Application of jasminum grandiflorum extract in preparation of medicine for treating and / or preventing hepatic encephalopathy
By using frangipani extract and oleuropein to lower blood and brain ammonia levels, the problems of adverse reactions and recurrence of existing drugs are solved, and safe and effective treatment and prevention of hepatic encephalopathy is achieved.
Patent Information
- Application Number
- CN202510825822.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-19
- Publication Date
- 2025-09-16
AI Technical Summary
Existing drugs for treating hepatic encephalopathy, such as lactulose and rifaximin, are often accompanied by adverse gastrointestinal reactions and are prone to recurrence. There is a lack of safe and effective drugs to lower blood ammonia and brain ammonia.
Frangipani flower extract and its main component oleuropein are used to prepare a drug for treating and/or preventing hepatic encephalopathy by lowering blood ammonia and brain ammonia levels and inhibiting astrocyte swelling.
Frangipani flower extract and oleuropein significantly reduce blood and brain ammonia levels, alleviate astrocyte swelling, have high safety and efficacy, and are suitable for the treatment and prevention of hepatic encephalopathy.
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Figure CN120643619A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the medical use of frangipani: frangipani flower extract and its main component oleuropein can significantly reduce blood ammonia and brain ammonia levels, and can be used to prepare drugs for treating and / or preventing hepatic encephalopathy and hyperammonemia-related diseases. Background Art
[0002] Hepatic encephalopathy (HE) is a clinical syndrome characterized by the accumulation of toxic substances in the body due to severe liver damage or portosystemic shunting, which affects central nervous system function. Its pathogenesis is complex, with ammonia poisoning being the most important. Typical pathological indicators include significantly elevated blood and brain ammonia levels, accompanied by brain pathological features such as astrocyte swelling and neuroinflammation [Chinese Medical Association Gastroenterology Branch, Hepatobiliary Disease Group, Chinese Expert Consensus on the Clinical Diagnosis and Treatment of Latent Hepatic Encephalopathy. Gastroenterology, Vol. 28, No. 12, 2023, 722-738].
[0003] The liver, a core organ of ammonia metabolism, converts ammonia into urea through the ornithine cycle, which is then excreted through the kidneys, thereby maintaining extremely low blood ammonia levels. Astrocytes play a key role in brain ammonia metabolism, converting ammonia to glutamine via glutamine synthetase, thereby maintaining brain ammonia homeostasis. In pathological conditions, when liver function is impaired, ammonia metabolism and clearance are impaired, leading to elevated blood ammonia levels and, consequently, brain ammonia levels. Excessive ammonia increases glutamine synthesis by astrocytes. The accumulation of glutamine within the cells increases intracellular osmotic pressure, triggering cell swelling. This swelling can directly lead to brain parenchymal edema, increased intracranial pressure, compression of neurons and blood vessels, and exacerbation of the pathological features of hepatic encephalopathy, such as cerebral ischemia and hypoxia. However, there is still a lack of effective treatments for hyperammonemia and hepatic encephalopathy, which is characterized by ammonia poisoning, resulting in a high mortality rate.
[0004] Although lactulose and rifaximin are currently first-line treatments for hepatic encephalopathy, they are often associated with adverse gastrointestinal effects, including abdominal distension, abdominal pain, and nausea, and are prone to relapse after discontinuation. Therefore, there is an urgent need to develop new, safe, and effective drugs that can lower blood and brain ammonia.
[0005] Jasminum grandiflorum, the dried buds of Jasminum grandiflorum L., a plant of the genus Jasminum in the family Oleaceae, has a slightly bitter taste, a neutral nature, and enters the liver meridian. It is known for relieving heart qi stagnation and pain, and relieving diarrhea and abdominal pain. Primarily produced in Guangdong, Yunnan, and Tibet, China, it is also widely cultivated worldwide and is listed in the first volume of the "Guangdong Province Standards for Traditional Chinese Medicine." It has a wide range of pharmacological effects, including antioxidant, anti-inflammatory, antiviral, anti-ulcer, and in vitro antibacterial properties, with particular efficacy against Stenotrophomonas maltophilia [Wei Fenghuan, A Jasminum Flower Extract, Its Preparation Method, and Application. Patent No.: ZL202110583833.5, granted on March 29, 2022]. Further research has revealed that Jasminum flower extract and its main component, oleuropein, significantly reduce blood and brain ammonia, suggesting that Jasminum flower has the potential to be used in the development of drugs for the treatment and / or prevention of hepatic encephalopathy. Summary of the Invention
[0006] In view of the above-mentioned defects in the prior art, the object of the present invention is to design and provide a use of a Jasmine flower extract in the preparation of a drug for treating and / or preventing hepatic encephalopathy.
