Berberine: Botanical Occurrence, Traditional Uses, Extraction Methods, and Relevance in Cardiovascular, Metabolic, Hepatic, and Renal Disorders

Berberine-containing plants have been traditionally used in different parts of the world for the treatment of inflammatory disorders, skin diseases, wound healing, reducing fevers, affections of eyes, treatment of tumors, digestive and respiratory diseases, and microbial pathologies. The physico-chemical properties of berberine contribute to the high diversity of extraction and detection methods. Considering its particularities this review describes various methods mentioned in the literature so far with reference to the most important factors influencing berberine extraction. Further, the common separation and detection methods like thin layer chromatography, high performance liquid chromatography, and mass spectrometry are discussed in order to give a complex overview of the existing methods. Additionally, many clinical and experimental studies suggest that berberine has several pharmacological properties, such as immunomodulatory, antioxidative, cardioprotective, hepatoprotective, and renoprotective effects. This review summarizes the main information about botanical occurrence, traditional uses, extraction methods, and pharmacological effects of berberine and berberine-containing plants.


INTRODUCTION Berberine
Berberine(5,6-dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium) Figure 1, is a nonbasic and quaternary benzylisoquinoline alkaloid, a relevant molecule in pharmacology and medicinal chemistry. Indeed, it is known as a very important natural alkaloid for the synthesis of several bioactive derivatives by means of condensation, modification, and substitution of functional groups in strategic positions for the design of new, selective, and powerful drugs (Chen et al., 2005).

TRADITIONAL USE OF BERBERINE-CONTAINING SPECIES
In the Berberidaceae family, the genus Berberis comprises of ∼450-500 species, which represent the main natural source of berberine. Plants of this genus are used against inflammation, infectious diseases, diabetes, constipation, and other pathologies . The oldest evidence of using barberry fruit (Berberis vulgaris) as a blood purifying agent was written on the clay tablets in the library of Assyrian emperor Asurbanipal during 650 BC (Karimov, 1993). In Asia, the extensive use of the stem, stem bark, roots, and root bark of plants rich in berberine, particularly Berberis species, has more than 3000 years of history. Moreover, they have been used as raw material or as an important ingredient in Ayurvedic and traditional Chinese medicine (Birdsall, 1997;Kirtikar and Basu, 1998;Gupta and Tandon, 2004;Kulkarni and Dhir, 2010). In Ayurveda, Berberis species have been traditionally used for the treatment of a wide range of infections of the ear, eye, and mouth, for quick healing of wounds, curing hemorrhoids, indigestion and dysentery, or treatment of uterine and vaginal disorders. It has also been used to reduce obesity, and as an antidote for the treatment of scorpion sting or snakebite (Dev, 2006). Berberine extracts and decoctions are traditionally used for their activities against a variety of microorganisms including bacteria, viruses, fungi, protozoa, helminthes, in Ayurvedic, Chinese, and Middle-Eastern folk medicines (Tang et al., 2009;Gu et al., 2010).
In Yunani medicine, Berberis asiatica has multiple uses, such as for the treatment of asthma, eye sores, jaundice, skin pigmentation, and toothache, as well as for favoring the elimination of inflammation and swelling, and for drying ulcers (Kirtikar and Basu, 1998). Decoction of the roots, and stem barks originating from Berberis aristata, B. chitria, and B. lycium (Indian Berberis species), have been used as domestic treatment of conjunctivitis or other ophthalmic diseases, enlarged liver and spleen, hemorrhages, jaundice, and skin diseases like ulcers (Rajasekaran and Kumar, 2009). On the other hand, the use of decoction of Indian barberry mixed with honey has also been reported for the treatment of jaundice. Additionally, it has been reported the use of decoction of Indian barberry and Emblic myrobalan mixed with honey in the cure of urinary disorders as painful micturition (Kirtikar and Basu, 1998). Numerous studies dealing with its antimicrobial and antiprotozoal activities against different types of infectious organisms (Vennerstrom et al., 1990;Stermitz et al., 2000;Bahar et al., 2011) have been assessed so far. Moreover, it has been used to treat diarrhea (Chen et al., 2014) and intestinal parasites since ancient times in China (Singh and Mahajan, 2013), and the Eastern hemisphere, while in China it is also used for treating diabetes (Li et al., 2004).
Nowadays, a significant number of dietary supplements based on plants containing berberine (Kataoka et al., 2008) are used for reducing fever, common cold, respiratory infections, and influenza (Fabricant and Farnsworth, 2001). Another reported use for berberine-containing plants is their application as an astringent agent to lower the tone of the skin. Also, positive effects were observed on the mucous membranes of the upper respiratory tract and gastrointestinal system with effects on the associated ailments (Chen et al., 2014;Yu et al., 2016).
In southern South America leaves and bark of species of the genus Berberis are used in traditional medicine administered for mountain sickness, infections, and fever (San Martín, 1983;Houghton and Manby, 1985;Anesini and Perez, 1993).

