Cherokee rose root extract with anti-inflammatory effect as well as extraction method and application of cherokee rose root extract

Abstract

The invention discloses an extraction method of a cherokee rose root extract with an anti-inflammatory effect. The extraction method comprises the following steps: alcohol extraction: removing an alcohol solvent; filtering the obtained product by using a nano ceramic membrane and an ultrafiltration membrane in sequence to obtain a clear solution; the pore diameter of the nano ceramic membrane is 30 to 500 nm; the aperture of the ultrafiltration membrane is 5-50KD; enriching and capturing the clarified liquid by adopting a high-performance preparative liquid chromatography system: loading a sample on the clarified liquid, leaching by adopting pure water, and eluting by adopting ethanol to obtain an eluent; a preparative column adopted by the high-performance preparative liquid chromatography system is C18YE filler; and concentrating and drying the eluent to obtain the cherokee rose root extract with the anti-inflammatory effect. According to the extraction method, the anti-inflammatory effect of the cherokee rose root extract can be well improved, and the prepared cherokee rose root extract has an excellent treatment effect on gynecological diseases and has the effect equivalent to or better than that of a gynecological Qianjin capsule in the aspect of inflammation inhibition.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine processing, and more specifically, relates to a golden cherry root extract with anti-inflammatory effect, an extraction method and application thereof. Background Art

[0002] Golden Rosa is the dried mature fruit of the golden rosa (Rosa laevigata Michx.) of the Rosaceae family. It was first recorded in "Shu Materia Medica" and is also known as white jade belt, downhill tiger, thorn vine thorn, thorny mantis tree, toad mantis tree, sugar jar, three-leaf le, etc. It is distributed in southern my country. The whole body of the golden rosa plant is a treasure. In addition to the fruit, its roots, leaves, and flowers are all recorded as medicines in ancient herbal books of different ages. At present, the fruit and root of golden rosa are commonly used in compound medicines. The medicine based on golden rosa root (i.e. golden rosa root) has multiple effects and can be used for the treatment of hepatitis, chronic enteritis, nephritis, gallstones, intermittent fever and other diseases, which can enhance physical fitness and improve immunity. In addition, golden rosa root is also helpful for cardiovascular health, can prevent atherosclerosis, and reduce the incidence of cardiovascular disease. At present, golden rosa root has been made into a variety of compound medicines and is widely used in clinical practice, such as gynecological Qianjin tablets, Sanjin tablets, Jinji capsules and tablets and other Chinese patent medicines.

[0003] So far, a variety of chemical components have been isolated from the root of Rosa Laevigata, mainly including triterpenoid saponins, flavonoids, flavanols, glycosides, diphenylene and diphenylene glycosides, steroids and steroidal saponins, fatty acids, etc. "Research Progress on Chemical Components and Pharmacological Actions of Rosa Laevigata Root, Long Xiaoqin, Guangxi University of Chinese Medicine" discloses that the root of Rosa Laevigata contains a variety of chemical components, including triterpenoid saponins, flavonoids, glycosides, flavanols, steroids, fatty acids, carbohydrates, etc., and has anti-inflammatory, antibacterial, anti-tumor, immune function enhancement, antioxidant, hypoxia resistance, anti-arrhythmia and other pharmacological effects.

[0004] At present, the research on the pharmacological effects of golden cherry root mainly focuses on anti-inflammatory aspects. For example, the gynecological Qianjin capsule containing golden cherry root extract has a good inhibitory effect on patients with uterine inflammation (such as chronic pelvic inflammatory disease, chronic endometritis, chronic cervicitis, etc.). Golden cherry root has many functions and complex ingredients, and the extraction process is crucial to the anti-inflammatory effect of golden cherry root extract. In the preparation method of gynecological Qianjin capsule, the extraction process of golden cherry root, one of its main raw materials, is water extraction. However, the golden cherry root extraction method is relatively extensive, and the extraction of anti-inflammatory related active substances in the raw material golden cherry root and the removal of impurities are limited.

[0005] Therefore, how to optimize the extraction process of the root of Sakura rosa-sinensis and improve the anti-inflammatory activity of the root extract of Sakura rosa-sinensis has become a technical problem that needs to be solved urgently. Summary of the invention

[0006] In view of the above-mentioned existing technical problems, the primary purpose of the present invention is to provide a method for extracting a root extract of the golden cherry with anti-inflammatory effect. The extraction method can better improve the anti-inflammatory effect of the root extract of the golden cherry and has excellent effects in improving gynecological diseases such as chronic pelvic inflammatory disease.

