Research progress on pharmacological action and effective drug carrier of berberine

Yue Liu, Bai-Jie Ren, Xin-Ying Zou, Jing-Yi Lu, Lei Wang, Dong-Hui Gao

Department of Periodontics,Hospital of Stomatology,Jilin University,Changchun 130021,Jilin,China

Keywords:Berberine Pharmacological effects Drug carrier

ABSTRACT Berberine(BBR) is an isoquinoline alkaloid that can be extracted from the traditional Chinese medicine Huang Lian. It has anti-inflammatory, anti-cancer, protection of nerves, hypoglycemic, blood lipid, anti-oxidation, antibacterial and other effects. It can be used clinically to treat chronic colitis, bacterial vaginitis, rheumatoid arthritis, breast cancer, liver cancer, Alzheimer's disease, diabetes, obesity and other common diseases. This paper reviews the pharmacological effects of berberine and the research progress of effective drug carriers in order to provide new ideas for the clinical application of berberine.

Berberine(BBR) , the molecular formula is C20H19NO5, molecular weight is 353.36 g/mol, is the main isoquinoline alkaloids in the stems and roots of many berberine plants, yellow or orange crystalline powder, with weak special odor and characteristic bitter taste, which can be extracted with alcohol in neutral medium or under the condition of adding acetic acid.Slightly soluble in ethanol, methanol and water [1].The absorption of BBR in the intestine is poor, most of the oral dose stays in the gastrointestinal cavity and eventually excrete with feces; after absorption from the gastrointestinal tract, it can be widely distributed in various organs of the human body, but the concentration in plasma is low; BBR absorbed parts of the human body can be transformed into multiple metabolites by o - demethylation, deethyleneization, reduction and binding to glucuronic acid and sulfuric acid [2].A large number of animal and clinical data in recent years have shown that BBR exhibit a broad spectrum of pharmacological effects, including anti-inflammatory, antibacterial, anti-tumor, neuroprotective, hypoglycemic, lipid-lowering effects, and have been used to treat a range of diseases, especially for the treatment of anti-tumor and diabetes.But because there are still some limitations BBR its clinical application, the most important of which are its poor water solubility and poor gastrointestinal absorption, and low bioavailability (about 5%), are classified as IV drugs. Furthermore, BBR has the firstpass effect, which is the second reason for its low bioavailability [3].Considering the above problems, the use of effective drug carriers to reduce these limitations and improve the efficacy of BBR is crucial. In this paper, the main pharmacological effects of BBR and the latest effective drug carriers are reviewed.

1.Pharmacological effects of berberine

1.1Inhibition of inflammatory response

1.1.1 Inhibition of the release of proinflammatory factors

A study in vitro showed that BBR inhibited the lethal cytokines in CD4+T cells of colon propria of chronic colitis mice, mainly Th1 related interferon - γ and Th17 related interleukin-17a [4].In addition, BBR has the effect of improving the intestinal epithelial tight junction injury induced by proinflammatory cytokines.This process is regulated by cytokines such as Th2 cytokine interleukin-13 (IL-13), tumor necrosis factor - α and interferon - γ [5].In addition, BBR with a concentration of 10.0 nmol / l can inhibit the production of IL-8 in rectal mucosa in rats with colitis induced by trinitrobenzene sulfonic acid, thus playing a beneficial role in the healing of intestinal mucosa [5].

In the process of oxidative stress, mitochondria produce excessive reactive oxygen species (ROS), which can damage islet cells and promote their apoptosis so as to reduce insulin secretion, eventually induce or worsen insulin resistance and diabetes, and also lead to diabetic nephropathy, retinopathy, neuropathy and other chronic complications. Some scholars collected samples of vaginal epithelium and secretion before and 1 month after BBR treatment of bacterial vaginitis (BV), and used Western blot method to detect the expression level of oxidative stress related proteins.The results showed that the oxidative stress of vaginal secretions after BBR treatment was significantly decreased, manifested by the increased activity of superoxide dismutase (SOD) and catalase (CAT), and the decrease of malondialdehyde (MDA) and H2O2levels[6].

