Chemistry and Pharmacology of Bergenin or Its Derivatives: A Promising Molecule

Bergenin is a glycosidic derivative of trihydroxybenzoic acid that was discovered in 1880 by Garreau and Machelart from the rhizomes of the medicinal plant Bergenia crassifolia (currently: Saxifraga crassifolia—Saxifragaceae), though was later isolated from several other plant sources. Since its first report, it has aroused interest because it has several pharmacological activities, mainly antioxidant and anti-inflammatory. In addition to this, bergenin has shown potential antimalarial, antileishmanial, trypanocidal, antiviral, antibacterial, antifungal, antinociceptive, antiarthritic, antiulcerogenic, antidiabetic/antiobesity, antiarrhythmic, anticancer, hepatoprotective, neuroprotective and cardioprotective activities. Thus, this review aimed to describe the sources of isolation of bergenin and its in vitro and in vivo biological and pharmacological activities. Bergenin is distributed in many plant species (at least 112 species belonging to 34 families). Both its derivatives (natural and semisynthetic) and extracts with phytochemical proof of its highest concentration are well studied, and none of the studies showed cytotoxicity for healthy cells.


Introduction
The rapid development of the chemistry of natural products has led to the isolation of a variety of secondary metabolites. In particular, bergenin, also known as ardisic acid B, bergenit, bergenitol, cuscutin, peltophorin, and vakerin [1,2]. Its name is derived from where it was isolated, i.e., from the ornamental and medicinal plant Bergenia crassifolia L. (currently: Saxifraga crassifolia L.-Saxifragaceae), and it was obtained from the rhizomes of this plant [1,2]. Although originally obtained from a small plant distributed in the temperate regions of North-Central Asia, mainly in Russia, the bergenin molecule has been found in several plant species distributed worldwide. To date, bergenin has been obtained from different species of different families (Table 1).

