 1:10.

[0024] Preferably, in step S4, the drying is freeze-drying. Further preferably, the freeze-drying temperature is -18 to -20°C.

[0025] Preferably, in step S3, the ethanol concentration during elution is 60-70%.

[0026] Preferably, in step S4, the concentration temperature is 45 to 50° C. and the vacuum degree is -0.15 to -0.05 MPa.

[0027] Furthermore, the present invention also seeks to protect the Herba Lycopodiellae extract obtained by extracting the Herba Lycopodiellae extract using the extraction method of the Herba Lycopodiellae extract.

[0028] Furthermore, the present invention also seeks to protect the use of the above-mentioned Eleutherococcus chinensis extract in the preparation of drugs for anti-inflammatory or treatment of gynecological diseases.

[0029] Furthermore, the above-mentioned Eleutherodactyla extract can achieve the purpose of treating gynecological diseases by reducing the levels of inflammatory factors TNF-α, IL-1β, IL-18, and IL-6 in serum, or by reducing the levels of transforming growth factor TGF-β1 and vascular endothelial growth factor VEGF.

[0030] More specifically, the extract of Eleutherodactyla truncatula has a more excellent inhibitory effect on vascular endothelial growth factor VEGF.

[0031] More specifically, the gynecological diseases described in the present invention include but are not limited to uterine inflammation, such as chronic pelvic inflammatory disease, chronic endometritis, chronic cervicitis, etc.

[0032] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for extracting the Herba Lycopodii extract, and the extraction method can well improve the anti-inflammatory effect of the Herba Lycopodii extract. Compared with the Gynecological Qianjin Capsule, the Herba Lycopodii extract provided by the present invention can not only better reduce the damage to the spleen, thymus and uterus; it can also better reduce the levels of TNF-α, IL-1β, IL-18, IL-6, TGF-β1 and VEGF in serum, especially has an extremely excellent inhibitory effect on vascular endothelial growth factor VEGF. The Herba Lycopodii extract has a better effect in improving gynecological diseases such as chronic pelvic inflammatory disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The sample state diagrams are the extract, alcohol removal solution, clarified solution (500nm), retentate (500nm), clarified solution (50KD) and retentate (50KD).

[0034] Figure 2 Prepare chromatograms for the liquid phases of the loading solution, eluent, elution solution, and regeneration solution.

[0035] Figure 3 This is the sample status diagram of clarified liquid (50KD), loading liquid, eluent, eluting liquid and regeneration liquid.

[0036] Figure 4 It is the HPLC chromatogram of the extract, alcohol removal solution, clarified solution (500nm), clarified solution (50KD), loading solution, eluent, elution solution, regeneration solution, and combined solution (elution solution and regeneration solution) lyophilized powder.

[0037] Figure 5 It is the characteristic peak quantity transfer diagram of each chromatographic peak in Example 1.

[0038] Figure 6 The following are the observation pictures of the uterus morphology of rats in each experimental group.

[0039] Figure 7 This is the effect of each experimental group on the spleen, thymus and uterus indexes of CPID model rats.

[0040] Figure 8 These are the HE staining results of the uterus of rats in each experimental group.

[0041] Fig. 9 These are the effects of each experimental group on the concentrations of inflammatory factors IL-6, TNF-α, IL-1β, and IL-18 in the serum of CPID model rats.

[0042] Fig.10 This is the effect of each experimental group on the levels of TGF-β1 and VEGF in the serum of CPID model rats. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0044] Example 1 Extraction method of the extract of the Herba Lysimachiae

[0045] (1) Pretreatment of medicinal materials: One kilogram of medicinal materials of P. truncatum was taken as the research object, and the medicinal materials of P. truncatum were crushed, and the mass after crushing was weighed as 1 kg; the relevant data and information of P. truncatum are shown in Table 1 below.

[0046] Table 1

[0047] sample Weighing mass (g) batch number Suppliers Thousand-pound medicinal materials 1000 Y005-2104006 Qianjin Pharmaceutical

[0048] (2) Extraction: 1 kg of crushed Herba Lycopodiellae was extracted with 70% ethanol by volume, with a solid-liquid ratio of 1:10 (kg / L). The mixture was heated in a water bath until it was slightly boiling, about 90°C, and the extraction was continued for 1 hour under slight boiling conditions. The extraction was repeated twice to obtain Herba Lycopodiellae extract (batch number: QJB-TQ-210719). The data and information related to the extraction steps are shown in Table 2 below.