EXTRACTION METHODS
Berberine, a quaternary protoberberine alkaloid (QPA) is one of the most widely distributed alkaloid of its class. Current studies suggest that isolation of the QPA alkaloids from their matrix can be performed using several methods. The principles behind these methods consist of the interconversion reaction between the protoberberine salt and the base. The salts are soluble in water, stable in acidic, and neutral media, while the base is soluble in organic solvents. Thus during the extraction procedure, the protoberberine salts are converted in their specific bases and further extracted in the organic solvents (Marek et al., 2003;Grycová et al., 2007). In the case of berberine, the classical extraction techniques like maceration, percolation, Soxhlet, cold or hot continuous extraction are using different solvent systems like methanol, ethanol, chloroform, aqueous, and/or acidified mixtures. Berberine's sensitivity to light and heat is the major challenge for its extraction. Hence, exposure to high temperature and light could lead to berberine degradation and thus influencing its matrix recovery. In his study Babu et al. (2012) demonstrated that temperature represent a crucial factor in both extraction and drying treatments prior extraction. The yield of berberine content in C. fenestratum stem tissue samples was higher in case of samples dried under the constant shade with 4.6% weight/weight (w/w) as compared to samples dried in oven at 65 • C (1.32% w/w) or sun drying (3.21% w/w). As well hot extraction procedure with methanol or ethanol at 50 • C gave lower extraction yields when compared with methanol or ethanol cold extraction at −20 • C. Thus, berberine content in   Torres et al., 1992 Berberis iliensis Popov -Young shoots Karimov andShakirov, 1993 Roots Dzhalilov et al., 1963 Berberis integerrima Bunge.
Large solvent volumes and long extraction time represent other drawbacks of conventional extraction methods (Mokgadi et al., 2013). For example, Rojsanga and Gritsanapan (2005) used maceration process to extract 100 g of C. fenestratum plant material with a total volume of 3,200 mL solvent (80% ethanol) over a period of 416 h. Furthermore, in a different study, Rojsanga et al. (2006) used several classical extraction techniques like maceration, percolation, and Soxhlet extraction to extract the berberine from C. fenestratum stems. This time even if the extracted plant material was in a lower amount than the previous study (30 vs. 100 g), large solvent volumes (2,000 mL for maceration, 5,000 mL for percolation, and 600 mL for Soxhlet extraction) over long time periods (7 days for maceration and 72 h for Soxhlet extraction) were employed (Rojsanga and Gritsanapan, 2005;Rojsanga et al., 2006).
Even though conventional methods are widely used in berberine extraction, a number of other different methods have been developed lately. This led to an improved extraction efficiency, a decreased extraction time and solvents' volumes used in the extraction. Thus, ultrasound assisted solvent extraction (USE), microwave-assisted solvent extraction (MAE), ultrahigh pressure extraction (UPE), and supercritical fluid extractions (SFE), pressurized liquid extraction (PLE) have been successfully used as alternative extraction techniques with better results when compared with classical extraction methods.
Ultrasonically and microwave-assisted extraction are considered green, simple, efficient, and inexpensive techniques (Alupului et al., 2009). Teng and Choi (2013) extracted berberine from Rhizome coptidis by optimized USE. Using response surface methodology, they identified that the optimal extraction conditions were 59% ethanol concentration, at 66.22 • C within 46.57 min. A decrease in the extraction time (39.81 min) was obtained by Chang (2013). He used the combination of ionic liquids solutions as green solvents with USE to extract berberine from Coptis chinensis in order to apply an environmentally friendly approach (Chang, 2013). Moreover, in their study, Xu et al. (2017) compared several extraction tehniques like USE, distillation, and Soxhlet extraction in order to establish an high-efficient method for phellodendrine, berberine, and palmatine extraction from fresh Phellodendron bark (Cortex phellodendri). In the case of berberine, the combination of simple or acidified solvent (water, ethanol, and methanol) with the adjustment of the specific setting characteristics to each extraction type enabled them to determine the highest extraction yield. They concluded that the use of USE and hydrochloric acid-acidified methanol were the most efficient in extracting berberine. The USE extraction yield was significantly higher when compared to distillation and Soxhlet extraction, with values of ∼100 mg/g toward 50 and 40 mg/g berberine, respectively (Xu et al., 2017).
The important reduction in organic solvent and extraction time determined the increasing interest in MAE, too. Lately, MAE was used as a green and cost-effective alternative to conventional methods. Using central composite design, Satija et al. (2015) successfully optimized the MAE parameters in terms of irradiation power, time, and solvent concentration to extract berberine form Tinospora cardifolia. They compared two classical extraction techniques like maceration and Soxhlet extraction with MAE under optimized conditions (60% irradiation power, 80% ethanol concentration, and 3 min extraction time). The results showed that MAE extraction had the highest yield of berberine content with 1.66% (w/w) while Soxhlet and maceration had 1.04 and 0.28% (w/w), respectively. Their study is emphasizing the dramatic time reduction in case of MAE (3 min) when compared with Soxhlet extraction (3 h) and maceration (7 days) together with solvent and energy consumption (Satija et al., 2015).
Another novel extraction technique considered to be environmentally friendly is UPE. The interest toward this extraction technique is increasing because it presents several advantages toward classical extraction techniques like increased extraction yields, higher quality of extracts, less extraction time, and decreased solvent consumption (Xi, 2015). These are achieved at room temperature by applying different pressure levels (from 100 to 600 MPa) between the interior (higher values) and the exterior of cells (lower values) in order to facilitate the transfer of the bioactive compounds through the plant matrices in the extraction solvent (Liu et al., 2006(Liu et al., , 2013. In the study regarding berberine content in Cortex phellodendri, Guoping et al. (2012) made a comparison between UPE, MAE, USE, and heat reflux extraction techniques. They observed that the higher extraction yield and the lower extraction time was obtained in case of UPE with 7.7 mg/g and 2 min extraction time toward reflux, USE and MAE with 5.35 mg/g and 2 h, 5.61 mg/g and 1 h. and 6 mg/g and 15 min, respectively (Guoping et al., 2012).
