Use of active ingredient hypericin in hypericum perforatum in preparation of anti-migraine drugs

Abstract

The application discloses application of an active ingredient hypericin in Hypericum perforatum in preparation of anti-migraine drugs, relates to the technical field of medicines, and identifies the active ingredient hypericin that can enter the brain from Hypericum perforatum extract, further combines BV2 cell in-vitro anti-inflammatory experiments, and analyzes blood rheology indexes of cerebral blood vessels and blood stasis changes after drug administration of rats, and confirms that the hypericin has good anti-migraine effect. The application provides definite experimental basis for the application of the active ingredient in anti-migraine drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of hypericin, an active ingredient in St. John's wort, in the preparation of anti-migraine drugs. Background Technology

[0002] Migraine is a common chronic neurological disorder, belonging to the category of primary headache. Its global prevalence is approximately 15%, with about 200 million patients in my country, making it the second leading cause of disability. Clinically, it is characterized by periodic, recurrent attacks of unilateral or bilateral throbbing moderate to severe headaches, often accompanied by gastrointestinal symptoms such as nausea and vomiting, as well as sensory abnormalities such as photophobia and phonophobia, severely impacting patients' quality of life. Currently, commonly used migraine medications include nonsteroidal anti-inflammatory drugs (NSAIDs), opioid analgesics, and ergotamine. While these drugs can relieve symptoms in the short term, they generally have limitations such as significant adverse reactions and high relapse rates. Therefore, developing highly effective, low-toxicity anti-migraine drugs with clear mechanisms of action and related treatment strategies has become a crucial area requiring breakthroughs in this field.

[0003] Hypericin, the core active ingredient of St. John's wort, has been shown to possess various pharmacological activities, including antidepressant, antiviral, and neuroprotective effects. However, research on its systemic biological activity in treating migraines remains limited. Although some studies suggest that hypericin may exert its potential effects through the regulation of oxidative stress, neuroinflammation, and the 5-HT system, its direct efficacy against migraines lacks sufficient literature reports, limiting its development and application as an anti-migraine drug. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide the application of hypericin, an active ingredient in St. John's wort, in the preparation of anti-migraine drugs.

[0005] The technical solution of the present invention is as follows: The application of hypericin, an active ingredient in St. John's wort, in the preparation of anti-migraine drugs, characterized in that the structural formula of hypericin is as follows: .

[0006] Preferably, the hypericin can inhibit the release of TNF-α, IL-6 and NO.

[0007] Preferably, the drug comprises an active ingredient and a pharmaceutically acceptable carrier.

[0008] Preferably, the hypericin is extracted from St. John's wort.

[0009] Preferably, the activity screening method for hypericin includes the following steps: S1: Take the medicinal material of St. John's wort, add ethanol solution, reflux and extract to obtain the ethanol extract of St. John's wort; S2: After administering St. John's wort extract to rats by gavage, brain tissue was analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to identify the brain-entering components, determine potential active substances, and screen for the highest content of hypericin. S3: Hypericin was used for anti-inflammatory analysis of BV2 cells and cerebral hemodynamic analysis.

[0010] Preferably, in step S2, the liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QFS-MS) conditions are as follows: A Waters ACQUITY UPLC BEH T3 column (100 mm × 2.1 mm, 1.8 μm) was used; flow rate: 0.3 mL / min; column temperature: 40 °C; injection volume: 2 μL. Binary gradient elution: A: 0.1% formic acid solution, B: acetonitrile; 0-3 min, 5% B; 3-8 min, 5% B increases to 10% B; 8-20 min, 10% B increases to 20% B; 20-30 min, 20% B increases to 43% B; 30-40 min, 40-43% B increases to 70% B; 43-47 min, 95% B decreases to 5% B; 47-50 min, maintains at 5% B, % is volume fraction.

[0011] Preferably, in step S2, the mass spectrometry conditions in the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method are as follows: It employs an electrospray ionization source with positive / negative ion mode detection; detection method: multiple reaction detection; relative molecular mass m / z: 50-1250 Da; ion source temperature: 550℃; ion source injection pressure: 5.5 kV; atomizer and auxiliary gas pressure: 55 psi; curtain gas pressure: 30 psi; de-clustering voltage: 100 V; collision energy: 10 V.

[0012] Preferably, in step S3, the anti-inflammatory analysis of BV2 cells involves evaluating the effect of hypericin on the release of TNF-α, IL-6, and NO from lipopolysaccharide-induced BV2 cells within a safe concentration range for its effects on BV2 cell proliferation and cytotoxicity.

