Shiyu Huangab and
Gangliang Huang*a
aKey Laboratory of Carbohydrate Science and Engineering, Chongqing Normal University, Chongqing 401331, China
bSchool of Chemistry and Chemical Engineering, Southwest University, Chongqing 400700, China. E-mail: huangdoctor226@163.com
First published on 14th July 2025
The extraction methods of berberine hydrochloride include water, acidified water, lime milk, and ethanol extraction. The water extraction method is mainly a decoction method, whereas the alcohol extraction method mainly includes microwave, ultrasonic and cable reflux extraction. In the alcohol extraction method, the solvent has low restriction and can be used repeatedly, making the operation simple and increasing the extraction rate; however, the equipment investment for ultrasonic extraction and other methods is substantial. This method can shorten the extraction time and achieve a higher extraction rate; however, it has disadvantages such as high solvent cost, large solvent amount, difficult recovery, hidden danger of safety in operation, the extraction process involving heating steps, and high energy consumption. Thus, it is not suitable for industrial mass production. The extraction processes of sour water and lime milk are relatively simple and inexpensive, which are commonly used in industry at present; however, the lime milk method consumes a considerable amount of lime milk. Therefore, considering production maneuverability and cost, the acid–water impregnation method is easier for industrial implementation. Berberine has many functions, including antibacterial effects, anti-inflammatory effects, detoxification, and lowering of blood sugar and blood fat levels. To clarify the hypoglycemic mechanism of berberine, a better understanding of its pharmacological effects is helpful, providing a basis for the rational application of berberine in the treatment of type 2 diabetes.
Among them, Coptis chinensis, Berberis vulgaris L. and Scutellaria baicalensis are the most abundant in berberine. It was reported that the content of berberine in the bark and roots was more abundant than that in other parts. The content of berberine in different parts was investigated, and its highest content was found in Berberis roots (1.6–4.3%). The altitude at which plants grow may also affect the berberine content in plants. In one study, it was found that plants grown at low altitudes contained more berberine than those grown at high altitudes. In addition, through phytochemical analysis, it was found that berberine was rich in alkaloids, with a content as high as 1.43%. The difference in berberine content among different varieties of the same genus was also investigated: the content of Berberis asiatica was higher, 4.3%, followed by Berberis lycium (4.0%). The quantification of Tinospora cordifolia and Tinospora sinensis contents yielded 0.3192% and 0.0967% (w/w), respectively.1 Seasonal changes will also affect the content of berberine. The yield of berberine was the highest in summer, 2.8% in root, 1.8% in stem bark and 1.9% in winter.
Although there are several types of compounds produced by plants, most of them are considered to be secondary metabolites, and the physiological and developmental factors of plants significantly influence their production. Callus culture has become the focus of research on the in vitro production of metabolites. Different kinds and concentrations of auxin, 3% sucrose and 0.7% Agar corn, and different concentrations of Skoog (MS) Agar were used as explants to establish an excellent plant callus culture system with high contents of small leaf tin fruit and berberine. The content of berberine was higher in the medium containing 5 μM α-naphthylacetic acid. After 40 μM tyrosine was added to the MS medium, the yield of berberine was increased by 3.19-fold. Therefore, this method can be used for the mass harvest of berberine. Such callus cultures could also be used to initiate suspension cultures of T. cordifolia for studies on berberine.2
The main plant sources of berberine include Coptidis rhizoma and Cortex phellodendri. Effects of environmental factors on berberine content: (1) different soil types can affect the absorption and accumulation of nutrients by plants, thus affecting the berberine content. For example, soil rich in organic matter contributes to the synthesis and accumulation of berberine. (2) Climate factors such as temperature, humidity and light will also affect the content of berberine. Appropriate temperature and light conditions can promote its synthesis, while extreme weather conditions may inhibit its synthesis. (3) Planting methods and fertilization will also affect its content. Reasonable fertilization and planting management can improve the berberine content. Effects of biological factors on berberine content: (1) there are genetic differences between different varieties of Coptis chinensis and Phellodendron amurense, which will affect the berberine content. Selecting high-content varieties for planting can increase the yield. (2) Pests and diseases can affect the growth and development of plants, which in turn affect the synthesis and accumulation of berberine. Effective pest control measures can reduce this negative impact.