Super critical fluid extraction is another environmentally friendly efficient technique used in phytochemical extraction. Because the extraction is performed in the absence of light and oxygen, the degradation of bioactive compounds is reduced. Also, the inert and non-toxic carbon dioxide used as a main extraction solvent in combination with various modifiers (e.g., methanol) and surfactants (e.g., Tween 80) at lower temperatures and relatively low pressure, allows the efficient extraction of bioactive compounds (Liu et al., 2006;Farías-Campomanes et al., 2015). In case of berberine extraction from the powdered rhizome of Coptis chinensis Franch, the highest recovery of berberine was obtained when 1,2-propanediol was used as a modifier of supercritical CO 2 (Liu et al., 2006).
Pressurized liquid extraction, also known as pressurized fluid extraction, pressurized solvent extraction, and accelerated solvent extraction (ASE) is considered a green technology used for compounds extraction from plants (Mustafa and Turner, 2011). Compared with conventional methods, PLE increases the extraction yield, decreases time and solvent consumption, and protects sensitive compounds. In their study, Schieffer and Pfeiffer (2001) compared different extraction techniques like PLE, multiple USE, single USE, and Soxhlet extraction in order to extract berberine from goldenseal (Hydrastis canadensis). When compared in terms of extraction yield the results are comparable, ∼42 mg/g berberine, except single USE with slightly lower content (37 mg/g berberine). Big differences were observed in the extraction time, PLE requiring only 30 min for a single sample extraction compared to 2 h for multiple extraction techniques or 6 h for Soxhlet extraction (Schieffer and Pfeiffer, 2001).    When referring to berberine extraction from biological samples, the extraction process is relatively simple and involves several steps like sample mixing with extraction solvents (e.g., methanol, acetone, acetonitrile), vortex, centrifugation followed by supernatant evaporation under nitrogen stream (Table 3). Other extraction techniques like solid phase extraction (SPE) can also be applied.
UV-Vis spectrophotometry can be considered as one of the most rapid detection methods for berberine quantitative analysis from plant extracts. Based on the Beer-Lambert law, berberine concentration can be determined according to its absorption maxima at 348 nm. Joshi and Kanaki (2013) quantified berberine in Rasayana churna samples in the range of 2-20 µg/mL, the interference with other compounds being avoided by the specific isolation of the alkaloid fraction (Joshi and Kanaki, 2013).
Next, high-performance liquid chromatography (HPLC) is a versatile, robust, and widely used technique for the qualitative and quantitative analysis of natural products (Sasidharan et al., 2011). This approach is widely used in berberine identification and quantification. Generally, the choices of stationary phase in berberine separation are variants of C18-based silica column (Table 3) with a mobile phase consisting of simple or acidified solvents like water, methanol, or acetonitrile, used as such or in combination with phosphate buffers. Normally, the identification and separation of berberine can be accomplished using either isocratic or gradient elution system. Berberine identification is further accomplished using high sensitivity UV or DAD (diode array detectors) detectors. For example, Shigwan et al. (2013) developed in his study a reverse phase HPLC method with photodiode array detection (PDA) to quantify berberine from Berberis aristata and B. tinctoria. They used a Unisphere-C18 column (5 µm, 4.6 × 150 mm) with an isocratic gradient of acidified water (with 0.1% trifluoroacetic acid) and acetonitrile (60:40, v/v) to elute berberine within 5 min. The developed method was reproducible, validated, precise, and specific for berberine quantification (with a concentration range between 0.2 and 150 µg/mL; Shigwan et al., 2013).
Two other commonly used techniques in berberine quantification are thin layer chromatography (TLC) and high performance thin layer chromatography (HTPLC). Sometimes, these methods are preferred over HPLC, offering the possibility of running several samples simultaneously along with the use of small amount of both samples and mobile phases (Samal, 2013). For these reasons, Samal (2013) used an HPTLC method to quantify berberine from A. mexicana L. using toluene and ethyl acetate (9:3, v/v) as mobile phases, and a silica gel plate as stationary phase, they developed a simple, rapid, and cost-effective method for berberine quantification. The LOD (0.120 µg) and LOQ (0.362 µg) of the method are in accordance with high-quality requirements.
Following the same principles (small sample volume, high separation efficiency, and short analysis time), capillary electrophoresis (CE) was successfully used in berberine analysis. Du and Wang (2010) used CE with end-column electrochemiluminescence (ECL) detection for berberine analysis in both tablets and Rhizoma coptidis. Using a 4 min analysis time, a small sample volume (3.3 nL) and a LOD of (5 × 10 −9 g/mL), the developed method proved to be highly sensitive and with good resolution (Du and Wang, 2010).
Besides UV, HPLC, HTPLC, TLC, and CE, other detection methods like liquid chromatography coupled with mass spectrometry (LC/MS) are currently employed to quantify berberine in biological fluids. Generally, it is considered a powerful technique for the analysis of complex samples because it offers rapid and accurate information about the structural composition of the compounds, especially when tandem mass spectrometry (MS n ) is applied. For example, Xu et al. (2015) developed a sensitive an accurate LC-MS/MS method to determine berberine and other seven components in rat plasma using multiple reactions monitoring (MRM) mode. Compounds separation was optimized using six different types of reversephase columns, and two different mobile phases (methanolwater and acetonitrile-water with different additives). Additives like formic acid, acetic acid, and ammonium acetate were added in different concentrations as follows: 0.1, 0.5, 1, and 2% for formic acid, 0.1, 0.5, 1, and 2% for acetic acid and 0.0001, 0.001, 0.01 mol/L for ammonium acetate. The method was also tested in terms of specificity, linearity, lower limit of quantification (LLOQ), precision, accuracy, and stability (Xu et al., 2015).

