Neuroprotective effect and preparation methods of berberine

Berberine (BBR) is a natural alkaloid, which has played an important role in the field of medicine since its discovery in the late 19th century. However, the low availability of BBR in vivo prevents its full effect. In recent years, a large number of studies confirmed that BBR has a protective effect on the nervous system through various functions, yet the issue of the inability to systematically understand the protection of BBR on the nervous system remains a gap that needs to be addressed. Many existing literature introductions about berberine in neurodegenerative diseases, but the role of berberine in the nervous system goes far beyond these. Different from these literatures, this review is divided into three parts: preparation method, mechanism, and therapeutic effect. Various dosage forms of BBR and their preparation methods are added, in order to provide a reasonable choice of BBR, and help to solve the problem of low bioavailability in treatment. More importantly, we more comprehensively summarize the mechanism of BBR to protect the nervous system, in addition to the treatment of neurodegenerative diseases (anti-oxidative stress, anti-neuroinflammation, regulation of apoptosis), two extra mechanisms of berberine for the protection of the nervous system were also introduced: bidirectional regulation of autophagy and promote angiogenesis. Also, we have clarified the precise mechanism by which BBR has a therapeutic effect not only on neurodegenerative illnesses but also on multiple sclerosis, gliomas, epilepsy, and other neurological conditions. To sum up, we hope that these can evoke more efforts to comprehensively utilize of BBR nervous system, and to promote the application of BBR in nervous system protection.


Introduction
Berberine (BBR) was first discovered from the bark of Xanthoxylon clava.In the past, BBR was isolated from plants such as Berberidaceae, Ranunculaceae, Rutaceae, Menispermaceae, Papaveraceae, Loganiaceae, and Rhamnaceae.Nowadays BBR can be synthesized artificially (Zhang and Shen, 2023).Numerous studies on the pharmacological effects and associated mechanisms of BBR have been carried out recently in many countries.These studies have revealed that BBR has unique impacts and pharmacological activities on the cardiovascular system, nervous system, and endocrine system.These effects can lower blood lipids and protect the cardiovascular system, produce anti-anxiety effects by influencing brain neurotransmitters, and improve insulin sensitivity in the treatment of diabetes.The application potential of BBR in both the prevention and treatment of cardiovascular, cerebrovascular, nervous system, and other important disorders cannot be ignored (Hu and Mo, 2017).
As the worldwide population ages, hundreds of thousands of elderly people suffer from neurological issues.The majority of medications have adverse effects for central nervous system disorders (Dording and Boyden, 2019).But as a natural alkaloid, BBR has fewer adverse effects and offers some benefits in the medical management of central nervous system disorders.Researchers reported that BBR can penetrate the blood-brain barrier, reduce the permeability of the blood-brain barrier, and protect the integrity of the blood-brain barrier, thus maintaining the homeostasis of the central nervous system (Ma et al., 2010;Song et al., 2022) and is more suitable for brain diseases.Through its unique regulation mechanism, BBR have a great effect on regulating the nervous system (Ding et al., 2021).Moreover, BBR has a ameliorative impact on neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, and Huntington's disease) and psychiatric diseases (depression, anxiety, schizophrenia).To better investigate and develop the use of BBR in central nervous system illness, we elucidated the extraction method of BBR and dosage forms of BBR, as well as its pharmacological effects on inflammation, oxidative stress, apoptosis, autophagy and angiogenesis were described.In addition, it summarizes how BBR affects various signaling pathways to regulate the processes of central nervous system diseases.

Physicochemical properties and pharmacokinetics of BBR
BBR is a quaternary amine alkaloid with unique physical and chemical properties (Li et al., 2020).The molecular formula of BBR is C 20 H 18 NO 4 .BBR is a faint yellow acicular crystal, the melting point is 204.8-205.4°C.(The chemical structural formula of BBR is seen in Figure 1).BBR has high solubility in hot water, slight solubility in cold water and cold ethanol, and almost insoluble in chloroform and ether.Under physiological conditions, BBR mainly exists in ionized form and is easy to self-accumulate in the acidic environment of the gastrointestinal tract.The solubility at pH 1.2 (HCl) is 1/20 of that at pH 7.0 (Spinozzi et al., 2014).Due to the lipophobic nature of BBR, its passage through intestinal cells is obstructed.Its effective permeability coefficient in rat intestinal mucosa was 0.178 × 10 −4 cm s -1 , which confirmed its low permeability (Chen et al., 2011;Zhou, J. X. et al., 2022).
BBR is poorly soluble.BBR that is absorbed in by the gut can also be expelled back into the intestinal lumen by the action of P-glycoprotein (P-gP).BBR is the substrate of the efflux transporter P-gP, which makes BBR difficult to be absorbed orally and shows a very low bioavailability (<1%) in both animals and humans.Chen et al. studied that the absolute bioavailability of oral pathway in rats was 0.68% (Chen et al., 2011).In addition, P-gP inhibitors could increase the absorption of BBR by 6 times in rats, indicating that P-gP contributed to intestinal malabsorption of BBR (Pan et al., 2002).Liu et al. discovered that after the rats were given BBR intragastric administration, approximately 1/2 of the BBR was eliminated through the gastrointestinal tract, while the remaining 1/2 was excreted through the intestinum tenue, resulting in the oral absolute bioavailability of rats is 0.36% (Liu et al., 2010).It was found through experimental determination that first pass elimination of the intestine of BBR was the main obstacle to its oral absolute bioavailability.The liver's significant extraction and distribution of BBR might have led to its low concentration in rat plasma.After oral administration, BBR can be widely distributed in liver, kidney, brain, heart and other parts (Tan et al., 2013), and can penetrate the blood-brain barrier and rapidly distribute to the thalamus (Wang et al., 2005).A large amount of scientific evidence shows that BBR is metabolized by demethylation, glucuronidation and/or sulfonation (Ahmed et al., 2015), and the pharmacological activity of the metabolite is consistent with BBR (Wang et al., 2017).In both human and rats, the active metabolites of BBR are mainly divided into the following four kinds: berberrubine (M1), thalifendine (M2), demethyleneberberine (M3), jatrorrhizine (M4) (Liu et al., 2009).One study discovered that M1, M3, and M4 can directly cross the blood-brain barrier to perform a neuroprotective effect (Pan et al., 2024), but whether M2 can cross the blood-brain barrier remains unknown.In addition, the metabolites of BBR are all P-gP substrates, and the order of binding strength is as follows: M1 < BBR < M4 < M2 < M3.The higher the binding affinity, the more difficult the transport of the compound is (Zhang et al., 2019).Therefore, the oral bioavailability of M1 may be better than BBR.Not only that, BBR metabolites also showed higher concentrations in plasma, and the lipophilicity of M1 was higher than BBR (Wang et al., 2015), this suggests that the study of the metabolically active components of berberine may provide an opportunity to further improve the efficacy of BBR.Finally, in terms of excretion, BBR is mainly excreted through urine and bile in the form of metabolites.In addition, BBR has high safety, rarely significant adverse reactions in clinical trials, and its metabolite structure can remain relatively stable after entering the brain, suggesting that it can play a therapeutic role in nervous system diseases (Wang, X. J. et al., 2024).et al., 2015), and the specific content of BBR extraction method is shown in Table 1.