[0049] Table 2

[0050] Extraction solution batch number Extraction volume (L) Sample volume (mL) Solid content (mg / mL) Extraction rate (%) QJB-TQ-210719 15.2 50 7.7 11.7

[0051] (3) Alcohol removal: 15.2 L of the extract with batch number QJB-TQ-2010719 was placed in a 20 L rotary evaporator, concentrated to 3 L (without alcohol taste) at 65 ° C and a vacuum degree of -0.095 MPa, and then diluted to 10 L with water to obtain the alcohol removal solution with batch number QJB-NS-210720; the relevant data and information of the alcohol removal step are shown in Table 3 below.

[0052] Table 3

[0053]

[0054] (4) Nano-ceramic membrane filtration: 10.0 L of the alcohol solution was taken and passed through a 500 nm ceramic membrane. 4.5 L of pure water was added for top washing, a total of 2 times, 2.25 L each time, and 1 L of 10% ethanol was used for top washing. A total of 15.7 L of clarified liquid (500 nm) with batch number QJB-500nm-CQ and 2.0 L of retained liquid (500 nm) with batch number QJB-500nm-JL were collected; the membrane filtration operation steps, membrane filtration step-related data and information are shown in Tables 4 and 5, respectively.

[0055] Table 4

[0056]

[0057]

[0058] Table 5

[0059] batch number Volume(L) Sample volume (mL) Solid content (mg / mL) QJB-500nm-CQ 15.7 50 4.2 QJB-500nm-JL 2.0 50 17.7

[0060] (5) Ultrafiltration membrane filtration: 15.7 L of the clarified liquid (500 nm) of batch number QJB-500nm-CQ was filtered through a 50KD ultrafiltration membrane (temperature controlled below 50°C, pressure controlled at 0.1-0.25 MPa), and 1 L of pure water was used for top washing to obtain 17.6 L of the clarified liquid (50KD) of batch number QJB-50KD-CQ and 1.5 L of the retentate (50KD) of batch number QJB-50KD-JL. The data and information related to the ultrafiltration membrane filtration step are shown in Table 6 below.

[0061] Table 6

[0062] batch number Volume(L) Sample volume (mL) Solid content (mg / mL) QJB-50KD-CQ 17.6 50 3.8 QJB-50KD-JL 1.5 50 3.15

[0063] (6) Preparation: 17.6 L of the clarified liquid (50KD) of batch number QJB-50KD-CQ was taken for liquid phase preparation using a high performance preparative liquid chromatography system (the chromatographic column filler was PS-NVP). The process steps for liquid phase preparation are shown below, and the method is shown in Table 7. Figure 1 The sample status diagrams are the extract, alcohol removal solution, clarified solution (500nm), retentate (500nm), clarified solution (50KD) and retentate (50KD). Figure 2 Prepare chromatograms for the liquid phases of the loading solution, eluent, elution solution, and regeneration solution.

[0064] ① Loading: Take 17.6L of the clarified liquid (50KD) with batch number QJB-50KD-CQ for loading to obtain the loading liquid;

[0065] ② Elution: Use pure water for elution, 3BV, to obtain the eluent;

[0066] ③ Elution: 60% ethanol was used for elution, 3 BV, and the eluent was obtained;

[0067] ④ Regeneration: Use 90% ethanol for regeneration, 9.8BV, to obtain the regeneration solution.

[0068] Figure 3 This is the sample status diagram of clarified liquid (50KD), loading liquid, eluent, eluting liquid and regeneration liquid.

[0069] Table 7

[0070] Time Flow rate (ml / min) A％ 0 30 100 588 30 100 588 40 100 602 40 100 616 40 100 670 20 100

[0071] (7) Liquid phase analysis

[0072] ①Instrument: High performance liquid chromatography analyzer

[0073] ②Sample: the whole process (extract solution, alcohol removal solution, clarified solution (500nm), clarified solution (50KD), loading solution, eluent, elution solution, regeneration solution, combined solution (elution solution and regeneration solution) lyophilized powder)

[0074] ③Analytical column: Tnature C18 (4.6*250mm, 5um, 100A)

[0075] ④Injection volume: 10uL

[0076] ⑤Mobile phase: A: ACN; B: 0.1% phosphoric acid water

[0077] ⑥Analysis method:

[0078] Time V(ml / min) A％ B％ 0 1.5 5 95 3 1.5 5 95 27 1.5 30 60 40 1.5 95 5 46 1.5 95 5

[0079] Figure 4It is the HPLC chromatogram of the extract, alcohol removal solution, clarified solution (500nm), clarified solution (50KD), loading solution, eluent, elution solution, regeneration solution, and combined solution (elution solution and regeneration solution) lyophilized powder.