Ultrasonic-assisted solvent extraction (USE) is considered a green, simple, efficient and cost-effective technology. Compared with the distillation method and Soxhlet extraction method to extract berberine from fresh Phellodendron bark, an efficient method was established. The yield of berberine extracted by USE was the highest, which was about 100 mg g−1. The yield of berberine by distillation and Soxhlet extraction was 50 mg g−1 and 40 mg g−1, respectively. In addition, the effective matrix recovery of berberine was shown when using methanol acidified with hydrochloric acid. Another study also obtained a relatively high yield of berberine in Rhizome coptidis, and the optimal extraction conditions were 59% ethanol, 66 °C and 45 min. Meanwhile, USE, with a green solvent, ionic liquid solutions, reduced the extraction time (40 min) for berberine in Coptis chinensis. A non-toxic, environmentally-friendly mixed solvent of water and glycerol was used to extract berberine from B. vulgaris root bark using the USE technique. Berberine concentration and DPPH radical scavenging activity of the extracts (RSA IC50) varied with temperature, glycerol concentration, and ultrasound power. A high berberine yield of 145.5 μg mL−1 (80 °C, 50%, 144 W) and RSA IC50 of 58.88 μg mL−1 (80 °C, 30%, 720 W) were obtained under different optimum conditions.4
A single-factor experimental method is used to explore the range of optimal extraction conditions for ultrasonic and microwave collaborative extraction of berberine, and then optimize the conditions by the response surface method. The results show that the best extraction conditions are as follows: extraction solvent, 0.05 mol per L H2SO4; material-to-liquid ratio of 15 g mL−1; ultrasonic time of 10 min; microwave time of 3 min; microwave power of 600 W, and the xanthin extraction rate of 11.43%. Compared with traditional ethanol immersion extraction and sulfuric acid immersion extraction, ultrasonic-microwave collaborative extraction saves a significant amount of time, and the extracted flavinin is no different from that extracted by traditional methods.
With the assistance of an ultraviolet-visible spectrophotometer, a single-factor test analysis is carried out by the ultrasonic-assisted extraction process. The maximum influence level of each factor is as follows: 70% ethanol concentration, material–liquid ratio of 1:
25 (g
:
mL), 180 W ultrasonic power, and ultrasonic treatment for 30 minutes at 50 °C ultrasonic temperature. At the same time, in order to further investigate the reliability and stability of the best process, the orthogonal experiment is used, which shows that the process has good stability and high reliability. This provides experimental support for the extraction of berberine hydrochloride. Through the study of ultrasonic-assisted and enzymatic-assisted extraction processes, the optimum process conditions for the combined enzymatic hydrolysis-ultrasound extraction of berberine hydrochloride were investigated. HPLC was used to determine the content of berberine hydrochloride in the lower foot of Huanglian. Through this method, the reliability of this test and the stability of instrument operation are effectively verified, and the purity of ultrasonic-assisted extraction, enzymatic paralysis-assisted extraction and enzymatic hydrolysis-ultrasound combined extraction of berberine hydrochloride products is further analyzed and evaluated simultaneously. At the same time, as a quality control of the extraction process, specific components can be analyzed more accurately.
The extraction method of berberine was explored, and the difference between Soxhlet and ultrasonic extraction was mainly compared. The results showed that the optimal conditions for Soxhlet extraction are 100 mL of ethanol solvent and extraction for 3 hours. The optimal conditions for ultrasound extraction are 100 mL of ethanol solvent and ultrasound extraction for 1 hour, which is divided into three 20-minute intervals. The extraction efficiency of the ultrasound method is 9.6% higher than that of the traditional Soxhlet extraction method, with higher repeatability. The method is simpler and easier to operate than the Soxhlet extraction method. The effect of ultrasonic extraction is better than that of traditional Soxhlet extraction methods.
The ultrasonic method is better than the immersion method for extracting berberine from huanglian. Within 30 minutes, the extraction rate of berberine increases with the increase in ultrasound treatment time, and there is a peak. When the ultrasound treatment time is 30 minutes, the extraction rate is 8.12%. Huanglian powder is soaked in a sulfuric acid solution with a concentration of 0.094 mol L−1, and then treated with ultrasonic waves. The extraction rate of berberine is high. For berberine in huanglian, ultrasonic extraction at a frequency of 20 kHz is appropriate. Huanglian powder is soaked for 24 hours, and then treated with ultrasound, resulting in a relatively high extraction rate of berberine.
High temperatures and light can lead to the automatic degradation of berberine, and the matrix recovery of these extraction methods varies significantly due to its sensitivity to temperature and light. The influence of temperature for extracting berberine in Coscinium fenestratum stem tissue samples was investigated and a 4.6% weight/weight (w/w) yield was observed in the case of samples dried under the constant shade, which was higher than from samples dried in an oven at 65 °C (1.32% w/w) or sun drying (3.21% w/w). Hot and cold methanol or ethanol were also applied to compare the matrix recovery yield of berberine. The yield in the shade-dried samples was 4.6% (w/w) for the methanolic cold extraction and 1.29% (w/w) for the methanolic hot extraction.
It is worth noting that the extractant affects not only the extraction rate but also the activity of microorganisms. The bark extracted from the root bark of Chinese cabbage yields better medicinal food value. Among different solvents, ethanol has the highest extraction rate (173.36 mg g−1), followed by water (24.54 mg g−1), n-hexane (11.88 mg g−1) and acetone (6.56 mg g−1). Bacteriostatic experiments revealed that ethanol and water extracts exhibited strong antibacterial activity against Staphylococcus aureus (S. aureus), even at concentrations as low as 12.5 mg mL−1, even after several dilutions, while n-hexane and acetone extracts did not show such strong activity. The difference in inhibition rate and yield may be attributed to the higher content of polyphenols and/or alkaloids in water and ethanol extracts than in acetone and n-hexane extracts. It must be noted that acetone and n-hexane are non-polar solvents and therefore have low solubility in polyphenols and/or alkaloids.6 Another study also found that the ethanol extract of Berberis root collected from Meduvrhi and Kiza (Croatia) had significant inhibitory activity against S. aureus ATCC6538 (minimum inhibitory concentration (MIC) was 25 mg mL−1). Later studies found that the minimum inhibitory concentration of alkaloids extracted from the roots against S. aureus ATCC25923 was 5–10 mg mL−1.