The dosage form of BBR
BBR has a broad application prospect and is mainly used in the form of hydrochloride in clinic.However, due to the low solubility of hydrochloride, its popularization and use are limited.At the same time, the bitter taste of BBR and other reasons also make it difficult to swallow, affecting the compliance of patients.Therefore, the design of different dosage forms has been endless, including liposomes, β-cyclodextrin inclusion complex, dropping pills, microspheres, microemulsions, solid lipid nanoparticles, targeting drug delivery system, etc. (Wu, J. D. et al., 2013), the specific contents are shown in Table 2.
The aforementioned list includes all possible dose forms, preparation methods, and administration characteristics of berberine; nevertheless, the ones that may be useful in neurological illnesses are of greater significance.Most neurological diseases, including multiple sclerosis, Parkinson's disease, Alzheimer's disease, epilepsy, and cerebral infarction, require long-term medication.The benefits of slow-release dosage forms include decreased frequency of administration, stable drug concentration, and fewer side effects related to peak blood drug concentration (Andrade, 2015).Therefore, berberine is more suitable for long-term drug use when the dosage form is liposome, microsphere, nanoemulsion, and nanoparticles.In addition, the poor water solubility and low bioavailability of berberine is also a problem that must be paid attention to.Wang et al. 's study showed that when Berberine is prepared as an inclusion compound of β-cyclodextrin, specific intermolecular interactions can be formed between Berberine Hydrochloride and βcyclodextrin, and this dosage form greatly improves the solubility of berberine, thereby improving its bioavailability (Wang et al., 2020).Furthermore, the drug absorption velocity and absorption rate are enhanced when the dose forms are microspheres and nanoparticles because of their small particle sizes, which enhances bioavailability.The creation of berberine dosage forms is crucial for the nervous system's application, and suitable dosage forms enable berberine to fulfill its therapeutic function and enhance its healing effect.
2 Pharmacological mechanism of neuroprotective effect of BBR

Anti-oxidative stress
Oxidative stress occurs because of an excessive accumulation of reactive oxygen species (ROS), leading to an imbalance between oxidants and antioxidants, which in turn leads to the production of many neurological diseases (Li et al., 2020).During oxidative stress, an overabundance of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) can lead to lipid peroxidation, protein oxidation, protein nitration, and sugar cooxidation.This can cause damage to the plasma membrane of nerve tissue, destruction of the cytoskeleton, and mutation of nucleic acids (Barnham et al., 2004;Uttara et al., 2009).The generation of ROS is related to glutathione (GSH), superoxide dismutase (SOD) and other chemicals (Li, H. Y. et al., 2024).Once ROS is excessive and inhibits the antioxidant activity of cells, oxidative stress will occur (Cheng et al., 2022), the mechanism of BBR against oxidative stress is shown in Figure 2. BBR has the potential to reduce ROS production and protect nerve cells from oxidative damage.Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a primary producer of ROS, and BBR can alleviate oxidative stress by decreasing the production of NADPH oxidase (Ma et al., 2018).BBR effectively inhibits lipid peroxidation while enhancing glutathione content and superoxide dismutase activity in cells (Sadeghnia et al., 2017).BBR can effectively decrease the generation of ROS in both cytoplasmic and mitochondrial cells (Sun Y. et al., 2017).This reduction is likely due to the activation of Adenosine monophosphate (AMP)-activated protein kinase (AMPK) and sirtuin1 (SIRT1)/forkhead box O1 (FOXO1) pathways (Li, H. R., 2024).The antioxidative stress activity of BBR is also associated with the transcription factor PPAR, which is a transcription factor induced by ligands.Activation of PPARs can play an antioxidant role, And BBR can activate PPARδ to clear ROS and exert neuroprotective effects (Shou et al., 2022).BBR may also exert antioxidant effects by lowering inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and boosting HO-1.

