A saponin HZ extracted from twelve hours of she medicine and its application in treating ulcerative colitis

Abstract

The present application relates to a kind of saponins HZ extracted from shi medicine twelve hours and its preparation method and application.Twelve hours saponins HZ is prepared using the following method: with shi medicine twelve hours as raw material, using impregnation extraction, extraction, microporous resin MCI column chromatography separation to obtain extract Fr.B4, extract Fr.B4 is purified by Sephadex LH-20, obtain FrB4-1~FrB4-6 6 components, component FrB4-4 is separated by semi-preparative high performance liquid chromatography (CH3CN:H2O=30:70, 3.0mL / min) to obtain twelve hours saponins HZ.Twelve hours saponins HZ has significant effect in the treatment of ulcerative colitis, and can be further used for developing safe, effective, controllable traditional Chinese medicine anti-inflammatory, ulcerative colitis new drug.

Description

Technical Field

[0001] This invention relates to a saponin HZ extracted from the twelve hours of She medicine, its preparation method, and its application in a drug for treating ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a nonspecific, chronic inflammatory bowel disease affecting the mucosa and submucosa of the rectum and colon. The main clinical symptoms include abdominal pain, diarrhea, and bloody or purulent stools. Current research does not fully elucidate the pathogenesis of UC, but it is generally believed to be related to environmental diversity, genetic susceptibility, immune abnormalities, and inflammatory factors. UC is characterized by its long duration, recurrent episodes, and extensive lesions, and it is highly prevalent in young adults, making it considered one of the most difficult-to-treat diseases in modern society. With changes in modern lifestyles, the incidence of UC is gradually increasing globally.

[0003] Currently, the main treatments for ulcerative colitis (UC) include medication and surgery. Surgical treatment is often used in the later stages of UC and carries a high risk of mortality. Medication is the most common treatment for UC, and the main drugs include aminosalicylic acid derivatives, glucocorticoids, immunosuppressants, antibacterial and anti-infective drugs, and biologics. However, the efficacy of these drugs is unstable, most only provide symptomatic relief, have many adverse reactions, limited applicability, and some drugs, such as biologics, are expensive and cannot be used long-term. Therefore, finding a drug that is accurate, safe, has few adverse reactions, is affordable, and reliable is of significant value.

[0004] my country boasts abundant resources of traditional Chinese medicine products. Screening for novel bioactive compounds from natural plants or exploring new biological applications of discovered compounds has become an important approach to new drug development. *Clematis florida* Thumb. var. *plena* D. Don, a plant in the Ranunculaceae family, is a dried tuberous root commonly used by the She ethnic minority in Fu'an, Ningde, Fujian Province. In She medicine, it is called "Twelve Hours" and has long been used to treat injuries and swelling, possessing the effects of promoting blood circulation, dispelling wind and dampness, and relieving pain. In July 2018, the applicant of this invention applied for a patent concerning an extract of *Clematis florida* Thumb., Fr.B4, its preparation method, and its application. This patent utilizes maceration extraction, extraction, and microporous resin MCI column chromatography to obtain the extract Fr.B4. This extract Fr.B4 has therapeutic effects on rheumatoid arthritis and can be used in the preparation of drugs for treating rheumatoid arthritis. However, the extract Fr.B4 is a mixture of multiple compounds, each with different pharmacological effects and functions. Further research and refinement by those skilled in the art are needed to obtain natural bioactive components with different pharmacological effects.

[0005] Twelve-Hour Saponin HZ is a natural bioactive component extracted from the Twelve-Hour Saponin of She Medicine, which has multiple functions such as anti-inflammatory and antioxidant effects. Summary of the Invention

[0006] The purpose of this invention is to provide a saponin HZ extracted from the Twelve Hours of She Medicine, its extraction method, and its application in a drug for treating ulcerative colitis. Because this saponin is mentioned multiple times in the specification of this invention, for ease of writing, the inventors have named the target saponin Twelve Hours Saponin HZ.

[0007] The objective of this invention is achieved through the following technical solution: a method for extracting twelve-hour saponins HZ from She medicine, comprising the following steps:

[0008] (1) Preparation of maceration extract: Using She medicine twelve-hour period as raw material, She medicine twelve-hour period is macerated and extracted with polar solvent to obtain maceration extract;

[0009] (2) Preparation of crude extract: Disperse the impregnated extract in water to obtain crude extract;

[0010] (3) Preparation of total saponin extract: The crude extract was extracted sequentially with petroleum ether, ethyl acetate and n-butanol, and then the n-butanol extracts were combined and the n-butanol was removed by vacuum distillation to obtain total saponin extract.

[0011] (4) Preparation of extract Fr.B4: The total saponin extract was separated by microporous resin MCI column chromatography with methanol-water as the eluent for gradient elution. The eluted fraction of Fr.B4 with a mass fraction of 70% methanol was collected, concentrated under reduced pressure and dried to obtain the extract Fr.B4.