[0007] The second object of the present invention is to provide a method for extracting a Rosa rugosa root extract to obtain the Rosa rugosa root extract.

[0008] The third object of the present invention is to provide the use of the above-mentioned Cherry Blossom Root Extract in the preparation of drugs for anti-inflammatory or treatment of gynecological diseases.

[0009] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0010] A method for extracting a golden cherry root extract having an anti-inflammatory effect comprises the following steps:

[0011] (1) Alcohol extraction: extract the root of Rosa rugosa with alcohol, and then remove the alcohol solvent;

[0012] (2) Filtration: The product obtained in step (1) is filtered using a nano-ceramic membrane and an ultrafiltration membrane in sequence to obtain a clarified liquid; the pore size of the nano-ceramic membrane is 30 to 500 nm; the pore size of the ultrafiltration membrane is 5 to 50 kD;

[0013] (3) Enrichment and capture:

[0014] The clarified liquid is enriched and captured by using a high performance preparative liquid chromatography system: the clarified liquid is loaded with a sample, then washed with pure water, eluted with ethanol, and the liquid eluted with ethanol is collected to obtain an eluent; the preparation column used in the high performance preparative liquid chromatography system is a C18YE filler;

[0015] (4) The eluate is concentrated and dried to obtain the Cherry Blossom Root Extract with anti-inflammatory effect.

[0016] The present invention first uses an alcohol solvent to extract the components in the root of golden cherry, so that the anti-inflammatory active ingredients such as flavonoids in the root of golden cherry can be better extracted from the crude drug. Subsequently, the alcohol extract is treated with a nano-ceramic membrane with a specific pore size. The nano-ceramic membrane with a specific pore size 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 golden cherry root extract. Further, the present invention uses an ultrafiltration membrane with a specific pore size for further filtration. The ultrafiltration membrane with a specific pore size can effectively filter out the anti-inflammatory active ingredients such as flavonoids in the root of golden cherry, and can further remove other non-anti-inflammatory active substances in the root of golden cherry, such as polysaccharides, proteins and other substances. The selective separation of anti-inflammatory active ingredients such as total flavonoids in the root of golden cherry can be achieved by combining the nano-ceramic membrane with the above-mentioned specific pore size and the ultrafiltration membrane. In addition, the combination of nano-ceramic membrane + ultrafiltration membrane can be used to perform two-stage screening and separation of the alcohol extract, which not only avoids the problem of macromolecular substances clogging the membrane pores and low extraction efficiency caused by direct 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.

[0017] 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 root extract of golden cherry. The inventors found through research that, compared with other conventional preparative columns, the present invention uses a specific preparative column filler: C18YE filler, which has a very strong adsorption capacity for anti-inflammatory active ingredients such as flavonoids in the extract, thereby playing an excellent enrichment and capture role.

[0018] Through the optimization of the above process and in combination with other process conditions, the extract of the root of golden cherry extracted by the extraction method has excellent therapeutic effect on gynecological diseases. The root of golden cherry 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. In addition, the root of golden cherry extracted by the extraction method of the present invention also has a good solid recovery rate, which is as high as more than 90%.

[0019] Preferably, in the step (1), the alcohol extraction is: mixing the root of Sakura rosa-sinensis with ethanol, and extracting in a water bath to obtain the root of Sakura rosa-sinensis alcohol extract.

[0020] More preferably, in step (1), the alcohol solvent is removed by evaporation and concentration. More specifically, the evaporation and concentration temperature is 60 to 70°C and the vacuum degree is -0.12 to -0.08 MPa. More preferably, the evaporation and concentration temperature is 65°C and the vacuum degree is -0.095 MPa.

[0021] Preferably, the volume concentration of the ethanol is 70-90%. More preferably, the volume concentration of the ethanol is 70%.

[0022] Preferably, the temperature of the water bath is 80-90° C. More preferably, the temperature of the water bath is 85° C.

