Amide alkaloids NFB, their preparation methods and applications

Abstract

This invention discloses an amide alkaloid, NFB, its preparation method, and its applications, belonging to the field of medicinal chemistry. This novel amide alkaloid, NFB, was isolated from *Piper nigrum*, a plant in the genus *Piper*, and its specific structure is as follows: In vitro pharmacodynamic evaluation showed that this novel amide alkaloid, NFB, can alleviate neuroinflammatory responses after modeling injury by inhibiting the M1 phenotype of microglia and promoting their conversion to the M2 phenotype. In vivo pharmacodynamic evaluation also indicated that NFB can regulate microglial polarization after MCAO / R in SD rats to alleviate neuroinflammatory responses, while simultaneously improving neurological function and exerting a neuroprotective effect. This novel amide alkaloid, NFB, can be used to prepare drugs for regulating microglial M1 / M2 phenotype polarization, drugs for inhibiting neuroinflammatory responses, and / or drugs for treating ischemic stroke.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry. More specifically, this invention relates to an amide alkaloid, NFB, its preparation method, and its applications. Background Technology

[0002] Ischemic stroke (IS), a core central nervous system disease, is caused by cerebrovascular lesions leading to acute occlusion of the arteries supplying the brain, resulting in local brain ischemia and hypoxia, and ultimately, the rapid onset of corresponding neurological deficits. Current treatment strategies for ischemic stroke focus on vascular recanalization and preventing further neuronal damage. Mechanical thrombectomy and thrombolysis with recombinant tissue plasminogen activator are widely used clinically as the "gold standard" for achieving vascular recanalization. However, due to the limited treatment window of only 4.5 hours and the accompanying ischemia-reperfusion (I / R) injury, preventing and mitigating I / R injury and improving post-stroke functional recovery are key research areas.

[0003] Among the numerous pathological mechanisms of ischemic stroke, secondary neuroinflammation is a key factor determining the functional prognosis after stroke. Microglia, as the first line of defense in the central nervous system, briefly exhibit an M2 anti-inflammatory phenotype in the early stages of ischemia, but inevitably develop towards a destructive M1 pro-inflammatory polarization in the later stages. This leads to the continuous expansion of neurological damage after ischemic stroke and hinders the initiation of repair. Therefore, regulating the M1 / M2 phenotype polarization balance of microglia is considered an important strategy for treating ischemic stroke.

[0004] In recent years, amide alkaloids derived from natural products have attracted widespread attention due to their diverse biological activities. Various amide alkaloids have been isolated from plants of the Piper genus, and they have been reported to possess varying degrees of activity, including antioxidant, antitumor, anti-inflammatory, antibacterial, and immunomodulatory effects. For example, from Piper spp. (… Piper sarmentosum Pipersarmenoids AC, an amide alkaloid isolated from Roxb., exhibits inhibitory activity against LPS-induced NO production in BV2 cells, and its IC50 value is [not specified]. 50 The values ​​were 9.36, 12.53, and 10.77 μM, respectively. Furthermore, some N-cinnamylpyrrole alkaloids can affect neuroinflammation and oxidative stress. The representative compound PB-1 showed inhibitory activity in LPS or OGD / R-induced neuroinflammation and oxidative stress models, and alleviated cerebral ischemia-reperfusion injury in the MCAO / R model.

[0005] However, existing amide alkaloids still have shortcomings in the treatment of ischemic stroke: (1) Most known amide alkaloids have weak anti-neuroinflammatory activity, IC50, and low cerebral infarction. 50The values ​​are mostly at the micromolar level or above, which makes it difficult to meet the needs of drug development that is highly efficient and low in toxicity; (2) Existing studies are mostly focused on single indicators such as the inhibitory effect of compounds on NO release, and lack in-depth and systematic research on the core mechanism of regulating the M1 / M2 phenotypic polarization of microglia. Therefore, developing an amide alkaloid with a novel structure, strong activity, clear mechanism and neuroprotective effect by regulating microglia polarization is of great significance for the treatment of ischemic stroke. Summary of the Invention

[0006] One object of the present invention is to provide an amide alkaloid NFB, its preparation method and application, so as to at least solve the above-mentioned problems.

[0007] To achieve the objectives and other advantages of this invention, an amide alkaloid NFB is provided, having the following structure: .

[0008] The present invention also provides a method for preparing the above-mentioned amide alkaloid NFB, comprising the following steps: 1) Take the raw material of false betel leaves and branches, extract it by heating and reflux with ethanol solution, combine the extracts and concentrate under reduced pressure to obtain the extract; 2) After suspending the extract in water, extract with ethyl acetate and collect the ethyl acetate fraction; 3) The ethyl acetate extract was separated by macroporous resin column chromatography, and gradient elution was performed using an ethanol-water system. The 80:20 and 85:15 eluent fractions were collected and combined. 4) The fraction obtained in step 3) was subjected to MCI column chromatography with gradient elution of methanol-water to separate fraction AK and collect fraction E; 5) Separate fraction E by dextran gel column chromatography, elute with 50% methanol-water solution to obtain fractions E1-E9, and collect fraction E5; 6) Separate fraction E5 by RP-C18 reversed-phase column chromatography with gradient elution in a methanol-water system to obtain fractions E5a-E5o and collect fraction E5e. 7) Separate fraction E5e by silica gel column chromatography, eluting sequentially with petroleum ether-acetone system and dichloromethane-methanol system to obtain fractions E5e1-E5e13, and collect fraction E5e8. 8) The E5e8 fraction was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) using methanol-water as the mobile phase, and the retention time was collected. t R The fraction with a content of 32.1 min yielded the compound NFB.

[0009] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned amide alkaloid NFB or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0010] The present invention also provides the use of the above-mentioned amide alkaloid NFB or pharmaceutical composition in the preparation of a drug for regulating the M1 / M2 phenotypic polarization of microglia.

[0011] Preferably, the regulation involves inhibiting microglia from polarizing to the M1 pro-inflammatory phenotype and promoting their polarization to the M2 anti-inflammatory phenotype.

[0012] The present invention also provides the use of the above-mentioned amide alkaloid NFB or pharmaceutical composition in the preparation of a medicament for inhibiting neuroinflammatory responses.

[0013] The present invention also provides the use of the above-mentioned amide alkaloid NFB or pharmaceutical composition in the preparation of a medicament for treating ischemic stroke.