[0012] (5) Preparation of Twelve-Hour Saponin HZ: The extract Fr.B4 was purified by Sephadex LH-20 to obtain 6 components FrB4-1 to FrB4-6. Component FrB4-4 was separated by semi-preparative high performance liquid chromatography (CH3CN:H2O=30:70, 3.0mL / min) to obtain Twelve-Hour Saponin HZ.

[0013] The polar solvent is one or a mixture of methanol, ethanol, or water.

[0014] The specific operation method of step (1) is as follows: using She medicine twelve hours as raw material, after crushing She medicine twelve hours, add 6 to 8 times the weight of raw material in methanol or ethanol for maceration and extraction according to the weight-volume ratio. Extract for a total of 3 times, each time for 7 days. After that, combine the extracts, recover the solvent, and obtain the macerated extract.

[0015] The specific operation method of step (2) is as follows: disperse the maceration extract in water at a weight-volume ratio of 1 to 5 times to obtain crude extract.

[0016] The specific operation method of step (3) is as follows: the crude extract is first extracted with petroleum ether three times and the aqueous phase is collected; then the aqueous phase is extracted with ethyl acetate three times, and the aqueous phase after ethyl acetate extraction is extracted with n-butanol three times. The n-butanol extracts are combined and the n-butanol is recovered under reduced pressure to obtain the total saponin extract.

[0017] The specific operation method of step (4) is as follows: the total saponin extract is subjected to microporous resin MCI column chromatography, and water, 20% methanol, 50% methanol, 70% methanol, 90% methanol and methanol are used as eluents in sequence for gradient elution. The elution fraction Fr.B4 in 70% methanol is collected, concentrated under reduced pressure and dried to obtain extract Fr.B4.

[0018] The specific operation method for step (5) is as follows:

[0019] The extract Fr.B4 was subjected to Sephadex LH-20 column chromatography with methanol as the eluent. Each 50 mL sample was collected, concentrated under reduced pressure, and the same samples were combined to obtain 6 samples from FrB4-1 to FrB4-6. The sample FrB4-4 was separated by semi-preparative high performance liquid chromatography to obtain the compound Twelve-Hour Saponin HZ.

[0020] The solvent used in the semi-preparative high-performance liquid chromatography method is a mixture of CH3CN and H2O, with a volume ratio of CH3CN to H2O of 30:70, and the flow rate is set to 3.0 mL / min.

[0021] The present invention also provides a twelve-hour saponin HZ prepared by the method described above.

[0022] The application of the aforementioned twelve-hour saponin HZ in the preparation of drugs for treating ulcerative colitis.

[0023] Animal experiments have shown that the above-mentioned twelve-hour saponin HZ has a significant therapeutic effect on experimental ulcerative colitis. The experimental methods and results are as follows:

[0024] SPF-grade C57BL / 6 mice were used in the experiment. Appropriate numbers of mice were randomly selected as the normal control group, model group, 5-aminosalicylic acid treatment group, and low and high dose groups of Twelve-Times-Saponin HZ. The normal control group had free access to purified water, while the other groups were induced to develop the ulcerative colitis model with 2.5% sodium dextran sulfate (DSS). Appropriate doses were administered via gavage. General conditions such as body weight and feces were observed. The DAI disease activity index was scored, and the levels of inflammatory cytokines, colon length and mass were measured. Colon tissue histopathological sections were examined. Data were processed using statistical methods. Results showed that Twelve-Times-Saponin HZ could improve the disease activity index in animals with ulcerative colitis, reverse colon length shortening, restore normal intestinal barrier permeability, and normalize cytokine levels in colon tissue. HE staining and light microscopic observation of mouse colon tissue sections showed that Twelve-Times-Saponin HZ could reduce the degree of intestinal damage in mice with ulcerative colitis.

[0025] In this invention, the preparation of pharmaceutical formulations involves adding appropriate excipients to the above-mentioned Twelve Time Saponins HZ to prepare solid dosage forms such as tablets, capsules, granules, lozenges, chewable tablets, and dispersible tablets, as well as semi-solid dosage forms such as gels. It can also be prepared as liquid dosage forms such as oral liquids and compound preparations.

[0026] Compared with existing technologies, the advantages of this invention are as follows: the twelve-hour saponin HZ obtained by the method provided by this invention has a significant effect in treating ulcerative colitis and can be further used to develop safe, effective, and controllable new traditional Chinese medicine anti-inflammatory and anti-ulcerative colitis drugs. The refined twelve-hour saponin HZ can be used to prepare drugs of various dosage forms, and has the advantages of convenient detection, small dosage, convenient administration, and high efficacy, thus possessing broad market prospects. Attached Figure Description

[0027] Figure 1 This is a structural diagram of the twelve-hour saponin HZ isolated in Example 1.

[0028] Figure 2This is the 1H NMR spectrum of saponin HZ in Example 3.

[0029] Figure 3 This is the carbon NMR spectrum of saponin HZ in Example 3.

[0030] Figure 4 This is the NMR mass spectrum of saponin HZ in Example 3.

[0031] Figure 5 This is a graph showing the changes in mouse weight in Example 4.