ANTIOXIDANT EFFECT
Under normal conditions, the body maintains a balance between the antioxidant and pro-oxidant agents (reactive oxygen species-ROS and reactive nitrogen species-RNS; Rahal et al., 2014).
The imbalance between pro and antioxidants occurs in case of increased oxidative stress (Bhattacharyya et al., 2014).
The oxidative stress builds up through several mechanisms: an increase in the production of reactive species, a decrease in the levels of enzymes involved in blocking the actions of pro-oxidant compounds, and/or the decrease in free radical scavengers (Pilch et al., 2014).
An experimental study demonstrated the effect of berberine on lipid peroxidation after inducing chemical carcinogenesis in small animals (rats). An increase in LPO (lipid peroxidation) was observed after carcinogenesis induction, but also its significant reversal after berberine administration (30 mg/kg). Berberine shows therefore at least partial antioxidant properties, due to its effect on lipid peroxidation (Thirupurasundari et al., 2009).
Other mechanisms involved in the antioxidant role of berberine are: ROS/RNS scavenging, binding of metals leading to the transformation/oxidation of certain substances, freeoxygen removal, reducing the destructiveness of superoxide ions and nitric oxide, or increasing the antioxidant effect of some endogenous substances. The antioxidant effect of berberine was comparable with that of vitamin C, a highly-potent antioxidant (Shirwaikar et al., 2006;Ahmed et al., 2015).
The increase in blood sugar leads to oxidative stress not by generating oxygen reactive species but by impairing the antioxidant mechanisms. Administration of berberine to rats with diabetes mellitus increased the SOD (superoxide dismutase) activity and decreased the MDA (malondialdehyde) level (marker of lipid peroxidation). This antioxidant effect of berberine could explain the renal function improvement in diabetic nephropathy (Liu et al., 2008b).
The oxidative stress plays an important role in the pathogenesis of many diseases. The beneficial effect of berberine is presumed to reside mostly in its antioxidant role.