[0014] The present invention has at least the following beneficial effects: This invention provides a novel amide alkaloid, NFB, with a novel structure, significant activity, clear mechanism, and well-validated in vitro and in vivo pharmacodynamics. It inhibits neuroinflammatory responses by regulating the M1 / M2 phenotypic polarization of microglia, thereby alleviating neuronal damage, cerebral infarction, and cerebral edema following ischemia-reperfusion injury, and improving neurological function. It possesses the potential to be a candidate drug for the treatment of ischemic stroke and has good clinical application prospects and commercial development value. Specifically: First, the compound NFB of this invention exhibits significant anti-neuroinflammatory activity. In vitro Griess assay results show that NFB has a dose-dependent inhibitory effect on LPS-induced NO release from BV-2 microglia, with an IC50 value of [missing information]. 50 The value is as low as 1.16 µM, which is superior to most previously reported similar compounds (such as pipersarmenoids AC with an IC50 value as low as 1.16 µM). 50 The values ​​were 9.36, 12.53, and 10.77 µM, respectively, demonstrating higher anti-inflammatory activity. Meanwhile, MTT assay results showed that NFB did not exhibit significant cytotoxicity in the 0.1–10 µM concentration range, exhibiting a good safety window and providing a safety guarantee for its further drug development.

[0015] Secondly, the compound NFB of this invention can effectively regulate the M1 / M2 phenotypic polarization of microglia. In vitro RT-PCR and Western blot experiments consistently showed that in two neuroinflammation models induced by LPS and OGD / R, NFB dose-dependently reduced the mRNA and protein expression levels of M1 polarization markers (iNOS, CD16, CD86) and pro-inflammatory factors (IL-1β, IL-6, TNF-α), while simultaneously increasing the expression levels of M2 polarization markers (CD206, Arg-1, YM-1) and anti-inflammatory factors (IL-4, IL-10, TGF-β). These results indicate that NFB restores the M1 / M2 polarization balance by inhibiting microglia polarization towards the M1 pro-inflammatory phenotype and promoting their transformation towards the M2 anti-inflammatory phenotype, thereby alleviating neuroinflammatory responses at their source. This provides a novel drug intervention strategy for the treatment of ischemic stroke.

[0016] Third, the compound NFB of this invention exhibits significant in vitro neuroprotective effects. OGD / R injury experiments showed that NFB treatment significantly increased the survival rate of injured BV-2 microglia in a concentration-dependent manner. Further SH-SY5Y neuronal protection experiments demonstrated that NFB-treated microglia conditioned medium (MCM) significantly improved the survival rate of SH-SY5Y neurons after OGD / R injury, suggesting that NFB indirectly protects neurons by inhibiting microglia-mediated neuroinflammation, thus possessing potential neuroprotective value.

[0017] Fourth, the compound NFB of this invention exhibits excellent pharmacodynamic effects in an in vivo ischemic stroke model. In vivo pharmacodynamic evaluation using the SD rat MCAO / R model showed that: (1) NFB significantly reduced the mNSS neurological deficit score in rats, with the high-dose group (3 mg / kg) showing better efficacy than the positive control drug butylphthalide (NBP 80 mg / kg); (2) NFB significantly reduced the infarct volume, with the infarct rate in the high-dose group decreasing from 38.79% in the model group to 11.12%, showing better efficacy than the positive control drug NBP; (3) NFB effectively reduced cerebral edema, with the brain water content in the high-dose group decreasing from 84.41% in the model group to 81.12%; (4) NFB improved the behavioral function of rats after ischemia and shortened the time rats experienced sensation and touch on the affected limb in the tape experiment. These results fully demonstrate that NFB has a definite anti-ischemic stroke effect at the whole animal level.

[0018] Fifth, the compound NFB of this invention can alleviate neuronal damage after ischemia-reperfusion and regulate microglial activation. Immunofluorescence experiments confirmed that NFB can significantly increase the number of NeuN-positive neurons in the ischemic cerebral cortex and reduce TUNEL-positive apoptotic cells and TUNEL-positive cells. + / NeuN + The number of co-localized positive cells; reduced Iba-1 + It activates the number of microglia and promotes the transformation of microglia from an amoeboid activated morphology to a resting state; it reduces Iba-1. + / iNOS + (M1 type) colocalized cell count, increased Iba-1 + / Arg-1 + (M2 type) colocalized cell number. These results further validate, from a histological perspective, the mechanism by which NFB exerts its neuroprotective effect by regulating microglial M1 / M2 polarization.

[0019] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0020] Figure 1 The effect of compound NFB on LPS-induced NO release in BV-2 cells; Figure 2 The effect of compound NFB on LPS-induced BV-2 cell survival; Figure 3 The effect of compound NFB on OGD / R-induced BV-2 cell damage; Figure 4 The effect of compound NFB on the activity of SH-SY5Y cells after LPS-induced BV-2 microglia were treated with conditioned medium; Figure 5 The effect of compound NFB on the activity of SH-SY5Y cells damaged by OGD / R after being exposed to conditioned medium containing LPS-induced BV-2 microglia. Figure 6 The study investigated the effects of compound NFB on the levels of pro-inflammatory factors and M1 marker mRNA in LPS-induced BV-2 microglia. Figure 7 The effect of compound NFB on the mRNA levels of anti-inflammatory factors and M2 markers in LPS-induced BV-2 microglia; Figure 8 The effect of compound NFB on the mRNA levels of pro-inflammatory factors and M1 markers in OGD / R-induced BV-2 microglia; Figure 9 The effect of compound NFB on the mRNA levels of anti-inflammatory factors and M2 markers in OGD / R-induced BV-2 microglia; Figure 10The effect of compound NFB on the levels of polarization markers and pro-inflammatory cytokine proteins in LPS-induced BV-2 microglia; Figure 11 The effect of compound NFB on the levels of polarization markers and pro-inflammatory cytokine proteins in OGD / R-induced BV-2 microglia; Figure 12 The results of the modified neurological deficit scores of mNSS in SD rats were 1 h and 24 h after ischemia-reperfusion. Figure 13 These are representative images of TTC staining (a) and a quantitative map of cerebral infarction volume (b); Figure 14 This is the experimental result of detecting cerebral edema using the wet-dry weight method; Figure 15 These are the results of the modified behavioral tape experiment; Figure 16 The effect of compound NFB on neuronal damage in the ischemic cerebral cortex of MCAO / R rats; Figure 17 The effect of compound NFB on the activation of microglia in the ischemic cerebral cortex of MCAO / R rats; Figure 18 The effect of compound NFB on the expression of iNOS, an M1-type marker, in the ischemic cerebral cortex of MCAO / R rats; Figure 19 The effect of compound NFB on the expression of Arg-1, an M2-type marker, in the ischemic cerebral cortex of MCAO / R rats; Figure 20 The compound NFB affects the levels of pro-inflammatory factors and M1 marker mRNA in the ischemic cerebral cortex. Figure 21 The effect of compound NFB on the levels of anti-inflammatory factors and M2 marker mRNA in the ischemic cerebral cortex. Figure 22 The effect of compound NFB on the expression level of M1 / M2 polarization marker proteins in the ischemic cerebral cortex. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, so that those skilled in the art can implement it based on the description.