[0013] Preferably, in step S3, the cerebral hemodynamic analysis involves: after inducing a migraine rat model by intraperitoneal injection of nitroglycerin, anesthetizing the rats at predetermined time points, and using a laser speckle contrast imaging system to monitor changes in cerebral hemodynamics in real time.

[0014] The beneficial effects of this invention are as follows: By identifying hypericin, an active ingredient that can penetrate the brain, from the extract of St. John's wort, and further combining this with in vitro anti-inflammatory experiments on BV2 cells, as well as analysis of cerebral hemorheological parameters and congestion changes in rats after administration, this invention confirms that hypericin has a good anti-migraine effect. This invention provides clear experimental evidence for the application of this active ingredient in anti-migraine drugs. Attached Figure Description

[0015] Figure 1 The image shows the total ion chromatogram of the components entering the brain after administration of St. John's wort in positive ion mode. In the figure, a: blank methanol; b: blank brain tissue; c: brain tissue after administration of St. John's wort.

[0016] Figure 2 The figure shows the total ion chromatogram of the negative ion mode of the components entering the brain after administration of St. John's wort. In the figure, a: blank methanol; b: blank brain tissue; c: brain tissue after administration of St. John's wort.

[0017] Figure 3 Ion chromatograms were extracted in a positive ion mode to obtain the brain-entering components of St. John's wort after administration.

[0018] Figure 4 Ion flow chromatogram of negative ion mode of components entering the brain after administration of St. John's wort.

[0019] Figure 5 Figure 1 shows the in vitro anti-inflammatory effect of BV2 cells. A: Effect of CCK-8 activity; B: Levels of inflammatory factors. In the figure, #: At the same treatment time, compared with the normal group, P<0.05, n=6. ##: At the same treatment time, compared with the normal group, P<0.01, n=6. *: At the same treatment time, compared with the model group, P<0.05, n=6. **: At the same treatment time, compared with the model group, P<0.01, n=6.

[0020] Figure 6 This is a hemorheological diagram of cerebral blood vessels. Detailed Implementation

[0021] The application of hypericin, an active ingredient in St. John's wort, in the preparation of anti-migraine drugs, characterized in that the structural formula of hypericin is as follows: .

[0022] Preferably, the hypericin can inhibit the release of inflammatory factors, specifically inhibiting the release of TNF-α, IL-6 and NO.

[0023] Preferably, the drug comprises an active ingredient and a pharmaceutically acceptable carrier, which may be one or more of a filler, disintegrant, flavoring agent, or lubricant, and is not limited thereto.

[0024] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0025] 1. Preparation of St. John's wort alcohol extract 1000g of St. John's wort herb was extracted twice with 10 times its volume of 75% ethanol under reflux for 1 hour each time, and the total extract was collected. The extract was concentrated using a rotary evaporator and evaporated to dryness to obtain 136g of St. John's wort ethanol extract, with an extraction rate of 13.6%. For animal administration, the extract was dissolved in 0.5% CMC-Na (6.3g / mL gavage solution), aliquoted, and stored at 4℃ for subsequent experiments.

[0026] 2. Identification of brain-penetrating components After administering St. John's wort extract to rats by gavage, brain tissue was analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to identify the brain-penetrating components, determine potential active substances, and screen for the highest content of hypericin. Liquid chromatography conditions: A Waters ACQUITY UPLC BEH T3 column (100 mm × 2.1 mm, 1.8 μm); flow rate: 0.3 mL / min; column temperature: 40℃; injection volume: 2 μL. Binary gradient elution (A: 0.1% formic acid water, B: acetonitrile): 0-3 min, 5% B; 3-8 min, 5% B → 10% B; 8-20 min, 10% B → 20% B; 20-30 min, 20% B → 43% B; 30-40 min, 40-43% B → 70% B; 43-47 min, 95% B → 5% B; 47-50 min, 5% B → 5% B, % is volume fraction.

