Preparation method of stringy stonecrop herb general flavone and application of stringy stonecrop herb general flavone in preparation of medicine for treating inflammatory bowel disease
By enriching and extracting total flavonoids from Sedum sarmentosum, the problem of unsatisfactory efficacy of Sedum sarmentosum extract has been solved. The prepared total flavonoids from Sedum sarmentosum showed significant alleviating effects in a mouse model of ulcerative colitis, which was superior to traditional extracts and has good potential for clinical application.
Patent Information
- Application Number
- CN202511378723.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-25
- Publication Date
- 2025-11-21
AI Technical Summary
In the existing technology, the efficacy of Sedum sarmentosum extract in treating inflammatory bowel disease is not ideal. The key active components and their efficacy have not been elucidated in depth, which limits its drug application in inflammatory bowel disease.
A method for enriching and extracting total flavonoids from Sedum sarmentosum is provided, comprising ultrasonic extraction and macroporous adsorption resin elution, to prepare high-content total flavonoids from Sedum sarmentosum, and to identify the main flavonoid components therein.
The prepared total flavonoids from Sedum sarmentosum had a significant alleviating effect on ulcerative colitis in mice, with bioactivity significantly superior to that of the total extract of Sedum sarmentosum and comparable to that of the clinical positive drug 5-aminosalicylic acid, showing good prospects for clinical application.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method for preparing total flavonoids from Sedum sarmentosum and their application in the preparation of drugs for treating inflammatory bowel disease. Background Technology
[0002] Ulcerative colitis (UC) is an idiopathic chronic inflammatory bowel disease affecting the colonic mucosa and submucosa. Clinical manifestations include recurrent episodes of diarrhea, bloody and mucous stools, and abdominal pain. Epidemiological studies show a rising global incidence rate, with approximately 15% of severely affected patients requiring bowel resection. To alleviate the heavy burden of ulcerative colitis treatment, there is an urgent need for more novel drugs to mitigate its impact on public health.
[0003] Sedum sarmentosum Bunge is a herbaceous plant belonging to the Crassulaceae family, with total flavonoids accounting for 5%-8% of its active ingredients. In traditional Chinese medicine, it is used to clear heat and dampness, detoxify and reduce swelling, primarily treating damp-heat jaundice and carbuncles. Although there are some studies on the use of Sedum sarmentosum extract in the treatment of enteritis, its efficacy is not ideal compared to clinically proven drugs. The key active components and their effects are not fully elucidated, limiting its application in inflammatory bowel disease. Therefore, there is an urgent need to improve the therapeutic effect of Sedum sarmentosum on inflammatory bowel disease through modern science and technology, promoting the modernization of traditional Chinese medicine. Summary of the Invention
[0004] The purpose of this invention is to provide a method for preparing total flavonoids from *Sedum sarmentosum* and its application in the preparation of drugs for treating inflammatory bowel disease, thereby solving the problems existing in the prior art. This invention independently developed a method for enriching and extracting total flavonoids from *Sedum sarmentosum*. This method can extract high levels of total flavonoids from *Sedum sarmentosum* and identify the main flavonoid components. Animal ulcerative colitis model experiments showed that the total flavonoids from *Sedum sarmentosum* prepared in this invention have a significant alleviating effect on ulcerative colitis in mice. Furthermore, it verified that the bioactivity of this total flavonoids in treating inflammatory bowel disease is significantly better than that of the total extract of *Sedum sarmentosum*, and comparable to the activity of the clinical positive drug 5-aminosalicylic acid, demonstrating good clinical application prospects. This aligns with the guiding principles of improving the quality of traditional Chinese medicine in my country, promotes the research and development of innovative traditional Chinese medicines, and provides a new method for the treatment of inflammatory bowel disease.
[0005] To achieve the above objectives, the present invention provides the following solution:
[0006] This invention provides a method for preparing total flavonoids from Sedum sarmentosum, comprising the following steps:
[0007] (1) Mix Sedum sarmentosum with ethanol solution and extract with ultrasonication to obtain Sedum sarmentosum extract;
[0008] (2) The extract of Sedum sarmentosum was eluted with macroporous adsorption resin in sequence using water, 10% ethanol solution, 30% ethanol solution, 50% ethanol solution, 70% ethanol solution and 95% ethanol solution as eluents. The eluents of 30% ethanol solution and 50% ethanol solution were collected and combined, and concentrated under reduced pressure to obtain the total flavonoids of Sedum sarmentosum.