[0022] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0023] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0024] Example 1: Preparation of compound NFB.

[0025] The preparation method of amide alkaloid NFB includes the following steps: 10.8 kg of raw material from the branches and leaves of *Prunella vulgaris* was extracted by heating and reflux with 95% ethanol as solvent three times, with each extraction lasting 3 hours. The ethanol extracts were combined and concentrated under reduced pressure to obtain 1.4 kg of extract. The extract was then suspended in water and extracted with ethyl acetate to obtain 400.0 g of the ethyl acetate fraction.

[0026] The ethyl acetate extract was separated by macroporous resin column chromatography with gradient elution using ethanol-water systems (30:70, 50:50, 80:20, 85:15). Subsequently, the 80:20 and 85:15 eluent fractions were combined and then subjected to MCI column chromatography with gradient elution using methanol-water systems (30:70~100:0) to obtain a total of 12 fractions of AK.

[0027] Fraction E (15.4 g) was separated by dextran gel column chromatography, eluted with 50% methanol-water solution, yielding nine fractions E1-E9. Fraction E5 (2.4 g) was subjected to RP-C18 reversed-phase column chromatography, using a gradient elution with a methanol-water system (30:70-70:30) to obtain fractions E5a-E5o. Fraction E5e (438.2 mg) was separated by silica gel column chromatography (200-300 mesh), using a gradient elution with a petroleum ether-acetone system (5:1-2:1) and a dichloromethane-methanol system (40:1-4:1) to obtain fractions E5e1-E5e13. Fraction E5e8 (138.6 mg) was purified by semi-preparative reversed-phase high-performance liquid chromatography to obtain compound NFB (methanol-water, 40:60, 110.0 mg). t R =32.1 min).

[0028] The chemical structure of compound NFB is shown below: .

[0029] Compound NFB: white powder; HR-ESI-MS m / z 232.0969 [M + H] + (calcd. for 232.0968,C 13 H 14 NO3). 1H NMR (DMSO- d 6, 400 MHz); δ H 8.97 (1H, d, J = 3.0 Hz, NH-1), 7.58 (1H,dd, J = 5.4, 1.8 Hz, H-3′), 7.28 (2H, t, J = 7.8 Hz, H-6 / 8), 7.21 (2H, d, J = 7.8Hz, H-5 / 9), 7.19 (1H, t, J = 7.8 Hz, H-7), 6.60 (1H, dt, J = 3.0, 1.8 Hz, H-4′),6.40 (1H, dd, J = 5.4, 1.8 Hz, H-2′), 2.84 (2H, t, J = 7.8 Hz, H-3), 2.48 (2H, t, J = 7.8 Hz, H-2), 13 C NMR (DMSO- d 6, 100 MHz): δ C 172.3 (C-1), 171.6 (C-1′), 153.8(C-3′), 141.0 (C-4), 128.4 (C-6 / 8), 128.3 (C-5 / 9), 126.0 (C-7), 123.3 (C-2′), 82.0 (C-4′), 36.8 (C-2), 30.4 (C-3).

[0030] Example 2: In vitro pharmacodynamic evaluation of compound NFB.

[0031] I. Griess method for determining NO content.

[0032] BV-2 microglia increased at a rate of 2.5 × 10⁻⁶. 4 Cells were seeded at a density of 1 / 2 wells in 96-well plates, with three groups: a control (CON), an LPS group, and an LPS + drug group. Cells adhered for 24 h and were then observed to have reached a density of over 80%. A concentration gradient of 16 compounds (NFB) was established (0.1, 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg / L). µ M) and LPS (final concentration 100 ng / mL) were reacted together for 24 h, 150µ L / well. Take 50 μL of a 96-well plate and sequentially sample 50 μL of each well. µ Add a new 96-well plate to each well, and add 50 ml of Griess reagent. µ L / well, incubated in an incubator for 15 min, and the absorbance OD value was measured at 540 nm wavelength using a microplate reader.

[0033] Test results as follows Figure 1 As shown, compared to the LPS group, <0.01, <0.001; compared with the blank group, ### P <0.001. Compared with the CON group, the NO content in the LPS group was increased, and NO release was inhibited in a dose-dependent manner after NFB treatment. NFB at 2 µ At a concentration of M, the NO inhibition rate reaches approximately 50%, and its IC50 value is... 50 The value is 1.16 µ M. indicates that NFB has potential anti-neuroinflammatory activity.

[0034] Furthermore, during the separation and purification process in Example 1, compounds 1-5 were isolated from adjacent fractions E5f8 and F, as shown below. These compounds are all cinnamamide derivatives, sharing a common structural feature: a left-side benzene ring connected to a right-side five-membered nitrogen-containing heterocycle (pyrrole and its derivatives) via acrylamide / propionamide. In the LPS-induced BV-2 microglia NO release model, compounds 1-4 did not show significant inhibitory activity, with an IC50 of [missing value]. 50 All values ​​are greater than 50 μ M; Although compound 5 exhibits activity, IC 50 The value is only 8.12 μ M, but with low activity.

[0035] Compound NFB also contains a left-side aromatic benzene ring and an amide bond, belonging to a derivative with a cinnamic amide skeleton. It belongs to the same structural family as compounds 1-5, making the activity comparison between them more convincing: with the shared benzene ring-amide backbone remaining unchanged, the difference in the type of heterocycle and substituents on the right side leads to a huge disparity in biological activity. The right side of compounds 1-5 consists of a pyrrole ring or a derivatized five-membered nitrogen heterocycle, while the right side of NFB is... α , β -Unsaturated γ - Lactone ring. Although both are five-membered heterocycles, they differ significantly in electronic effects, steric effects, and hydrogen bonding interactions. This is the core reason why NFB activity is far superior to compounds 1-5. The specific structural differences are reflected in the following three aspects: First, their electrophilic responses are fundamentally different. The right side of NFB... α , β -Unsaturated γ - The lactone fragment is highly electrophilic; its lactone carbonyl group forms a conjugated system with the intracyclic double bond, and the carbonyl carbon... β - Carbon atoms can all serve as electrophilic centers, readily undergoing Michael addition reactions or lactone ammonolysis reactions with nucleophilic amino acid residues containing thiol or amino groups on target proteins under physiological conditions, thereby achieving reversible or irreversible covalent binding to the target. The pyrrole rings in compounds 1-3 are relatively electron-rich heterocycles, making it difficult for them to form strong interactions with amino acid residues near the target site, and also making covalent binding difficult. The pyrrolidone rings in compounds 4-5 belong to lactam structures, although in the structure of compound 5... α , β Unsaturated lactams can also act as Michael addition acceptors, but because the nitrogen atom in lactams has a stronger electron-donating conjugation effect than the oxygen atom in lactones, the electrophilicity of its carbonyl carbon is significantly weaker than that of the lactone carbonyl group. Simultaneously, the diacyl substitution on the nitrogen atom of lactams results in extremely low ring-opening ability, making it more susceptible to N-amide bond decomposition in pyrrolidones, leading to the catabolic metabolism of compounds 4 and 5. Therefore, although compound 5 may undergo Michael addition, its steric effect, electronic effect, and reactivity are all lower than NFB, making it difficult to achieve efficient target weak inter-binding or covalent binding.