Anti-neuroinflammation
Neuroinflammation is the inflammation of nerve tissue, resulting from various sources such as traumatic brain damage and autoimmune.Although neuroinflammation can start in different trigger sites, it all has one thing in common: microglia and astrocytes are constantly activated (Perry and Holmes, 2014), Figure 3 shows how BBR works to reduce neuroinflammation.BBR can prevent neuroinflammation and possibly be used as a candidate drug to treat illnesses of the central nervous system that are caused by inflammation.BBR may provide neuroprotective effects by decreasing the generation of several neurotoxic compounds by activated microglia (Nam et al., 2010), BBR has been observed to effectively suppress the activation of NF-κB and the phosphorylation of Akt, p38, and extracellular regulated protein kinases (ERK).It is suggested that BBR may hinder the inflammatory response of microglia by reducing the PI3K/Akt and mitogen-activated protein kinase (MAPK) pathway (Wan, J. S. and Zhang, M. R., 2018).BBR inhibits the pro-inflammatory reaction by stimulating AMPK in BV-2 microglia.Additionally, BBR notably reduces the expression of iNOS and COX-2 in BV-2 microglia induced by LPS or interferon (IFN)-γ.It also hinders the synthesis of nitric oxide.Furthermore, BBR has the ability to normalise inflammatory factors.It can effectively decrease the generation of pro-inflammatory cytokines TNF-α and IL-1β, as well as the manifestation of IL-6 in BV2 cells that have been activated by LPS (Lu et al., 2010;Zhang et al., 2016a).BBR also decreases the production of inflammatory  factors and GFAP, while suppressing Sphk1/S1P signalling and stimulating CREB signalling (Cheng et al., 2022).

Regulation of apoptosis
Nerve cell injury can cause apoptosis to some extent, resulting in the loss of nervous system function.Apoptosis is one of the important autostable mechanisms in multicellular organisms (Li, M. and Lin, J., 2014).BBR exerts anti-apoptosis effects on nerve cells by inhibiting caspase-3, which acts as an executor of apoptosis, and increasing the expression ratio of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and pro-apoptotic protein Bcl-2 associated X (Bax).BBR modulates the activity of many proteins involved in autophagy, such as Microtubule-associated protein 1A/1B-light chain 3 (LC3), Beclin-1, and p62.It also affects the function of apoptosis regulating proteins, including caspase 3, caspase 8, caspase 9, poly ADP-ribose polymerase (PARP), and Bcl-2/Bax, which play a role in inhibiting neuronal apoptosis (Zhang et al., 2016b).Liang et al. found that BBR can inhibit the cytotoxicity of Aβ25-35 on neuron cells, reduce the activity of various caspase proteins, increase Bcl-2/Bax, and have anti-apoptotic effects (Liang et al., 2017).Most studies (Kim et al., 2014a;Kim et al., 2014b;Simões Pires et al., 2014;Hsu et al., 2012) believe that BBR can regulate the activity of subunit p55γ promoter and activate PI3K/Akt pathway by enhancing PI3K kinase, and then inhibit the activity of Bad, a positive regulator of apoptosis, and reduce the production of pro-apoptotic caspase-3 (Hu et al., 2012).But Simoes et al. propose that the Akt/GSK3β pathway, ERK1/2 pathway, and JNK pathway play an important part in the neuroprotective action of BBR against apoptosis (Simões Pires et al., 2014).However, in addition to the anti-apoptosis effect of BBR, when it comes to the specific therapeutic effect of BBR on some nervous system diseases, it also has the pro-apoptotic effect of inducing disease cell apoptosis.For example, in the treatment of glioblastoma, BBR inhibits ER stress-induced apoptosis in T98G cells by generating ROS and mitochondria-dependent mechanisms (Eom et al., 2010), BBR can also induce apoptosis of U251 and U87 cells by significantly inhibiting the activation of ERK1/2 pathway (Rauf et al., 2021) (Figure 4).

Regulation of autophagy
Upregulation of autophagy is a promising treatment approach for a range of neurodegenerative disorders (Menzies et al., 2017).BBR, as an autophagy modulator, affects autophagy by affecting targets or pathways associated with autophagy (AMPK, mTOR, MAPK, Beclin-1, and SIRT1) (Figure 5).Furthermore, the effect of BBR depends on the environment, and its regulation of autophagy is bidirectional, not only promoting autophagy, but also inhibiting autophagy (Mohammadinejad et al., 2019).AMPK/mTOR is a basic modulator of autophagy, and BBR can regulate AMPK/mTOR through transcriptional mechanism.BBR significantly increases AMPK activity through the production of ROS and the elimination of AMPKα1 (Fan et al., 2015;Issat et al., 2011).BBR inhibits PI3K activity, significantly downregulates AKT phosphorylation in a manner that depends on the dosage, and inhibits the phosphorylation of mTOR, p70 ribosome S6 protein kinase (Thr389) and S6 (Ser235/236), promoting the early initiation of autophagy (Huang et al., 2021;Yi et al., 2015).BBR upregulates autophagy by regulating mitogen-activated protein kinase (MAPK) (Sun et al., 2015).Beclin-1 has binding sites for Vps34 and Bcl-2.When autophagy is induced, Beclin-1 separates out from Bcl-2 and binds to Vps34, enhancing the binding force between Beclin-1 and Vps34, which inducing autophagy (Fleming et al., 2011).BBR can upregulate the expression of Beclin-1 and promote its binding to Vps34.Interestingly, BBR can also inhibit autophagy and produce neuroprotective effects by making Bcl-2 and Beclin-1 bind continuously (Ding, S. et al., 2021).SIRT1 can activate autophagy and promote the expression of genes associated with autophagy by The ratio of phospholipid to cholesterol was 3:1, the ratio of drug to lipid was 1: 15, the mass concentration of phospholipid was 30 g/L, and the pH of external aqueous phase was 7.0 It has remarkable sustained release properties in vitro Wang et al. (2013) The liposomes were stable and reliable when the ratio of BBR hydrochloride to polysorbate 80 was 1:0.4 and the ratio of soybean lipid (soybean phosphate) was 1:20 The dosage of BBR was 0.030g, βcyclodextrin was 2.0g, the inclusion time was 90min The antibacterial ability of liposome is stronger than BBR alone, which can reduce drug dose and stimulation, prolong drug action time and improve drug efficiency, which has great economic and practical value in pharmaceutical engineering Li et al. (2003) It was prepared by saturated aqueous solution method and orthogonal test method.The optimum process was as follows: temperature was 80 °C, inclusion 2h, and the ratio of host and guest molecules was 4:1 (g/g) It can improve the solubility of insoluble drugs, improve bioavailability and cover up odors Qi, (2010) Dropping pill drug is heated and mixed with the matrix, insoluble condensing agent is dropped, and the molten drop shrinks into a pill in the condensate and then condenses into a solid state PEG1000+PEG4000 (1:1) was used as the matrix, the drug-matrix was 1:4, the material temperature was 95 °C, the dimethylsilicone oil was used as the coolant, the coolant temperature was 5 °C, the drip diameter was 3mm, the drip rate was 50 drops/min, the drop distance was 6 cm This process provides a reference method for reforming some insoluble drug dosage forms Chen and Zhou, L.