[0032] Figure 6 This is a graph showing the DAI disease activity index score in Example 4.

[0033] Figure 7 This is a diagram showing the results of colon length measurement in mice in Example 4.

[0034] Figure 8 This is a graph showing the results of the mouse serum inflammatory cytokine assay in Example 4.

[0035] Figure 9 This is a graph showing the results of the determination of antioxidant biochemical indicators in mouse serum in Example 4.

[0036] Figure 10 This is a diagram showing the results of a mouse colon tissue pathological examination in Example 4.

[0037] Figure 11 This is a diagram showing the effects of NF-κB signaling pathway proteins in mouse colon tissue in Example 4.

[0038] Figure 12 This is an immunofluorescence analysis diagram of NF-κB in mouse colon tissue from Example 4. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:

[0040] A method for extracting twelve-hour saponins HZ from She medicine, comprising the following steps:

[0041] (1) Preparation of maceration extract: Using She medicine twelve-hour period as raw material, She medicine twelve-hour period is macerated and extracted with polar solvent to obtain maceration extract;

[0042] (2) Preparation of crude extract: Disperse the impregnated extract in water to obtain crude extract;

[0043] (3) Preparation of total saponin extract: The crude extract was extracted sequentially with petroleum ether, ethyl acetate and n-butanol, and then the n-butanol extracts were combined and the n-butanol was removed by vacuum distillation to obtain total saponin extract.

[0044] (4) Preparation of extract Fr.B4: The total saponin extract was separated by microporous resin MCI column chromatography with methanol-water as the eluent for gradient elution. The eluted fraction of Fr.B4 with a mass fraction of 70% methanol was collected, concentrated under reduced pressure and dried to obtain extract Fr.B4.

[0045] (5) Preparation of Twelve-Hour Saponin HZ: Fr.B4 was separated using Sephadex LH-20 with methanol as the eluent to obtain 6 components from FrB4-1 to FrB4-6. Component FrB4-4 was separated using semi-preparative high-performance liquid chromatography (CH3CN:H2O=30:70, 3.0mL / min) to obtain Twelve-Hour Saponin HZ.

[0046] The polar solvent is one or a mixture of methanol, ethanol, or water.

[0047] The specific operation method of step (1) is as follows: using She medicine twelve hours as raw material, after crushing She medicine twelve hours, add 6 to 8 times the weight of raw material in methanol or ethanol for maceration and extraction according to the weight-volume ratio. Extract for a total of 3 times, each time for 7 days. After that, combine the extracts, recover the solvent, and obtain the macerated extract.

[0048] The specific operation method of step (2) is as follows: disperse the maceration extract in water at a weight-volume ratio of 1 to 5 times to obtain crude extract.

[0049] The specific operation method of step (3) is as follows: the crude extract is first extracted with petroleum ether three times and the aqueous phase is collected; then the aqueous phase is extracted with ethyl acetate three times, and the aqueous phase after ethyl acetate extraction is extracted with n-butanol three times. The n-butanol extracts are combined and the n-butanol is recovered under reduced pressure to obtain the total saponin extract.

[0050] The specific operation method of step (4) is as follows: the total saponin extract is subjected to microporous resin MCI column chromatography, and water, 20% methanol, 50% methanol, 70% methanol, 90% methanol and methanol are used as eluents in sequence for gradient elution. The elution fraction of 70% methanol, Fr.B4, is collected, and then concentrated under reduced pressure and dried to obtain the extract Fr.B4.

[0051] The specific operation method of step (5) is as follows: extract Fr.B4 is subjected to Sephadex LH-20 column chromatography with methanol as the eluent. One 50 mL sample is collected, concentrated under reduced pressure, and the same sample is combined to obtain 6 samples from FrB4-1 to FrB4-6. The sample FrB4-4 is separated by semi-preparative high performance liquid chromatography (CH3CN:H2O=30:70, 3.0 mL / min) to obtain Twelve-Hour Saponin HZ.

[0052] The 20% methanol, 50% methanol, 70% methanol, and 90% methanol are respectively methanol aqueous solutions with a mass fraction of 20%, 50%, 70%, and 90%.

[0053] The present invention will be further described below with reference to specific embodiments:

[0054] Example 1:

[0055] The 12-hour root of Ningde Fu'an Shecaotang was purchased as raw material. After the raw material was crushed into coarse powder, 20 kg was taken and 150 L of methanol was added for extraction. The extraction was carried out 3 times, each time for 7 days. The extracts were combined and the methanol was recovered under reduced pressure to obtain 878 g of methanol-impregnated extract.

[0056] The obtained impregnation extract was dispersed in 1.2 L of water to obtain a crude extract.

[0057] The crude extract was first extracted three times with petroleum ether, 1.2 L each time. The aqueous phase was then extracted three times with ethyl acetate, 1.2 L each time. The aqueous phase after extraction was then extracted three times with n-butanol, 1.2 L each time. The n-butanol extracts were combined, and the solvent was recovered under reduced pressure to obtain 313.4 g of total saponin extract.