[0023] Preferably, the solid-liquid ratio of the root of golden cherry to ethanol is 1:8 to 10. Further preferably, the solid-liquid ratio of the root of golden cherry to ethanol is 1:8.

[0024] Preferably, in step (2), the pore size of the nano-ceramic membrane is 300-500 nm. More preferably, the pore size of the nano-ceramic membrane is 500 nm.

[0025] Preferably, in step (2), the pore size of the ultrafiltration membrane is 40-50 KD. Further preferably, the pore size of the ultrafiltration membrane is 50 KD.

[0026] Preferably, in step (4), the concentration temperature is 40 to 50°C, and the concentration vacuum is -0.15 to -0.05Mpa. More preferably, the temperature is 45°C, and the vacuum is -0.08Mpa.

[0027] Preferably, in step (4), the drying is freeze-drying. More specifically, the freeze-drying temperature is -18 to -20°C.

[0028] Preferably, in step (3), the specifications of the preparation column C18YE filler are 30um, 100A, and 1.5kg.

[0029] Preferably, in step (3), the ethanol concentration during elution is 60-70%.

[0030] Furthermore, the present invention also claims protection for a method for extracting a Rosa rugosa root extract having anti-inflammatory effects.

[0031] Furthermore, the present invention also seeks to protect the use of the above-mentioned Cherry Blossom Root Extract in the preparation of drugs for anti-inflammatory or treatment of gynecological diseases.

[0032] Furthermore, the above-mentioned golden cherry root extract can achieve the purpose of anti-inflammatory or treatment of gynecological diseases by reducing the level of inflammatory factors in serum, or by reducing the level of transforming growth factor and vascular endothelial growth factor. More specifically, the inflammatory factors are TNF-α, IL-1β, IL-18, IL-6; the transforming growth factor is TGF-β1; and the vascular endothelial growth factor is VEGF.

[0033] Preferably, 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.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention provides a method for extracting a root extract of golden cherry with anti-inflammatory effect, and the extraction method can improve the anti-inflammatory activity of the root extract of golden cherry. Compared with the gynecological Qianjin capsule, the root extract of golden cherry can not only better reduce the damage of the spleen, thymus and uterus; but also better reduce the levels of inflammatory factors, transforming growth factor and vascular endothelial growth factor. The root extract of golden cherry obtained by the extraction method has a better effect on improving gynecological diseases such as chronic pelvic inflammatory disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The sample status diagram is 500nm-clarified liquid, 500nm-retentate, 50KD-clarified liquid, and 50KD-retentate.

[0037] Figure 2 Prepare chromatograms for the liquid phases of the loading solution, eluent, elution solution, and regeneration solution.

[0038] Figure 3 This is a sample status diagram of 50KD-clarified liquid, loading liquid, eluent, elution liquid and regeneration liquid.

[0039] Figure 4 This is the full-wavelength PDA detection spectrum.

[0040] Figure 5 It is the liquid chromatogram of the extract, alcohol removal solution, 500nm-clarified solution, 50KD-clarified solution, 50KD-retentate solution, loading solution, eluent, elution solution, regeneration solution and freeze-dried eluent.

[0041] Figure 6 It is the characteristic peak quantity transfer diagram of each chromatographic peak in Example 1.

[0042] Figure 7 The following are the observation pictures of the uterine morphology of rats in each experimental group.

[0043] Figure 8This is the effect of each experimental group on the spleen, thymus and uterus indexes of CPID model rats.

[0044] Fig. 9 These are the results of HE staining of the uterus of rats in each experimental group.

[0045] Fig.10 This is the effect of each experimental group on the concentration of inflammatory factors in the serum of CPID model rats.

[0046] Fig.11 This is the effect of each experimental group on the levels of TGF-β1 and VEGF in the serum of CPID model rats.

[0047] Fig.12 This is the liquid preparation chromatogram when PS-NVP is used as the liquid preparation filler.

[0048] Fig.13 This is a comparison chart of sample flowthrough when PS-NVP is used as the liquid phase to prepare the filler. DETAILED DESCRIPTION

[0049] 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.

[0050] Example 1 A method for extracting a rosa oleifera root extract having anti-inflammatory effects

[0051] 1. Preparation process:

[0052] (1) Pretreatment of medicinal materials: The root of Golden Cherry was crushed. The relevant data and information of the root of Golden Cherry are shown in Table 1.