CARDIOVASCULAR EFFECTS OF BERBERINE Effect on Cardiac Contractility
The beneficial effect of berberine in cardiac failure was demonstrated in a study on 51 patients diagnosed with NYHA (New York Heart Association) III/IV cardiac failure with low left ventricular ejection fraction (LVEF) and premature ventricular contractions and/or ventricular tachycardia. These patients received tablets containing 1.2 g berberine/day, together with conventional therapy (diuretics, ACEI-angiotensin-convertingenzyme inhibitors, digoxin, nitrates) for 2 weeks. An increase in LVEF was observed in all patients after this period, but also a decrease in the frequency and complexity of premature ventricular contractions. The magnitude of the beneficial effect was in direct proportion with the plasma concentration of berberine (Zeng, 1999).

The Cardioprotective Effect During Ischemia
Berberine can provide cardio-protection in ischemic conditions by playing various roles at different levels: modulation of AMPK (AMP-activated kinase) activity, AKT (protein kinase B) phosphorylation, modulation of the JAK/STAT (Janus kinase/signal transducers and activators of transcription) pathway and of GSK3β (glycogen synthase kinase 3β; Chang et al., 2016). AMPK is an important enzyme playing an essential role in cellular metabolism and offering protection in ischemic conditions by adjusting the carbohydrate and lipid metabolism, the function of cell organelles (mitochondria, endoplasmic reticulum) and the apoptosis (Zaha et al., 2016).
Berberine activates the PI3K (phosphoinositide 3kinase)/AKT pathway which is considered a compensatory mechanism limiting the pro-inflammatory processes and apoptotic events in the presence of aggressive factors. The activation of this pathway is associated with a reduction of the ischemic injury through the modulation of the TLR4 (toll-like receptor 4)-mediated signal transduction (Hua et al., 2007).
Several supporting data indicate that the JAK2/STAT3 signaling plays an important role in cardioprotection against ischemia-reperfusion injury (Mascareno et al., 2001).
GSK3β is a serine/threonine protein-kinase, an enzyme involved in reactions associated to important processes at the cellular level: metabolization, differentiation, proliferation, and apoptosis. Berberine inhibits this kinase, thereby exercising its cardioprotective effect (Park et al., 2014).