[0036] Second, there is a significant difference between the chiral center and the molecular spatial configuration. The right-side ring of NFB contains... α , β - The unsaturated lactone conjugated system, with atoms on the ring essentially in the same plane, exhibits a rigid near-planar conformation, effectively restricting the conformational freedom of the molecule. Simultaneously, the 4′ carbon atom of this ring is a chiral center, and its specific stereoconfiguration endows the molecule with a clear stereoscopic effect, enabling it to achieve a precise "lock-and-key" spatial match with the target protein's active pocket. In contrast, compound 1 has its chiral center located on a saturated pyrrolidine ring, connected to a hydroxyl group. The pyrrolidine ring has a certain spatial conformation, but its spatial environment differs significantly from that of NFB. Compounds 2-5 completely lack a chiral center and the specific stereo orientation possessed by NFB, making precise spatial matching difficult when performing chiral recognition with target proteins.

[0037] Third, the hydrogen bond donor-acceptor combination patterns differ. In the NFB molecule, the NH group in propionamide can act as a strong hydrogen bond donor, and the carbonyl oxygen group in the lactone ring can act as an excellent hydrogen bond acceptor. The two form a fixed spatial arrangement and can simultaneously participate in a multiple hydrogen bond network within the target protein pocket, greatly enhancing the binding stability of the target complex. However, compounds 1-5 do not have free NH, lack a strong hydrogen bond donor, and do not have a hydrogen bond synergistic effect of equal strength as NFB.

[0038] In summary, NFB retains the benzene ring and amide bond on the left side, while replacing the five-membered nitrogen heterocycle on the right side with one containing an acetylamino group. α , β - Unsaturated lactone ring. It is this structural modification that endows the NFB molecule with a strong electrophilic covalent reaction site, good steric efficiencies, and a better synergistic hydrogen bond network, ultimately enhancing the IC50 of NFB. 50 The value reached 1.16 μ M is far superior to compounds 1-5 (IC). 50 >50 μ M may be only 8.12 μ The above comparison results fully demonstrate that, under the premise that the cinnamamide core remains unchanged, replacing the five-membered nitrogen heterocycle on the right side with an acetamino-substituted unsaturated lactone ring is the decisive structural factor that enables NFB to acquire potent anti-neuroinflammatory activity. This also points to a clear direction for chemical modification for subsequent drug optimization and candidate compound development based on this type of skeleton.

[0039] II. MTT assay for cytotoxic activity.

[0040] Take the remaining 100% of the NO content in the Griess method for NO content detection. µ For the L-type 96-well plate, add 10g of MTT reagent to each well. µ Incubate in an incubator for 2 hours, discard the supernatant, and add 150 ml of DMSO. µ L / well, placed on a shaker at minimum shaking speed and timed for 5 min. OD value was measured at 490 nm using a microplate reader to determine the maximum non-toxic concentration of NFB.

[0041] Cell viability was detected using the MTT assay, and the results are as follows: Figure 2 As shown, compared to the LPS group, <0.001. NFB in 0.1-10 µ No significant cytotoxicity was observed within the M concentration range; from 15 µ M began to exhibit significant toxic activity, and cell viability decreased to 74%. Based on these results, and considering the effect of NFB on NO release in the previous step, the compound concentration gradient was set at 1, 5, and 10. µ M will conduct subsequent experiments.

[0042] III. Methods for detecting the effect of NFB on the activity of BV-2 cells after OGD / R injury.

[0043] BV-2 cells were grown at a rate of 2.5 × 10⁻⁶. 4 The cells were seeded at a density of 1000 μL in 96-well plates, with three groups: a control group (CON), an LPS group, and an LPS + drug group. After 24 h, different concentrations of NFB (1, 5, 10 g / L) were added to the drug groups. µ M), 200 µNFB pretreatment was performed on each well. After 2 h, the medium was replaced with ischemic-glucose medium containing different concentrations of the drug. The culture plate was then placed in an anoxic chamber, and a mixed gas (95% N2, 5% CO2) was introduced at a flow rate of 20 L / min for 5 min. OGD treatment was then performed in an incubator for 4 h. After 4 h, the 96-well plate was removed, the supernatant was discarded, and the medium was replaced with 200 L / well of complete medium containing the drug. µ L / well, reoxygenated for 24 h, and OD value was detected at 490 nm using a microplate reader.

[0044] The results are as follows Figure 3 As shown, compared to the LPS group, <0.01, <0.001; compared with the blank group, ### P <0.001. After OGD / R modeling, a large number of cells died, and their survival rate dropped to 40%. However, NFB treatment significantly improved cell survival rate in a concentration-dependent manner, indicating that NFB can exert a certain degree of neuroprotective effect.

[0045] IV. Detection methods for the neuroprotective effect of NFB on SH-SY5Y neurons.

[0046] (1) The SH-SY5Y human neuroblastoma cell line is a subline obtained from the SK-N-SH neural cell line after three cloning processes, and is widely used in neuronal cell biology research. LPS induces BV-2 cells to produce a large number of pro-inflammatory mediators. Therefore, in this study, SH-SY5Y cells were treated with conditioned medium (MCM) after LPS stimulation of microglia to evaluate whether NFB has a protective effect on neural cells.

[0047] Log-phase SH-SY5Y cells were harvested at 3.0 × 10⁻⁶ cells per cell line. 4 / wells were seeded into 96-well plates, with blank control, blank drug control, LPS, and LPS + NFB (1, 5, 10 wells). µ There are eight groups in total (M). The cells adhered to the wall for 24 h, the supernatant was removed, and the collected MCM of the corresponding group was replaced. The cells were incubated in an incubator for 24 h, and their OD values ​​were measured at 490 nm.

[0048] The results are as follows Figure 4 As shown, compared to the LPS group, <0.01, <0.001; compared with the blank group, ### P<0.001. Conditioned medium without LPS showed no significant toxicity to SH-SY5Y cells; conditioned medium treated with LPS reduced the survival rate of nerve cells to approximately 64%; while conditioned medium treated with LPS + NFB significantly improved the survival rate of SH-SY5Y cells in a dose-dependent manner, suggesting that NFB can alleviate nerve cell damage by inhibiting microglia-mediated neuroinflammation.