1.1.3 Inhibition of inflammatory corpuscle activation pathway

Inflammatory corpuscles are composed of a variety of proteins, which participate in the body's natural immune defense response, and have the functions of promoting the maturation of cytokine precursors and inducing programmed cell death.NLRP3 of these inflammatory bodies play a particularly important role in the inflammatory response of the body. In addition, NOD like receptors can also form inflammatory bodies after activation, which can recognize some inflammatory signals in cells. A study in vitro showed that BBR could inhibit TLR4 / MyD88 dependent signaling pathway, inhibit NF-κB activation, significantly reduce the expression of NLRP3 inflammatory bodies, and down regulate the inflammatory response of human umbilical vein endothelial cells induced by lipopolysaccharide [7].

1.2 Anti-tumor effects

1.2.1 Regulation of cellular signaling pathways

Some scholars have used a systemic pharmacological binding molecular docking method to verify the therapeutic potential and related molecular mechanisms of BBR for liver cancer in vitro.The docking method shows that the binding of BBR and protein kinase (Akt) may lead to the inhibition of Akt activity and confirm that BBR downregulates the expression of phosphorylated Akt and PI3K in MHCC97-H and HepG2 cells, and inhibits cell proliferation, migration and PI3K in a dose-dependent manner. The results showed that BBR inhibited the occurrence and development of liver cancer by inhibiting PI3K / Akt pathway [8].

1.2.2 Induction of cell cycle arrest

Studies have examined the antitumor effects of BBR on three human embryonic rhabdomyosarcoma (RMS) cell lines (ERMS1、KYM1 and RD).After treatment with 10 μM BBR, the number of ersm1 cells in G1 phase increased significantly and inhibited them from entering S/ G2 phase;KYM1 cells were blocked in G1 phase; the G1 cell cycle of RD cells stagnated. Therefore, BBR significantly inhibited the cell cycle of all RMS cells in the G1 stage and strongly inhibited the growth of rhabdomyosarcoma cells [9].

Tsarevitch Ivan sat on the Gray Wolf s back and took Helen the Beautiful in his arms, and the Wolf began run- fling more swiftly than fifty horses, across the three times nine countries, back to the Tsardom of Tsar Afron. The nurse and ladies-in-waiting of the Tsarevna hastened to the Palace, and the Tsar sent many troops to pursue them, but fast as they went they could not overtake the Gray Wolf.

1.2.3 Inhibition of tumor vascular endothelial growth factor

Some scholars have explored the effect of BBR on tumor angiogenesis and related mechanisms through reverse screening and molecular docking experiments. The results showed that BBR could bind to and inhibit the activity of VEGF receptor 2, block the activation of Akt / mTOR / P70S6K signal pathway, and then inhibit tumor angiogenesis, providing a new choice for anti-tumor angiogenesis lead compounds [10].

1.2.4 Inhibition of apoptosis protease activation

Apoptosis is mainly mediated by caspase family, and cytochrome c is released and activated by mitochondria during apoptosis. Caspase-1, caspase-8 and caspase-9 are often used to measure the level of apoptosis. It has been shown that BBR can activate the precursors of caspase-8 and caspase-9 to induce apoptosis of oral cancer cells, which may be related to the change of expression level of FasL, the ligand of cancer cell death receptor [11].Caspase-1 is an important target gene in the inflammatory pathway mediated by the nod like receptor (NLR) family protein (NLRP3). BBR can inhibit the level of caspase-1 in a dose-dependent manner, regulate the expression of NLRP3 inflammatory related genes in human breast cancer cells (MDA-MB231), and inhibit human breast cancer [12].