[0009] Optionally, in step (1), the ethanol solution is a 70% ethanol solution.
[0010] Optionally, in step (1), the mass ratio of the creeping sedum to the ethanol solution is 1:10.
[0011] Optionally, in step (1), the conditions for ultrasonic extraction are: 500W, 40KHz, 3 extractions, 1 hour each time.
[0012] Optionally, in step (2), during the elution process, the extract of Sedum sarmentosum is dissolved in a 70% ethanol solution before elution.
[0013] Optionally, in step (2), the elution volume of the water is 40L, the elution volume of the 10% ethanol solution is 90L, the elution volume of the 30% ethanol solution is 90L, the elution volume of the 50% ethanol solution is 100L, the elution volume of the 70% ethanol solution is 75L, and the elution volume of the 95% ethanol solution is 90L.
[0014] The present invention also provides total flavonoids of Sedum sarmentosum prepared by the above preparation method.
[0015] The present invention also provides the use of the above-mentioned total flavonoids from Sedum sarmentosum in the preparation of a medicament for treating inflammatory bowel disease.
[0016] Optionally, the inflammatory bowel disease includes ulcerative colitis.
[0017] The present invention also provides a medicament for treating inflammatory bowel disease, the medicament comprising the above-mentioned total flavonoids from Sedum sarmentosum and a pharmaceutically acceptable carrier.
[0018] The present invention discloses the following technical effects:
[0019] This invention independently developed a method for enriching and extracting total flavonoids from *Sedum sarmentosum*. This method can extract a high content of total flavonoids from *Sedum sarmentosum* (flavonoid content 70.19%) and identify the main flavonoid components. Animal ulcerative colitis model experiments showed that the total flavonoids from *Sedum sarmentosum* prepared in this invention have a significant alleviating effect on ulcerative colitis in mice. Furthermore, it verified that the bioactivity of this total flavonoids in treating inflammatory bowel disease is significantly better than that of the total extract of *Sedum sarmentosum*, and comparable to the activity of the clinical positive drug 5-aminosalicylic acid, demonstrating good clinical application prospects. This aligns with the guiding principles of improving the quality of traditional Chinese medicine in my country, promotes the research and development of innovative traditional Chinese medicine, and provides a new method for the treatment of inflammatory bowel disease. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0021] Figure 1 HPLC chromatogram of total flavonoids from Sedum sarmentosum;
[0022] Figure 2 Structural diagram of flavonoid components in total flavonoids of Sedum sarmentosum;
[0023] Figure 3 The curves showing the change in body weight of mice in each group (1) and the DAI index (2) are shown.
[0024] Figure 4 The results of colon length detection for each group of mice are shown; (1) shows the anatomical diagram of the colon of each group of mice, and (2) shows the statistical results of the colon of each group of mice.
[0025] Figure 5 Colonoscopy images of mice in each group;
[0026] Figure 6 HE-stained sections of colon tissue from each group of mice;
[0027] Figure 7 The results of inflammatory factors detection in the colon tissue of mice in each group are as follows: (1) is interleukin-6 (IL-6), (2) is interleukin-1β (IL-1β), and (3) is tumor necrosis factor-α (TNF-α).
[0028] Figure 8The results of the comparison between total flavonoids and total extract of Sedum sarmentosum in the treatment of ulcerative colitis are as follows: (1) the change in colon and rectum length of mice in each group, (2) the disease activity index of mice in each group, (3) the change in body weight of mice in each group, and (4) the relative expression level of interleukin-1β in intestinal tissue of mice in each group. Detailed Implementation
[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.
[0035] Example 1: Preparation of total flavonoids from Sedum sarmentosum
[0036] a. Crush 2.5 kg of Sedum sarmentosum, add 10 times the amount of 70% ethanol solution and extract by ultrasonication (500W, 40KHz) three times, 1.0 hour each time. Combine the extracts, concentrate under reduced pressure and recover the ethanol to obtain 303 g of Sedum sarmentosum extract.