[0049] (2) Further, using an OGD / R-induced SH-SY5Y cell model, the culture supernatant of LPS-induced BV-2 cells was used to intervene in the damaged nerve cells, and the protective effect of NFB was evaluated. SH-SY5Y cells in logarithmic growth phase were used at 3.0 × 10⁻⁶ cells / cells. 4 / wells were seeded into 96-well plates, with blank control group, OGD / R group, OGD / R + LPS group, and OGD / R + LPS + NFB group (1, 5, 10 wells). µ (M) A total of six groups were established. After 24 hours, the supernatant was aspirated. Except for the blank and blank drug groups, which were replaced with complete culture medium, the other groups were replaced with ischemic-glucose-deficient culture medium to establish the OGD / R model. After 4 hours, the culture plates were removed, the culture medium was discarded, the OGD / R groups were replaced with complete culture medium, and the other groups were given the corresponding MCM, 200 mg / L. µ L / well, reoxygenated for 24 h, OD value detected at 490 nm.

[0050] The results are as follows Figure 5 As shown, compared to the LPS group, <0.01, <0.001; compared with the blank group, ### P <0.001. Compared with the control group, cell viability in the OGD / R group decreased to 69%; cell survival rate further decreased after LPS intervention; while the addition of NFB significantly improved nerve cell survival rate in a concentration-dependent manner, suggesting that NFB can alleviate microglia-mediated inflammatory response after OGD / R and exert a neuroprotective effect.

[0051] V. RT-PCR experiment.

[0052] Step 1: Establish a cell model First, an LPS-induced neuroinflammation model of BV-2 microglia was established. Logarithmic growth phase cells were harvested at a concentration of 5.0 × 10⁻⁶ cells. 5 / wells were seeded into 6-well plates, and after 24 h of adherent growth, pre-incubation with the drug was performed for 2 h. Then, LPS (final concentration 100 ng / mL) was added and incubated for 4, 6, and 24 h. The OGD / R-induced microglial cell damage model also used logarithmic growth phase cells at 5.0 × 10⁶ cells / well. 5 / wells were seeded into cell culture dishes, and after 24 h of adherent growth, the cells were pre-incubated with drugs for 2 h, followed by OGD modeling for 4 h, and then replaced with complete culture medium. The cells were reoxygenated for 6, 12, and 24 h respectively, in preparation for subsequent RNA extraction.

[0053] Step 2: Extraction of total RNA from cells Discard the DMEM in the cell culture dish, wash twice with PBS buffer, then add 1 mL of Trizol and vigorously pipette to lyse the cells. Incubate on ice for 10 min. Then add 200 mL of chloroform to each tube. µ The liquid phase was vigorously shaken in a vortex and allowed to stand for 10 minutes to separate. Then, a low-temperature centrifuge was set to 12000 rpm, 4 °C, and centrifuged for 20 minutes. After centrifugation, the liquid separated into three layers; 200 ml of the upper aqueous phase was collected. µ L, add 300 ml of isopropanol at a ratio of 1:1.5. µ Invert the tube 10 times slowly and let it stand for 10 minutes to precipitate the RNA. Centrifuge again at 12000 rpm, 4 °C, for 10 minutes. Discard the supernatant to obtain the RNA precipitate. Add 1 mL of 75% ethanol-DEPC water to wash the precipitate and centrifuge at 7500 rpm, 4 °C, for 10 minutes. Repeat the previous step and centrifuge again, discarding the supernatant. Finally, add enzyme-free water to dissolve the precipitate. Then, measure its concentration to prepare for subsequent reverse transcription and amplification.

[0054] Step 3: Reverse Transcription and Amplification Prepare the relevant reagents according to the reverse transcription kit; the reaction system is 20... µ The resulting mixture was reverse transcribed into cDNA using a 96-well rapid thermal cycler, and the sample was stored at -20 °C.

[0055] Subsequently, the corresponding amplification reagents were prepared using the amplification kit, and 10 amplification units were established according to the instructions. µ The L amplification system used primer sequences designed and validated on NCBI and synthesized by Shanghai Sangon Biotech. The amplification program was then set up on a real-time quantitative PCR instrument to amplify the target gene.

[0056] The effects of compound NFB on the levels of polarization markers and inflammatory mediator mRNA in LPS-induced BV-2 microglia are as follows: Figure 6 , Figure 7 As shown, compared to the LPS group, <0.05, <0.01, <0.001; compared with the blank group, ## P <0.01, ### P<0.001. Among them, the positive control drug minocycline (MINO) = 30. µ M. Compared with the control group, the LPS group had higher levels of pro-inflammatory factors (IL-1). β IL-6, TNF-α α The mRNA levels of M1 markers (CD16, CD86, iNOS) were significantly increased in the LPS group; NFB treatment showed a dose-dependent decrease in the expression of these markers. Conversely, the expression of anti-inflammatory factors (IL-4, IL-10, TGF-β) was significantly increased in the LPS group. β The mRNA levels of M1 polarization markers (CD206, Arg-1, YM-1) were significantly lower in the control group than in the control group, while NFB treatment increased their expression levels. These results suggest that NFB can inhibit M1 phenotype polarization in microglia and promote their conversion to the M2 phenotype, thereby alleviating LPS-induced neuroinflammation.

[0057] The effects of compound NFB on the levels of polarization markers and inflammatory mediator mRNA in OGD / R-induced BV-2 microglia are as follows: Figure 8 , Figure 9 As shown, compared to the OGD / R group, <0.05, <0.01, <0.001; compared with the blank group, ## P <0.01, ### P <0.001, MINO of positive drug = 30 µ Compared with the control group, the levels of M1 markers and pro-inflammatory cytokines mRNA in microglia were increased after OGD / R; the expression of M2 marker Arg-1 and anti-inflammatory cytokine IL-4 showed no significant difference from the control group, while the expression of CD206, YM-1, IL-10, and TGF-β were significantly higher. β The expression of NFB was upregulated. After NFB treatment, the levels of M1 type and pro-inflammatory factor mRNA decreased in a concentration-dependent manner, while the levels of M2 type and anti-inflammatory factors increased significantly, indicating that NFB can inhibit the polarization of microglia to M1 type after OGD / R and promote their polarization to M2 type, thereby alleviating the inflammatory response after injury.

[0058] VI. Western-Blot Protein Immunoblotting Experiment Method.