Despite this, the method also has disadvantages, including the need for a large amount of solvent, long extraction time and increasing extraction costs. For instance, berberine was extracted from 800 g of the powder and stem bark by a 2.5 L methanol thermal extraction (50 °C, 3 h).7 It was also extracted from 100 g of C. fenestratum plant material by soaking in 3.2 L of ethanol (80%) for 16 h.
In order to improve the selectivity and effectiveness of the extraction process, some green, simple and efficient technologies such as microwave-assisted solvent extraction (MAE), ultrasound-assisted solvent extraction (USE), ultra-high pressure extraction (UPE), supercritical fluid extraction (SFE), pressurized liquid extraction, enzymatic extraction, aqueous two-phase extraction have been successfully developed (Fig. 2).
Microwave-assisted solvent extraction (MAE) is a green and economical traditional extraction method that can increase intracellular temperature, release berberine from broken cells, and reduce the use of organic solvents and extraction time. The relatively high yield of berberine content at 1.66% from Tinospora cardifolia was achieved using MAE under optimized conditions (60% irradiation power, 80% ethanol concentration, and an extraction time of 3 minutes), while only 1.04% and 0.28% were obtained from Soxhlet and maceration, respectively. Compared with Soxhlet extraction (3 h) and impregnation (7 d), MAE significantly shortened the extraction time (3 min) and reduced solvent and energy consumption.
Ultra-high pressure extraction (UPE) is another environment-friendly extraction technology. Its principle is to use different pressure levels inside and outside the cell (high value) and extracellular (low value) at room temperature to promote the transfer of bioactive substances through the plant matrix in the extraction solvent, thereby improving the extraction rate and quality, shorten the extraction time and reduce the solvent consumption. One study compared UPE, MAE, USE, and heat reflux extraction to extract berberine from C. phellodendri, and UPE exhibited the highest extraction yield (7.7 mg g−1) and the shortest extraction time (2 min), followed by MAE (6 mg g−1 and 15 min), USE (5.61 mg g−1 and 1 h) and reflux (5.35 mg g−1 and 2 h).
Supercritical fluid extraction (SFE) is an environmentally friendly and efficient technology that can reduce the degradation of bioactive substances in the absence light and oxygen. Furthermore, inert and non-toxic carbon dioxide is used as the main extraction solvent of SFE, and various modifiers (such as methanol) and surfactants (such as Tween 80) are combined at a low temperature and relatively low pressure to effectively extract bioactive compounds. The recovery rate of berberine in the rhizome of Coptis chinensis was the highest when 1,2-propanediol was used as the modifier of supercritical CO2.
Pressurized liquid extraction is considered a green technology for extracting sensitive compounds from various plants, offering advantages such as improving the extraction rate, shortening the time, reducing solvent consumption and so on. The extraction of berberine from Canadian narcissus by four extraction methods, including PLE, multiple use, single use and Soxhlet extraction, was studied. The extraction time of PLE was shorter (30 min), while the extraction times of multiple extraction (2 h) and Soxhlet extraction (6 h) were longer.
The principle of enzymatic extraction of berberine is to use cellulase to convert cellulose into soluble glucose, destroy the cell wall and release berberine. This method has the advantages of being time-efficient, low consumption and convenient post-processing, and is beneficial to the environment. Nevertheless, the activity conditions of the corresponding enzymes are complex, and the high selectivity of the enzyme limits its application in the treatment of different plants containing berberine.8
A magnetic ionic liquid (MILs) aqueous two-phase system (MILATPs), consisting of five choline ionic liquids (MILS) containing piperidinyl anions, was synthesized by mixing with a series of inorganic salts. The MIL-ATPs coupled with HPLC-UV analysis were used to quantify berberine hydrochloride in Rhizoma coptidis, and a high partition coefficient of berberine (127.68) was observed with precision values (RSD%) of 1.40% and 2.83% for intra-day (n = 6) and inter-day (n = 3), respectively. The limits of detection (LOD) and quantification (LOQ) for berberine were 0.023 mg L−1 and 0.077 mg L−1, respectively. This method also yielded a high content of berberine (123.95 mg g−1) from the raw material of Rhizoma coptidis. After removing berberine hydrochloride with D101 resin, the recovery rate is 99.8%, which can be recycled.
Rosin-based polymer microspheres (RBPM) with a clean surface, narrow particle size distribution, mesopic structure and excellent thermal stability were prepared using ethylene glycol maleate as a cross-linker and methacrylic acid as a functional monomer. The results exhibited that the dynamic adsorption capacity of RBPM on total alkaloids was 612.4 mg g−1, and the separation capacity of total alkaloids increased from 34.2% to more than 91.0%.