[0037] b. Dissolve the extract of Sedum sarmentosum in 70% ethanol (6L), add HPD-300 macroporous adsorption resin (1kg), evaporate the solvent under reduced pressure, and then dry-load the sample onto an HPD-300 macroporous adsorption resin (6.5kg) column (inner diameter: 15cm, height: 97cm). Elute sequentially with H2O (40L), 10% ethanol (90L), 30% ethanol (90L), 50% ethanol (100L), 70% ethanol (75L), and 95% ethanol (90L). Collect the eluent and label it as AF fraction. Concentrate under reduced pressure and recover the ethanol.
[0038] c. The total flavonoid content in fraction AF was determined by colorimetry. The determination process is as follows:
[0039] ① Preparation of reference solution: Accurately weigh 1.0 mg of rutin reference standard, place it in a 10 ml volumetric flask, add 10 ml of 70% ethanol, sonicate until dissolved, cool, dilute to the mark with 70% ethanol, shake well to obtain 0.1 mg / ml rutin reference solution;
[0040] ② Calculation of the standard curve: Accurately measure 0.0 (blank), 10.0, 20.0, 40.0, 60.0, 80.0, and 100.0 μg / ml of rutin reference solution and place them in 1.0 ml volumetric flasks respectively. Add solvent to make up to 1 ml, mix well, and add 100 μL of the above reference solution to each well of a 96-well plate, with 3 replicates per group. Add 20 μL of 5% sodium nitrite solution sequentially, mix well, and let stand for 6 min (in the dark). Add 20 μL of 10% aluminum nitrate solution, mix well, and let stand for 6 min. Add 80 μL of 4% sodium hydroxide solution, shake well, and let stand for 15 min. After the reaction is terminated, immediately measure the absorbance (OD value) at 525 nm using a microplate reader. Plot the standard curve with absorbance (A) as the ordinate and concentration (C) as the abscissa, and perform linear regression analysis to obtain the regression equation: A = 0.002C + 0.0413, r 2 =0.999, A and C are in the range of 0.0589 to 0.2445 mg / ml -1 It exhibits good linearity within the given range.
[0041] ③ Determination of total flavonoid content in each fraction: Accurately weigh 100 mg of fractions A-F into 1 ml volumetric flasks, add 1 ml of 70% ethanol, sonicate to dissolve, and dilute to the mark to obtain the test solution. Accurately pipette 100 μL of the test solution from fraction A-F into 96-well plates, with 3 replicates per group. Following the established colorimetric procedure of the standard curve described above, after the reaction is completed, measure the absorbance (OD value) using an ELISA reader.
[0042] The concentration is calculated by substituting the absorbance (OD value) of the measured sample into the standard curve equation:
[0043]
[0044] In the formula, C: concentration obtained from the standard curve (mg / mL), V: total volume of sample extract (ml), N: dilution factor, and M: sample mass (g).
[0045] Finally, the flavonoid contents of fractions AF were determined to be 0.0475%, 3.39%, 65.85%, 80.48%, 7.105%, and 12.7%, respectively. The eluents of fractions C and D were combined, concentrated under reduced pressure, and ethanol was recovered to obtain total flavonoids from *Sedum sarmentosum* (37.3 g). Its flavonoid content was 26.18 g, 70.19%. The chemical composition of the total flavonoids from *Sedum sarmentosum* was analyzed by high-performance liquid chromatography (HPLC), and the results are as follows: Figure 1 As shown. This invention further identified 29 major flavonoids in the total flavonoids of *Sedum sarmentosum*, and the results are as follows. Figure 2 As shown.