[0059] 1. Establishment of cell model Cells in the logarithmic growth phase were harvested at a concentration of 5.0 × 10⁻⁶ 5 / Vessicles were seeded into medium and large dishes to establish LPS and OGD / R models, respectively. The remaining subsequent operations were the same as in "V. RT-PCR Experiment, Step 1, Establishing Cell Models". The incubation time for LPS was 12 and 24 h, and for OGD, it was incubated for 24 h after 4 h of modeling, followed by total protein extraction.

[0060] 2. Extraction of total protein from cells Prepare lysis buffer in advance according to experimental needs. Discard the supernatant from the cell culture dish, add 1 mL of PBS to wash the cells, and repeat the operation twice. Then add 150 mL of PBS to each tube. µ Cells were scraped off using a cell scraper to obtain L of lysis buffer, and the resulting cell lysis buffer was transferred to Eppendorf tubes and placed on ice for 30 min of lysis, vortexing for 5-10 seconds every 5 min. Then, the cells were centrifuged at 15000 rpm, 4 °C for 20 min using a low-temperature high-speed centrifuge. After centrifugation, the supernatant was aspirated, and SDS loading buffer was added and mixed well. The protein was then boiled in a metal bath at 100 °C for 10 min and stored at -80 °C. Protein concentration was determined using a BCA protein assay kit: each group had 3 replicates, and 15 μL of protein was added to the blank wells. µ L sterile water, add 12 to the sample well µ L sterilized water + 3 µ L protein solution, add 100 ml to each well. µ L working solution, incubated at 37℃ with shaking for 20 min, and the OD value at 562 nm was detected by microplate reader.

[0061] 3. Electrophoresis, electroporation, blocking, and incubation with primary and secondary antibodies for imaging. The concentration of SDS-polyacrylamide gel (8% or 10%) was determined based on the molecular weight (KD) of the target protein, and the gel was prepared using a one-step PAGE color gel electrophoresis kit. The prepared SDS gel was incubated at room temperature for at least 60 min, and electrophoresis was performed at a constant voltage of 80 V until the molecular weight standard was completely separated. The PVDF membrane was activated with anhydrous methanol for 30 s and then immersed in the electroporation buffer. The membrane was assembled in a "sandwich" structure (filter paper-gel-PVDF membrane-filter paper) and electroporated at a constant current of 300 mA for 110 min. After electroporation, the PVDF membrane was blocked with protein-free rapid blocking buffer and incubated on a side-shaking incubator for 1 h. Primary antibodies were prepared using Western blotting buffer at the following dilution ratios: Arg-1 (1:3000), CD86 (1:3000), iNOS (1:1000), CD206 (1:1000), and IL-1. β (1:1000) β-actin (1:20000). Incubate overnight on a side-shaking incubator in a light-protected box at 4°C. The primary antibody is recovered the next day, and the membrane is washed three times with TBST. Fluorescent secondary antibodies are prepared with 5% skim milk powder: rabbit anti (1:10000) and mouse anti (1:20000), and incubated for 1 h in the dark. After washing the membrane three times with TBST, it is developed using an Odyssey near-infrared laser imaging system.

[0062] The effects of NFB on the expression of polarization markers and inflammatory mediator proteins in microglia after LPS modeling were verified by Western blotting. Results are as follows: Figure 10 As shown, compared with the CON group, the LPS group had higher levels of the M1 biomarker iNOS, CD86, and the pro-inflammatory factor IL-1. β The expression levels of M2 markers CD206, Arg-1, and anti-inflammatory factor IL-10 were significantly increased, while the expression of M2 markers CD206, Arg-1, and anti-inflammatory factor IL-10 was significantly decreased. NFB treatment dose-dependently reduced the expression of M1 and pro-inflammatory mediator proteins, while increasing the expression of M2 and anti-inflammatory mediator proteins.

[0063] The effect of NFB on the expression of polarization proteins in microglia after OGD / R modeling was then verified. The results are as follows: Figure 11 As shown, compared with the CON group, the levels of M1 biomarkers iNOS, CD86, and the pro-inflammatory factor IL-1 were significantly higher after OGD / R. β The expression levels of M2 protein were significantly increased, while the expression of M2 markers CD206 and Arg-1 decreased. NFB treatment reduced M1 protein expression in a dose-dependent manner, while increasing M2 protein expression.

[0064] Example 3: In vivo pharmacodynamic evaluation of compound NFB.

[0065] I. Establishment of the MCAO / R model in SD rats.

[0066] SD rats were randomly divided into five groups of 12 rats each, including the sham operation group, the model group (MCAO / R), the low-dose NFB group (0.5 mg / kg), the high-dose NFB group (3 mg / kg), and the positive control drug butylphthalide (NBP) group (80 mg / kg).

[0067] A rat model of acute middle cerebral artery ischemia-reperfusion (MCAO / R) injury was established using the suture occlusion method. The specific procedure is as follows: (1) Prepare surgical instruments (such as suture needles, sterile syringes, surgical scissors, forceps, arterial clamps, etc.) and materials (such as iodine, 75% medical alcohol, heating pads, sutures, etc.) before the operation; (2) Weigh the rats and calculate the anesthetic dose and drug dosage based on the weight. After intraperitoneal injection of anesthesia, fix the rats in a supine position on the surgical board. (3) Disinfect the skin on the right side of the neck with iodine, shave the hair on the right side of the midline of the neck, make an incision of about 2-3 cm, bluntly separate the neck muscles and fascia, and expose the common carotid artery (CCA). (4) Carefully separate the fascia around the CCA and the vagus nerve to expose the CCA, internal carotid artery (ICA), and external carotid artery (ECA). (5) Clamp the CCA and ICA with arterial clamps, ligate the distal end of the ECA, cut a small opening after threading the suture at the proximal end, insert the suture plug, release the ICA arterial clamp, and send the suture plug through the CCA and ICA to the origin of the middle cerebral artery (MCA), ligate and fix it, and release the CCA arterial clamp. (6) Suture the wound, disinfect with povidone-iodine, place the rat in a warming cage, and gently pull out the suture plug after 1 hour of ischemia to achieve reperfusion; (7) Neurological function scores were assessed 1 h and 24 h after reperfusion, and behavioral tests were completed 24 h later before subsequent experiments were conducted.

[0068] II. mNSS Neurological Function Score.

[0069] Using the modified mNSS neurological function scoring model, a comprehensive assessment of motor, reflex, sensory, balance, and other abilities was conducted on rats in the sham-operated group, model group, and drug-treated group. Twelve rats were in each group, and their scores were subsequently tallied. Specific evaluation criteria are shown in Table 1 below.