Functional magnetic adsorbents have good biocompatibility and unique physical and chemical properties. An aptamer is an oligonucleotide or peptide molecule that can be bound to a specific target molecule with high selectivity, affinity, and specificity, comparable to or better than antibodies, enabling biosensors based on fluorescence intensity detection, electrochemical changes, or color changes. Aptamer-functionalized Fe3O4 magnetic nanoparticles were prepared and used as a SPE adsorbent to extract berberine from the C. phellodendri. Under the optimal conditions (pH = 7.5, Mg2+ concentration of 5 mmol mL−1, incubation temperature of 30 °C, desorption time of 5 min and elution solvent of acetonitrile (2.5 mL)), the purity of berberine extracted from C. phellodendri was as high as 98.7% compared with that of 4.85% in the extract, indicating that aptamer-functionalized Fe3O4 MNPs-based SPE method was very effective for berberine enrichment and separation from a complex herb extract. Furthermore, berberine was separated from nine different concentrations of a single C. phellodendri extract to demonstrate the applicability and reliability of this technique, and the relative recoveries of the spiked solutions in all samples were between 95.4 and 111.3%, with relative standard deviations ranging between 0.57 and 1.85% (Table 1).
Extraction methods | Advantages | Disadvantages |
---|---|---|
Maceration; Soxhlet; percolation; reflux | Mature methods with inexpensive equipment and large-scale extraction | Large solvent volumes, long extraction time, low extraction recovery and purity |
Molecularly imprinted polymer extraction | Environmentally friendly technique with high selectivity, affinity, specificity, extraction yield and purity | High-cost and rigorous condition limit its use for large-scale extraction |
Nano strategies | Novel method with high selectivity, affinity, specificity, extraction yield and purity | High costs and rigorous conditions limit its use for large-scale extraction |
With the development of modern science and technology, new modern separation methods, each with its own advantages and disadvantages have emerged, including PLE, USE, MAE, UPE, and SFE. These methods improve the extraction rates of berberine, shorten the extraction time, and are more suitable for industrial production. Enzymatic extraction has almost no effect on the extraction of original plant contents; however, the cost of biological enzymes is very high; hence, it is only suitable for low-dose research in the laboratory. However, the whole process of supercritical CO2 extraction does not require organic solvents, and there is no residual solvent; however, it increases the equipment investment and operation difficulty of the process.
However, the GLUT4 transport function of patients with type 2 diabetes is low, which leads to an increase in blood glucose. The experimental results of Yan et al. showed that berberine could improve the state of insulin resistance (IR), enhance the expression of PI3-K and GLUT4 in target tissues, increase the circulation speed of GLUT4 in cells, and improve the affinity and binding capacity of the insulin receptor in hepatocyte membranes.1
Some studies have shown that the anti-inflammatory effect of berberine can inhibit the development of diabetes. It was found that the levels of inflammatory factors (CPR, IL-6, TNF-α and IL-1) in the serum of diabetic rats treated with berberine decreased significantly, while the level of adiponectin increased significantly.2
Oxidative stress occurs when the body is subjected to various harmful stimuli, and the excessive production of highly reactive molecules leads to an imbalance between the oxidative and antioxidant systems, which induces and mediates the apoptosis of pancreatic cells and destruction of islets, resulting in an increase in blood sugar levels.3 It has been found that berberine has a beneficial therapeutic effect on oxidative stress. It can not only significantly reduce the level of malonic acid in the serum and liver tissue of diabetic rats and increase the activities of superoxide dismutase, glutathione peroxidase, catalase and glutathione, but also up-regulates the gene expression of positive transcription elongation factor B and regulates blood glucose through antioxidation.4
Studies have shown that small doses of berberine can significantly increase glucose uptake in HepG2 cells without insulin dependence, showing a good hypoglycemic effect.5
The experimental study found that berberine can promote the repair of islet β cells in diabetic rats, increase the content of insulin in blood glucose, facilitate the transport of glucose, and thus reduce blood glucose levels.6
The results of an experimental study have shown that berberine has a beneficial hypoglycemic effect, which can increase glucose metabolism by stimulating glycolysis or inhibit glucose heterogeneity by inhibiting glucose oxidation in mitochondria, thereby assisting in the regulation of blood sugar levels.7 Another study showed that berberine can increase the level of fasting blood glucose in diabetic rats by inhibiting hepatocyte gluconeogenesis.