J. (2008)
The ratio of drug and matrix (PEG6000) is controlled at 1:4, the drop rate in the drip process is controlled at 40 or 50 drops/min, the temperature of the liquid is 70 or 85-90 °C, the temperature in the middle of the condensing tube is 6-8 °C, the temperature in the bottom of the condensing tube is −2 °C, the drop distance is 5 or 7cm, and the diameter of the dropper is 1.2-1.5 mm or 2 mm.The height of condensing column is 90-100 cm The prepared dropping pills have small dissolution time, good appearance quality and small difference in pill weight, which meet the quality requirements of dropping pills.The method has certain value for the industrial production and clinical application of dropping pills

Promote angiogenesis
Angiogenesis is achieved by stimulating the growth of blood vessels, which can promote the survival of neurons, improve brain plasticity and restore nerve function (Yang and Torbey, 2020), Figure 6 shows the mechanism of BBR promoting angiogenesis.BBR activates AMPK signaling pathway and has a vital function in promoting angiogenesis.BBR also promotes alternative activation (anti-inflammatory M2) polarization of microglia and inhibits classical activation (pro-inflammatory M1) polarization through AMPK signaling pathway activation to promote angiogenesis (Zhu et al., 2019).Zhu et al. found that BBR activated Akt and subsequently increased angiogenesis by activating miR-29b expression in vitro and in vivo (Zhu et al., 2017).Vascular  2023)'s study showed that BBR could promote the expression of VEGF and induce angiogenesis.By activating the HIF-1α/VEGF signal transduction pathway, BBR can increase the expression of microvascular density, VEGF and hypoxia inducible factor-1α (HIF-1α), promote angiogenesis, and thus achieve neuroprotective effects (Liu et al., 2017).Mechanism of BBR against oxidative stress.Anti-neuroinflammatory mechanism of BBR.
Frontiers in Pharmacology frontiersin.org09 3 BBR and central nervous system diseases 3.1 Cerebrovascular diseases