Data Collection
The bibliographic research methodology was adopted and conducted using the databases available for institutional access, i.e., Google Scholar, Medline, Pubmed and-SciFinder. Regarding the latter, the research was carried out both using keywords and by searching for similarities with the chemical structure of the molecule bergenin. The following terms were used as descriptors of the bibliographic research: "bergenin", "plant extracts", "in vitro assays", "in vivo assays", "natural sources", "chemical aspects", "derivatives", "antimalarial activity", "antileishmanial activity", "trypanocidal activity", "antiviral activity", "antibacterial activity", "antifungal activity", "anti-inflammatory activity", "antinociceptive activity", "antiarthritic activity", "antiulcerogenic activity", "antidiabetic/antiobesity", "antiarrhythmic activity", "anticancer activity", "antioxidant activity", "hepatoprotective activity", "neuroprotective activity", "cardioprotective activity", and using the Boolean operators "AND" and "OR". A total of 6550 papers, 6 master's dissertations, and 2 doctoral theses were found, of which 210 papers, 2 master's dissertations, and 2 doctoral theses published in the period from 1880 to 2022 were selected. The inclusion criteria were articles written in Portuguese, Spanish, or English, and their abstracts were available. Doctoral theses and master's dissertations were also included. Papers, dissertations, and theses that studied biological and pharmacological activities of bergenin, natural derivatives of bergenin, semisynthetic derivatives of bergenin, and plant extracts with phytochemical confirmation that bergenin is one of the constituents were selected. The exclusion criteria were studies that did not refer to at least one of the research themes and papers that had been retracted.
It was discovered in 1880 by Garreau and Machelart [92]. The first proposals on the structure of bergenin were provided by Tschitschibabin et al. (1929). First, a chemical structure with only two rings and one aliphatic chain was proposed. Later, in 1950, the structure was revised by Shimokoriyama and proposed with three rings of six members [92,93] (Figure 1).
The biosynthesis of bergenin is directly related to the biosynthesis of gallic acid, which originates in the combination of erythrose-4-phosphate with phosphoenolpyruvate, which leads to the formation of 3-dehydroquinic acid. Dehydration of the latter leads to the formation of 3-dehydroshikimic acid, which in turn is converted to gallic acid through oxidation and enolization reactions. Other alternative routes would be the degradation of the side chain of hydroxycinnamic acids or through the condensation of an acetyl-CoA group with three malonyl-CoA units. Regarding the C-glycosylation step, previous experiments have shown contrasting results since the use of isotopically labeled [7-14 C] benzoic acid and hydroxycinnamic acids indicated that Cglycosylation would occur in C6-C3 compounds, to the detriment of the C6-C1 derivative such as benzoic acid [95]. On the other hand, the use of the molecule labeled D-[U- 14 C] glucose in the presence of labeled gallic acid indicated that the C-glycosylation step occurs preferentially in the C6-C1 derivative [96]. Subsequently, the condensation of the glycosidic portion with the carboxylic acid of the gallic acid portion occurs, which leads to the formation of the lactone portion observed in the bergenin structure. Finally, more recent studies have suggested that the C-glycosylation step occurs prior to the Omethylation of the phenolic hydroxyl of the gallic acid-derived portion by Omethyltransferases [97]. Several bergenin derivatives have been isolated from plants, but not all showed marked biological activities. The main substances derived from bergenin with this type of potential are demethylated analogs, or those which are esterified with phenolic acids (e.g., gallic acid). Among the natural derivatives of bergenin, the following stand out: riverbergenin A isolated from the trunk of R. hypocrateriformis [18], norbergenin from the leaves of A. japonica [79], 11-O-galloylbergenin from the rhizome of A. gigantifolia [81] [36,102]. In addition to these, some semisynthetic derivatives have been designed in order to increase their pharmacological potential, for which several The biosynthesis of bergenin is directly related to the biosynthesis of gallic acid, which originates in the combination of erythrose-4-phosphate with phosphoenolpyruvate, which leads to the formation of 3-dehydroquinic acid. Dehydration of the latter leads to the formation of 3-dehydroshikimic acid, which in turn is converted to gallic acid through oxidation and enolization reactions. Other alternative routes would be the degradation of the side chain of hydroxycinnamic acids or through the condensation of an acetyl-CoA group with three malonyl-CoA units. Regarding the C-glycosylation step, previous experiments have shown contrasting results since the use of isotopically labeled [7-14 C] benzoic acid and hydroxycinnamic acids indicated that C-glycosylation would occur in C 6 -C 3 compounds, to the detriment of the C 6 -C 1 derivative such as benzoic acid [95].
On the other hand, the use of the molecule labeled D-[U-14 C] glucose in the presence of labeled gallic acid indicated that the C-glycosylation step occurs preferentially in the C 6 -C 1 derivative [96]. Subsequently, the condensation of the glycosidic portion with the carboxylic acid of the gallic acid portion occurs, which leads to the formation of the lactone portion observed in the bergenin structure. Finally, more recent studies have suggested that the C-glycosylation step occurs prior to the O-methylation of the phenolic hydroxyl of the gallic acid-derived portion by O-methyltransferases [97].
Several bergenin derivatives have been isolated from plants, but not all showed marked biological activities. The main substances derived from bergenin with this type of potential are demethylated analogs, or those which are esterified with phenolic acids (e.g., gallic acid). Among the natural derivatives of bergenin, the following stand out: riverbergenin A isolated from the trunk of R. hypocrateriformis [18], norbergenin from the leaves of A. japonica  [36,102]. In addition to these, some semisynthetic derivatives have been designed in order to increase their pharmacological potential, for which several modifications have been proposed. Figure 2 lists all the natural and semisynthetic derivatives of bergenin cited in this review.

Antimalarial Activity
Bergenin, or its derivatives and extracts containing bergenin, have been reported to have antimalarial activity in vitro and in vivo studies. To date, about 15 in vitro and 5 in vivo studies on this topic have been published (