[0007] In order to achieve the above object, the present invention adopts the following technical solutions:
[0008] In one aspect, the present invention provides the use of oleuropein in the preparation of a medicament for treating and / or preventing hyperammonemia-related diseases.
[0009] In the application, the hyperammonemia-related disease is hepatic encephalopathy.
[0010] The application of oleuropein can reduce blood ammonia and brain ammonia levels.
[0011] In the application, the oleuropein can reduce the swelling of astrocytes in brain tissue.
[0012] In a second aspect, the present invention provides the use of a frangipani flower extract in the preparation of a medicament for treating and / or preventing diseases related to hyperammonemia.
[0013] In the application, the hyperammonemia-related disease is hepatic encephalopathy.
[0014] In the application, the frangipani flower extract is obtained by extracting the frangipani flower with ethanol.
[0015] The application of the frangipani flower extract can reduce blood ammonia and brain ammonia levels.
[0016] In the application, the frangipani flower extract can reduce the swelling of astrocytes in brain tissue.
[0017] In a third aspect, the present invention provides a pharmaceutical composition for treating and / or preventing diseases related to hyperammonemia, comprising oleuropein or frangipani flower extract, and pharmaceutically acceptable excipients.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] The present invention creatively uses the frangipani flower extract and its main component oleuropein to prepare a drug for lowering blood ammonia and brain ammonia levels, which can be used to treat and / or prevent hepatic encephalopathy, has high safety and significant efficacy. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is the HPLC chromatogram of the frangipani flower extract, where Peak 1 is oleuropein;
[0021] Figure 2 The results of the effects of frangipani flower extract and oleuropein on reducing blood and brain ammonia levels in mice induced by LPS-D-GalN are shown in Figure 1, wherein (A) is the blood ammonia concentration result graph, and (B) is the brain ammonia concentration result graph;
[0022] Figure 3 The figure shows the morphology of astrocytes and GFAP protein expression in the hippocampus of mice;
[0023] Figure 4 The results show that frangipani and oleuropein reduce LPS-D-GalN-induced astrocyte swelling in the hippocampus of mouse brain tissue, where (A) shows the mean fluorescence intensity of GFAP, and (B) shows the mean fluorescence area of GFAP. ns indicates no statistical difference. DETAILED DESCRIPTION
[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0025] Example 1: Preparation of Frangipani Flower Ethanol Extract
[0026] Weigh 80.00 g of Jasminum grandiflorum powder (purchased from Guangzhou Zhixin Pharmaceutical Co., Ltd. and identified as Jasminum grandiflorum L., a plant of the genus Jasminum in the Oleaceae family, by a professor at the Department of Traditional Chinese Medicine Identification at Southern Medical University) and place it in a 2-L flask. Add 600-1000 mL of 30-90% ethanol and extract under reflux for 1 hour. Repeat twice. The extracts are filtered, combined, and evaporated in a 60°C water bath to obtain an ethanol extract of Jasminum grandiflorum (JEE). Freeze-dry to obtain lyophilized JEE powder, which is then stored dry. Analyze the JEE using HPLC to quantify oleuropein content.
[0027] HPLC analysis conditions: C 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm, with a flow rate of 0.8 mL / min, an injection volume of 10 μL, and a column temperature of 25°C. The detection wavelengths for oleuropein were 240 nm and chlorogenic acid were 320 nm. The flow gradient and gradient elution program are shown in Table 1.
[0028] Table 1 Mobile phase gradient elution program
[0029]
[0030] The results are as follows Figure 1 As shown, the relative retention time of the oleuropein peak in the frangipani flower extract is 39.5 minutes, with a resolution of 2.04. Further spectral purity analysis of the two peaks using a five-point method confirmed that the oleuropein peaks were single, demonstrating that the optimized chromatographic conditions met the requirements for quantitative analysis of oleuropein. Analysis and calculation of the oleuropein content in three parallel JEE test solutions revealed a value of 201.6 ± 3.8 μg / mg, representing 20% of the JEE lyophilized powder content.