Transient cerebral ischemia, ischemiareperfusion, stroke and cerebral infarction
Cerebral ischemia affects all ages, from newborns to the elderly population, and is a major cause of death and morbidity (Dietz et al., 2022).Hydrogen peroxide (H 2 O 2 ) is a highly active molecule in the oxidation process.The imbalance between the antioxidant system and the oxidative system is participated in neurodegeneration and ischemic brain injury.The pharmacological mechanism of BBR in treating cerebrovascular diseases is presented in Figure 7. BBR can enhance the activity of NSCs damaged by H 2 O 2 , improve the damaged morphology of cells, and improve the proliferation inhibition induced by H 2 O 2 , resulting in brain tissue reconstruction (Ye, A. L. et al., 2015).Intraperitoneal injection of BBR solution could upregulate the expression levels of phosphorylatioed protein kinase B (p-Akt), phosphorylated glycogen synthase kinase-3 (p-GSK3) and cAMP-response element binding protein (p-CREB).It also reduces the expression of nuclear factor-kappaB (NF-κB), and plays a protective role in brain tissue after cerebral ischemia (Zhang, X. L., 2013).BBR reduces the outflow of potassium ions from ischemic neurons, inhibits neuronal apoptosis in hippocampal CA1 region after ischemia, and thus protects ischemic brain tissue (Wang et al., 2004).Chai et al. discovered that BBR can specifically bind to poly(A) signal in order to control the RB1 mRNA.This prevents the degradation of RB1 mRNA and the increase in Rb protein levels during ischemia reperfusion damage, thereby regulating the release of transcription factors, blocking cell cycle, inhibiting apoptosis and promoting cell survival (Chai et al., 2014).BBR can activate the sphingosine-1-phosphate (S1P)/hypoxia inducible factor-1 (HIF-1) pathway, which is conducive to improving neuronal cell damage caused by ischemia and hypoxia (Zhang Z. et al., 2016).On the basis of the research conducted by Hu et al. (Hu et al., 2012), BBR enhances the activity of PI3K p55γ promoter during cerebral ischemia-reperfusion, resulting in increased Akt activity and decreased caspase-3 activity, thereby exerting an anti-ischemic  Frontiers in Pharmacology frontiersin.org10 apoptosis effect.BBR enhances the expression of peroxidase proliferator activating receptor (PPARγ) in ischemia-reperfusion injury, which may be related to the decrease of recombinant DNA methyltransferase 1 (DNMT1) and recombinant DNA methyltransferase 3A (DNMT3a) expression and the decrease of PPARγ promoter methylation in ischemia-reperfusion injury (Pang et al., 2018).In addition, BBR can also achieve neuroprotective effect by inhibiting inflammatory response.The study of Yoo et al. found that the protective effect of BBR on the brain of ischemiareperfusion injured gerbils is also related to the inhibition of COX-2 expression, prostaglandin E2 generation and its antiinflammatory mechanism (Yoo et al., 2008).BBR can reduce the neuroinflammatory response by down-regulating the expression of metastasis-associated lung adenocarcinoma transcript 1 (Malat1) and high mobility group box 1 (HMGB1).It protects neuronal cells from cerebral ischemia-reperfusion injury (Cao et al., 2020).BBR downregulates the levels of proinflammatory cytokines iNOS, COX-2, IL-1β, IL-6 and TNF-α, and upregulates the expression of antiinflammatory cytokine IL-10 through targeting MAPK pathway and AMPK independent mode, alleviates the inflammatory response caused by ischemia reperfusion in rats, exerts the neuroprotective effect in brain.In addition, BBR can also carry the exosome miR-182-5p to injured neurons and play a neuroprotective role by inhibiting neuroinflammation and improving brain injury after ischemic stroke (Ding et al., 2023).Moreover, BBR inhibits ischemic neuronal death by reducing the activity of type 1 N- Mechanism of BBR promoting angiogenesis.Effects of BBR on cerebrovascular diseases.
Frontiers in Pharmacology frontiersin.org11 methyl-D-aspartate receptor (NMDA), reducing the excessive excitatory amino acids produced by ischemic stimulation and Ca 2+ inflow caused by oxygen free radicals (Yoo et al., 2006).BBR also inhibits central sympathetic nerves by blocking αadrenergic receptors, affecting cerebral blood flow supply in stroke patients (Benaissa et al., 2009).Autophagy is an important catabolic process in lysozyme and an essential pathway for survival in ischemic stroke (Li, H. Y. et al., 2024).For cerebral infarction, BBR can effectively reduce blood lipid levels, inhibit the expression of oxidized low-density lipoprotein (ox-LDL) and matrix metalloproteinase-9 (MMP-9), inhibit the growth of carotid atherosclerotic plaque, reduce plaque area, improve plaque stability, and improve the long-term neurological score of cerebral infarction patients (Chai, M. J. et al., 2017a).BBR can Effects of BBR on neurodegenerative diseases.Neuroprotective pathway of BBR.
increase the level of serum catalase (CAT) and reduce the expression of Malondialdehyde (MDA) in patients with acute cerebral infarction, thereby improving the neurological deficits (Chai, M. J. et al., 2017b).BBR reduce the serum IgG level of patients with acute cerebral infarction, which may inhibit the humoral immune response in the acute phase of cerebral infarction.BBR alleviates inflammatory response in rats with acute cerebral infarction, and its mechanism may be related to activation of Wnt/β-catenin signaling pathway (Yao, Y. et al., 2023).Xi et al. suggested that BBR can protect ischemic neurons by reducing the content of E-Selectin and intercellular call adhesion molecule-1 (ICAM-1) in the plasma of patients with cerebral infarction (Xi, G. M. et al., 2003).Additionally, BBR can also reduce ischemia-reperfusion induced cerebral infarction (Zhou et al., 2008).

Cerebrovascular disease in patients with diabetes and hyperhomocysteinemia
Diabetic peripheral neuropathy (DPN) is the most common chronic complication of diabetes mellitus (DM).Hyperhomocysteinemia (HHcy) can also lead to DPN.BBR improves nerve conduction velocity in diabetic peripheral neuropathy patients.The combination of mecobalamine with DPN can improve the symptoms and nerve conduction velocity of DPN patients, and has significant clinical efficacy (Ye, A. L. et al., 2015).Zhao et al. found through their research (Zhao, X. L. et al., 2006) that BBR can reduce the damage of brain cells, improve the compensatory function of brain cells and reduce blood sugar by alleviating mitochondrial calcium overload and release mitochondrial Cyt-C, thus playing a protective role in the brain tissue of diabetic rats.

Multiple sclerosis
Multiple sclerosis (MS) is an autoimmune disease characterized by immune imbalance, central nervous system inflammatory response, and myelin destruction.Experimental autoimmune encephalomyelitis (EAE) is the recognized animal model of this disease (Kroenke et al., 2008).In the inflammatory response of EAE, Th1 cells and Th17 cells are activated, and the secretion of inflammatory cytokines (INF-γ, IL-6, IL-17) respectively increases.BBR can directly act on the JAK/STAT signaling pathway and selectively inhibit the differentiation of Th1 and Th17 cells.BBR indirectly affects the function of Th1 and Th17 cells by affecting the expression and function of costimulatory molecules and the production of IL-6, which is due to inhibition of NF-κB activity in CD11b (+) APC (Qin et al., 2010).In addition, BBR can also inhibit the expression and activity of MMP-9, inhibit the migration of T cells to the central nervous system, and protect the blood-brain barrier, thereby reducing the inflammatory infiltration of the central nervous system, alleviating the disease of EAE mice, slowing down the inflammatory response and reducing the incidence of demyelination (Ma et al., 2010).