Insufficient insulin secretion in diabetic patients will lead to an increase in plasma FFAs and TG concentrations, which will affect glucose metabolism, and high FFAs can also cause triglyceride accumulation in β-cells and β-cell apoptosis, resulting in further deterioration of blood glucose. It has been found that berberine can improve insulin resistance induced by free fatty acids, reduce the expression of PCSK9 in HepG2 cells and affect the level of blood cholesterol. Berberine can help statins play a lipid-lowering role.8,9
AMPK is one of the key molecules in the regulation of biological energy metabolism, and it is essential for the body to maintain blood glucose balance by activating the AMPK protein pathway to improve energy metabolism and regulate blood glucose.10 Berberine is a natural monomer in medicinal food, which has a good affinity for AMPK. Berberine may have a synergistic effect on blood glucose regulation by acting with AMPK. Berberine may improve insulin resistance by activating the AMPK pathway and coordinating blood glucose regulation by combining with other pathways.11
Berberine promotes the expression of SIRT1 by increasing the expression level of SOD. SIRT1 is a deacetylase that triggers the deacetylation of forkbox O (FOXO) factor in oxidative stress and stimulates the transcription of FOXO target genes including SOD.15 Berberine can also inhibit oxidative stress by up-regulating the mRNA content of SOD in diabetic mice.16 The MIR-106b/SIRT1 pathway is involved in the role of berberine in reducing oxidative stress in diabetic mice.17
NADPH oxidase is another source of increased ROS by up-regulating the contents of various glycosylation products, fatty acids and glucose. Berberine inhibits its expression, thus reducing oxidative stress.18 In addition, berberine reduced the production of ROS by inhibiting the expression of NADPH oxidase 2max 4 (NADPH oxidase 2max 4).19
AMPK is also considered one of the anti-diabetic mechanisms of berberine, as its activation has a negative effect on the regulation of NADPH oxidase and a positive effect on the up-regulation of CD36 expression.20 The correlation between berberine down-regulation of NADPH oxidase and activation of AMPK was studied. The results showed that the activation of AMPK was not only related to the down-regulation of NADPH oxidase but also to the up-regulation of SOD expression.21 The correlation between berberine down-regulation of NADPH oxidase and activation of AMPK was studied. It showed that the activation of AMPK was not only related to the down-regulation of NADPH oxidase, but also related to the up-regulation of SOD expression.22
Uncoupling protein 2 (UCP2) plays a negative role in the regulation of reactive oxygen species (ROS) production and oxidative stress. The increased expression of UCP2 induced by berberine inhibited the production of ROS in the kidney or adipose tissues, but the up-regulated UCP2 also inhibited the insulin secretion of islet β-cells. Berberine can increase the expression of UCP2 in the artery, but the expression of berberine in hepatocytes is opposite to that of berberine. Therefore, it is necessary to further explore the relationship between berberine regulation of UCP2 and specific tissues.23
The Nrf2 pathway is also involved in berberine inhibiting oxidative stress and improving the condition of diabetes. Berberine can stimulate the expression of Nrf2, activate the expression of antioxidant enzymes, increase the levels of GSH and SOD, and inhibit the production of ROS by activating P38, AMPK and PI3K/Akt signal pathways.
Berberine has been shown to strongly affect carbohydrate metabolism. The compound also protects pancreatic β cells and increases the sensitivity of peripheral tissue to insulin by inducing the activities of GLUT-1, GLUT-4 and insulin type 1 (Ins-1) receptors. It also stimulates glycolysis and reduces insulin resistance by polarizing macrophages, inducing lipolysis and increasing energy consumption (by losing weight and limiting insulin resistance caused by obesity). In the liver, berberine inhibits the FOX01, SREBP1 and ChREBP pathways, as well as HNF-4 α (hepatocyte nuclear factor 4 α) mRNA, which hinders the process of gluconeogenesis. In the gut, it blocks glucosidase, which reduces glucose absorption. Its interference with intestinal flora reduces the level of monosaccharides and inhibits the development of diabetic complications.24
The introduction of a glycosylation group at the berberine 9-O position can improve the antidiabetic activity of the compound; however, the compound is unstable. A glycosylated berberine derivative (1) was designed and synthesized to provide stable physicochemical properties by introducing a triazole spacer into the berberine structure. The antidiabetic and cytotoxic effects of these compounds on HepG2 cells were tested. The results showed that the cytotoxicity of berberine derivative modified by glucose, galactose and mannose (2a–c) was lower, and the hypoglycemic activity of compounds 2c and 2d was higher than that of berberine.25
A series of disaccharide-modified berberine derivatives with potential for the treatment of type 2 diabetes (2) were designed and synthesized by Wang and colleagues. An anti-diabetic investigation of the synthesized compounds was performed in a zebrafish model using a fluorescently labelled glucose analog 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) as a glucose tracker. The results showed that compound 2e modified with berberine 9-O disaccharide had the highest antidiabetic activity. In addition, the dose-dependent study of compound 2e confirmed that the derivative could significantly promote 2-NBDG uptake by zebrafish larvae and eyes, achieving a hypoglycemic effect.26 It was found that the introduction of disaccharides at the 9-O position resulted in a higher hypoglycemic activity than monosaccharides.