EFFECTS ON THE ENDOTHELIUM
Berberine induces endothelial relaxation by increasing NO production from arginine through the activity of eNOS (endothelial nitric oxide synthase) which is considered a key element in the vasodilation process. Besides increasing the NO level, it also up-regulates eNOS mRNA. Furthermore, berberine facilitates the phosphorylation of eNOS and its coupling to HSP 90 (heat shock proteins), which consequently increases NO production .
Moreover, berberine reduces endothelial contraction by reducing COX-2 expression. Any imbalance in COX 1 or 2 activity may alter the ratio between prothrombotic/antithrombotic and vasodilator/vasoconstrictor effects .
The beneficial effect of berberine on the TNFα-induced endothelial contraction was also recorded, as well as an increase in the level of PI3K/AKT/eNOS mRNA (Xiao et al., 2014).

THE ROLE OF BERBERINE IN ATHEROSCLEROSIS
Atherogenesis is a consequence of high blood lipid levels and is associated with inflammatory changes in the vascular wall. Berberine interferes with this process by up-regulating the expression of SIRT1 (silent information regulator T1) and by inhibiting the expression of PPARγ (peroxisome proliferatoractivated receptor-γ). SIRT1 is a NAD-dependent deacetylase. The SIRT1 enzyme has many targets (PPARγ , p53), all playing different roles in atherogenesis (Chi et al., 2014).

The Role of Berberine in Lipid Metabolism
The effects of berberine on lipid metabolism are also the consequence of its effects on LDL cholesterol receptors. On one hand, these receptors are stabilized by an extracellular signalregulated kinase (ERK)-dependent pathway, and on the other, berberine increases the activity of LDL receptors through the JNK pathway (Cicero and Ertek, 2009).
Moreover, berberine has an effect on ACAT (cholesterol acyltransferases), a class of enzymes that transform cholesterol into esters, thus playing an essential role in maintaining cholesterol homeostasis in different tissues. There are two types of ACAT enzymes, ACAT1, and ACAT2. ACAT1 is a ubiquitous enzyme, while ACAT2 can be found only in hepatic cells and enterocytes. Berberine influences the activity of ACAT2 without an effect on ACAT1, therefore reducing the intestinal absorption of cholesterol and decreasing its plasmatic level (Chang et al., 2009;Wang et al., 2014).
The hypolipidemic effect of berberine is also a result of its action on PCSK9 (proprotein convertase subtilisin kexin 9). This enzyme can attach itself to LDL receptors, leading to a decrease in LDL metabolization and an increase in its blood level (Xiao et al., 2012).
In a clinical trial, 63 patients with dyslipidemia were randomly divided in three groups. The first group was treated with berberine (1,000 mg/day), the second with simvastatin (20 mg/day) and the third with a combination of berberine and simvastatin. The authors reported a 23.8% reduction in LDL-C levels in patients treated with berberine, a 14.3% reduction in those treated with simvastatin and a 31.8% LDL-C reduction in the group treated with both simvastatin and berberine. This result demonstrates that berberine can be used alone or in association with simvastatin in the treatment of dyslipidemia (Kong et al., 2008).

THE ROLE OF BERBERINE IN GLUCOSE METABOLISM
Many studies demonstrated that berberine lowers blood sugar, through the following mechanisms: -Inhibition of mitochondrial glucose oxidation and stimulation of glycolysis, and subsequently increased glucose metabolization (Yin et al., 2008a). -Decreased ATP level through the inhibition of mitochondrial function in the liver, which may be the probable explanation of gluconeogenesis inhibition by berberine (Xia et al., 2011). -Inhibition of DPP 4 (dipeptidyl peptidase-4), a ubiquitous serine protease responsible for cleaving certain peptides, such as the incretins GLP1 (glucagon-like peptide-1) and GIP (gastric inhibitory polypeptide); their role is to raise the insulin level in the context of hyperglycemia. The DPP4 inhibition will prolong the duration of action for these peptides, therefore improving overall glucose tolerance (Almasri et al., 2009;Seino et al., 2010).
Berberine has a beneficial effect in improving insulin resistance and glucose utilization in tissues by lowering the lipid (especially triglyceride) and plasma free fatty acids levels . The effect of berberine (1,500 mg day) on glucose metabolism was also demonstrated in a pilot study enrolling 84 patients with type 2 diabetes mellitus. The effect, including on HbA1c, was comparable to that of metformin (1,500 mg/day), one of the most widely used hypoglycemic drugs. In addition, berberine has a favorable influence on the lipid profile, unlike metformin, which has barely any effect (Yin et al., 2008b).