Glioma
Due to its invasive nature, molecular signaling, and location in the central nervous system, glioma is one of the most perplexing cancers (Kundu et al., 2019).BBR-mediated apoptosis blocks the AMPK/mTOR/ULK1 pathway and reduces tumor growth in glioblastoma multiforme (GBM) cells in vivo (Wang et al., 2016).Target of neuroprotective effect of BBR.
Frontiers in Pharmacology frontiersin.org13 BBR inhibits tumor growth and inhibits the expression of p-ERK1/ 2 and Ki-67 in glioma cells (Sun et al., 2018b).Neuroinflammatory cytokines such as IL-1 secreted by glioma cells are believed to have an impact on the genesis and development of tumors (Nasrollahzadeh et al., 2020).BBR inhibits the activation of inflammatory cytokine caspase-1 through the ERK1/2 signaling pathway, inhibits glioma cells, and subsequently produces IL-1 and IL-18.BBR has a specific anti-proliferation effect on glioma cells, and treatment with BBR can also reduce the cellular motility of U251 and U87 cells and induce cell apoptosis.BBR induces apoptosis of T98G cells in glioblastoma by mediating endoplasmic reticulum stress (Eom et al., 2010).BBR inhibits TGF-β1/SMAD2/3 signaling pathway and affects the proliferation, migration, invasion and apoptosis of glioma cells (Jin et al., 2022).Moreover, BBR has the potential to reverse the mechanism of epithelial-mesenchymal metastasis, which is a hallmark of tumor invasion (Tong et al., 2019).BBR inhibits tumor development by regulating neuroblastoma cell differentiation, stem cell function, and inducing cell death (Rauf et al., 2021).

Epilepsy
Epilepsy is one of the most widespread neurological diseases in the world.According to the study of Ghanem et al. (Ghanem et al., 2021), BBR can significantly reduce the activity of hypoxia inducible factor-1α (HIF-1α), transforming growth factor-β1 (TGF-β1), histone deacetylase (HDAC) and neuronal restrictive silencing factor NRSF gene expression levels in epileptic mice, and increase the level of Brain-derived neurotrophic factor (BDNF).Intraperitoneal injection of BBR in the mouse model of maximal electroshock-induced seizures can reduce the duration of the seizure of the tonic hind limb extension, resist the seizure of convulsions, and reduce the mortality (Bhutada et al., 2010).The study of Zhang et al. showed that BBR can alleviate pentylenetetrazole (PTZ)induced seizures, potentially protect zebrafish from further seizures, and use BBR can restore abnormal neuron firing in zebrafish larvae during seizures.In addition, BBR also suppresses the inflammatory response caused by epilepsy (Zhang et al., 2020).Sedaghat et al. found that BBR prevents the loss of hippocampal CA3 neurons and prevents the development of abnormal mossy fiber sprouting (MFS), which is a basic element of the action circuit in chronic epilepsy (Sedaghat et al., 2017).These data suggest that BBR also exerts a neuroprotective effect by alleviating Status epilepticus (SE) and spontaneous recurrent seizures (SRS) in the hippocampal model of epilepsy.

Neurodegenerative diseases
With the increase of the elderly population, age-related diseases such as neurodegenerative diseases are becoming more and more common, posing a threat to human health (Heemels, 2016).Specifically, neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's syndrome, etc.The pathogenesis of neurodegenerative diseases has several common features, such as oxidative damage and mitochondrial dysfunction.Among them, oxidative stress refers to excessive accumulation of ROS, which can lead to mitochondrial dysfunction and ultimately induce neurodegenerative diseases.BBR significantly reduces ROS production in cytoplasm and mitochondria (Caspersen et al., 2005;Poprac et al., 2017), Moreover, BBR also alleviates neurodegenerative diseases by regulating neuroinflammation and autophagy (Figure 8).

Alzheimer's disease
Alzheimer's disease (AD) is a neurological illness characterized by memory loss and cognitive impairment, accounting for around 60%-80% of all dementia cases (Rostagno, 2022).Aβ deposition is a crucial factor in the development of Alzheimer's disease (Huat et al., 2019).BBR can improve the effect of AD through various mechanisms, including inhibiting the hyperphosphorylation of Tau protein, inhibiting the production of Aβ, and inhibiting four key enzymes (acetylcholinesterase, butylcholinesterase and two isomers of monoamine oxidase) in the pathogenesis of AD.BBR can reduce the phosphorylation level of Tau protein, which may be related to its activation of the Akt/glycogen synthase kinase three signaling pathway (Durairajan et al., 2012).BBR can reverse the increase of malondialdehyde content and the decrease of SOD activity induced by calyculin A. Calyculin A-induced tau hyperphosphorylation is decreased by increasing protein phosphatase 2A (PP2A) activity and decreasing glycogen synthase kinase 3β (GSK-3β) activity (Yu et al., 2011).BBR can also enhance autophagy activity and promote autophagy clearance of tau through Class III PI3K/beclin-1 pathway (Chen et al., 2020).BBR significantly reduces Aβ, possibly by down-regulating the phosphorylation of amyloid precursor protein (APP) through activation of PI3K/Akt/GSK3 pathway, and inhibiting the production of Aβ 42 by inhibiting PERK/eIF2α/BACE1 signaling pathway (Wu et al., 2021).BBR can also promote Aβ clearance by promoting the expression of insulin-degrading enzyme (IDE) (Zhu, F. Q. et al., 2010).Inhibition of AChE and BChE has been shown to be a key target for effective management of AD by alleviating cholinergic deficiency and improving neurotransmission, and BBR has inhibitory effects on both AChE and BChE (Greig et al., 2005;Muñoz-Torrero, 2008).Not only that, BBR can also activate macrophages and increase their phagocytic function, increase the production of interleukin, (IL)-1 and can be used as a neuroprotective agent against AD (Kumazawa et al., 1984;Panahi et al., 2013).Long-term administration of BBR increases the expression of IL-1β and iNOS in the hippocampus of AD mice and improves memory impairment (Ji and Shen, 2012).