[0046] Example 2: Pharmacodynamic test of total flavonoids from Sedum sarmentosum
[0047] This embodiment uses a mouse ulcerative colitis model to evaluate the alleviating effect of the total flavonoids from *Sedum sarmentosum* prepared in Example 1 on mouse colitis through drug intervention experiments. The specific experimental steps are as follows: After acclimatizing C57 mice for 2-3 days, the experimental animals were randomly divided into groups (n=6): low-dose and high-dose total flavonoids from *Sedum sarmentosum* treatment groups (50, 200 mg / kg); 5-aminosalicylic acid treatment group (500 mg / kg); normal control group (normal drinking water); and model group (3% DSS drinking water). The treatment groups received 3% DSS drinking water while simultaneously being administered total flavonoids from *Sedum sarmentosum* or 5-aminosalicylic acid by gavage, while the normal control group received an equal volume of physiological saline by gavage. Mouse weight was recorded daily, and the disease activity index (DAI) was scored. Nine days after modeling, the mice were euthanized, and their tissues were collected.
[0048] Mouse body weight, DAI index, and pathological examination results are as follows: Figure 3-5 As shown, treatment with total flavonoids from *Sedum sarmentosum* effectively alleviated the weight loss and increased disease activity index in mice induced by DSS. Figure 3), and improved the phenomenon of colorectal shrinkage in mice. Figure 4 Colonoscopy results showed that total flavonoids from *Sedum sarmentosum* could effectively alleviate colonic ulcers and bleeding in mice. Figure 5 ).
[0049] Further staining of mouse colon tissue with hematoxylin and eosin (HE) was performed, and the staining steps were as follows:
[0050] (1) Dewaxing and hydration: Xylene I → Xylene II → 100% ethanol → 95% ethanol → 80% ethanol → 70% ethanol → distilled water, each for 5 minutes.
[0051] (2) Stain with hematoxylin for 3-5 minutes.
[0052] (3) Differentiation and blueing: 1% hydrochloric acid ethanol (1-3 seconds) → rinse with running water for 15 minutes → Scott blueing solution (0.2% NaHCO3 + 0.3% MgSO4).
[0053] (4) Stain with 0.5% eosin Y (pH 4.6 ethanol solution) for 1-2 minutes.
[0054] (5) Dehydration and transparency: 95% ethanol → 100% ethanol I → 100% ethanol II → xylene I → xylene II for 2 minutes each.
[0055] (6) Sealing: Seal with neutral resin.
[0056] The results are as follows Figure 6 As shown, compared with the model group, total flavonoids from *Sedum sarmentosum* effectively reversed phenomena such as crypt structure distortion / disappearance, goblet cell reduction, crypt abscess, intraepithelial neutrophil infiltration (cryptitis), and submucosal edema. This indicates that total flavonoids from *Sedum sarmentosum* protected the integrity of the colonic tissue in ulcerative mice and effectively alleviated colonic inflammation.
[0057] Further qPCR was performed on mouse colon tissue. The specific experimental steps are as follows.
[0058] Weigh 20 mg of colon tissue into an EP tube, add 500 μL of Trizol and 4 zirconium beads to each tube, homogenize using a tissue homogenizer (the homogenization step can be ignored for cell pellets), mix well, incubate at room temperature for 5 min, centrifuge at 12000 rpm at 4°C for 5 min; add 100 μL of chloroform, mix well, incubate for 5 min, centrifuge at 12000 rpm at 4°C for 15 min; transfer the supernatant to a new EP tube, add 250 μL of isopropanol, mix well, incubate for 10 min, centrifuge at 12000 g at 4°C for 10 min, discard the supernatant; wash the pellet with 500 μL of cold 80% ethanol, centrifuge at 7500 g at 4°C for 5 min, dry at 4°C; dissolve in DEPC water, and determine the RNA concentration using an ONE nucleic acid quantification instrument; use The IIIOne-Tube RT SuperMix (+gDNA Remover) reverse transcription kit was used to obtain cDNA through reverse transcription according to the kit instructions. Real-time quantitative PCR was then performed according to the SupRealQ Ultra Hunter SYBR qPCR Master Mix (U+) instructions.
[0059] The results are as follows Figure 7 As shown, compared with the model group, the expression of inflammatory factors such as IL-1β, TNF-α, and IL-6 in the colon tissue of the total flavonoids intervention group of Sedum sarmentosum was significantly reduced. The above results indicate that the total flavonoids of Sedum sarmentosum can effectively alleviate DSS-induced colonic inflammation.