Khvostov and his colleagues synthesized a new berberine derivative 3 and analyzed its hypoglycemic effect. Biological tests show that the compound exhibits a highly significant hypoglycemic activity, which is attributed to an increase in insulin sensitivity after single and multiple uses. Obese type 2 diabetic (T2DM) mice exhibited improved glucose tolerance, decreased fasting insulin level and sensitivity, decreased total body weight and interscapular fat, and increased interscapular brown fat activity. All these effects were also histologically confirmed, with reduced steatosis in the liver and smaller fat droplets in brown adipose tissues.27
The hypoglycemic effect of berberine is also related to the activation of AMPK. The increase in glucose levels led to the inhibition of AMPK activity, and berberine could activate AMPK activity.33 When AMPK is inactivated, it can also inhibit the production of pro-inflammatory cytokines, such as COX2 and iNOS. On the other hand, berberine-activated P38 plays an important role in anti-inflammation. After treatment with berberine (400 mg kg−1) in female SD rats, GLUT4 was up-regulated and IR was decreased by activating the PI3K/AKT pathway and inhibiting MAPK pathway.34 Therefore, these effects of berberine are partly due to the bi-directional regulation of the AMPK signal pathway. The hypoglycemic effect of berberine is attributed to the inhibition of inflammatory polarization by interacting with TLR4 and interfering with the TLR4/MyD88/NF κ B signal pathway.35 The effect of berberine-inhibited pro-inflammatory cytokines on improving diabetes is also mediated by Nrf2, and Nrf2 promotes the expression of anti-inflammatory enzyme HO-1.36
The role of berberine in diabetes mellitus and insulin resistance by targeting the NF-κB pathway has also been reported.37 The nuclear transfer of NF-κB transcription factor is limited by berberine's stable IκB-α, which can induce the expression of pro-inflammatory cytokines such as IL-6, iNOS, COX-2 and TNF-α. In addition, it has been reported that berberine can protect HIT-T15 pancreatic γ cells from palmitic acid-induced apoptosis by up-regulating PPAR-β expression.38
Currently, studies indicate that berberine has certain potential in improving blood sugar control and liver function; however, its safety, adverse reactions and contraindications need to be scientifically evaluated. Pre-diabetes intervention: large-scale clinical trials (involving more than 2800 people) have shown that taking berberine for 18 consecutive months can reduce the risk of pre-diabetes progressing to diabetes by 41%, and the mechanism is related to activating AMP kinase, promoting glucose utilization and inhibiting intestinal glucose absorption. Synergistic hypoglycemic effect: some studies have found that berberine combined with metformin can significantly reduce glycosylated hemoglobin, but the improvement effect of berberine alone on glycosylated hemoglobin is not clear. Gastrointestinal adverse reactions: common gastrointestinal discomfort, such as nausea, vomiting and constipation, can be relieved after meals. Alternative drugs should be used cautiously: their hypoglycemic effect is weaker than that of mainstream drugs (such as metformin), and thus cannot be used as an alternative alone; long-term use may cause intestinal flora imbalance. Risk of hypoglycemia: the combination with hypoglycemic agents may increase the probability of hypoglycemia, which needs close monitoring. Improve the mechanism of liver fibrosis: by repairing the intestinal barrier (increasing the expression of tight junction proteins Occludin and ZO-1 by 83–89%), inhibiting the inflammatory pathway of TLR4/NF-κB and regulating the intestinal flora (increasing the abundance of short-chain fatty acid-producing bacteria), liver injury and fibrosis can be alleviated. In animal experiments, berberine reduced the deposition of liver collagen by 42.3%, and the safety window was wide (IC50 = 128.7 μM).
The effects of berberine on cognitive function in patients with diabetes include the inhibition of anti-inflammatory activity and improvement of insulin resistance. Berberine not only activated the PI3K/AKT/mTOR and MAPK signal pathways but also down-regulated the translocation of new PKC subtypes and NF-κB in neurons. In addition, berberine also inhibited the expression of amyloid precursor protein and BACE-1, and reduced the production of oligomer AB42.45
The relieving effect of berberine on diabetic and diabetic neuropathic pain is related to its inhibitory effect on oxidative stress and neuroinflammation, which may be mediated by the μ-opioid receptor (MOR). Berberine significantly inhibited lipid peroxidation, activity of reactive oxygen species (ROS) and catalase (CAT), and the tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) protein levels; however, it significantly increased the μ-opioid receptor (MOR) protein and mRNA levels.46
AMPK is beneficial to metabolism and mitochondria in many chronic diseases. It has been reported that AMPK has a therapeutic effect on mitochondrial dysfunction in cultured dorsal root ganglion (DRG) neurons. Berberine increased the expression of p-AMPK in STZ-induced diabetic rats, which prevented mitochondrial dysfunction and autophagy damage. In addition, berberine enhanced the endogenous antioxidant defense system mediated by Nrf2, thereby inhibiting neuronal injury and neuroinflammation.47
In addition, berberine can promote Nrf2-dependent NGF protein expression and neurite growth at a specific concentration, thereby treating diabetic neuropathy.48 In addition, berberine activates the PI3K-Akt signal transduction pathway in type 2 diabetic rats, which has the effect of anti-apoptosis and reducing cerebral ischemia/reperfusion injuries.49