[0080] (8) Concentration and freeze-drying:

[0081] ① Concentration: The eluent was concentrated under reduced pressure at 45°C and a vacuum degree of -0.08Mpa to a volume of about 100ml.

[0082] ② Freeze-drying: freeze the concentrated eluate in a -40°C refrigerator, put it into a freeze dryer after it is frozen, and perform freeze-drying at -18 to -20°C to obtain the Herba Lysimachiae extract.

[0083] The above scheme is a preferred embodiment of the present invention, which is used to reflect the specific implementation process of the scheme of the present invention, and does not mean that the present invention can only achieve the technical effect under the conditions of the above specific embodiments. The present invention can achieve the technical effect of the present invention under the conditions that the pore size of the nano-ceramic membrane is 30-500nm, the pore size of the ultrafiltration membrane is 5-50KD, the temperature during ultrafiltration membrane filtration is ≤50℃, and the pressure is 0.1-0.25MPa. Test Example 1 Determination of the solid mass of the extract of the radix quinquefolii

[0084] (1) Solid mass determination method: Take 10 ml of each of the extract, alcohol-removing solution, clarified solution (500 nm), retentate (500 nm), clarified solution (50 KD), retentate (50 KD), loading solution, rinsing solution, eluent, and regeneration solution obtained in Example 1 and dry them in an evaporating dish to measure the solid mass, and convert the solid mass in the total volume. Weigh the mass of two combined liquid freeze-dried powders (mix the loading solution + rinsing solution in Example 1, and mix the eluent + regeneration solution, and prepare according to the concentration and freeze-drying steps in Example 1). The mass data of the two combined liquid freeze-dried powders are shown in Table 8 below. During the preparation of the extract of the Herba Lysimachiae, the mass data of each step are shown in Table 9 below.

[0085] Table 8

[0086]

[0087] Table 9

[0088]

[0089]

[0090] Test Example 2 Process Control of Extracts from Rhizoma Coptidis

[0091] The diluent, clarified liquid (500nm), clarified liquid (50KD), eluent and lyophilized powder were subjected to HPLC analysis under the following conditions; the characteristic peak value was transferred to analyze the peak area obtained by integrating the characteristic peaks in the spectrum, and the peak area ratio of each process section was calculated.

[0092] Chromatographic conditions

[0093] Analytical column: Tnature C18 (4.6*250mm, 5um, 100A)

[0094] Injection volume: 10uL

[0095] Mobile phase: A: ACN; B: 0.1% phosphoric acid water

[0096] Analytical methods:

[0097] Time V(ml / min) A％ B％ 0 1.5 5 95 3 1.5 5 95 27 1.5 30 60 40 1.5 95 5 46 1.5 95 5

[0098] Figure 5 It is a characteristic peak value transfer diagram of each chromatographic peak in Example 1. The value transfer of the whole process of the preparation process of Qianjinba is shown in Table 10 below.

[0099] Table 10

[0100]

[0101]

[0102] From the above figure and table, we can see that:

[0103] ① The transfer rates of each characteristic peak in the Qianjinba dilution-500nm clarification process are all greater than 90%.

[0104] ②The overall chromatographic peak transfer rate of the 50KD clarification process was 87.07%.

[0105] ③ The transfer rates of the characteristic peaks of the preparation process are 99.16%-139.77%, and the high-efficiency preparation process is generally stable.

[0106] From the quality data, the recovery rate of the current process is relatively high, reaching more than 90%. In the preparation process, PS-NVP filler has a strong adsorption capacity for flavonoid components and has a good enrichment effect on the main components.

[0107] Test Example 3: Efficacy test of the extract of Eupatorium truncatum

[0108] (1) Experimental Materials

[0109] 183 SPF female SD rats, weighing 160-200 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., license number SYXK (Xiang) 2019-0009. Animal breeding environment: temperature 22-24 °C, humidity 60%-65%, day and night alternation, free food and water.