[0053] Table 1

[0054] sample Weighing mass (g) batch number Suppliers Golden cherry root medicinal material 1000.0 Y007-2101001 Qianjin Pharmaceutical

[0055] (2) Extraction: 1 kg of the crushed Cherry Blossom Root medicinal material was heated in a water bath with 70% ethanol by volume to a slight boiling temperature (about 85°C) for extraction. The extraction was continued for 1 hour under slight boiling conditions, and the extraction was performed 3 times in total. Among them, the solid-liquid ratio (kg / L) of the Cherry Blossom Root medicinal material and ethanol was 1:10 to obtain an extract. The relevant data and information of the extraction steps are shown in Table 2 below.

[0056] Table 2

[0057] Extraction solution batch number Extraction volume (L) Sample volume (mL) Solid content (mg / mL) Extraction rate (%) JYG-TQ-210727 26.8 50 5.15 13.8

[0058] (3) Alcohol removal: 26.8 L of the extract of batch number JYG-TQ-2010727 was placed in a 20 L rotary evaporator at 65 ° C and a vacuum degree of -0.095 MPa, and concentrated to 7 L (without alcohol taste). Then, water was added to dilute to 14 L to obtain a concentrated solution of batch number JYG-NS-210728 (the relevant data and information of the alcohol removal step are shown in Table 3 below).

[0059] Table 3

[0060]

[0061] (4) Nano-ceramic membrane filtration: 14.0 L of concentrated liquid was passed through a 500 nm ceramic membrane, and 4.5 L of pure water was added for top washing, a total of 3 times, 1.5 L each time, and 2.0 L of 10% ethanol was used for top washing. A total of 20.25 L of 500 nm-clarified liquid with batch number JYG-500nm-CQ and 1.03 L of 500 nm-retentate with batch number JYG-500nm-JL were collected; the membrane filtration operation steps, membrane filtration step-related data and information are shown in Tables 4 and 5, respectively.

[0062] Table 4

[0063] time Pressure (Br) Flux (mL / min) Remark 12:12 / 1200 Pure water flux 12:17 0.25 650 / 12:22 0.25 650 / 12:27 0.25 650 / 12:32 0.25 650 / 12:37 0.23 650 Add 1.5L of water 12:42 0.20 600 Add 1.5L of water 12:47 0.20 600 Add 1.5L of water 12:32 0.1 550 Add alcohol 2.0L 12:41 0 0 Finish

[0064] Table 5

[0065] batch number Volume(L) Sample volume (mL) Solid content (mg / mL) JYG-500nm-CQ 20.25 50 5.5 JYG-500nm-JL 1.03 50 8.2

[0066] (5) Ultrafiltration membrane filtration: 20.25 L of 500 nm-clarified liquid of batch number JYG-500 nm-CQ was filtered through a 50 KD membrane and top washed with 2 L of pure water. A total of 20.25 L of 50 KD-clarified liquid of batch number JYG-50KD-CQ and 2.5 L of 50 KD-retentate of batch number JYG-50KD-JL were collected. The data and information related to the ultrafiltration membrane filtration operation steps are shown in Tables 6 and 7 below, respectively.

[0067] Table 6

[0068]

[0069]

[0070] Table 7

[0071] batch number Volume(L) Sample volume (mL) Solid content (mg / mL) JYG-50KD-CQ 20.25 50 3.8 JYG-50KD-JL 2.5 50 9.8

[0072] (6) Enrichment and capture preparation: 20.25 L of 50KD-clarified liquid with batch number JYG-50KD-CQ was taken and liquid phase preparation was performed using a high performance preparative liquid chromatography system. During the preparation process, the process pressure was controlled to be <5 MPa. The process steps of the liquid phase preparation are shown below, and the method is shown in Table 8. In addition, the chromatographic column filler for the liquid phase preparation was C18YE filler (specifications: 30 um, 100 A, 1.5 kg). Figure 1 The sample status diagram is 500nm-clarified liquid, 500nm-retentate, 50KD-clarified liquid, and 50KD-retentate. Figure 2 Prepare chromatograms for the liquid phases of the loading solution, eluent, elution solution, and regeneration solution.