Another study evaluated the protective effect of berberine on diabetic encephalopathy through the SIRT1/ER stress pathway. The endoplasmic reticulum stress-related proteins (PERK, IRE-1A, EIF-2A, PDI and CHOP) were significantly reduced, and the SIRT1 protein expression was increased.50
One study also showed that berberine improves the irS-1 levels in the brain and restores the expressions of Glut-1 and Glut-3, which changes glucose homeostasis in the brain.51
In addition, the renal protective effect of berberine in DN progression may be due to its ability to inactivate the TLR4/NF-κB pathway, thereby relieving stZ-induced renal injury, inflammatory response and HG-induced apoptosis of podocytes.56 On the other hand, berberine can inhibit palmitic acid (PA)-induced activation of dynamic related protein 1 (Drp1) and help stabilize podocyte mitochondrial morphology, suggesting that berberine has a therapeutic effect on diabetic nephropathy.57 Another study showed that berberine reduced the protein and mRNA expression levels of TGF-β1, vimentin and α-SMA in DN rats.58 In addition, berberine inhibits the transfer of TGF-β1 from glomerular Mesangial cells to podocytes, thus protecting the function of podocytes, which may be one of the potential mechanisms of berberine's protective effect on diabetic nephropathy.59
Renal tubular epithelial to mesenchymal transition (EMT) and renal tubulointerstitial fibrosis are the main pathological changes of DN. The anti-fibrosis effect of berberine in the kidney can reduce podocyte apoptosis and inhibit EMT in DN. In the study of DN model KKAy mice and high glucose-induced renal tubular epithelial cell EMT,60 it was found that berberine could inhibit renal tubular epithelial cell EMT and renal interstitial fibrosis, and berberine-mediated EMT inhibition was achieved through the Notch/Snail pathway.61
The relationship between the protective effect of berberine on diabetic nephropathy and the podocyte injury induced by high glucose levels was discussed. Berberine can significantly enhance the activation of AMPK and protect AMPK-silenced podocytes from apoptosis induced by high glucose levels. Furthermore, berberine significantly increased the high glucose-elevated Unc-51-like autophagy-activating kinase 1 (ULK1) S317/S555 phosphorylation, Beclin-1 expression, the ratio of LC3II to LC3I expression and the number of autophagosome; however, it reduced ULK1 S757 phosphorylation in podocytes. In addition, berberine significantly reduced the inhibitory effect of compound C on podocyte autophagy. The protective effect of berberine on podocyte apoptosis induced by high glucose levels could be significantly alleviated by pretreatment with 3-methyladenine or baffinomycin A1. Therefore, berberine can promote the activation of AMPK, promote autophagy in podocytes and protect podocytes from glucose-induced injury. This may help to design new interventions for the treatment of diabetic nephropathy.62
Diabetic cardiomyopathy is a kind of diabetic heart disease, which refers to myocardial dysfunction without hypertension and coronary artery disease. Unless diabetic patients have hypertension and myocardial ischemia, there are few obvious clinical symptoms, which include mainly the left ventricular diastolic dysfunction. Berberine can stimulate glucose uptake and consumption in H9c2 cardiomyocytes, and has anti-apoptotic activity by increasing AMPK activity, thereby promoting the recovery of cardiac function. In addition, the hypoglycemic effect of berberine on differentiated cardiomyocytes may be related to changes in neutral lipid metabolism.64–66 Another study also reported that activation of the AMPK signaling pathway by berberine treatment could improve myocardial cell damage induced by high glucose, stimulate mitochondrial biogenesis, and restore autophagy flux in H9C2 cells.67 In addition, berberine can prevent diabetic cardiomyopathy by interfering with the metabolism of phosphatidylcholine (PCs), phosphatidylethanolamine (PEs) and sphingolipids (SMs).68 A study to investigate combination therapy to control hyperglycemia and hypertension simultaneously with diabetes was conducted. After 8 weeks of continuous administration of berberine at 100 mg per kg per day, blood glucose levels and blood pressure were decreased, and the function and expression of the BKCaβ1 subunit in cerebrovascular smooth muscle cells were increased.69
The effects of berberine on anti-diabetes and anti-osteoporosis have been documented. The purpose of this study was to observe the effect of berberine on bone disorders induced by experimental type 1 diabetes in rats. Londzin and his team conducted experiments on 3-month-old female rats dividing them into three groups: I-healthy control group, II-diabetic control group, and III-diabetic rats treated with berberine. Diabetes was induced by a single injection of streptozotocin. After a period of administration of berberine (50 mg per kg per day p. o.), biochemical indexes, such as serum bone turnover markers, bone mass and mineralization, histomorphometric parameters and mechanical properties, were studied. It was found that berberine could antagonize the effects of bone formation markers (osteocalcin) concentration, growth plate and cancellous bone microstructure parameters in diabetic rats; however, it could not improve bone mineralization and bone mechanical properties in diabetic rats.76
Wang et al. investigated the role of berberine in slowing the progression of diabetic retinopathy in diabetic patients treated with insulin. The results showed that insulin intervention could specifically stimulate the activities of hypoxia inducible factor-1 α and vascular endothelial growth factor in different types of retinal cells. Berberine can inhibit its activity in a dose-and time-dependent manner. Berberine inhibits the activity of AKT/mTOR and resumes the AKT/mTOR signalling pathway, thereby weakening the inhibitory effect of berberine on the expression of hypoxia inducible factor-1 α and vascular endothelial growth factor. Berberine inhibits the progression of diabetic retinopathy in experimental type I and type II diabetic mice treated with insulin. It is proven that berberine inhibits insulin-induced activation of retinal endothelial cells through the Akt/mTor/HIF-1α/VEGF pathways, thus improving insulin-induced diabetic retinopathy.79