[0070] Table 1. Detailed Rules for Neurological Function Scoring The mNSS score was used to assess the neurological function status of rats after ischemic stroke. The results are as follows: Figure 12 As shown, compared to the MCAO / R group, <0.001; compared with the sham surgery group, ### P <0.001; Butylphthalide (NBP = 80 mg / kg): Positive control. One h after reperfusion, the scores of all model groups were approximately 12 points, indicating severe neurological deficits. Twenty-four h after reperfusion, the scores in the NFB treatment groups significantly decreased, with the 3 mg / kg dose group showing better results than the positive control drug NBP (80 mg / kg). These results indicate that NFB can alleviate MCAO / R-induced neurological damage in rats.

[0071] III. TTC staining test for cerebral infarction.

[0072] TTC staining is used to assess tissue ischemia injury. TTC is a lipid-soluble, light-sensitive complex that can be reduced to insoluble red tribenzo[a]azine by dehydrogenases in living cells within the tissue. In ischemic areas, dehydrogenase activity is reduced or absent, preventing TTC reduction and resulting in a white appearance. After neurological function scoring, rats were anesthetized and euthanized. The brain was harvested, washed with PBS, flash-frozen in liquid nitrogen, and then briefly frozen at -80°C. Once the tissue had slightly hardened, the brain was sliced ​​into 6-7 pieces (1-2 mm thick) and incubated in a 1% TTC solution at 37°C in the dark for 30-40 minutes, turning the slices every 10 minutes. After staining, the slices were transferred to fixative and stored at room temperature in the dark. The brain slices were arranged sequentially and photographed. ImageJ software was used to calculate the infarct area and the cerebral infarction rate.

[0073] Normal brain tissue appears red after TTC staining, while infarcted areas appear white. Results are as follows: Figure 13 As shown, compared to the MCAO / R group, <0.01, <0.001; compared with the sham surgery group, ### P <0.001; NBP was a positive control. No infarction was observed in the sham surgery group, while the infarction rate in the modeling group reached 38.79%. The NFB treatment group significantly reduced the infarct area, with the high-dose group showing an infarction rate reduced to 11.12%, demonstrating superior efficacy compared to NBP.

[0074] IV. Experiment for detecting cerebral edema using the wet-dry weight method.

[0075] The water content in brain tissue was determined using the wet-dry weight method. Brain tissue was harvested and weighed immediately after scoring (i.e., wet weight). After TTC staining, brain slices were collected, dried in a 95 ℃ oven for 48 h, and weighed dry. The brain edema rate was calculated using the formula: (wet weight - dry weight) / wet weight × 100%.

[0076] The results are as follows Figure 14 As shown, compared to the MCAO / R group, <0.01; compared with the sham surgery group, ### P <0.001; NBP served as a positive control. Compared with the sham-operated group, the cerebral edema content in the MCAO / R group reached 84.41%, while the high-dose NFB group decreased to 81.12% after NFB treatment, comparable to the effect of NBP. This suggests that NFB can reduce cerebral edema after MCAO / R and exert a neuroprotective effect.

[0077] V. Behavioral tests.

[0078] A modified adhesive tape test was used to evaluate neurological function damage and recovery in rats after cerebral ischemia. A 1 cm wide and 3 cm long piece of transparent adhesive tape was cut, rolled into a sleeve, and placed on the forelimb of the rat on the opposite side of the ischemic side. The rat was placed in a cage and observed for 30 seconds. The time spent by the rat touching the sleeve with the other forelimb during the observation period was collected and counted.

[0079] The modified tape test was used to examine whether NFB could improve neurological deficits in MCAO / R rats. Results are as follows: Figure 15 As shown, compared to the MCAO / R group, <0.001; compared with the sham surgery group, ### P <0.001; NBP (80 mg / kg) served as a positive control. Compared to the sham group, rats in the model group did not touch the cuff within 30 seconds, while the time taken for rats in the NFB treatment group to consciously touch the cuff was reduced to 20.83 and 10.08 seconds, respectively. This suggests that NFB can repair neurological damage after MCAO / R.

[0080] VI. Immunofluorescence experiment.

[0081] Experimental methods: (1) Detection of the effect of NFB on neuronal damage in the ischemic cerebral cortex of MCAO rats. Rats were anesthetized and sacrificed. Brain tissue was washed with PBS and fixed in 4% paraformaldehyde at 4℃ for 24 h. It was then dehydrated sequentially with 20% and 30% sucrose solutions and stored at -80℃ for later use. Sections were prepared by cryostat (10 mm thick). µ Store at -80℃. Before use, warm to room temperature for 20 min, fix with acetone for 5 min, wash 3 times with TBST, and block with goat serum for 40 min. Incubate with primary antibody (diluted with TBST at a ratio of 1:200) overnight at 4℃, wash with TBST, add fluorescent secondary antibody (diluted with TBST at a ratio of 1:250), and incubate at room temperature in the dark for 1 h. Mount with 4',6-diamidinyl-2-phenylindole (DAPI), and take pictures using an EVOS M7000 fully automated microscopy imaging system. All images are statistically analyzed in ImageJ.

[0082] (2) To detect the effect of NFB on the activation of microglia in the ischemic cerebral cortex of MCAO rats. The subsequent experimental procedures were the same as those in (1).

[0083] (3) Detect the effect of NFB on the expression of the M1 marker iNOS in the ischemic cerebral cortex of MCAO rats. The subsequent experimental procedures are the same as above (1).

[0084] (4) Detect the effect of NFB on the expression of Arg-1, an M2 marker, in the ischemic cerebral cortex of MCAO rats. The subsequent experimental procedures were the same as those in (1).

[0085] Experimental results: (1) The effect of NFB on neuronal damage in the ischemic cortex of rats after MCAO / R injury was detected by using a double-labeled fluorescence colocalization method with NeuN-labeled mature neurons and TUNEL-labeled apoptotic cells. The results are as follows: Figure 16 As shown, compared with the sham group, the number of NeuN-positive cells in the cortical area of ​​the model group was significantly reduced, and the number of TUNEL-positive cells and TUNEL-positive cells were also significantly reduced. + / NeuN + The number of co-localized positive cells increased significantly, and NFB treatment significantly increased the number of NeuN-positive cells while decreasing TUNEL and TUNEL levels. + / NeuN + The number of colocalized cells suggests that NFB can inhibit neuronal damage following ischemia-reperfusion injury.

[0086] (2) Iba-1 was used + Immunofluorescence labeling was used to detect the effects of NFB on the morphology and abnormal activation of microglia in the ischemic cerebral cortex. Results are as follows: Figure 17 As shown, compared with the sham group rats, the number of activated microglia in the model group was significantly increased, and the morphology was amoebic (enlarged cell body, shorter and thicker synapses). After NFB treatment, the number of activated microglia was significantly reduced, the cell body was smaller and the number of synapses increased. The effect of the high-dose group was comparable to that of NBP, indicating that NFB can effectively reduce the activation of microglia in the ischemic cortex of MCAO / R rats.