[0073] ① Loading: Take 20.3 L of 50KD-clarified liquid with batch number JYG-50KD-CQ for loading to obtain loading liquid;

[0074] ② Elution: Use pure water for elution, 3BV, to obtain the eluent;

[0075] ③ Elution: 60% ethanol was used for elution, 4 BV, and the eluent was obtained;

[0076] ④ Regeneration: Use 90% ethanol for regeneration, 3BV, to obtain regeneration solution.

[0077] Figure 3 This is a sample status diagram of 50KD-clarified liquid, loading liquid, eluent, elution liquid and regeneration liquid.

[0078] Table 8

[0079]

[0080]

[0081] (7) Liquid phase analysis

[0082] ①Instrument: High performance liquid chromatography analyzer

[0083] ②Sample: the whole process (extracting solution, alcohol removal solution, 500nm-clarified solution, 50KD-clarified solution, 50KD-retentate solution, loading solution, eluent, eluent, regeneration solution, freeze-dried eluent)

[0084] ③Analytical column: Tnature C18 (4.6*250mm, 5um, 100A)

[0085] ④Injection volume: 10uL

[0086] ⑤Mobile phase: A: chromatographic acetonitrile (ACN); B: 0.1% phosphoric acid water

[0087] ⑥Analysis method:

[0088] Time V(ml / min) A％ B％ 0 1.5 5 95 5 1.5 10 90 35 1.5 20 80 40 1.5 30 70 50 1.5 90 10

[0089] Figure 4 This is the full-wavelength PDA detection spectrum. Figure 5 It is the liquid chromatogram of the extract, alcohol removal solution, 500nm-clarified solution, 50KD-clarified solution, 50KD-retentate solution, loading solution, eluent, elution solution, regeneration solution and freeze-dried eluent.

[0090] (8) Concentration and freeze-drying:

[0091] ① Concentration: Combine the prepared sample solution and eluent, and concentrate the combined solution, eluent, and regeneration solution under reduced pressure at 45°C and a vacuum degree of -0.08Mpa to a volume of about 200ml.

[0092] ② Freeze-drying: freeze the three portions of concentrated solution in a -40°C refrigerator, put them into a freeze dryer after they are frozen, and perform freeze-drying at -18 to -20°C, wherein the freeze-dried eluent is the extract of the root of golden cherry.

[0093] 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 solid-liquid ratio of golden cherry root and ethanol is 1:8-10, and the temperature of the water bath is 80-90℃.

[0094] Example 2 Determination of solid mass of components in each step of the extraction method of the root extract of golden cherry

[0095] (1) Solid mass determination method: Take 10 ml of each sample of the extract, concentrate, 500 nm-clarified solution, 500 nm-retentate, 50 KD-clarified solution, 50 KD-retentate, loading solution, eluent, elution solution, and regeneration solution obtained in Example 1, dry in an evaporating dish to measure the solid mass, and convert the solid mass in the total volume. The solid mass is directly measured for the lyophilized powder after combining the loading solution + eluent, the lyophilized powder of the elution solution, and the lyophilized powder of the regeneration solution. During the preparation process of the golden cherry root extract, the mass data of the components prepared in each step are shown in Table 9 below.

[0096] Table 9

[0097]

[0098] Example 3 Characteristic peak value transfer of each step in the extraction method of the root extract of golden cherry

[0099] The diluent, 500nm-clarified liquid, 50KD-clarified liquid, 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.

[0100] Instrument: High performance liquid chromatography analyzer

[0101] Analytical column: Tnature C18 (4.6*250mm, 5um, 100A)

[0102] Injection volume: 10uL

[0103] Mobile phase: A: chromatographic acetonitrile (ACN); B: 0.1% phosphoric acid water

[0104] Analytical methods:

[0105] Time V(ml / min) A％ B％ 0 1.5 5 95 5 1.5 10 90 35 1.5 20 80 40 1.5 30 70 50 1.5 90 10

[0106] Figure 6 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 Golden Sakura Root is shown in Table 10 below.

[0107] Table 10

[0108]

[0109] Depend on Figure 6 From the results in the table above, we can see that:

[0110] ① The transfer rates of each characteristic peak of the golden cherry root dilution-500nm clarification process were all greater than 90%.

[0111] ②The chromatographic peak transfer rate of the 50KD clarification process is not high. You can consider filtering the 50KD retentate and combining it with the clarified liquid for preparation.