[0058] The obtained total saponin extract was subjected to microporous resin MCI column chromatography, with water, 20% methanol, 50% methanol, 70% methanol, 90% methanol, and methanol as eluents in sequence. The eluent fractions of each eluent were collected, wherein the eluent fraction of water was designated as Fr. B1, the eluent fraction of 20% methanol was designated as Fr. B2, the eluent fraction of 50% methanol was designated as Fr. B3, the eluent fraction of 70% methanol was designated as Fr. B4, the eluent fraction of 90% methanol was designated as Fr. B5, and the eluent fraction of methanol was designated as Fr. B6.

[0059] The elution fraction of Fr.B4 in 70% methanol was subjected to solvent recovery under reduced pressure and freeze-dried to obtain a brownish-black gel-like substance, which is the extract Fr.B4. The extract Fr.B4 weighed 48.1g and was subsequently tested to contain 90% triterpenoid saponins.

[0060] The obtained Fr.B4 fraction was subjected to Sephadex LH-20 column chromatography with methanol as the eluent. Each 50 mL fraction was collected, concentrated under reduced pressure, and the same fractions were combined to obtain 6 fractions from FrB4-1 to FrB4-6. The fraction FrB4-4 was separated by semi-preparative high performance liquid chromatography (CH3CN:H2O=30:70, 3.0 mL / min) to obtain 4.8 g of Twelve-Hour Saponin HZ.

[0061] Example 2:

[0062] The 12-hour root of Ningde Fu'an Shecaotang was purchased as raw material. After the raw material was crushed into coarse powder, 5 kg was taken and 35 L of 80% ethanol aqueous solution was added for soaking and extraction. The extraction was carried out 3 times, each time for 7 days. The extracts were combined and the solvent was recovered under reduced pressure to obtain 222 g of soaking extract from ethanol aqueous solution.

[0063] The obtained impregnation extract was dispersed in 0.3 L of water to obtain a crude extract.

[0064] The crude extract was first extracted three times with petroleum ether, 0.3 L each time. The aqueous phase was then extracted three times with ethyl acetate, 0.3 L each time. The aqueous phase after extraction was then extracted three times with n-butanol, 0.3 L each time. The n-butanol extracts were combined, and the solvent was recovered under reduced pressure to obtain 78.3 g of total saponin extract.

[0065] The obtained total saponin extract was subjected to microporous resin MCI column chromatography, with water, 20% methanol, 50% methanol, 70% methanol, 90% methanol, and methanol as eluents in sequence. The eluent fractions of each eluent were collected, wherein the eluent fraction of water was designated as Fr. B1, the eluent fraction of 20% methanol was designated as Fr. B2, the eluent fraction of 50% methanol was designated as Fr. B3, the eluent fraction of 70% methanol was designated as Fr. B4, the eluent fraction of 90% methanol was designated as Fr. B5, and the eluent fraction of methanol was designated as Fr. B6.

[0066] The elution fraction of Fr.B4 in 70% methanol was subjected to solvent recovery under reduced pressure and freeze-dried to obtain a brownish-black gel-like substance, which is the extract Fr.B4. The extract Fr.B4 weighed 12.1g and was subsequently tested to contain 89% triterpenoid saponins.

[0067] The obtained Fr.B4 fraction was subjected to Sephadex LH-20 column chromatography with methanol as the eluent. One 50 mL fraction was collected, concentrated under reduced pressure, and the same fractions were combined to obtain 6 fractions from FrB4-1 to FrB4-6. The fraction FrB4-4 was separated by semi-preparative high performance liquid chromatography (CH3CN:H2O=30:70, 3.0 mL / min) to obtain 1.2 g of Twelve-Hour Saponin HZ.

[0068] Example 3:

[0069] The compounds obtained in Examples 1 and 2, namely, Twelve-Hour Saponin HZ, were identified. Twelve-Hour Saponin HZ is a white amorphous powder. Since the NMR and mass spectra of Twelve-Hour Saponin HZ obtained in Examples 1 and 2 are similar, only the spectrum of Twelve-Hour Saponin HZ obtained in Example 1 is provided below:

[0070] NMR analysis:1 H-NMR(400MHz,pyridine-d5):6.60(1H,br.s),6.18(1H,d),5.94(1H,overlap),5.86(1H,overlap),5.35(1H,br.s),5.02(1H,m),4.91(1H,d),4 .74-4.81(3H,m,overlap),4.37-4.67(10H,m,overlap),4.15-4.30(9H,m,overlap),3.91-4.12(5H,m,overlap),3.83(1H,br.s),3.75(1H,d),3 .65(1H,m),3.12(2H,m,overlap),2.02-2.20(4H,m,overlap),1.89-1.97(3H,m,overlap),1.61-1.82(3H,m),1.59(3H,br.s),1.52(1H,m),1.48 (3H,br.s),1.33-1.45(4H,m),1.14-1.32(6H,m),1.28(3H,s),1.03-1.07(5H,m,overlap),0.99(3H,s),0.82(6H,s),0.76-0.81(5H,m,overlap).