The formation of advanced glycation end products (AGEs) and the activation of AGEs-related signal pathways in the retina of diabetic patients lead to diabetic retinopathy. Wang et al. found that berberine (BBR), a natural compound, is an effective AGEs inhibitor, which can significantly inhibit the formation of AGEs and its related TLR4/STAT3/VEGF signal pathway in retinal endothelial cells, thus contributing to the treatment of diabetic retinopathy (DR).80
Berberine can also reduce intestinal glucose absorption, inhibit α-glucosidase and lower postprandial blood glucose levels, which is similar to the hypoglycemic effect of α-glucosidase inhibitors.84 In addition, berberine can also reduce the intestinal disaccharidase activity of diabetic mice and Caco-2 cells.85
A rat model of polycystic ovary syndrome by intraperitoneal injection of testosterone propionate was established. The experiments were divided into the model group, low-dose berberine group (BL), high-dose berberine group (BH), metformin (Met) group, and control group (CON). The morphology of the ovary, hormone level and glucose and lipid metabolism were measured. The UID-mRNA-SEQ of ovarian tissue was detected to explore the mechanism of berberine in promoting ovulation. Three biomarkers of endometrial receptivity were detected by the immunohistochemical method. The results showed that the number of vesicles increased and the number of corpus luteum decreased in the model group. A high-dose berberine intervention can reverse these changes. Berberine could also reduce the levels of serum luteinizing hormone (LH) and total cholesterol (TC) in PCOS rats. At the same time, berberine improved the impairment of abnormal oral glucose tolerance without affecting fasting insulin levels and homeostasis model assessment-insulin resistance (HOMA-IR). The ovarian protein expression of LHCGR and CYP19A1 and the mRNA expression in granulosa cells decreased in the model group, and the expression could be restored to a certain extent by the intervention of berberine. In the model group, the thickness of the endometrium decreased and the expression of integrin αvβ3 and lysophosphatidic acid receptor 3 (LPAR3) increased, which could be reversed by berberine. This suggests that berberine can promote ovulation in patients with PCOS, and its mechanism may be related to the up-regulation of LHCGR and CYP19A1. Berberine can also improve endometrial receptivity by down-regulating the expression of αvβ3 and LPAR3.97
Alcohol extraction methods include microwave extraction, (Soxhlet's) reflux extraction, ultrasonic extraction, flash extraction, etc., which have the advantages of low solvent restriction, repeated use, energy saving, environmental protection, high safety, simple operation and high extraction efficiency. However, ultrasonic extraction involves a large investment in equipment.98,99 With the improvement of methods and the exploration of conditions, new methods such as aqueous two-phase extraction, enzymatic extraction, supercritical CO2 extraction, liquid membrane extraction and ultra-high pressure water jet extraction have emerged, which have improved the berberine extraction rate, shortened the extraction time and are more suitable for industrial production.
Each extraction method has its own unique advantages. For example, the enzymatic extraction method has negligible effect on extracting the components of the original plant contents.100 The entire process of supercritical CO2 extraction does not utilize organic solvents and the extract has no residual solvent. Liquid membrane has a strong enrichment effect. It can be predicted that more advanced extraction technologies will emerge in the future, providing a reference for the extraction, production and clinical application of berberine.
Berberine has been used in clinics for its functions of clearing away heat and toxic materials, as well as resisting bacteria. It exhibits many pharmacological activities, including as anti-dysentery, anti-infectious protozoa and anti-tumor, along with lowering blood sugar, regulating blood lipids, lowering blood pressure and anti-arrhythmia.
One of the main functions of berberine is to activate the enzyme AMP-activated protein kinase (AMPK) in vivo. AMPK is a very powerful enzyme in the body, which is often called the “metabolic master switch”. AMPK is responsible for regulating the metabolism of the body at the cellular level. The currently used hypoglycemic medicinal food are officially approved in seven categories, namely, biguanidine α-glucosidase inhibitors, insulin sensitizers, secretagogues, as well as new DPP-4 inhibitors and SGLT-2 inhibitors, are oral medicinal food, including injectable hypoglycemic medicinal food, while berberine hydrochloride is not included in the regular hypoglycemic medicinal food. Berberine has unique advantages in multi-target regulation (hypoglycemic, lipid-regulating and anti-inflammatory) and diabetes prevention, but its clinical orientation is still mainly adjuvant therapy. Compared with traditional secretagogues, the risk of hypoglycemia is lower; compared with the DPP-4 inhibitor, it has a wider effect but less convenience. Compared with SGLT-2 inhibitors, the evidence of cardiac and renal hard endpoints is weak. It is necessary to strengthen dosage form improvement and long-term benefit verification in the future.
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