[0112] ③ The transfer rate of each characteristic peak of the preparation process is 88%-142%, and the high-efficiency preparation process is generally stable.

[0113] Judging from the quality data, in the current process, the recovery rate of each process stage can reach more than 90%. In the fraction processing process, the rotary evaporation of the preparation elution part is more troublesome, and large swollen bubbles hanging on the wall are easily generated in the rotary evaporation bottle, which is speculated to be related to the saponin components of the root of golden cherry. Overall, the preparation process of the single-flavor medicinal component of the root of golden cherry is stable. Impurities or insoluble substances are separated by pre-treatment methods such as centrifugation and clarification. The preparation process is repeatable and controllable, and can be scaled up.

[0114] Example 4 Pharmacological efficacy test of Cherry Blossom Root Extract

[0115] 1. Experimental Materials

[0116] (1) Experimental animals:

[0117] 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.

[0118] (2) Drugs:

[0119] 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.; Golden Cherry Root Extract was prepared and provided by Zhuzhou Qianjin Pharmaceutical Co., Ltd. according to Example 1. Each drug was made into an aqueous solution of a certain concentration with ultrapure water and administered intragastrically.

[0120] 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 McFarland turbidimeter tube (Yuezhu Medical Equipment No. 20140010, Zhuhai Beso Biotechnology Co., Ltd.), hemostatic sponge (Gan Medical Equipment No. 20182640140, Guangzhou Kuaikang Medical Equipment 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).

[0121] High-speed refrigerated centrifuge (Microfuge 20R, Beckman Coulter, Inc., USA), single-channel pipette (10-1000ul, Eppendorf), multi-function microplate reader (2021005559, BIOTEK / Synergy).

[0122] 2. Experimental methods

[0123] 2.1 Establishment of chronic pelvic inflammatory disease (CPID) model

[0124] Model rat construction: 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, the rats' vaginal openings were observed for congestion, redness, and swelling, and the estrus period was observed. After the estrus cycle of the rats was induced, 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 rat uterus 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.

[0125] The successfully modeled rats were divided into 4 groups according to the random number table method: model group, Jygina chinensis root extract (JYG) group, Kangfuyan capsule (KFY) group, and Fuke Qianjin capsule (FKQJ) group.

[0126] 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.

[0127] Blank group: The blank group used normal mice without treatment.

[0128] 2.2 Grouped Dosing

[0129] Golden cherry root extract group: dosage 310.93 mg kg -1 ·d -1 The model rats were given extracts from the roots of Cherry Blossom;

[0130] Kangfuyan capsule group: dosage 324.002 mg·kg -1 ·d -1 The model rats were given Kangfuyan capsules;

[0131] Gynecological Qianjin Capsule Group: dosage: 216.001 mg·kg -1 ·d -1 Gynecological Qianjin Capsule was administered to the model rats;

[0132] 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;

[0133] Sham operation group: Rats in the sham operation group were given the same amount of distilled water as the model group;

[0134] Blank group: The blank group was given the same amount of distilled water as the model group;

[0135] Each group was continuously administered with drug (or distilled water) for 21 days.

[0136] 2.3 Sample collection

[0137] 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.

[0138] 2.4 Body weight and organ index of rats

[0139] 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.

[0140] 2.5 Gross morphology and histopathological examination of rat uterus

[0141] 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, and the slices were dewaxed by xylene, dehydrated by alcohol gradient, and HE stained. After sealing with neutral gum, the pathological changes of rat uterine tissues were observed under a microscope. 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.

[0142] 2.6 Detection of TNF-α, IL-1β, IL-18, IL-6, TGF-β1 and VEGF levels in rat serum

[0143] 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.

[0144] 2.7 Statistical analysis

[0145] 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.

[0146] (3) Results

[0147] 3.1 Observation of rat uterine morphology

[0148] 21 days after modeling, the uteri of rats in the blank group, sham operation group, and model group were randomly selected to observe the morphology of the uteri with the naked eye. Figure 7 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 rats in the sham operation group, and the size, shape and color are normal. The uterus of rats in the model group is 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.