[0031] Example 2: Jasmine flower extract and oleuropein reduce blood ammonia levels and brain ammonia levels in mice induced by LPS-D-GalN
[0032] 1. Experimental Animals
[0033] C57BL / 6J male mice, 8–9 m, 28 ± 3 g, were purchased from the Laboratory Animal Care Center of Southern Medical University. Mice were housed at room temperature (20 ± 2°C) under a standard 12-h light / dark cycle with free access to food and water. Adaptive feeding was performed for 7 days before the experiment.
[0034] 2. Reagents and drugs
[0035] Blood ammonia test kit (Nanjing Jiancheng Bioengineering Institute);
[0036] Lipopolysaccharide (LPS, Escherichia coli (O111:B4), Sigma) was prepared with sterile saline to a 500 ng / mL solution and administered intraperitoneally at 5 μg / kg for model establishment.
[0037] D-(+)-Galactosamine hydrochloride (D-GalN, ≥99%, Sigma) was prepared with sterile saline to a 20 mg / mL solution and administered intraperitoneally at 200 mg / kg for model establishment.
[0038] Silybin capsules (Tianjin Tasly Shengte Pharmaceutical Co., Ltd.) were prepared with distilled water to 10 mg / mL and administered orally to mice at 100 mg / kg.
[0039] Oleuropein (Chengdu Yirui Biotechnology Co., Ltd.) was prepared with distilled water at 4 mg / mL and administered orally to mice at 40 mg / kg.
[0040] The ethanol extract freeze-dried powder (i.e., JEE freeze-dried powder) prepared in Example 1 was prepared into 20 mg / mL and 80 mg / mL solutions using distilled water, and then administered to mice orally at 200 mg / kg and 800 mg / kg, respectively.
[0041] 3. Main instruments
[0042] Full-wavelength microplate reader Multiskan GO, centrifuge, analytical balance, small vortex mixer, etc.
[0043] 4. Mouse grouping, drug administration, and modeling methods
[0044] Sixty C57BL / 6J male mice (8-9 months old, weighing 28±3g) were randomly divided into six groups after 7 days of adaptive feeding: a blank group (NC), a model group (MC), a positive drug group (PC), an oleuropein group (OL), a low-dose Jasmine flower extract group (LJEE), and a high-dose Jasmine flower extract group (HJEE). The NC and MC groups were gavaged with distilled water at a volume of 10 mL / kg; the PC group was gavaged with silybin (100 mg / kg silybin in aqueous solution); the OL group was gavaged with oleuropein (40 mg / kg oleuropein in aqueous solution); the LJEE group was gavaged with freeze-dried Jasmine flower extract powder (200 mg / kg Jasmine flower extract in aqueous solution); and the HJEE group was gavaged with freeze-dried Jasmine flower extract powder (800 mg / kg Jasmine flower extract in aqueous solution).
[0045] The mice were weighed and administered intragastrically daily for 28 days. One hour after the last dose, the blank group received an intraperitoneal injection of 0.9% saline at a dose of 10 mL / kg. The remaining groups received an intraperitoneal injection of a mixed solution of LPS (5 μg / kg) and D-GalN (200 mg / kg) 15 minutes later to establish the model. The mice were closely observed and sacrificed 6 hours after LPS / D-GalN injection. Blood samples and brain tissue were collected for testing of relevant indicators.
[0046] 5. Experimental testing methods and results:
[0047] (1) Detection of blood ammonia levels
[0048] The eyes of the mice treated with the above treatments were removed and blood was collected. The blood was placed in a centrifuge tube and centrifuged at 3000 rpm for 10 minutes at 4°C to collect serum. The ammonia level in the serum was measured using a kit and a microplate reader.
[0049] (2) Detection of brain ammonia levels
[0050] The mouse brain tissue after the above different treatments was taken, added with PBS at a ratio of 1:9 (w / v), homogenized, and centrifuged at 12000 rpm at 4°C for 10 min. The supernatant was taken as the test sample and measured according to the kit instructions.
[0051] The results of the effects of frangipani flower extract and oleuropein on blood ammonia and brain ammonia levels in ALI mice are shown in Table 2 below.
[0052] Table 2 Effects of Jasmine Flower Extract and Oleuropein on Lowering Blood and Brain Ammonia
[0053]
[0054] Compared with the blank group, # P<0.05, ## P < 0.01, ### P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.