[0071] 13 C-NMR(100MHz,pyridine-d5):176.4,143.8,122.8,105.8,104.7,103.9,102.1,100.8,95.1,88.4,80.4 ,77.9,77.9,77.7,76.4,75.9,75.2,74.9,74.7,73.3,73.2,72.2,72.2,72.0,71.9,71.3,70.0,69.8,69. 6,69.4,69.4,68.5,68.4,64.8,64.6,60.7,55.6,47.6,46.6,45.8,41.6,41.2,39.4,39.1,38.5,36.5,33.5,32.6,32.6,32.0,30.2,27.7,27.7,26.2,25.6,23.3,23.2,22.9,18.0,17.9,17.9,17.0,16.6,15.2.

[0072] Mass spectrometry analysis: ESI-MS (m / z): 1359.30 [M+Na] + The structural formula of the twelve-hour saponin HZ is as follows: Figure 1As shown, the 1H-NMR spectrum, 13C-NMR spectrum, and mass spectrum of the dodecahour saponin HZ are respectively as Figure 2 , 3 shown in Figure 4.

[0073] Example 4:

[0074] 1 Materials:

[0075] 1.1 Animals:

[0076] SPF-grade C57BL / 6 mice, male, 4 - 6 weeks old, body weight 18 - 22 g, were purchased from Wushi Laboratory Animal Trading Co., Ltd. The animal quality certificate number is: SCXK(Zhe)2019 - 0002. The whole process of animal experiment operation complied with the regulations of the Laboratory Animal Ethics Committee of Fujian Medical University (Animal Experiment Ethics Review Number: IACUC FJMU 2022 - 0025). They were housed in the SPF (specific pathogen-free) animal house of the Laboratory Animal Center of Fujian Medical University. Conditions: temperature (23 ± 2)°C, humidity (45 ± 2)%, and the experiment started after 3 days of adaptive feeding.

[0077] 1.2 Drugs and Reagents:

[0078] The dodecahour medicinal materials were purchased from Fuan Shecaotang in Ningde City, Fujian Province. The dodecahour saponin HZ used in this experiment was prepared by this laboratory. The dodecahour saponin HZ was the compound dodecahour saponin HZ obtained according to the method described in Example 1. 5-Aminosalicylic acid (5-ASA), purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a content of 99%, batch number: J1911040.

[0079] Dextran sulfate sodium (DSS, molecular weight: 35000 - 36000), purchased from MP Biomedicals, USA; mouse TNF-α, IL-10, IL-1β enzyme-linked immunosorbent assay kits, purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.; GSH, SOD, NO, and MDA detection kits (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.); universal tissue fixative, purchased from Wuhan Sevier Biotechnology Co., Ltd.

[0080] 1.3 Instruments:

[0081] 96-well culture plates (Corning Incorporated, USA); inverted microscope (Olympus, Japan); precision electronic balance (AND Corporation EK-182A, Japan); vertical (ultra) low-temperature freezer (Forma Scientific, USA); benchtop high-speed centrifuge (Beckman, USA); microplate reader (Thermo Scientific Multiskan FC, USA); electrophoresis apparatus (Beijing Liuyi Biotechnology Co., Ltd.); immunoblotting imaging system (Bio-Rad, USA); pathology slide machine and scanner (Leica Instruments, Shanghai).

[0082] 2 methods:

[0083] 2.1 Establishment and grouping of an acute colitis mouse model:

[0084] Fifty male C57BL / 6 mice were randomly divided into five groups: a normal control group, a model group, a 5-aminosalicylic acid (SAA) administration group, and low- and high-dose Twelve-Times-Saponin HZ administration groups, with ten mice in each group. A 2.5% DSS solution was prepared using ultrapure water. Except for the normal control group, all other groups were given free access to the 2.5% DSS solution for 7 consecutive days. The normal control group was given ultrapure water. After 7 days, all groups were given free access to ultrapure water. During the experiment, mouse weight, fecal characteristics, and fecal blood loss were observed and recorded daily. Fecal occult blood was detected using the benzidine method. Successful model establishment was considered when mice exhibited weight loss, diarrhea, fecal occult blood, or bloody stools.

[0085] 2.2 Animal administration:

[0086] From day 1 of modeling, mice in the 5-aminosalicylic acid group were administered 0.2 mL / 10 g of 25 mg / mL 5-aminosalicylic acid solution via gavage daily; mice in the low- and high-dose Twelve-Times Saponin HZ groups were administered 0.2 mL / 10 g of 5 mg / mL and 10 mg / mL Twelve-Times Saponin HZ solution via gavage daily, respectively; and mice in the normal and model groups were administered the same volume of 0.5% CMC-Na aqueous solution via gavage. Administered once daily at the same time for 10 consecutive days. The test drug was suspended in 0.5% CMC-Na.

[0087] 2.3 Observation indicators and detection methods:

[0088] 2.3.1 Animal fecal condition and weight measurement: The fecal condition of mice was observed and recorded daily, and their weight was measured once a day.

[0089] 2.3.2 Disease Activity Index (DAI) Assessment: The DAI of mice was assessed once a day according to the Disease Activity Index scoring criteria in the table below.