[0149] After the treatment, the abdominal cavity was opened and the pelvic condition was observed. The results showed that there was no obvious abnormality in the uterus and pelvic cavity of the blank group rats. Although there was a small amount of adhesion between the pelvic organs and the surrounding tissues of some rats in the sham operation group, there was no bleeding; there was no statistical difference between the blank group and the sham operation group. The model group showed swelling with pus accumulation inside the uterus, the uterine cavity tended to be transparent and edematous, and it adhered to the surrounding tissues and was difficult to separate. Compared with the model group, each drug group (JYG group, KFY group, FKQJ group) could significantly improve the pelvic adhesion of rats with pelvic inflammatory disease.

[0150] 3.2 Effects on spleen, thymus and uterus indexes in CPID model rats

[0151] Figure 8 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 sham operation 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 JYG group can significantly reduce the spleen, thymus and uterus indexes of the model rats, indicating that it significantly improves the damage to the immune function of the pelvic inflammatory disease model rats. In addition, the uterine index of the JYG group was also significantly lower than that of the FKQJ group, indicating that the extract of the golden cherry root has a significant effect on reducing uterine damage and regulating uterine swelling, and the effect is significantly better than that of the gynecological Qianjin capsule.

[0152] 3.3 Results of HE staining of rat uterus

[0153] Fig. 9The HE staining results of the uterus of rats in each experimental group are shown in the figure. The HE staining results show that the endometrium of the blank group is smooth and structurally intact, with only a small amount of neutrophil infiltration. Similarly, the uterine wall of the sham operation group is clearly layered; the endometrial glands are regular, the muscle fiber texture is clear; and the epithelial cells are arranged in a single layer of columns. In the model group, obvious neutrophil infiltration can be seen in the endometrium, severe tissue edema, and the endometrial thickness is significantly thinned. Inflammatory cells infiltrate, some of which penetrate deep into the muscle layer, and accumulate throughout the entire layer. In each drug-treated group (JYG group, KFY group, FKQJ group), the endometrial epithelial cells proliferate or fall off slightly, and the inflammatory cell infiltration is significantly reduced.

[0154] 3.4 Effects on the concentration of inflammatory factors in the serum of CPID model rats

[0155] Fig.10 The figure shows the effect of each experimental group on the concentration of inflammatory factors in the serum of CPID model rats. As shown in the figure, compared with the blank group, there was no significant difference in the levels of serum factors in the sham operation group (P>0.05), while the serum IL-6, TNF-α, IL-1β and IL-18 levels of the model group rats were significantly increased (P<0.01).

[0156] In addition to the positive control drug, compared with the model group, the JYG group can significantly reduce the concentration of various inflammatory factors in the serum of the model rats, indicating that it significantly improves the inflammatory response of the pelvic inflammatory disease model rats. In addition, the concentration of the inflammatory factor IL-1β in the JYG group was also significantly lower than that in the FKQJ group; the concentrations of the inflammatory factors IL-6 and TNF-α in the JYG group were comparable to those in the FKQJ group. This shows that the anti-inflammatory effect of the golden cherry root extract is superior to that of the Fuke Qianjin capsule.

[0157] 3.5 Effects on the levels of TGF-β1 and VEGF in the serum of CPID model rats

[0158] Fig.11 The effect of each experimental group on the levels of TGF-β1 and VEGF in the serum of CPID model rats. Transforming growth factor TGF-β1 and vascular endothelial growth factor (VEGF) are important factors regulating cell growth and differentiation, which can promote increased vascular permeability, extracellular matrix degeneration, endothelial cell migration, proliferation and angiogenesis, and have a great correlation with pelvic adhesion and tissue hyperplasia.

[0159] like Fig.11As 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 sham operation 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). In addition to the positive control drug, compared with the model group, the JYG group could significantly reduce the content of TGF-β1 and VEGF in the serum of the model rats; in addition, the levels of TGF-β1 and VEGF in the JYG group were equivalent to those in the FKQJ group.

[0160] Comparative test of different preparation processes

[0161] (1) The extraction method of the root extract of Sakura cerasifera in Example 1 is followed, except that in step (6), the preparative column filler used is PS-NVP.