[0027] Mass spectrometry conditions: Electrospray ionization (ESI) source, positive / negative ion mode detection. Detection method: Multiple reaction detection (MRM), relative molecular mass (m / z): 50-1250 Da, ion source temperature (TEM): 550℃, ion source injection pressure (IS): 5.5 kV, nebulizer (GS1) and auxiliary gas (GS2) pressure: 55 psi, curtain gas (CUR) pressure: 30 psi, declusing voltage (DP): 100 V, collision energy (CE): 10 V. Five SD rats were selected and fasted for 12 hours before the experiment, with free access to water. After gavage, the animals were sacrificed at 0, 0.5, 1, 2, and 4 hours, and brain tissue was collected and aliquoted into different centrifuge tubes. The brain tissue from each time point was divided into two portions: one for identification of brain-infiltrating components and the other for spatial mass spectrometry imaging analysis. Nine times the volume of physiological saline was added to the brain tissue, homogenized, and centrifuged at 12000 rpm for 15 min. The supernatant was collected. 500 μL of the brain tissue supernatant was added to 2 mL of methanol, vortexed for 3 min, centrifuged, and the supernatant was collected. The supernatant was dried, reconstituted with methanol, filtered through a membrane, and analyzed by LC-MS / MS.

[0028] Ion images of the brain-entering components of St. John's wort under positive and negative ion modes after drug administration are shown below. Figures 1-5 Seven compounds were identified: two phloroglucinols, one flavonoid, one organic acid, one phenylpropanoid, and two others. Hypericin was the most abundant component in the brain and is also its characteristic (representative) component; therefore, it is speculated to be the key active ingredient. Detailed identification information for each compound is listed in Table 1.

[0029] Table 1. Identification of chemical components of St. John's wort entering the brain.

[0030] 3. Anti-inflammatory analysis of BV2 cells The effects of hypericin (1.875-240 μM) on the proliferation and cytotoxicity of BV2 cells were evaluated using the CCK-8 assay, and the safe concentration of the drug was finally determined.

[0031] BV2 cells were cultured in 10% serum and divided into: a blank group (cultured in complete medium containing cells), a control group (cultured in cell-free complete medium), a model group (cultured in complete medium containing lipopolysaccharide, 1 μg / mL), a positive control group (cultured in medium containing a mixture of lipopolysaccharide and huperzine A, with a final lipopolysaccharide concentration of 1 μg / mL and huperzine A of 1 μM), and drug treatment groups (cultured in medium containing a mixture of lipopolysaccharide and hypericin, with a final lipopolysaccharide concentration of 1 μg / mL and hypericin concentrations of 7.5, 15, and 30 μM). Logarithmic growth phase cells were seeded in 96-well plates (5 × 10⁶ cells / well). 3 After culturing for 24 hours, the cell status was observed under a microscope. 100 μL of CCK-8 medium was added to each well, and the cells were incubated in the dark for color development. The OD value was measured at 450 nm. Six replicates were performed per group. Cell viability was calculated using the following formula: .

[0032] The results are as follows Figure 5As shown in Figure A, within the concentration range of 1.875-30 μM, hypericin had no significant effect on BV2 cell viability, with cell survival maintained at approximately 100%, showing no significant difference compared to the control group (P>0.05). When the drug concentration increased to 60 μM, hypericin exhibited significant cytotoxicity, with cell viability decreasing to below 70% (P<0.05), and pathological morphological changes such as cell swelling, cell membrane rupture, and cell structure destruction were observed. Furthermore, the model group treated with a final concentration of 1 μg / mL lipopolysaccharide did not significantly affect BV2 cell viability (P>0.05), remaining comparable to the control group. Based on these results, subsequent experiments used concentrations of 7.5, 15, and 30 μM for further investigation.

[0033] The effects of hypericin on the release of TNF-α, IL-6 and NO from lipopolysaccharide-induced BV2 cells are as follows: Figure 5 As shown in Figure B, compared with the control group, the release of inflammatory factors TNF-α, IL-6, and NO in the model group was significantly increased (P<0.01), indicating that the BV2 cell inflammation model was successfully established under 1 μg / mL lipopolysaccharide stimulation. Compared with the model group, all intervention groups of hypericin were able to inhibit the release of TNF-α, IL-6, and NO to varying degrees (P<0.05), showing a trend of recovery towards the control group, but their effects were lower than those of the positive drug group. The above results suggest that under the lipopolysaccharide-induced inflammatory environment, hypericin may enhance the defense of BV2 cells against external interference and has the ability to inhibit the release of inflammatory factors.

[0034] 4. Cerebral hemodynamic analysis Nine healthy female SD rats were selected and fasted for 12 hours before the experiment, with free access to water. They were then acclimatized for one week before the experiment and randomly divided into three groups (n=3 per group): a blank control group (intraperitoneal injection of an equal volume of physiological saline), a model group (intraperitoneal injection of nitroglycerin, 10 mg / kg), a positive control group (zolmitriptan, 0.25 mg / kg), and a hypericin administration group (100 mg / kg). Except for the blank control group, all other groups received intraperitoneal injections of nitroglycerin (10 mg / kg) on ​​days 1, 3, 5, 7, and 9; the blank control group received an equal volume of physiological saline, and the hypericin group received the drug via gavage on day 10. Nine days after nitroglycerin injection, the rats were anesthetized and their body temperature was maintained at 37°C on a constant-temperature heating platform.