[0110] Fuke Qianjin Capsules (0.4 g*48 capsules, national medicine standard Z20020024) were purchased from Zhuzhou Qianjin Pharmaceutical Co., Ltd.; Kangfuyan Capsules (0.4 g*48 capsules, national medicine standard Z20055634) were purchased from Shandong Buchang Shenzhou Pharmaceutical Co., Ltd.; and the extract of Qianjinba was prepared according to Example 1. Each drug was made into an aqueous solution of a certain concentration with ultrapure water and administered intragastrically.

[0111] Estradiol benzoate (2ml:4mg, veterinary drug number 220412511), Sichuan Jinke Pharmaceutical Co., Ltd.; PBS phosphate buffer (Cat.NO.P1010, PH 7.2-7.4, Solarbio), each bag was dissolved in 2L ultrapure water; Chloral hydrate (lot#:C12685890, Macklin), prepared into a 10% aqueous solution with ultrapure water, 0.5 Macklin turbidimeter tube (Yuezhu Medical Device No. 20140010, Zhuhai Beso Biotechnology Co., Ltd.), hemostatic sponge (Gan Medical Device No. 20182640140, Guangzhou Kuaikang Medical Device Co., Ltd.); rat TN F-α (M28015221), IL-1β (N15016990), IL-18 (N16016991), IL-6 (N21016918), TGF-β1 (N22016919) and VEGF (N23016920) ELISA kits were purchased from Wuhan Huamei Bioengineering Co., Ltd.; 4% paraformaldehyde universal tissue fixative (21200490, biosharp).

[0112] High-speed refrigerated centrifuge (Microfuge 20R, Beckman Coulter, Inc., USA), single-channel pipette (10-1000ul, Eppendorf), and multi-function microplate reader (2021005559, BIOTEK / Synergy).

[0113] (2) Experimental methods

[0114] 2.1 Establishment of chronic pelvic inflammatory disease (CPID) model

[0115] After one week of adaptive feeding, rats were subcutaneously injected with 0.2 mL of estradiol injection (estradiol benzoate injection for animals) once a day for 5-7 consecutive days. During the injection period, attention was paid to observe whether the vaginal opening of the rats was congested, red and swollen, and the estrus period was observed. After inducing the estrous cycle of the rats, the lower abdomen of the rats was disinfected with 75% alcohol under anesthesia, and a 2 cm longitudinal incision was made in the lower abdomen to expose the uterus. The uterus of the rats was scraped with a homemade curette. After two weeks, 0.3 mL of Ureaplasma urealyticum + Staphylococcus aureus + Escherichia coli (concentration 1*10 8 cfu / mL), and fill the needle hole with a piece of sterile hemostatic sponge (0.2*0.2cm), and insert a piece of sterile hemostatic sponge into the vagina to prevent the bacterial fluid in the abdominal cavity from flowing out.

[0116] The successfully modeled rats were divided into 4 groups according to the random number table method: model group, QJB extract group, Kangfuyan capsule (KFY) group, and Fuke Qianjin capsule (FKQJ) group.

[0117] Construction of sham-operated group (JSS group) rats: The construction of sham-operated group rats was the same as the operation of "model rat construction" in 2.1 above, the difference being that the sham-operated group rats were not infected with Mycoplasma + Staphylococcus aureus + Escherichia coli, and the inflammation model was not constructed.

[0118] Blank group: The blank group used normal mice without treatment.

[0119] 2.2 Grouped Dosing

[0120] The extract group of the herbaceous plant: the dosage was 39.96 mg kg -1 ·d -1 Giving the model rats the extract of Eleutherodactyla truncatula;

[0121] Kangfuyan capsule group: dosage 324.002 mg·kg -1 ·d -1 The model rats were given Kangfuyan capsules;

[0122] Gynecological Qianjin Capsule Group: dosage: 216.001 mg·kg -1 ·d -1 Gynecological Qianjin Capsule was administered to the model rats;

[0123] Model group: model rats were given distilled water, and the amount of distilled water was converted into the intragastric volume according to the body surface area of ​​the rats;

[0124] Sham operation group: Rats in the sham operation group were given the same amount of distilled water as the model group;

[0125] Blank group: The blank group was given the same amount of distilled water as the model group;

[0126] Each group was continuously administered with drug (or distilled water) for 21 days.