[0162] Fig.12 This is the liquid preparation chromatogram when PS-NVP is used as the liquid preparation filler. Fig.13 This is a comparison chart of sample flow rate when PS-NVP is used as the liquid phase to prepare the filler. Fig.12 and Fig.13 As shown in the figure, according to the results of liquid phase analysis, when the sample load reaches 30%, some of the flavonoids of the root of golden cherry have already flowed through, and when the sample load reaches 40%, all of the flavonoids of the root of golden cherry have begun to flow through. Therefore, when the sample load does not exceed 30%, PS-NVP can be used for the preparation of the root of golden cherry; when the sample load exceeds 40%, all of the flavonoids of the root of golden cherry have begun to be lost, and the anti-inflammatory active ingredients in the root of golden cherry extract are limited.

[0163] (2) The method for extracting the root of Sakura cerasifera in Example 1 was followed, except that the enrichment and capture preparation in step (6) was not performed, and the 50KD- clarified liquid was directly concentrated and freeze-dried to obtain the root of Sakura cerasifera extract.

[0164] The golden cherry root extract prepared by this process step contains more protein, pigment, oil and other components because it does not use the enrichment and capture process. Under the same medication conditions, its effect on the uterus, spleen and thymus indexes is worse than that of the golden cherry root extract in Example 1, and its reduction of 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 golden cherry root extract in Example 1.

[0165] (3) The extraction method of the root of golden cherry in Example 1 is followed, except that the ultrafiltration membrane filtration in step (5) is not performed, and the 500 nm-clarified liquid is directly enriched and captured for preparation.

[0166] The golden cherry root extract prepared by this process step contains more polysaccharides, proteins and other macromolecular substances 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 C18YE 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 golden cherry root 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 golden cherry root extract in Example 1.

[0167] (4) The extraction method of the root of Cherry Blossom in Example 1 is the same, except that in step (5), the pore size of the ultrafiltration membrane used is 100 KD.

[0168] The golden cherry root extract prepared by this process step also contains a certain amount of polysaccharides, proteins and other macromolecular substances. Although a specific preparation column was used for enrichment and capture, some other substances that can be combined with C18YE 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 golden cherry root extract in Example 1. 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 golden cherry root extract in Example 1, and is better than the golden cherry root extract in (3) above.

[0169] 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 a root extract of Cherry Blossoms with anti-inflammatory effect, characterized in that: The following steps are involved: (1) Alcohol extraction: extract the root of Rosa rugosa with alcohol, and then remove the alcohol solvent; (2) Filtration: The product obtained in step (1) is filtered using a nano-ceramic membrane and an ultrafiltration membrane in sequence to obtain a clarified liquid; the pore size of the nano-ceramic membrane is 30 to 500 nm; the pore size of the ultrafiltration membrane is 5 to 50 kD; (3) Enrichment and capture: The clarified liquid is enriched and captured by using a high performance preparative liquid chromatography system: the clarified liquid is loaded with a sample, then washed with pure water, eluted with ethanol, and the liquid eluted with ethanol is collected to obtain an eluent; the preparation column used in the high performance preparative liquid chromatography system is a C18YE filler; (4) The eluate is concentrated and dried to obtain the Cherry Blossom Root Extract with anti-inflammatory effect.

2. The extraction method according to claim 1, characterized in that In the step (1), the alcohol extraction is as follows: mixing the root of Sakura rosa-sinensis with ethanol, and extracting the mixture in a water bath to obtain the alcohol extract of the root of Sakura rosa-sinensis.

3. The extraction method according to claim 2, characterized in that The volume concentration of the ethanol is 70-90%.

4. The extraction method according to claim 3, characterized in that: The temperature of the water bath is 80-90°C.

5. The extraction method according to claim 4, characterized in that: The solid-liquid ratio of the golden cherry root to ethanol is 1:8-10.

6. The extraction method according to claim 1, characterized in that: In the step (2), the pore size of the nano-ceramic membrane is 300 to 500 nm.

7. The extraction method according to claim 1 or 6, characterized in that In the step (2), the pore size of the ultrafiltration membrane is 40 to 50 KD.

8. The extraction method according to claim 1, characterized in that In the step (4), the drying is freeze-drying.

9. The extract of the root of Cerasus cerasus with anti-inflammatory effect obtained by the method for extracting the root of Cerasus cerasus with anti-inflammatory effect according to any one of claims 1 to 8.

10. Use of the extract of the root of Cherry Blossom in claim 9 in the preparation of drugs for anti-inflammatory or treatment of gynecological diseases.

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