[0090] Table 1 DAI Scoring Criteria

[0091] Stool characteristics Body weight loss (%) bloody stool / occult blood Rating (points) normal none none 0 loose stool 6～10 Blood in feces 2 Between the two 11～15 Between the two 3 diarrhea >15 blood in stool 4

[0092] 2.3.3 Determination of inflammatory cytokine levels: At the end of the experiment, blood was collected from the orbital cavity. After the collected blood was allowed to stand for 30 min, it was centrifuged at 3000 rpm for 15 min to separate the serum. The levels of tumor necrosis factor (TNF-α), interleukin-10 (IL-10), and interleukin-1 (IL-1β) in the serum were determined using double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) according to the kit's operating requirements and procedures.

[0093] 2.3.4 Determination of antioxidant biochemical indicators: At the end of the experiment, blood was collected from the orbital cavity. After the collected blood was allowed to stand for 30 minutes, it was centrifuged at 3000 rpm for 15 minutes to separate the serum. The contents of nitric oxide (NO), malondialdehyde (MDA), reduced glutathione (GSH), and total superoxide dismutase (SOD) in the serum were determined according to the instructions and steps of the kit.

[0094] 2.3.5 Colonic histopathological examination: At the end of the experiment, colonic length was measured. A portion of the same proximal colon (near the cecum) was harvested from each mouse and fixed in a general-purpose tissue fixative for 24 hours. After decalcification, paraffin embedding, sectioning, and hematoxylin-eosin (HE) staining, the degree of colonic lesions in each group was compared and observed under a light microscope. Paraffin-embedded colonic tissue was cut into 5μm thick sections, dewaxed, repaired, blocked, incubated with antibodies, and counterstained with DAPI nuclei before observation and image acquisition under a fluorescence microscope. 2.3.6 Western blot detection of acute colitis-related protein expression: First, a protein lysis buffer (RIPA lysis buffer: protease inhibitor: phosphatase inhibitor = 100:1:1) was prepared. Digestive fluids, blood, and digestive products were washed from the colonic tissue with PBS. Protein lysis buffer was added at a ratio of 30mg tissue to 1000μL. The protein homogenizer was used on ice to ensure thorough contact between the lysis buffer and the tissue cells. After complete lysis, the cells were centrifuged at 10,000-14,000 rpm for 3-5 minutes, and the supernatant was collected to obtain a total protein solution from the colon tissue. The protein concentration of the total protein solution was determined using the BCA method, and Western blotting gel electrophoresis was performed to determine the expression levels of β-actin, IKKα / β, NF-κB P65, IKBα, p-IKKα / β, p-IκBα, and p-P65 proteins in the colon tissue of each group of mice.

[0095] 2.4 Statistical analysis:

[0096] One-way ANOVA was performed using SPSS 19.0 software. All experimental results are expressed as absolute values. P < 0.05 indicates a significant difference, and P < 0.01 indicates a highly significant difference.

[0097] 3. Results:

[0098] 3.1 General condition and weight changes:

[0099] When ulcerative colitis occurs, mice exhibit symptoms such as lethargy, apathy, huddling, anorexia, diarrhea, bloody stools, and weight loss. Compared to the control group, the body weight of mice in the DSS model group gradually decreased with the extension of the modeling time, with a significant decrease starting on day 7 (P < 0.01); while the body weight of mice in the 5-ASA positive drug and low- and high-dose administration groups of 12-hour saponin HZ showed significant improvement compared to the DSS model group starting on day 7 (P < 0.05, P < 0.01). See Figure 5 .

[0100] 3.2 DAI Disease Activity Index Score:

[0101] The disease activity index (DAI) scores on the last day of the experiment were compared. Mice in the control group had a DAI score of 0, indicating no disease. Compared to the control group, the DAI score of mice in the DSS model group was significantly increased (P < 0.01), indicating that DSS successfully induced colitis in mice. Compared to the DSS group, the DAI disease activity index scores of the low- and high-dose 5-ASA positive control group and the 12-hour saponin HZ group were significantly decreased (P < 0.01), and the effect of 12-hour saponin HZ on the disease activity index of mice was dose-related. See [link to relevant documentation]. Figure 6 .

[0102] 3.3 Results of colon length measurement:

[0103] Observation of colonic tissue in UC mice revealed that the colonic tissue of mice in the normal group was intact, with no obvious lesions or congestion on the serosal, mucosal, and muscular surfaces, and the color was normal. In the model group, the colonic tissue showed shrinkage and shortening, congestion, significant thinning of the intestinal mucosa and muscular layer, and a changed color, appearing reddish-brown. Some mice showed adhesion between the colon and surrounding tissues, accompanied by a foul odor. The 5-ASA positive drug group showed reduced lesions in the colonic tissue and no obvious congestion. The low- and high-dose groups of the 12-hour saponin HZ showed varying degrees of improvement in colonic tissue length, congestion, muscular layer thickness, and color compared to the model group. (See...) Figure 7 .