HEPATOPROTECTIVE EFFECT OF BERBERINE
The hepatoprotective effect of berberine was demonstrated on lab animals (mice), in which hepatotoxicity was induced by doxorubicin. Pretreatment with berberine significantly reduced both functional hepatic tests and histological damage (inflammatory cellular infiltrate, hepatocyte necrosis; Zhao et al., 2012).
The mechanism by which berberine reduces hepatotoxicity was also studied on CCl 4 (carbon tetrachloride)-induced hepatotoxicity. Berberine lowers the oxidative and nitrosamine stress and also modulates the inflammatory response in the liver, with favorable effects on the changes occurring in the liver. Berberine prevents the decrease in SOD activity and the increase in lipid peroxidation and contributes to the reduction in TNF-α, COX-2, and iNOS (inducible nitric oxide synthase) levels. The decrease in transaminase levels supports the hypothesis according to which berberine helps maintain the integrity of the hepatocellular membrane (Domitrović et al., 2011).

NEPHROPROTECTIVE EFFECT OF BERBERINE
The chronic kidney damage occurring in time in patients with HT (hypertension) and DM (diabetes mellitus) is well known; it is mainly due to the atherosclerosis of the renal artery, caused by inflammation and oxidative stress. The protective effect of berberine on kidneys was studied on 69 patients suffering from both HT and DM, with blood pressure and blood sugar levels controlled with conventional medication. The patients received 300 mg berberine/day for 24 months, with 2-week interruptions every 5 months. The authors recorded lower CRP (C-reactive protein), MDA and SOD levels after treatment, but without significant changes in creatinine, arterial pressure, or glycaemia levels. These results support the renal protective effect of berberine through its anti-inflammatory and antioxidant effects (Dai et al., 2015).
Another animal study tested the renoprotective effect of berberine after administration of HgCl 2 (mercury chloride). This substance induces hepato-renal damage by increasing the oxidative stress (increases lipid peroxidation and NO levels, and lowers glutathione and SOD levels as well as the activity of other protective enzymes). Administration of HgCl 2 increased the AST (aspartate aminotransferase), ALT (alanine aminotransferase), and ALP (alkaline phosphatase) levels, compared to the control group. However, pretreatment with berberine lowered these enzymes significantly. In addition, both urea and creatinine levels were significantly increased in the HgCl 2 group vs. the control group, and again pretreatment with berberine prevented these changes. Additionally, the authors recorded higher pro-oxidant and lower antioxidant levels in the intervention group. These data support the hepatic and renal protective effects of berberine. Other studies performed on animal models with CCl 4− induced hepatotoxicity demonstrated the same effect (Othman et al., 2014).
In addition, berberine can lower the nephrotoxicity caused by cisplatine. In an animal study, berberine was administered in progressive doses of 1, 2, 3 mg/kg, orally, for 2 successive days, starting 2 days after cisplatine administration. After the last doses of berberine, the animals were sacrificed and the kidneys were examined by the pathologist. The results showed significant histological improvement and a reduction in NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells), TNF α, COX2 an iNOS levels, all of which support the anti-inflammatory effect of berberine (Domitrović et al., 2013).