1.3 Protection of nerves

1.3.1 β amyloid hypothesis

Cerebral amyloidosis is a preliminary pathological change that begins many years before the onset of clinical symptoms in patients with neurological disorders such as Alzheimer's disease (AD) .Amyloid is a transmembrane protein expressed at high levels in the brain and in a variety of tissues. It is mainly concentrated in neuronal synapses and is metabolized in a rapid and highly complex manner by a series of sequence proteases, such as α- secretase ,β- secretase, and membrane γ- secretase complexes. The study found that BBR could reduce the content of β - amyloid by regulating the amyloid protein in human glioma H4 cells without cytotoxicity.BBR can inhibit the pathological state of Aβ amyloidosis, glial hyperplasia and improve cognitive impairment in AD transgenic mouse models.In AD rabbit model, BBR treatment can prevent hippocampal neurodegeneration, improve behavior disorder and reduce β - amyloid activity [13].

1.3.2 Inhibition of oxidative stress

Oxidative stress plays a key role in the progression of neurological disorders, which, together with chronic neuroinflammation, triggers an active vicious cycle that leads to irreversible neuronal dysfunction and cell death. The study found that BBR protects against β amyloid-induced cell death in rat cortical neurons by reducing malondialdehyde (MDA) and reactive oxygen species production [14]. Another scholar has studied the effect of BBR on motor neuron survival and axon regeneration after spinal cord root avulsion and transplantation in rats.The results showed that the expression of 4-hydroxynonaldehyde (4-HNE) and MDA decreased significantly after BBR treatment with 120 mg / kg and 240 mg / kg.4-HNE and MDA are the main end products of the peroxidation of polyunsaturated fatty acids in cell membrane, which are considered as biological markers of lipid peroxidation. These results clearly show that BBR can prevent oxidative stress induced by avulsion [14].

1.3.3 Inhibition of tau protein

Intracellular neurofibrillary tangles (NFTs) are a typical neuropathological lesion. They are composed of a highly phosphorylated form of microtubule associated proteins. Excessive phosphorylation will destroy its normal function of regulating axon transport and lead to the formation of soluble tau protein and toxic substances.Tau protein, a tubulin involved in a variety of cellular functions, can bind to formed microtubules, maintain microtubule stability, reduce the dissociation of tubulin molecules,and induce microtubule bundles. Hyperphosphorylated tau proteins in neuropathy are migrated into cells. Due to increased fragility of microtubules and disruption of axonal transport, hyperphosphorylated tau proteins form paired helical filaments, resulting in nerve damage and cell death. Some scholars have found that BBR therapy can effectively reduce the hyperphosphorylation of tau proteins induced by Carcinogen A [1].

1.4 Lower blood sugar

Some scholars used ELISA kit to determine the level of diabetic rats before and after BBR treatment, and blood / cerebrospinal fluid glucose content was detected by blood glucose meter.The results showed that the peripheral postprandial blood glucose was significantly increased after streptozotocin injection combined with high-glucose and high-fat diet in rats.BBR significantly reduced glucose level, and although the level of glucose in CSF was lower than that in serum, the change trend of glucose level was consistent with that in serum.The level of serum insulin in diabetic rats was significantly increased, and it was normalized by BBR treatment; the level of insulin in cerebrospinal fluid of diabetic rats was significantly reduced, and BBR treatment could improve its level. Moreover, glucose transport capacity in the hippocampus and cerebral cortex of DM rats is significantly hindered and BBR use can partially reverse this phenomenon [15].

