Use of rehmannia extract in preparation of a medicine for preventing and / or treating ulcerative colitis

Abstract

The application discloses application of a rehmannia extract in preparation of a medicine for preventing and / or treating ulcerative colitis. The rehmannia extract has a significant protective effect on DSS-induced acute colitis in mice, which is reflected in improvement of survival rate, reduction of body weight loss, reduction of inflammatory activity index and repair of colon structure and the like. The application provides a scientific basis and experimental basis for development of the traditional Chinese medicine rehmannia in medicines for ulcerative colitis and related inflammatory bowel diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of Rehmannia glutinosa extract in the preparation of drugs for the prevention and treatment of inflammatory bowel disease (IBD), especially ulcerative colitis (UC). It further relates to the quality consistency and in vitro cell protection differences of extracts obtained from different Rehmannia glutinosa preparation processes, and the use of Rehmannia glutinosa extract in the preparation of drugs for the prevention and / or treatment of DSS-induced acute colitis. Background Technology

[0002] Ulcerative colitis, a common chronic inflammatory bowel disease, has limited clinical treatment options and suffers from significant drug side effects and a high relapse rate. However, the active ingredients of natural Chinese herbal medicines, due to their multi-target and low-toxicity advantages, show unique potential in the adjunctive and alternative treatment of inflammatory bowel disease.

[0003] Rehmannia is the herb of the Scrophulariaceae family, Rehmannia glutinosa ( Rehmannia glutinosa Rehmannia glutinosa (Libosch.) refers to the fresh or dried tuberous roots of Rehmannia glutinosa. Depending on processing and usage, it can be divided into fresh and dried forms. Historical herbal and medical texts record that fresh Rehmannia glutinosa has hemostatic, heat-clearing, and fluid-generating effects, while dried Rehmannia glutinosa primarily has heat-clearing, blood-cooling, yin-nourishing, and fluid-generating effects. The Compendium of Materia Medica also records that fresh Rehmannia glutinosa can clear heat, generate fluids, cool blood, and stop bleeding; dried Rehmannia glutinosa can clear heat, cool blood, nourish yin, and generate fluids. In recent years, Rehmannia glutinosa extract has attracted attention in research on various inflammatory and immune-related diseases. Studies have shown that polysaccharides from raw and dried Rehmannia glutinosa can enhance the body's non-specific immune function, promote lymphocyte proliferation, and increase the phagocytic activity of macrophages, thus improving conditions such as infection and slow wound healing caused by weakened immune function. Furthermore, the iridoids in raw and dried Rehmannia glutinosa have been found to inhibit the release of inflammatory mediators, reduce tissue edema and pain, and have a regulatory effect on inflammatory diseases such as rheumatoid arthritis and allergic dermatitis.

[0004] However, systematic animal evidence and standardized preparations for UC are still insufficient. Summary of the Invention

[0005] The purpose of this invention is to provide new medicinal uses for Rehmannia glutinosa extract.

[0006] The novel pharmaceutical use of Rehmannia glutinosa extract provided by this invention is its application in the preparation of drugs for the prevention and / or treatment of inflammatory bowel disease (IBD).

[0007] Specifically, the use is the application of Rehmannia glutinosa extract in the preparation of medicaments for the prevention and / or treatment of ulcerative colitis.

[0008] The Rehmannia glutinosa extract was prepared by a method comprising the following steps: Fresh Rehmannia glutinosa was used as raw material, and extraction was performed using 60%-75% ethanol (v / v) as solvent. The extract was collected, concentrated under reduced pressure to obtain a thick paste, and then dried under vacuum to obtain Rehmannia glutinosa extract. In the above method, based on the moisture content of the medicinal materials converted to a dry basis, the mass ratio of Rehmannia glutinosa to ethanol with a volume fraction of 60%-75% can be 1:4.5-5.5; The extraction temperature can be 65-75℃, specifically 72℃, and the time can be 1-2h, specifically 1.5h. The extraction can be performed multiple times, specifically twice; The vacuum concentration can be achieved by combining the extracts and concentrating them under reduced pressure to a relative density of approximately 1.29. The vacuum drying can be performed until the moisture content is ≤2.0%, resulting in a light yellow to yellowish-brown Rehmannia glutinosa extract powder.

[0009] This invention provides a novel use of Rehmannia glutinosa extract in the preparation of drugs for the prevention and treatment of ulcerative colitis, with a recommended dosage range of 0.5-2 g / kg (by gavage, once daily). Figure 1 The results showed that the aqueous solution of this sample was light yellow (RAE), while the aqueous control solution was dark brown (RWE). In in vitro experiments, Rehmannia glutinosa extract (RAE) significantly reduced HT-DNA-induced inflammatory damage to THP-1-derived macrophages, improved cell morphology and activity, and its effect was superior to that of the aqueous extract (RWE). In an in vivo DSS-induced mouse colitis model, Rehmannia glutinosa extract significantly improved survival rate, slowed weight loss, restored colon length, improved histopathology, and reduced the disease activity index (DAI), with the best effect observed at a dose of 2 g / kg, demonstrating its application potential as a drug for the prevention and treatment of ulcerative colitis. Attached Figure Description

[0010] Figure 1 Comparison of the physical properties and solution appearance of Rehmannia glutinosa extract. A: Rehmannia glutinosa extract (RWE) powder; B: Rehmannia glutinosa extract (RAE) powder (70% ethanol, volume fraction); C: Aqueous solution of water extract (2g powder / 250mL water); D: Aqueous solution of ethanol extract (2g powder / 250mL water, 70% ethanol).