Parkinson's disease
Parkinson's disease (PD) is the second most common neurological disorder, affecting about seven million people worldwide (Pringsheim et al., 2014).Inhibition of Monoamine oxidase-B (MAO-B) has been proved to delay the onset of PD or reduce the symptoms of PD (Youdim et al., 2006).BBR and MAO-B are bound by hydrophobic interaction, and BBR may partially reduce the degradation of dopamine and the production of H 2 O 2 through the inhibition of MAO-B, thus alleviating the symptoms of PD.Kim et al. (Kim et al., 2014a) used 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine/probenecid (MPTP/P) mouse PD model to study the effects of BBR on dopamine consumption and shortterm memory of hippocampal neurogenesis.BBR was found to enhance motor balance and coordination by preventing damage to dopaminergic neurons, also improved short-term memory by inhibiting apoptosis of hippocampal cells.These data show that BBR treatment may serve as a potential therapeutic strategy to improve memory impairment and physical dysfunction in PD patients.In the study of Negahdar et al. (Negahdar et al., 2015) by using 6-hydroxydopamine (6-OHDA) -induced PD model, BBR could significantly improve lateral rotation behavior in PD rats, and could also prevent the reduction of the number of tyrosinehydroxylase (TH) -positive neurons in the group, providing a new strategy for PD treatment.It is worth noting that BBR can enhance the cytotoxicity caused by 6-OHDA, and the intraparitoneal injection of BBR for 21d in the 6-OHDA PD rat model can cause degeneration of dopaminergic neurons in the substrantia nigra (Kwon et al., 2010).Therefore, patients with PD should pay attention to the interaction between BBR and levodopa and avoid drug effects when using BBR.

Huntington's disease
Huntington's disease (HD) is an inherited neurodegenerative genetic disorder caused by the amplification (variable length) of CAG trinucleotide repeats in HTT, the gene encoding the Huntington protein, which accumulate in affected brain regions in an age-dependent manner, leading to late-onset neurodegeneration (Walker, 2007).In Jiang et al.'s study (Jiang W. et al., 2015), BBR has a protective effect on transgenic HD (N171-82Q) mice.When taken orally, BBR can effectively alleviate motor dysfunction and prolong the survival time of transgenic HD mice, and BBR can also promote the degradation of mutant Huntington protein by enhancing autophagy function.The autophagy-lysosome pathway also plays a crucial role in the clearance of the readily aggregated mutant Huntington protein (polyQ-HTT) (Martinez-Vicente et al., 2010), and induction of autophagy enhances the clearance of polyQ-HTT aggregates and reduces the toxicity of the mutant Huntington protein fragment (Floto et al., 2007).BBR can significantly trigger autophagy and remove polyQ-HTT aggregates, thereby significantly improving the neurophenotype of HD mice (Jiang W. et al., 2015;Fan et al., 2019).

Mental system disorders
A mental illness is a disorder of brain function caused by a variety of reasons that manifests in different forms as disorders in mental functions like cognition, behavior, will and emotion.

Anxiety
Anxiety is a state of excessive fear, characterized by motor tension, sympathetic overactivity, worry, and vigilance syndrome.BBR plays an anti-anxiety role by reducing the concentration of norepinephrine, dopamine and 5-hydroxytryptamine in the brain stem, and increasing the concentration of Vanillymandelic Acid (VMA) and 4-hydroxy-3-methoxy-phenylacetic acid (HVA).The anti-anxiety effect of 100 mg/kg BBR on mice is the same as that of 1 mg/kg diazepam and 2 mg/kg buspirone, and its anti-anxiety effect is related to accelerating the renewal rate of monoamine transmitters in the brain stem and reducing the activity of 5-HT-ergic systems (Peng et al., 2004).Yu et al. found that BBR improved anxiety in 5XFAD transgenic mice with AD (Yu, W. et al., 2021).Autonomic nervous system dysfunction and anxiety and other mental disorders often occur in women before and after menopause due to the fluctuation or reduction of sex hormone levels (Mulhall et al., 2018).BBR can also increase the content of equol in feces and serum and the ratio of equol to daidzein by enriching Lactobacillus, Bacteroides, Bifidobacterium and Akkermansia muciniphila in the intestine.Improved anxiety-like behavior in female ovariectomized rats.The regulatory effect of BBR was eliminated in germ-free animals, but the changes in microbiota, equol content and anxiety-like behavior of the animals receiving fecal microbiota transplantation were basically the same as those of the donors.This suggests that BBR can improve anxiety-like behaviors induced by decreased ovarian hormones by regulating intestinal microbiota and promoting equol conversion (Fang, Y., 2022;Fang et al., 2021).