[0087] (3) Activated microglia undergo phenotypic polarization. Classical M1 polarization, in particular, highly expresses its marker iNOS, thereby catalyzing NO production and promoting neurotoxicity, exacerbating the inflammatory response following brain injury. The number of pro-inflammatory microglia in the lesion area was counted by detecting Iba-1⁺ / iNOS⁺ double-positive cells using immunofluorescence co-localization. Results are as follows... Figure 18 As shown, compared with the sham group, the number of colocalized cells in the MCAO / R group was significantly increased, and the number of activated inflammatory cells was significantly reduced after NFB treatment, suggesting that NFB can inhibit the pro-inflammatory polarization of microglia to M1 type.

[0088] (4) Activated microglia polarized in the M2 type are the opposite of M1. They help reduce inflammation and promote nerve cell regeneration and repair by secreting anti-inflammatory factors (IL-4, IL-10) and highly expressing polarization markers such as Arg-1 and CD206. Therefore, using Iba-1⁺ / Arg-1 + Colocalization was used to assess the expression of Arg-1, an M2 polarization marker, in the ischemic cortex. Results are as follows: Figure 19As shown, compared with the sham group, the number of colocalizations in the model group was significantly reduced, and the number of colocalizations increased significantly after drug treatment, suggesting that NFB can promote the polarization of microglia to the M2 type and exert an anti-inflammatory effect.

[0089] VII. Animal tissue RT-PCR experiment.

[0090] The rats were euthanized, and their brains were harvested. After washing with PBS, the brains were cut along the midline into left and right hemispheres. The cortical tissue was dissected, flash-frozen in liquid nitrogen, and stored at -80°C. Approximately 20 mg of tissue was taken, and 1 mL of Trizol was added. The mixture was sonicated (100 W, on ice), allowed to stand for 10 min, and then 200 mL of Trizol was added. µ L-chloroform, the subsequent RNA extraction, reverse transcription and amplification procedures are the same as in "Example 2, In vitro pharmacodynamic evaluation of NFB, V. RT-PCR experiment".

[0091] The results are as follows Figure 20 , Figure 21 As shown, compared to the MCAO / R group, <0.01, <0.001; compared with the sham surgery group, ### P <0.001; NBP was a positive control. Compared with the sham-operated group, the MCAO / R group had a lower concentration of pro-inflammatory factor (IL-1). β IL-6, TNF-α α The mRNA levels of M1 markers (CD16, CD86, iNOS) were significantly increased after NFB treatment; pro-inflammatory factors (IL-1) were significantly increased after NFB treatment. β IL-6, TNF-α α The mRNA levels of anti-inflammatory factors (IL-4, IL-10, TGF-β) were significantly reduced in the MCAO / R group compared to the sham-operated group. β The mRNA levels of these markers were significantly increased; NFB treatment resulted in a dose-dependent increase in the expression of these markers.

[0092] 8. Animal tissue Western blotting.

[0093] Brain tissue processing and collection are the same as in "VII. Animal Tissue RT-PCR Experiment". Take approximately 30 mg of tissue and add 300... µ L lysis buffer was used for sonication followed by lysis on ice for 1 h (vortexing for 10 s every 5 min). The mixture was centrifuged at 15000 rpm for 20 min at 4 °C, and the supernatant was collected. 5×SDS loading buffer was added, and the protein was boiled at 100 °C for 10 min and stored at -80 °C. Protein concentration was determined by BCA method, and subsequent Western blot procedures were the same as in in vitro experiments.

[0094] The results are as follows Figure 22As shown, consistent with in vitro experimental results, compared with the sham group, the expression of M1 polarization markers in the model group rats was significantly increased, while the protein expression levels of M2 markers CD206, Arg-1, and the anti-inflammatory factor IL-10 were not significantly different from those in the sham group. After NFB treatment, M1 polarization expression was significantly decreased, while M2 polarization expression was significantly upregulated, with the high-dose group showing better results than the positive control group. This indicates that NFB can inhibit M1 polarization expression in the ischemic cortex and promote M2 polarization at the protein level, thus improving ischemia-reperfusion injury.

[0095] The number of devices and processing scale described herein are for the purpose of simplifying the description of this invention. Applications, modifications, and variations of the amide alkaloid NFB and its preparation methods and applications of this invention will be readily apparent to those skilled in the art.

[0096] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. Amide alkaloids NFB, characterized in that, It has the following structure: 。 2. The method for preparing the amide alkaloid NFB as described in claim 1, characterized in that, Includes the following steps: 1) Take the raw material of false betel leaves and branches, extract it by heating and reflux with ethanol solution, combine the extracts and concentrate under reduced pressure to obtain extract; 2) After suspending the extract in water, extract with ethyl acetate and collect the ethyl acetate fraction; 3) The ethyl acetate extract was separated by macroporous resin column chromatography, and gradient elution was performed using an ethanol-water system. The 80:20 and 85:15 eluent fractions were collected and combined. 4) The fraction obtained in step 3) was subjected to MCI column chromatography with gradient elution of methanol-water to separate fraction AK and collect fraction E; 5) Separate fraction E by dextran gel column chromatography, elute with 50% methanol-water solution to obtain fractions E1-E9, and collect fraction E5; 6) Separate fraction E5 by RP-C18 reversed-phase column chromatography with gradient elution in a methanol-water system to obtain fractions E5a-E5o and collect fraction E5e. 7) Separate fraction E5e by silica gel column chromatography, eluting sequentially with petroleum ether-acetone system and dichloromethane-methanol system to obtain fractions E5e1-E5e13, and collect fraction E5e8. 8) The E5e8 fraction was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) using methanol-water as the mobile phase, and the retention time was collected. t R The fraction with a content of 32.1 min yielded the compound NFB.

3. A pharmaceutical composition, characterized in that, It comprises a therapeutically effective amount of the amide alkaloid NFB as described in claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

4. The use of the amide alkaloid NFB as described in claim 1 or the pharmaceutical composition as described in claim 3 in the preparation of a drug for regulating the M1 / M2 phenotypic polarization of microglia.

5. The application as described in claim 4, characterized in that, The regulation involves inhibiting microglia from polarizing to the M1 pro-inflammatory phenotype and promoting their polarization to the M2 anti-inflammatory phenotype.

6. The use of the amide alkaloid NFB as described in claim 1 or the pharmaceutical composition as described in claim 3 in the preparation of a medicament for inhibiting neuroinflammatory responses.

7. The use of the amide alkaloid NFB as described in claim 1 or the pharmaceutical composition as described in claim 3 in the preparation of a medicament for treating ischemic stroke.

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