[0011] Figure 2 The protective effects of Rehmannia glutinosa extract on macrophages. A. Regulatory effect of Rehmannia glutinosa extract (RWE) on HT-DNA-induced macrophage damage; B. Regulatory effect of Rehmannia glutinosa extract (RAE) on HT-DNA-induced macrophage damage.

[0012] Figure 3To demonstrate the protective effect of Rehmannia glutinosa extract (RAE) against colitis in mice. A. Experimental flowchart; B. Survival curve; C. Percentage change in body weight; D. Colon length; E. Representative colon photographs; F. Colonic pathology H&E staining; G. Disease activity index (DAI) Detailed Implementation Example 1: Preparation of Rehmannia glutinosa extract Using fresh Rehmannia glutinosa as raw material, all solvents were measured on a dry basis according to the moisture content of the medicinal material. Two extractions were performed using approximately 70% ethanol by volume: 30 kg of fresh Rehmannia glutinosa was washed, chopped, and placed in an extraction tank. Approximately five times the dry weight of 70% ethanol was added each time, and extraction was carried out at 72 °C for approximately 1.5 h. The two extracts were collected separately (approximately 95 kg and 90 kg for the first and second extractions in the example batch). After filtration, the extracts were concentrated under reduced pressure to a relative density of approximately 1.29, yielding approximately 4.7 kg of a thick paste. This paste was then vacuum dried until the moisture content was ≤2.0% (the example batch used vacuum drying, yielding 2.66 kg of extract powder with a residual moisture content of 1.58%), forming a light yellow to yellowish-brown Rehmannia glutinosa ethanol extract (RAE), which was used for subsequent quality control and formulation development.

[0013] Fresh Rehmannia glutinosa was used as raw material. After passing inspection, impurities were removed, and the raw material was set aside. Water was added to the pretreated Rehmannia glutinosa raw material at 8 times its weight, and the mixture was extracted three times at 60℃ for 2 hours each time. The extracts were collected separately, combined, and filtered while hot to remove insoluble impurities, yielding a clear filtrate. The filtrate was concentrated under reduced pressure to obtain a thick paste-like concentrate. After filtering to remove residue, the concentrate was spray-dried to obtain a uniform Rehmannia glutinosa extract (RWE) with a light brown to brownish-red color. This extract is intended for subsequent formulation development or quality research.

[0014] Example 2: Investigating the protective effect of macrophages in vitro In in vitro cell experiments, THP-1 cells were first induced to adhere and differentiate by PMA 50 ng / mL for 10 h. They were then divided into four groups: blank control, HT-DNA model, HT-DNA + RWE (0.5 mg / mL), and HT-DNA + RAE (0.5 mg / mL). An inflammation / damage model was constructed by transfecting HT-DNA at 2 µg / mL with Lipofectamine 3000 for 2 h, and the morphology was observed under a phase-contrast microscope (see [link to cell experiments]). Figure 2 ).in, Figure 2 In the treatment group A (HT-DNA + RWE, 0.5 mg / mL), RWE showed that it could improve the cell morphology induced by HT-DNA to a certain extent, with the spreading and monolayer continuity being restored compared with the model group, but problems such as local gaps and poor adhesion still existed; compared with the same dose, Figure 2The addition of HT-DNA+RAE (0.5 mg / mL) further enhanced the above-mentioned improvement effects, resulting in more complete cell spreading, more continuous monolayers, and better morphological integrity than the RWE group. The results suggest that both RAE and RWE exhibited anti-inflammatory and protective activities under the same dosage conditions, but the regulatory effect of RAE was stronger than that of RWE.

[0015] Example 3: DSS Acute Colitis Experiment To systematically evaluate the protective effect of Rehmannia glutinosa extract against DSS-induced ulcerative colitis in mice, a multi-parameter monitoring method was used to objectively measure the drug effect from multiple perspectives, including physiological, behavioral, and tissue morphology aspects.

[0016] 3.1 Experimental Materials Eight-week-old SPF-grade male C57BL / 6 mice were used. The modeling reagent was dextran sulfate sodium salt (DSS) (2% dissolved in sterile drinking water). The Rehmannia glutinosa extract (RAE) prepared in Example 1 was administered in two dose groups, 0.5 g / kg and 2 g / kg, dissolved in physiological saline, and administered by gavage once a day. Other experimental materials and daily feeding supplies were also used.