IMMUNOMODULATORY EFFECT OF BERBERINE
The immunomodulatory effect of berberine was demonstrated in many experimental and clinical contexts.
In an experimental autoimmune myocarditis model, berberine contributed to mitigate the cardiac damage by: limiting the rise in anticardiac myosin antibodies, modulating the activity of certain STATs and blocking Th1 and Th2 cell differentiation, which play an important role in the pathogenesis of myocarditis .
Experimental autoimmune neuritis is an experimental animal model equivalent to the Guillain-Barre syndrome in humans. This neurologic syndrome is characterized by autoimmune injury of the peripheral nervous system. The beneficial effect of berberine on this animal model resided in its influence on cellular and humoral immunity through the inhibition of lymphocyte proliferation (especially CD4), and the decrease in pro-inflammatory cytokines (IL-6 and TNF α; Li et al., 2014).
Experimental autoimmune encephalomyelitis is an established model of multiple sclerosis. Multiple sclerosis is a one of the most common diseases of the central nervous system (CNS) and involves neurodegenerative and inflammatory processes, and autoimmune demyelination (Ransohoff et al., 2015). The blood-brain barrier permeability and changes in matrix metalloproteinase (MMP) levels in the cerebrospinal fluid and brain were studied using this model (Ma et al., 2010). MMPs may be involved in demyelination and their activity in tissues depends on the balance between their level and their tissue inhibitors. MMP2 and MMP9 are the main endoproteinases involved in the migration of lymphocytes in CNS and in altering the BBB (blood brain barrier) (Avolio et al., 2003). Berberine has a beneficial effect in experimental autoimmune encephalomyelitis by inhibiting the activity of MMP9, reducing BBB permeability and, consecutively, by decreasing the inflammatory cellular infiltration of the CNS (Ma et al., 2010).
The current therapy used for inflammatory bowel diseases, including glucocorticoids and immunosuppressive agents, has a low level of safety. The effect of berberine was studied in combination with 5-ASA (5-aminosalicylic acid) vs. 5-ASA alone using an experimental animal model with DSS (dextran sulfate sodium)-induced colitis. The authors analyzed the level of proinflammatory cytokines in the animal gut. A decrease in COX2, IL6, and IL23 mRNA levels was observed in animals treated only with 5-ASA, whereas animals treated with both 5-ASA and berberine had a reduction in mRNA levels for COX2, IL6, IL23 as well as for TNF alfa and IL12b. This beneficial effect could partially be attributed to the inhibition of NF-kB and the reduction in JAK2 phosphorylation (through the influence on the JAK/STAT pathway) by both 5-ASA and berberine ; Figure 3).
Another study demonstrated that berberine increases the corticosteroid level in rats with experimentally-induced colitis. This engendered the theory that its beneficial effect may also be attributed to the increase in endogenous glucocorticoid levels, compounds with well-known therapeutic effect in inflammatory bowel disease (Minaiyan et al., 2011).

CONCLUSION
A review of the available scientific literature shows that the traditional medical uses of berberine-containing plants have been evaluated by modern pharmacological studies. Different species of berberine-rich plants have multiple pharmacological and therapeutic actions, such as antioxidant and immunomodulatory effects, protective action on the cardiovascular system, liver and kidney, endothelial relaxation, regulator on glucose metabolism and atherosclerosis, which can all be explained by the presence of berberine as well as other phyto constituents (when dealing with berberinecontaining plant extracts). Moreover, the effects of berberine vary according to its origin (different plants or pharmaceutical products) and its concentration, depending on the very diverse extraction and detection techniques already described. Over time, modern extraction techniques were increasingly preferred to classical ones. Since classical methods are generally time-and solvent-consuming processes, modern extraction techniques such as USE, MAE, UPE, SFE, and PLE are seen as better alternatives to overcome these limitations. Furthermore, berberine, due to its antioxidant and anti-inflammatory effects, has several clinical applications in many disorders, from inflammatory conditions to the metabolic syndrome. However, there are some traditional uses that have not yet been completely elucidated, and further studies are needed. Therefore, extensive studies on the potential of plants containing berberine that have shown aforementioned pharmacological activities should go through additional in vitro and in vivo studies.

AUTHOR CONTRIBUTIONS
MN, AM, JE, and RP have conceived and designed the structure of the manuscript, data collection, and drafting, as well as its revision. CB, GC, and AB have critically reviewed the manuscript. All authors have seen and agreed on the final version of the manuscript.