[0127] 2.3 Sample collection

[0128] After continuous administration for 21 days, the rats were fasted but not watered for 24 h. After anesthesia with 10% chloral, blood was collected from the abdominal aorta and allowed to stand at room temperature. After the blood was clearly stratified, the blood was rotated at 3000 r·min. -1 Centrifuge for 15 minutes, take the supernatant as serum, store in -80℃ refrigerator for later use, and detect the levels of TNF-α, IL-1β, IL-18, IL-6, TGF-β1 and VEGF in the serum of rats in each group according to the instructions of the kit. Take the uterine tissue of rats in each group, and observe the morphology and color of the uterine tissue with naked eyes. Take about 200 mg of uterine tissue from the same side of rats in each group and fix it in 4% paraformaldehyde for later use.

[0129] 2.4 Body weight and organ index of rats

[0130] During the experiment, the body weight of rats in each group was recorded weekly. After the rats were killed, the uterus, thymus, and spleen of the rats were removed, cleaned with PBS phosphate buffer, dried with filter paper, and weighed to calculate their organ index, organ index = organ weight (g) / rat body weight (g) × 100.

[0131] 2.5 Gross morphology and histopathological examination of rat uterus

[0132] The bilateral uterine tissues of rats were taken for macroscopic morphological observation; the right uterine tissues were taken, sliced ​​after dehydration, wax immersion, embedding, and baking at 60℃ for 3h. The slices were dewaxed with xylene, dehydrated with alcohol gradient, stained with HE, sealed with neutral gum, and placed under a microscope to observe the pathological changes of rat uterine tissues. CaseViewer software was used to observe the results of HE staining of each uterus, the integrity of the endometrium, the state and arrangement of each cell, and the edema and inflammatory cell infiltration.

[0133] 2.6 Detection of TNF-α, IL-1β, IL-18, IL-6, TGF-β1 and VEGF levels in rat serum

[0134] Serum samples were taken, and the levels of serum TNF-α, IL-1β, IL-18, IL-6, TGF-β1 and VEGF in rats in each group were detected according to the instructions of the kit.

[0135] 2.7 Statistical analysis

[0136] SPSS25.0 statistical software was used to organize the data. The measurement data were expressed as x±s. The LSD test was used for pairwise comparison. The difference was statistically significant when P<0.05. Compared with the normal group, ##P<0.01, compared with the model group, *P<0.05, **P<0.01.

[0137] (3) Experimental results

[0138] 3.1 Observation of rat uterine morphology

[0139] 21 days after modeling, the uteri of rats in the blank group, sham operation group, and model group were randomly selected and the morphology of the uteri was observed with the naked eye. Figure 6 The following are the observation pictures of the uterine morphology of rats in each experimental group. The results show that there is no difference between the left and right sides of the uterus of the sham-operated rats, and the size, shape and color are normal. The uterus of the model group is thick, swollen and has residual fluid, the fallopian tubes and ovaries are dilated, and there is fat tissue adhesion at the pinhole; there is also adhesion in the pelvic cavity, indicating that the model is successful.

[0140] After the treatment, the abdominal cavity was opened and the pelvic condition was observed. The blank group showed no obvious abnormalities in the uterus and pelvic cavity. Although some rats in the sham group had a small amount of adhesion between the pelvic organs and the surrounding tissues, there was no bleeding; there was no statistical difference between the blank group and the sham group. The model group showed swelling due to pus accumulation inside the uterus, and the uterine cavity tended to be transparent and edematous, and adhered to the surrounding tissues into a mass, which was difficult to separate. Compared with the model group, each drug group could significantly improve the pelvic adhesion of rats with pelvic inflammatory disease.

[0141] 3.2 Effects on spleen, thymus and uterus indexes in CPID model rats

[0142] Figure 7 The effect of each experimental group on the spleen, thymus and uterus index of CPID model rats. As shown in the figure, there was no statistical difference in the organ index between the JSS group and the blank group (P>0.05), proving that the surgical process had no effect on the experimental results. Compared with the blank group, the spleen, thymus and uterus indexes of the model group rats were significantly increased, indicating that the CPID model rats had certain immune damage (spleen and thymus enlargement), accompanied by uterine effusion and swelling. In addition to the positive control drug, the QJB group can significantly reduce the spleen, thymus and uterus indexes of the model rats, and the effect is better than the FKQJ group, indicating that it significantly improves the damage to the immune function of the pelvic inflammatory disease model rats, and also regulates the swelling of the uterus.