[0055] The results are as follows Figure 2 The results showed that both frangipani flower extract and oleuropein can significantly reduce blood ammonia and brain ammonia levels, and have a significant improvement effect on the symptoms of hyperammonemia-related diseases.
[0056] Example 3: Effects of Jasmine Flower Extract and Oleuropein on Reducing LPS-D-GalN-Induced Astrocyte Swelling in Mouse Brain Tissue
[0057] Immunofluorescence staining method for glial fibrillary acidic protein (GFAP) in the hippocampus of mouse brain tissue:
[0058] The brain tissues of the mice in different groups in Example 2 were taken separately, and the astrocyte proliferation reaction in the hippocampus of the brain tissue of the mice in different groups was analyzed according to the steps of frozen sectioning, fixation, antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, sealing, observation under an inverted fluorescence microscope and recording the immunofluorescence images of each tissue.
[0059] The specific process of frozen section production is as follows:
[0060] (1) Tissue pretreatment: After fixation with 4% paraformaldehyde, the specimens were dehydrated using a gradient sucrose treatment, followed by immersion in 15% and 30% sucrose / PBS solutions for 24 hours each. The endpoint of dehydration was determined by the natural sedimentation of the tissue. After dehydration, the specimens were rinsed three times with pre-cooled PBS to remove surface sucrose crystals. When removing excess liquid with filter paper, care was taken to maintain the integrity of the tissue morphology.
[0061] (2) Embedding and freezing: Position the trimmed tissue sample in a special mold with the cut surface facing down. Slowly inject OCT embedding medium until the tissue is completely immersed. Immediately place it in dry ice for rapid freezing. During the freezing process, the mold angle needs to be adjusted to ensure uniform solidification of the embedding medium and avoid the formation of bubbles that affect the quality of the slices.
[0062] (3) Optimization of the slicing process: Pre-cool the microtome to -20°C, add a small amount of embedding agent to the sample holder, place the embedded tissue in the sample holder for quick freezing, and start slicing after the embedding agent solidifies. Select a thickness of 5-15 μm.
[0063] (4) Slice storage: Use electrostatic adsorption to attach the slices to pre-treated adhesive slides and store at -80°C for long-term storage or -20°C for short-term use. Before storage, ensure that the slices are completely dry to avoid crystal formation.
[0064] Tissue section fixation is as follows:
[0065] Brain tissue sections were baked at 37°C for 10 minutes to allow the tissue to adhere tightly to the slide. The sections were then immersed in freshly prepared 4% paraformaldehyde / PBS (pH 7.4) fixative and fixed at room temperature for 30 minutes to maintain tissue structural integrity. After fixation, the sections were transferred to PBS rinse buffer and rinsed three times (5 minutes each). After each rinse, the sections were gently shaken at a 45° angle to remove any residual liquid.
[0066] Antigen retrieval is as follows:
[0067] Completely immerse the fixed tissue sections in preheated EDTA antigen retrieval buffer and place them in a constant temperature water bath maintained at 95±1°C for 10 minutes for heat-induced antigen retrieval. After the retrieval is completed, allow the sections to cool naturally to room temperature (about 1 hour) in the retrieval solution to avoid sudden cooling and tissue shedding. The cooled sections are again placed in PBS rinse solution for three 5-minute washes to ensure complete removal of the retrieval solution residue. The temperature and time parameters of the entire antigen retrieval process must be strictly controlled to ensure the antigen-antibody binding efficiency of subsequent immunostaining experiments.
[0068] The closure process is as follows:
[0069] Place the antigen-retrieved tissue sections in a constant humidity incubation chamber. Use a hydrophobic immunohistochemistry pen to draw a blocked area along the tissue edge (5 mm from the tissue edge). Evenly cover the tissue surface with 5% normal goat serum (dissolved in 0.1% Triton X-100 / PBS) and incubate at 37°C for 60 minutes to block nonspecific binding sites. Keep the incubation chamber sealed during the blocking process to prevent serum evaporation and crystallization.
[0070] Primary antibody incubation is as follows:
[0071] Dilute the astrocyte marker anti-GFAP (1:500) in PBST buffer containing 5% BSA. Apply the diluted primary antibody solution dropwise to the tissue area and incubate in a humidified chamber at 4°C for 16-18 hours. The next day, remove the sections and rinse three times with PBS for 5 minutes each, using a shaking rinse method. Remove any remaining liquid from the edges with filter paper.