1.5 Lower blood lipids

Excessive differentiation or proliferation of adipocytes will lead to the increase of their size or quantity, expansion of adipose tissue and obesity. The differentiation and proliferation of adipocytes are regulated by a series of factors or molecules, including Gal-3, a member of the gals family that binds carbohydrates or glycoproteins.The extracellular Gal-3 regulates the interaction between cells; the intracellular Gal-3 is related to cell survival and death; while the nuclear Gal-3 may affect gene expression.The study found that Gal-3 was positively correlated with obesity. In obese adults or children, elevated serum levels of Gal-3, which may affect its secretion from cells, were found to be associated with increased body fat. Other in vitro experiments showed that BBR inhibited the differentiation and proliferation of mouse preadipocytes isolated from epididymal white adipose tissue, during which Gal-3 expression levels were significantly downregulated [16]. Moreover, adenosine monophosphate (AMP)-dependent protein kinases (AMPK) play a key regulatory role in lipid metabolism and regulate redox balance.BBR can activate AMPK, reduce lipid production by activating mitogen-activated protein kinases and extracellular regulatory protein kinase pathways.

2.Effective drug carrier of Berberine

2.1 Mixed Micelles

Micellar nanocarriers made from a mixture of polymers and phospholipids have small particle sizes, usually > 5nm and < 100nm,are biocompatible and biodegradable drug carriers, have high loading capacity, and can spontaneously aggregate at pathological tumor sites in vivo. The micelle preparation of the drug can not only protect the drug from the potential inactivation effect in the biological environment, but also reduce the toxic and side effects on the unexpected tissues to the greatest extent, improve the permeability of the drug to the physiological barrier, so as to effectively increase the tissue distribution of the drug.Some scholars have found that lipid-based nanocarriers, amphiphilic mixed micelles (Mic) composed of polymeric phospholipid conjugates and PEG- succinate of tocopherols, are considered effective ways to improve BBR delivery in tumors.Mixed micelles in this experiment significantly enhanced the pro-apoptotic effect of BBR on prostate cancer in vitro models and the overall anticancer efficacy [17].

2.2 Chylomicrons

Some scholars have prepared bioactive cells containing BBR, and carried out sufficient in vitro experiments. The intestinal permeability was evaluated by using non crosslinked intestinal sac model and Caco-2 cell model, and the absorption and distribution of bioactive cells were detected by laser scanning confocal microscope. The permeability of loaded chylomicrons in BBR was 10.5 times higher than that in free BBR. CACO-2 study showed significant improvement in chylomicrometer permeability and cell uptake.In addition,the fluorescence of chylomicrocytes loaded with BBR was increased by 2 times. Therefore, Chylomicron could be used as an effective drug carrier to improve the bioavailability of BBR [18].

2.3 Oleate complex

It is found that the high water solubility and limited permeability of BBR hinder its local clinical application. Therefore, some scholars prepared BBR oleate complex (bbr-ol) by simple precipitation technology. The saturated solubility of bbr-ol in n-octanol increased by 251 times, which proves that compared with free drugs, the lipid solubility of the complex increased.Compared with the free BBR, the release rate of bbr-ol complex was slower and lasted longer. In addition, the in vitro permeation study of rat skin showed that compared with free BBR, the penetration of ion on BBR-OL complex was enhanced. In vivo studies of healthy rats confirmed that confocal microscopy revealed that local application of BBR-OL-rich hydrogels had better skin permeability and deposition than free BBR [19].

2.4 Long-circulating liposomes

BBR has good antitumor effect in vitro. However, intravenously administered BBR solution has adverse effects on cardiovascular system. There are studies to prepare common and polyethylene glycol (PEG) modified long-cycle BBR liposomes, respectively, and to evaluate their efficacy and safety as potential antitumor agents.As a result, BBR liposomes have uniform morphology, storage stability and sustained release properties in vitro. Compared with BBR solution, BBR liposomes significantly increased the retention time of BBR in circulation. In tumor bearing mice, BBR liposomes selectively increased the concentration of BBR in liver, spleen, lung and tumor, and decreased its distribution in heart and kidney.Importantly, long-term administration of BBR liposomes has been shown to be effective and safe in inhibiting tumor growth in PEGmodified long-circulating liposomes[20].