Depression
Depression is a relatively common disease with the highest suicide rate in psychiatric departments, mainly manifested by low mood, anxiety, insomnia, loss of appetite, and inconcentration (Malhi and Mann, 2018).Norepinephrine, serotonin, and dopamine are substrates for monoamine oxidase (MAO), and MAO inhibitors have antidepressant activity, BBR may regulate the levels of brain biogenic amines (norepinephrine, serotonin, and dopamine) by interacting with adrenaline receptors, 5-HT, dopamine, and MAO, and exert antidepressant-like effects in various depression models.Moreover, the antidepressant like effect of BBR in Forced Swimming Test (FST) involves interaction with the L-arginine-NO-cGMP pathway (Peng et al., 2007;Kulkarni and Dhir, 2008;Kulkarni and Dhir, 2007).Organic cation transporters (OCTs) and plasma membrane monoamine transporters (PMAT) are the most efficient transporters for uptake of 5-HT, NE and other biogenic amine neurotransmitters (Daws et al., 2013).Sun et al.'s study found that BBR can play an antidepressant role by inhibiting OCT2 and OCT3 (Sun et al., 2014).The study of Yi et al. showed that BBR also exerts antidepressant effects by regulating the miR-34a-synaptotagmin1/Bcl-2 axis (Yi et al., 2021).BBR may also inhibit NF-κB signaling pathway and its downstream targets such as proinflammatory cytokines and iNOS exert antidepressant effects (Liu et al., 2017).The HPA axis is an important part of the neuroendocrine system and is closely related to depression.The activation of HPA axis is manifested by increased secretion of corticotrophin-releasing factor (CRF) in the hypothalamus.Then the pituitary adrenocorticotrophin (ACTH) release.BDNF has been shown to be a key contributor to antidepressant effects (Erickson et al., 2012), and excessive plasma corticosterone (CORT) is also an important trigger for depressive episodes (Gong et al., 2019).BBR combined with ginsenoside can upregulate the expression level of plasma BDNF and downregulate the levels of CORT and ACTH (Shen et al., 2016;Zhang et al., 2021).BBR can also significantly reduce the expression of CRF in hypothalamus, and significantly improve the depressive behavior of rats with chronic morphine withdrawal (Lee et al., 2012;Gao et al., 2024).

Schizophrenia
Schizophrenia is a chronic and severe mental illness in which dopamine-mediated neurotransmission plays a crucial role in psychiatric and nervous system disorders.The prolyl oligopeptidase (POP) family of enzymes are cytoplasmic serine peptidases, and the activity of POP is reduced in depression and increased in psychiatric disorders such as mania and schizophrenia (Maes et al., 1995).BBR inhibits POP in a concentration dependent manner, thereby exerting anti schizophrenia effects (Tarrago et al., 2007).TetrahydroprotoBBRs (THPBs), a derivative of BBR, separated from Chinese herbal medicine compound, through its unique D2 receptor antagonist and agonist activity of D1, play a role of resistant schizophrenia (Chu et al., 2008;Kulkarni and Dhir, 2010).Ghotbi et al. created a rat schizophrenia model by administering MK-801 (NMDA receptor antagonist) (Ghotbi Ravandi et al., 2019), and the results showed that BBR had neuroprotective effects on rats with MK-801-related behavioral defects, suggesting that BBR had anti-schizophrenic effects (Shayganfard, 2023).

Conclusion
BBR is widely present in the roots, rhizomes, stems, or bark of many traditionally used herbs, and has a wide range of physiological activities, especially in neuroprotection, but its bioavailability is low, so the study of BBR dosage forms is very important to improve bioavailability.This review summarizes the extraction methods, dosage forms, pharmacological effects, and protective effects of BBR on the central nervous system, and summarizes a large number of studies, indicating that BBR can directly or indirectly regulate various intracellular molecules and signaling pathways (Figures 9, 10) , thereby improving nervous system diseases.Such as cerebrovascular disease, multiple sclerosis, glioma, epilepsy, AD, PD, HD, anxiety, depression, and schizophrenia.
At present, the complex network mechanism of BBR is not fully understood, which may be due to the interaction with multiple targets or with proteins that are involved in many pathways.In order to further study the pharmacological mechanism of berberine, it can be revealed by exploring the possible protein targets directly acting on berberine (some researches on the targets of direct BBR action are shown in Table 3).Researches on the targets of direct BBR action can not only reveal the mechanism of therapeutic effect of BBR from a fundamental perspective, but also provide new strategies for the design of drug combinations.However, there are few studies on the direct targets of BBR for the treatment of neurological diseases, so it is important to further explore BBR, which will help clarify the multi-active mechanism of BBR's neuroprotective effect and its corresponding biological effects.
FIGURE 1Chemical structure of BBR.

FIGURE 4
FIGURE 4Regulation of apoptosis mechanism of BBR.

FIGURE 10
FIGURE 10 Matrix metalloproteinase-9MPTP/P1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid BBR extraction methods mainly include Acid water extraction, Alkali water extraction and Alcohol extraction, of which acid water extraction and alkali water extraction are simple, low cost, but still have problems such as: low extraction rate, easy to corrode equipment, unsafe, not eco-friendly.Alcohol extraction methods include Microwave-assisted extraction, (Soxhlet) reflux extraction, Flash extraction, Ultrasonic extraction, etc., which not only has low solvent restriction and can be used repeatedly, but also can save energy and environmental protection, high safety, simple operation, and high extraction efficiency.However, ultrasonic extraction methods require large investment in equipment (Yang, Q. Z.

TABLE 1
Different extraction methods of BBR.
Sun, X. M. et al. (1996),Sun, X. M. et al. (1996), Zhang, Z. W. and Sun, X. M. (1995) Supercritical CO 2 extraction The supercritical fluid is contacted with BBR to dissolve it fully, and then the supercritical fluid CO 2 is changed into a gas by reducing pressure and heating up, and the BBR is precipitated Compared with the traditional solvent extraction method, it has the advantages of low temperature, high speed, high efficiency, good pharmacological effect and lower toxicity Liu et al. (2006) (Continued on following page)

TABLE 1 (
Continued) Different extraction methods of BBR.

TABLE 2 (
Continued) Different dosage forms of BBR.

TABLE 2 (
Continued) Different dosage forms of BBR.
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TABLE 3
Targets of direct BBR action.