[0143] 3.3 Results of HE staining of rat uterus

[0144] Figure 8 The HE staining results of the uterus of rats in each experimental group are shown in Figure 2. Figure 8The results of HE staining showed that the endometrium in the blank group was smooth and structurally intact, with only a small amount of neutrophil infiltration. Similarly, the uterine wall of the JSS group was clearly layered; the endometrial glands were regular, the muscle fiber texture was clear; and the epithelial cells were arranged in a single layer of columns. In the model group, obvious neutrophil infiltration was observed in the endometrium, severe tissue edema, and the endometrial thickness became thinner, with inflammatory cell infiltration, some of which penetrated deep into the muscle layer and accumulated throughout the entire layer. The endometrial epithelial cells in each drug-treated group were slightly proliferated or shed, and the inflammatory cell infiltration was significantly reduced.

[0145] 3.4 Effects on the concentration of inflammatory factors in the serum of CPID model rats

[0146] Fig. 9 The effects of each experimental group on the concentrations of inflammatory factors IL-6, TNF-α, IL-1β, and IL-18 in the serum of CPID model rats. Fig. 9 As shown in the results, compared with the blank group, there was no significant difference in the serum levels of various factors in the JSS group (P>0.05), while the serum IL-6, TNF-α, IL-1β and IL-18 levels in the model group were significantly increased (P<0.01); except for the KFY group, compared with the model group, the QJB group could significantly reduce the concentrations of inflammatory factors IL-6, TNF-α, IL-1β, and IL-18 in the serum of the model rats, especially the inflammatory factor IL-18. The above data show that the extract of Rhizoma Coptidis can significantly improve the inflammatory response of the pelvic inflammatory disease model rats, and the anti-inflammatory effect of the QJB group is significantly better than that of the FKQJ group.

[0147] 3.5 Effects on the levels of TGF-β1 and VEGF in serum of CPID model rats

[0148] Transforming growth factor TGF-β1 and vascular endothelial growth factor VEGF are important factors regulating cell growth and differentiation. They can promote increased vascular permeability, extracellular matrix degeneration, endothelial cell migration, proliferation and angiogenesis, and are highly correlated with pelvic adhesions and tissue hyperplasia.

[0149] Fig.10 The effects of each experimental group on the levels of TGF-β1 and VEGF in the serum of CPID model rats are shown in Figure 2. Fig.10As shown in the figure, compared with the blank group, there was no significant difference in the levels of TGF-β1 and VEGF in the serum of the rats in the JSS group (P>0.05), while the levels of TGF-β1 and VEGF in the serum of the rats in the model group were significantly increased (P<0.01); except for the positive control drug, compared with the model group, the QJB group could significantly reduce the level of TGF-β1 in the serum of the model rats, and its effect was not significantly different from that of FKQJ. The QJB group had a very excellent effect in reducing VEGF, and the inhibitory effect of the QJB group on VEGF was significantly better than that of the KFY group and the FKQJ group.

[0150] Comparative test of different preparation processes

[0151] (1) The extraction method of the Herba Lycopodiellae in Example 1 was followed, except that the enrichment and capture preparation in step (6) was not performed, and the clarified liquid (50 KD) was directly concentrated and freeze-dried to obtain the Herba Lycopodiellae extract.

[0152] The extract of Herba Lycopodiellae prepared by this process step contains more protein, pigment, oil and other components because it does not adopt the enrichment and capture process. Under the same medication conditions, its effect on the uterus, spleen and thymus index is worse than that of the Herba Lycopodiellae extract in Example 1, and its effect on reducing the levels of inflammatory factors TNF-α, IL-1β, IL-18, IL-6, transforming growth factor TGF-β1 and vascular endothelial growth factor VEGF in serum is worse than that of the Herba Lycopodiellae extract in Example 1.

[0153] (2) The extraction method of the radix serrata in Example 1 was followed, except that the ultrafiltration membrane filtration in step (5) was not performed, and the clarified liquid (500 nm) was directly enriched and captured for preparation.