[0060] Example 3: Comparison of the efficacy of total extracts and total flavonoids from Sedum sarmentosum.
[0061] Given the limited reports on the effects of *Sedum sarmentosum* on relieving enteritis, this embodiment uses a mouse ulcerative colitis model to compare the efficacy of total extracts and total flavonoids of *Sedum sarmentosum* in treating colitis. The mouse ulcerative colitis model was constructed as in Example 2. The total extract of *Sedum sarmentosum* was prepared by ultrasonic extraction (500W, 40kHz) three times with 1.0 hour each time, adding 10 times the volume of 70% ethanol solution. The extracts were combined, concentrated under reduced pressure, and the ethanol was recovered to obtain the total extract of *Sedum sarmentosum*. The dosage was 100 mg / kg (a low dose determined in the preliminary experiment). The preparation method of total flavonoids of *Sedum sarmentosum* was the same as in Example 1, with a dosage of 50 mg / kg (a low dose determined in the preliminary experiment). A model group and a normal control group were also set up. Mouse weight was recorded daily, and disease activity index scores were calculated. Nine days after modeling, mice were euthanized, and tissue samples were collected to detect the colorectal length ratio and perform qPCR on the colon tissue.
[0062] The results are as follows Figure 8 As shown, compared with the model group, both the total extract of *Sedum sarmentosum* (100 mg / kg) and the total flavonoids of *Sedum sarmentosum* (50 mg / kg) could reverse the phenomena of colonic shrinkage, weight loss, increased disease activity index, and increased relative interleukin-1β content in mice. However, the total flavonoids of *Sedum sarmentosum* showed a more significant effect in alleviating enteritis in mice than the total extract. Furthermore, the dosage of total flavonoids of *Sedum sarmentosum* was 50 mg / kg, lower than the dosage of the total extract (100 mg / kg). This indicates that, compared with the simply prepared total extract of *Sedum sarmentosum*, the total flavonoids of *Sedum sarmentosum* prepared by the specific process of this invention have a more significant therapeutic effect on colitis, demonstrating good medicinal value.
[0063] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A method for preparing total flavonoids from Sedum sarmentosum, characterized in that, Includes the following steps: (1) Mix Sedum sarmentosum with ethanol solution and extract with ultrasonication to obtain Sedum sarmentosum extract; (2) The extract of Sedum sarmentosum was eluted with macroporous adsorption resin in sequence using water, 10% ethanol solution, 30% ethanol solution, 50% ethanol solution, 70% ethanol solution and 95% ethanol solution as eluents. The eluents of 30% ethanol solution and 50% ethanol solution were collected and combined, and concentrated under reduced pressure to obtain the total flavonoids of Sedum sarmentosum.
2. The preparation method according to claim 1, characterized in that, In step (1), the ethanol solution is a 70% ethanol solution.
3. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of the creeping sedum to the ethanol solution is 1:
10.
4. The preparation method according to claim 1, characterized in that, In step (1), the conditions for ultrasonic extraction are: 500W, 40KHz, 3 extractions, 1 hour each time.
5. The preparation method according to claim 1, characterized in that, In step (2), during the elution process, the extract of Sedum sarmentosum is dissolved in a 70% ethanol solution before elution.
6. The preparation method according to claim 1, characterized in that, In step (2), the elution volume of water is 40L, the elution volume of 10% ethanol solution is 90L, the elution volume of 30% ethanol solution is 90L, the elution volume of 50% ethanol solution is 100L, the elution volume of 70% ethanol solution is 75L, and the elution volume of 95% ethanol solution is 90L.
7. Total flavonoids of Sedum sarmentosum prepared by the preparation method according to any one of claims 1-6.
8. The use of the total flavonoids of Sedum sarmentosum according to claim 7 in the preparation of a medicament for treating inflammatory bowel disease.
9. The application according to claim 8, characterized in that, The inflammatory bowel disease includes ulcerative colitis.
10. A drug for treating inflammatory bowel disease, characterized in that, The drug comprises the total flavonoids of Sedum sarmentosum as described in claim 7 and a pharmaceutically acceptable carrier.