[0035] The results of hemorheological parameters and congestion changes in rat cerebral blood vessels after administration of hypericin are shown in the figure. Figure 6The cerebral hemorheological parameters of the control group rats remained relatively stable throughout the observation period. The model group showed significant cerebral vasodilation and cerebral hemorrhage 60 minutes after modeling, a phenomenon that persisted until 90 minutes. Treatment with hypericin reduced the degree of cerebral vasodilation and cerebral hemorrhage to varying degrees, indicating that this component can effectively alleviate cerebral vasodilation and cerebral hemorrhage in migraine model rats.

[0036] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. The application of hypericin, an active ingredient in St. John's wort, in the preparation of anti-migraine drugs, characterized in that... The structural formula of hypericin is as follows: 。 2. The application of hypericin, the active ingredient in St. John's wort as described in claim 1, in the preparation of anti-migraine drugs, characterized in that, Hypericin can inhibit the release of TNF-α, IL-6 and NO.

3. The application of hypericin, the active ingredient in St. John's wort as described in claim 1, in the preparation of anti-migraine drugs, characterized in that... The drug comprises an active ingredient and a pharmaceutically acceptable carrier.

4. The application of hypericin, the active ingredient in St. John's wort as described in claim 1, in the preparation of anti-migraine drugs, characterized in that, The hypericin is extracted from St. John's wort.

5. The application of hypericin, the active ingredient in St. John's wort as described in claim 1, in the preparation of an anti-migraine drug, characterized in that, The activity screening method for hypericin includes the following steps: S1: Take the medicinal material of St. John's wort, add ethanol solution, reflux and extract to obtain the ethanol extract of St. John's wort; S2: After administering St. John's wort extract to rats by gavage, brain tissue was analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to identify the brain-entering components, determine potential active substances, and screen for the highest content of hypericin. S3: Hypericin was used for anti-inflammatory analysis of BV2 cells and cerebral hemodynamic analysis.

6. The application of hypericin, the active ingredient in St. John's wort as described in claim 1, in the preparation of an anti-migraine drug, characterized in that, In step S2, the liquid chromatography conditions in the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method are as follows: A Waters ACQUITY UPLC BEH T3 column (100 mm × 2.1 mm, 1.8 μm) was used; flow rate: 0.3 mL / min; column temperature: 40 °C; injection volume: 2 μL. Binary gradient elution, A: 0.1% formic acid solution, B: acetonitrile; 0-3 min, 5% B; 3-8 min, 5% B increases to 10% B; 8-20 min, 10% B increases to 20% B; 20-30 min, 20% B increases to 43% B; 30-40 min, 40-43% B increases to 70% B; 43-47 min, 95% B decreases to 5% B; 47-50 min, maintains at 5% B, where % is volume fraction.

7. The application of hypericin, the active ingredient in St. John's wort according to claim 1, in the preparation of an anti-migraine drug, characterized in that, In step S2, the mass spectrometry conditions in the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method are as follows: It employs an electrospray ionization source with positive / negative ion mode detection; detection method: multiple reaction detection; relative molecular mass m / z: 50-1250 Da; ion source temperature: 550℃; ion source injection pressure: 5.5 kV; atomizer and auxiliary gas pressure: 55 psi; curtain gas pressure: 30 psi; de-clustering voltage: 100 V; collision energy: 10 V.

8. The application of hypericin, the active ingredient in St. John's wort as described in claim 1, in the preparation of an anti-migraine drug, characterized in that, In step S3, the anti-inflammatory analysis of BV2 cells is performed by evaluating the effect of hypericin on the release of TNF-α, IL-6 and NO from lipopolysaccharide-induced BV2 cells within the safe concentration range for its effects on the proliferation and cytotoxicity of BV2 cells.

9. The application of hypericin, the active ingredient in St. John's wort as described in claim 1, in the preparation of an anti-migraine drug, characterized in that, In step S3, the cerebral hemodynamic analysis involves inducing a migraine rat model by intraperitoneal injection of nitroglycerin, anesthetizing the rats at predetermined time points, and using a laser speckle contrast imaging system to monitor changes in cerebral hemodynamics in real time.

Images