2.5 Superdeformed vesicles

BBR is a natural compound, with antioxidant, anti-inflammatory and other effects, can be used to treat vitiligo, and can be loaded into a suitable carrier to improve the efficacy.Some scholars have developed superdeformed vesicles loaded with BBR as adjuvant in the treatment of vitiligo. In this experiment, the ability of vesicles to promote the penetration of BBR was tested, and the antioxidative and photoprotective effects of keratinocytes and fibroblasts were studied in vitro, and the melanin production after vesicular therapy was further evaluated.The superdeformed vesicles are small in size, evenly dispersed and negatively charged, which can absorb a large amount of BBR and promote its penetration into the skin [21].

2.6 Co-crystallization

Berberine chloride (BCL) can be used as anhydrous compound, monohydrate, dihydrate and tetrahydrate.Therefore, during the production and storage of BBR tablets, when the ambient humidity changes, the higher the humidity, the more unstable the solid state of BBR tablets will be.It was found that a 1:1 co-crystallization (BCLCA) composed of Berberine chloride and citric acid could keep the thermal stability, solubility,dissolution rate and compressibility of tablets, and showed better stability to the change of humidity.Therefore, BCL-CA is a good alternative crystal form and can be used to BBR tablet fabrication [22].

2.7 Oil-in-water self-emulsifying system

Because of the disadvantages of low water solubility and low bioavailability in BBR, the clinical effect of BBR was limited. The results showed that BBR oil-in-water self-emulsifying system (SNE) could improve the bioavailability of BBR and enhance the effect of BBR on acute myeloid leukemia.The results showed that the release rate of BBR SNE was slower than that of BBR solution.The relative oral bioavailability of the system in rabbit model was 3.41 times higher than that of BBR.Furthermore, the monolayer transport of Caco-2 cells showed that the system could enhance the permeability of BBR and prevent its diffusion.Importantly, in mv4-11 transplanted leukemic mice, the mice treated with BBR SNE had longer survival time than those treated with BBR [23].

2.8 Dendrimers

Dendrimers (PAMAM) have stable molecular structure, monodispersity and good surface function, so they have the potential to release anticancer drugs / bioactive substances. The anticancer activity of G4 PAMAM dendrimer delivery BBR, on MCF-7 and MDA-MB-468 breast cancer cells was significantly higher. In the study of plasma level in albino rats, the half-life (T1 / 2) and the area under the drug time curve of BBR were significantly increased, T1 / 2 was 14.33 h, while the T1 / 2 of BBR alone was 6.7 h [24].

2.9 Chitosan

The low bioavailability and short biological half-life of BCL could adversely affect the protective effects of BBR on osteoarthritis.Some scholars have successfully developed a new type of chitosan microspheres loaded with BBR by the method of ionic crosslinking. The chitosan loaded with BBR is spherical and can pack a large number of BBR (100.8 ± 2.7 mg / g). These microspheres also show good release characteristics. BBR loaded chitosan microspheres significantly inhibited sodium nitroprusside-stimulated chondrocyte apoptosis as well as cytoskeletal remodeling, thereby enhancing mitochondrial membrane potential and maintaining nuclear morphology.Therefore, chitosan microspheres loaded with BBR have stronger anti apoptotic activity in the treatment of osteoarthritis, and compared with the same concentration of free BBR, the microspheres significantly inhibit the protein expression levels of nitroprusside induced hemolysin-3, hemolysin-5 and matrix metalloproteinase-13 in rat articular chondrocytes [25].

3.Conclusion

Since the absorption effect of oral BBR is poor, and after intramuscular injection, drugs can be distributed to human tissues and organs in a short time, but still can not maintain blood drug concentration for a long time.Therefore, in order to effectively improve the bioavailability of BBR, scholars at home and abroad have carried out a series of studies on the pharmacological function and effective drug carrier of BBR in recent years, and achieved some research results.However, the current research on BBR drug carriers is still in the animal experiment stage, and the related research on clinical application and long-term efficacy is still lacking. Further research is needed to make the BBR clinical drug effect more effective.