[0104] 3.4 Results of serum inflammatory cytokine assay:

[0105] In the normal control group, mice had low levels of TNF-α and IL-1β in their serum and high levels of IL-10. In the model group, rats showed varying degrees of increase in IL-1β and TNF-α levels and a decrease in IL-10 levels. However, after drug treatment, compared with the model group, all drug-treated groups showed varying degrees of decrease in the levels of TNF-α and IL-1β, and an increase in IL-10 levels. The 5-ASA-positive drug group and the high-dose group of Twelve-Hour Saponin HZ, in particular, showed a significant effect in reducing IL-1β and TNF-α levels and increasing IL-10 levels. (See...) Figure 8 .

[0106] 3.5 Results of serum antioxidant biochemical marker assay:

[0107] Oxidative stress is generally considered to be the regulation between oxidation and antioxidants in cells. In the pathogenesis of ulcerative colitis (UC), on the one hand, intestinal inflammation can promote excessive ROS production by immune cells; on the other hand, excessive ROS expression can damage intestinal epithelial cells, further aggravating intestinal inflammation. The results showed that NO activity and MDA content were significantly upregulated in the colon homogenate of mice in the DSS model group, while SOD and GSH expression levels were significantly downregulated compared to the control group (P < 0.01), indicating a significantly increased level of oxidative stress in the model group. However, when UC mice were pretreated with 12-hour saponin HZ and 5-ASA, GSH content and SOD activity significantly increased (P < 0.05 or P < 0.01), while NO and MDA activity levels significantly decreased (P < 0.05), and the degree of adjustment of oxidative stress levels by 12-hour saponin HZ was dose-dependent. See [link to relevant documentation]. Figure 9 .

[0108] 3.6 Results of colonic histopathological examination

[0109] When colonic ulcers occur, the intestinal mucosal glands are disordered, the intestinal mucosal epithelium is partially damaged, the mucosa is partially defective, multiple ulcers are scattered, crypts and goblet cells are lost over a large area, and numerous inflammatory cell infiltrations are visible in the mucosa and submucosa. Colonic tissue sections were stained with hematoxylin-eosin (HE), and colonic tissue was observed under a microscope. In the Control group, the colonic mucosa of mice was intact, without necrosis or shedding, the villi were neatly arranged, and the muscular layer structure was normal. Compared with the Control group, the DSS model group showed significant loss of glandular structure, inflammatory cell infiltration, swollen and shortened intestinal villi, a significant reduction in goblet cells, and a thinner mucosal layer that adhered to patches or was even absent. In the 5-ASA positive control group and the Twelve-Time Saponin HZ administration group, compared with the model group, the degree of intestinal wall structural disorder was reduced, the number of inflammatory cell infiltrations in the colonic mucosa was significantly reduced, and the pathological damage was improved to varying degrees, with the high-dose Twelve-Time Saponin HZ group showing the most significant improvement. The study suggests that 12-hour saponin HZ can reduce the degree of intestinal damage in mice with ulcerative colitis. (See...) Figure 10 .

[0110] 3.7 Effects of NF-κB signaling pathway proteins in mouse colon tissue

[0111] Western blot analysis was used to quantitatively analyze the expression of proteins related to the NF-κB signaling pathway. Compared with the control group, the expression levels of NF-κB P65, p-P65, p-IκBα, and p-IKKα / β proteins in the colon tissue of mice in the DSS model group were significantly increased (P < 0.01), while the expression level of IκBα protein was significantly decreased (P < 0.01), and the expression level of IKKα / β protein remained unchanged. Compared with the DSS model group, the expression levels of NF-κB in the colon tissue of mice in the 5-ASA positive drug group were significantly increased. The expression levels of NF-κB65, p-P65, p-IκBα, and p-IKKα / β proteins were significantly decreased, while the expression level of IκBα protein was significantly increased (P < 0.01). Compared with the DSS model group, the expression levels of NF-κB65, p-P65, p-IκBα, and p-IKKα / β proteins in the colon tissue of mice in both the low- and high-dose administration groups of Twelve-Hour Saponin HZ decreased to varying degrees, while the expression level of IκBα protein increased to varying degrees. Specifically, the expression of NF-κB65, p-P65, p-IκBα, and p-IKKα / β proteins in the colon tissue of mice in the high-dose administration group and the expression of p-IκBα and p-IKKα / β proteins in the colon tissue of mice in the low-dose administration group were significantly decreased (P < 0.01), while the expression of IκBα in the colon tissue of mice in the high-dose administration group was significantly increased (P < 0.01), showing a dose-dependent effect. See Figure 11 .

[0112] Simultaneously, immunofluorescence analysis of mouse colon tissue sections was performed to observe the expression level and distribution of NF-κB BP65 in the colon tissue. DAPI staining of cell nuclei showed a blue color under UV excitation, while NF-κB positive expression was indicated by red light from the corresponding fluorescein label. In the control group, the colon tissue endothelial cells were densely and uniformly distributed, with low NF-κB positive expression. Compared to the control group, the colon tissue endothelial cells in the DSS group were sparse and diffuse, with significantly increased NF-κB positive expression. Compared to the DSS group, the colon tissue endothelial cells in the 5-ASA positive drug and the low- and high-dose administration groups of Twelve-Times-Saponin HZ were more densely and uniformly distributed, while NF-κB positive expression decreased to varying degrees. (See...) Figure 12 .