[0154] The extract of Herba Lycopodiellae prepared by this process step contains more macromolecular substances such as polysaccharides and proteins because it is not filtered by ultrafiltration membrane. Although a specific preparation column is used for enrichment and capture later, some other substances that can be combined with PS-NVP filler are also enriched and captured. Under the same medication conditions, its effect on the uterus, spleen, and thymus indexes is worse than that of the Herba Lycopodiellae extract in Example 1, and its effect on reducing the levels of inflammatory factors TNF-α, IL-1β, IL-18, IL-6, transforming growth factor TGF-β1, and vascular endothelial growth factor VEGF in serum is worse than that of the Herba Lycopodiellae extract in Example 1.

[0155] (3) The extraction method of the radix serrata in Example 1 is different in that: in step (5), the pore size of the ultrafiltration membrane used is 100 KD.

[0156] The Herba Lysimachiae extract prepared by this process also contains a certain amount of polysaccharides, proteins and other macromolecular substances. Although a specific preparation column was used for enrichment and capture later, some other substances that can be combined with PS-NVP filler were also enriched and captured. Under the same medication conditions, its effect on the uterus, spleen, and thymus indexes is worse than that of the Herba Lysimachiae extract in Example 1. Its effect on reducing the levels of inflammatory factors TNF-α, IL-1β, IL-18, IL-6, and transforming growth factor TGF-β1 and vascular endothelial growth factor VEGF in serum is worse than that of the Herba Lysimachiae extract in Example 1, but better than the Herba Lysimachiae extract in (2) above.

[0157] The foregoing examples are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to require the widest possible range that can be imagined, and the embodiments presented herein are demonstrated by the applicant's actual test results. Therefore, the applicant's intention is that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges therein, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A method for extracting the extract of the Herba Lycopodii, characterized in that: The following steps are involved: S1. Mixing the 100-pound hyssop and an alcohol solvent, performing alcohol extraction, and concentrating the alcohol extract to remove the alcohol solvent to obtain an alcohol-free solution; S2. The alcohol removal liquid is first filtered using a nano-ceramic membrane and then filtered using an ultrafiltration membrane to obtain a clarified liquid; S3. The clarified liquid is loaded with a high-performance preparative liquid chromatography system, then rinsed with pure water, eluted with ethanol, and the liquid after elution with ethanol is collected to obtain an eluent; the preparative column of the high-performance preparative liquid chromatography system is PS-NVP; S4. The eluate is concentrated and dried to obtain the Herba Lycopodii Fructus extract.

2. The method for extracting the extract of the Herba Lycopodii flavescentis according to claim 1, characterized in that: In the step S2, the pore size of the nano-ceramic membrane is 30-500 nm; preferably, the pore size of the nano-ceramic membrane is 300-500 nm.

3. The method for extracting the Herba Lycopodiellae extract according to claim 1 or 2, characterized in that: In the step S2, the pore size of the ultrafiltration membrane is 5 to 50 KD; preferably, the pore size of the ultrafiltration membrane is 40 to 50 KD.

4. The method for extracting the Herba Lycopodiellae extract according to claim 1, characterized in that: In the step S2, during the ultrafiltration membrane filtration, the temperature is controlled to be ≤50°C and the pressure is controlled to be 0.1-0.25MPa.

5. The method for extracting the extract of the Herba Lycopodiellae according to claim 1, characterized in that: In the step S1, the alcohol solvent is ethanol with a volume concentration of 70 to 90%.

6. The method for extracting the Herba Lycopodiellae extract according to claim 1 or 5, characterized in that: In the step S1, the alcohol extraction is carried out in a water bath, and the temperature of the water bath is 80-90°C.

7. The method for extracting the extract of the Herba Lycopodii flavescentis according to claim 6, characterized in that: The solid-liquid ratio of the basil and the alcohol solvent is 1:8-10.

8. The method for extracting the Herba Lycopodiellae extract according to claim 1, characterized in that: In the step S4, the drying is freeze-drying.

9. The Herba Lycopodiellae extract obtained by the method for extracting the Herba Lycopodiellae extract according to any one of claims 1 to 8.

10. Use of the extract of the Herba Lysimachiae as claimed in claim 9 in the preparation of drugs for anti-inflammatory or treatment of gynecological diseases.

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