[0072] The fluorescent secondary antibody incubation (protect from light) is as follows:
[0073] After primary antibody incubation, place the tissue sections in a light-proof humidified chamber and add the fluorescently labeled secondary antibody working solution dropwise. Incubate at 37°C in the dark for 120 minutes. Wash three times (5 minutes each) with pre-chilled PBS using a shaking rinse method, protecting the sections from light throughout the wash process. Finally, remove any remaining liquid from the edges of the sections with filter paper and proceed immediately to subsequent mounting procedures or store at 4°C in the dark for no more than 4 hours.
[0074] The details of the sealing film are as follows:
[0075] Evenly apply 20 μL of anti-fluorescence quenching mounting solution containing 4',6-diamidino-2-phenylindole (DAPI) to the tissue slide. Slowly cover the slide with a coverslip at a 45° angle, ensuring that the mounting solution is evenly spread and no bubbles remain. After the mounted sample is placed in a light-proof box to stabilize for 10 minutes, immunofluorescence images of each tissue are observed and recorded under an inverted fluorescence microscope.
[0076] Image J analysis is as follows:
[0077] Import the image and separate the colors "Image→Color→Split Channels", select the image corresponding to the positive signal color, select the protein-positive area "Image→Adjust→Threshold", select the appropriate algorithm to allow the software to identify all protein-positive areas, and calculate the average positive staining optical density (mean density) = IOD / Area "Analyze→measure→mean".
[0078] Glial fibrillary acidic protein (GFAP) is a specific marker of astrocytes. GFAP expression directly reflects the activation state of astrocytes, and its upregulation is often accompanied by cell swelling and reactive proliferation. Therefore, to evaluate the inhibitory effects of frangipani and oleuropein on astrocyte hyperactivation, GFAP expression was assayed in mouse brain tissue sections.
[0079] The results are as follows Figure 3 and Figure 4 As shown, compared with the NC group, the fluorescence signal intensity and positive protein expression area of GFAP protein in the hippocampus of mice in the MC group were significantly increased (P<0.001), indicating that LPS / D-GalN-induced mouse astrocytes were highly activated and proliferated, with pathological features such as a significant increase in cell size, rounded cell bodies, and swollen and thickened processes. Compared with the MC group, the fluorescence signal intensity and positive protein expression area of GFAP protein in the hippocampus of mice in the drug groups were significantly decreased (P<0.05), indicating that both the JEE and OL groups can inhibit LPS / D-GalN-induced mouse astrocyte proliferation. Furthermore, compared with the NC group, the GFAP positive protein expression area of the three treatment groups was not significantly different (P>0.05), indicating that both frangipani flower extract and oleuropein can inhibit LPS / D-GalN-induced mouse hippocampal astrocyte hyperactivation and restore it to normal levels.
[0080] In summary, both frangipani flower extract and oleuropein significantly reduced blood and brain ammonia levels in mice with acute liver injury and alleviated pathological conditions such as astrocyte swelling caused by hyperammonemia. Frangipani flower extract and oleuropein have the potential to lower blood and brain ammonia and could be used to treat hyperammonemia.
Claims
1. Use of oleuropein in the preparation of a medicament for treating and / or preventing hyperammonemia-related diseases.
2. The use according to claim 1, characterized in that The hyperammonemia-related disease is hepatic encephalopathy.
3. The use according to claim 1, characterized in that The oleuropein can lower blood and brain ammonia levels.
4. The use according to claim 1, wherein The oleuropein can reduce the swelling of astrocytes in brain tissue.
5. Use of Jasmine flower extract in the preparation of drugs for treating and / or preventing diseases related to hyperammonemia.
6. The use according to claim 5, characterized in that The hyperammonemia-related disease is hepatic encephalopathy.
7. The use according to claim 5, characterized in that The frangipani flower extract is obtained by extracting the frangipani flower with ethanol.
8. The use according to claim 5, characterized in that The frangipani flower extract can reduce blood ammonia and brain ammonia levels.
9. The use according to claim 5, characterized in that The frangipani flower extract can reduce the swelling of astrocytes in brain tissue.
10. A pharmaceutical composition for treating and / or preventing hyperammonemia-related diseases, characterized in that: The invention comprises oleuropein or frangipani flower extract, and pharmaceutically acceptable excipients.