[0113] The above results suggest that Twelve-Hour Saponin HZ can improve the symptoms of DSS-induced acute colitis in mice by inhibiting the phosphorylation of IKKα / β protein kinase, thereby inhibiting the hydrolysis of the NF-κB / IκB complex and reducing the phosphorylation levels of NF-κB and IκB proteins.

[0114] The inventors of this invention also used semi-preparative high-performance liquid chromatography (HPLC) to separate the five components (FrB4-1, FrB4-2, FrB4-3, FrB4-5, and FrB4-6, excluding FrB4-4), to obtain other corresponding components. These components were then subjected to experimental therapeutic effects on ulcerative colitis. The results showed that, except for Twelve-Hour Saponin HZ, the other components did not exhibit any therapeutic effect on ulcerative colitis. However, the above experimental results indicate that Twelve-Hour Saponin HZ obtained in this invention has a significant effect in treating ulcerative colitis and can be further used to develop safe, effective, and controllable new traditional Chinese medicine anti-inflammatory and anti-ulcerative colitis drugs.

[0115] Finally, the above embodiments are merely examples for clearly illustrating the present invention and are not intended to limit the implementation. Those skilled in the art can make other variations based on the above description, and any obvious variations or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. The application of a twelve-hour saponin HZ in the preparation of a drug for treating ulcerative colitis, characterized in that: The preparation method of the twelve-hour saponin HZ includes the following steps: (1) Preparation of the maceration extract: Using the She medicine twelve-hour period as raw material, the She medicine twelve-hour period was macerated and extracted with a polar solvent to obtain the maceration extract; (2) Preparation of crude extract: Disperse the impregnated extract in water to obtain crude extract; (3) Preparation of total saponin extract: The crude extract was extracted sequentially with petroleum ether, ethyl acetate and n-butanol, and then the n-butanol extracts were combined and the n-butanol was removed by vacuum distillation to obtain total saponin extract. (4) Preparation of extract Fr.B4: The total saponin extract was separated by microporous resin MCI column chromatography, and gradient elution was performed with methanol-water as the eluent. The eluted fraction of 70% methanol was collected, concentrated under reduced pressure and dried to obtain extract Fr.B4. (5) Preparation of Twelve-Hour Saponin HZ: The extract Fr.B4 was subjected to... Sephadex LH-20 was purified to obtain 6 components from FrB4-1 to FrB4-6. Component FrB4-4 was separated by semi-preparative high-performance liquid chromatography to obtain the Twelve Time Saponin HZ. The structure of the twelve-hour saponin HZ is as follows:

2. The application according to claim 1, characterized in that: The polar solvent is one or a mixture of methanol, ethanol, or water.

3. The application according to claim 1, characterized in that: The specific operation method of step (1) is as follows: using She medicine twelve hours as raw material, after crushing She medicine twelve hours, add 6 to 8 times the weight of raw material in methanol or ethanol for maceration and extraction according to the weight-volume ratio. Extract for a total of 3 times, each time for 7 days. After that, combine the extracts, recover the solvent, and obtain the macerated extract.

4. The application according to claim 1, characterized in that: The specific operation method of step (2) is as follows: disperse the maceration extract in water at a weight-volume ratio of 1 to 5 times to obtain crude extract.

5. The application according to claim 1, characterized in that: The specific operation method of step (3) is as follows: the crude extract is first extracted with petroleum ether three times and the aqueous phase is collected; then the aqueous phase is extracted with ethyl acetate three times, and the aqueous phase after ethyl acetate extraction is extracted with n-butanol three times. The n-butanol extracts are combined and the n-butanol is recovered under reduced pressure to obtain the total saponin extract.

6. The application according to claim 1, characterized in that: The specific operation method of step (4) is as follows: the total saponin extract is subjected to microporous resin MCI column chromatography, and water, 20% methanol, 50% methanol, 70% methanol, 90% methanol and methanol are used as eluents in sequence for gradient elution. The elution fraction Fr.B4 of 70% methanol is collected, concentrated under reduced pressure and dried to obtain extract Fr.B4.

7. The application according to claim 1, characterized in that: The specific operation method for step (5) is as follows: Extract Fr.B4 was subjected to Sephadex LH-20 column chromatography was used, with methanol as the eluent. Each 50 mL sample was collected, concentrated under reduced pressure, and the same samples were combined to obtain 6 samples from FrB4-1 to FrB4-6. The sample FrB4-4 was separated by semi-preparative high performance liquid chromatography to obtain the Twelve Time Saponin HZ. The solvent used in the semi-preparative high-performance liquid chromatography method is a mixture of CH3CN and H2O, with a volume ratio of CH3CN to H2O of 30:70, and the flow rate is set to 3.0 mL / min.

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