[0017] 3.2 Grouping and Processing Mice were randomly divided into 4 groups: a normal control group of 6 mice and 10 mice in each of the other groups. Normal control group (NC): Free access to water, no intervention; DSS model group: 2% DSS was allowed to be drunk freely for 7 days to induce colitis, and 0.2ml of physiological saline was administered by gavage to each animal daily; Low-dose RAE group: RAE 0.5g / kg, 0.2ml / animal was administered by gavage daily during the same period as DSS; High-dose RAE group: RAE 2g / kg, 0.2ml / animal administered orally daily during the same period as DSS. All treatments were implemented simultaneously from the first day of the experiment, and purified water was provided for maintenance after the modeling was completed.

[0018] 3.3 Key Evaluation Indicators Survival rate: Dynamic monitoring throughout the process, and plotting survival curves to reflect the survival status of different treatment groups.

[0019] Weight change: Measure weight daily, calculate and analyze percentage changes in weight.

[0020] Colon length and appearance: At the end of the experiment, colon samples were collected, representative photographs were taken, and colon length was measured.

[0021] Colon pathology: At the end of the experiment, the colon was collected, H&E stained, and photographed.

[0022] Disease Activity Index (DAI): The disease activity index is calculated daily based on scores such as weight loss, stool characteristics, and fecal occult blood.

[0023] 3.4 Experimental Results according to Figure 3 The experiment was conducted according to the experimental flowchart shown in Figure A. Figure 3 B represents the survival curves of each group of mice, from... Figure 3 As shown in B, the survival rate of mice in the DSS model group was significantly reduced, while the RAE treatment group showed improved survival, especially the 2g / kg group, in which all mice survived, demonstrating a significant improvement effect.

[0024] Figure 3 C shows the changes in body weight of mice in each group. Among them, the body weight of mice in the DSS group continued to decrease, while the decrease in body weight in each RAE-treated group was significantly reduced, especially the body weight curve of the 2g / kg group, which was closest to the normal control group.

[0025] Figure 3 Figures D and E show that the colon in the DSS group was significantly shortened and had obvious inflammation, while the colon length in the RAE intervention group (especially 2g / kg) was significantly restored, the appearance was close to normal, and the inflammatory manifestations were reduced.

[0026] Figure 3 F showed that the DSS group had a large number of inflammatory cell infiltrations in the colon tissue and the intestinal epithelial barrier was destroyed. The RAE treatment group could significantly improve the DSS-induced intestinal pathological changes, significantly reduce inflammatory cell infiltration, and restore the intestinal epithelial structure.

[0027] Figure 3 G showed that the disease activity index (DAI) in the DSS group continued to rise, reflecting the worsening of the condition; compared with the DSS group, the DAI in the RAE 0.5g / kg and 2g / kg groups decreased significantly, with the improvement being particularly prominent at higher doses.

[0028] The above experimental results show that Rehmannia glutinosa extract has a significant protective effect against DSS-induced acute colitis in mice, manifested in improving survival rate, reducing weight loss, decreasing inflammatory activity index, and repairing colonic structure. It is effective within the dosage range of 0.5–2 g / kg, with the 2 g / kg dose showing particularly outstanding efficacy. This finding provides a scientific basis and experimental evidence for the development of Rehmannia glutinosa as a traditional Chinese medicine for ulcerative colitis and related inflammatory bowel diseases.

Claims

1. The use of Rehmannia glutinosa extract in the preparation of drugs for the prevention and / or treatment of inflammatory bowel disease.

2. Application of Rehmannia glutinosa extract in the preparation of drugs for the prevention and / or treatment of ulcerative colitis.

3. The application according to claim 1 or 2, characterized in that, The Rehmannia glutinosa extract was prepared by a method comprising the following steps: Fresh Rehmannia glutinosa was used as raw material, and ethanol (volume fraction 60%-75%) was used as solvent for extraction. The extract was collected, concentrated under reduced pressure to obtain a thick paste, and then dried under vacuum to obtain Rehmannia glutinosa extract.

4. The application according to claim 3, characterized in that, Based on the moisture content of the medicinal materials converted to a dry basis, the mass ratio of Rehmannia glutinosa to ethanol with a volume fraction of 60%-75% is 1:4.5-5.

5.

5. The application according to claim 3, characterized in that, The extraction temperature is 65-75℃, and the time is 1-2 hours.

6. The application according to claim 3, characterized in that, The extraction is performed multiple times, specifically twice.

7. The application according to claim 3, characterized in that, The vacuum concentration involves combining the extracts and concentrating them under reduced pressure to a relative density of approximately 1.

29.

8. The application according to claim 3, characterized in that, The vacuum drying process involves drying the sample to a moisture content of ≤2.0%, resulting in a light yellow to yellowish-brown powder of Rehmannia glutinosa extract.

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