Antimalarial Activity
Bergenin, or its derivatives and extracts containing bergenin, have been reported to have antimalarial activity in vitro and in vivo studies. To date, about 15 in vitro and 5 in vivo studies on this topic have been published (Table 2).
Singh et al. [39] reported the in vitro antimalarial activity of the crude extract and fractions derived from F. virosa against Plasmodium falciparum using the nucleic acid dye SYBR Green I-based fluorescence assay (MSF). In the study, different concentrations of the extract (0.1 to 100 µg/mL) were incubated with chloroquine-sensitive (3D7) and chloroquineresistant (K1) strains of P. falciparum, and subsequently, the plates were examined at 485.20 nm excitation and 530.20 nm emission for fluorescence units (RFUs) per well using a fluorescence reader (FLX800, BIOTEK). The findings indicated that the extracts/fractions significantly inhibited the sensitive (3D7) and resistant (K1) strains of P. falciparum. The crude extract presented an IC 50 of 2.35 µg/mL for 3D7 and 4.73 µg/mL for K1, while the fractions presented an IC 50 of 1.73 to 8.61 µg/mL for 3D7 and 2.32 to 20 µg/mL for K1 against 5.5 and 2.54 nM of the positive control (chloroquine), respectively [39]. Other studies with the same approach, using extracts with bergenin as one of the constituents, had satisfactory results in vitro: ethanolic and methanolic extract of stems and leaves of H. balsamifera against a culture of BHz 26/28 of P. falciparum resistant to chloroquine, IC 50 ranging from 8.37 to 49.65 µg/mL [13]; ethanolic extract of the rhizome of W. bignonia against strains of P. falciparum RKL-9 2, IC 50 of 5 µg/mL [15]; ethanolic extract of B. ciliata rhizome against P. falciparum strains RKL-9 and MRC-2, IC 50 of 5 µg/mL [15]; methanolic extract of D. Sanza-Minika stem bark against P. falciparum K1, IC 50 of 0.6 µg/mL [36].
The IC 50 of pure bergenin isolated from different parts of plant species (Table 2) was determined against P. falciparum in vitro, with the following results: 2.41-14.1 µg/mL [37,38,40,50,58,103]. The mechanism of action by which bergenin inhibits the growth of the parasite in vitro is not yet well known, but it is suggested that it is triggered by the inhibition of polymerization of the heme group of the parasite in a similar way to the mechanism of action of artemisinin since both are lactones of the same family [39,104]. In other words, in the first step, bergenin is activated by heme or a free iron (II) ion and produces free radicals and cytotoxic species. In the second step, these species react with the specific protein associated with the parasite's membrane, thus causing its death [104].
11-O-Galloylbergenin, a bergenin derivative isolated from the root of B. ligulata, was tested against P. falciparum and showed significant activity against a resistant P. falciparum strain (CQS D10) and exhibited good activity at low concentrations, with an IC 50 value of 2.34 µg/mL against a value of 28.07 nM for CQ (chloroquine) as a positive control [105]. These results are similar to the findings of a study in which 11-O-galloylbergenin was isolated from all parts of the plants of the species M. philippensis [49]. Other derivatives of natural bergenin had their IC 50 determined against P. falciparum, namely, 4-O- 50 3.9 µg/mL) [36], and 11-O-p-hydroxybenzoylnorbergenin (IC 50 of 4.9 µg/mL) [36].
The in vivo antimalarial activity of bergenin was also evaluated ( Table 2). Experimental models have used animals such as rats and mice infected with P. berghei [106]. P. berghei is a parasite that infects rodents and was discovered in the 90s in Congo. Since then, it has been widely used for experimental infections, which are considered models for biological and therapeutic studies [106]. Thirteen mice experimentally infected with P. berghei, with parasitemia of 35%, were treated with 800 mg/kg/day (100 µL twice daily) of bergenin (isolated from the leaves of R. aesculifolia) for 4 to 6 days intragastrically, and the findings showed that bergenin decreased parasitemia from 35 to 27%. These data indicate that bergenin suppresses the growth of P. berghei in vivo [6]. Singh et al. [39] experimentally induced P. berghei infection intraperitoneally in mice and, after 7 days of infection, treated with bergenin (isolated from F. virosa leaves) at concentrations of 25 to 100 mg/kg for 8 days, it caused an 85.13% suppression of parasitemia on the eighth day at a concentration of 100 mg/kg bergenin. These results are similar to the findings of Gorky et al. [15].
On the other hand, Da Silva et al. [107], in vitro experiments using bergenin (isolated from H. balsamifera leaves) against P. falciparum, found no activity (Table 2), but in vivo experiments found moderate efficacy in inhibiting the growth of P. berghei (IC 50 of 146.87 mg/kg), which suggests that more studies should be done to determine the concentrations for successful parasitic inhibitions. Table 2. Summary of studies regarding the antimalarial activity of bergenin and its derivatives. In vitro tests were carried out against P. falciparum and in vivo (in mice) against P. berghei.

Antileishmanial Activity
The antileishmanial activity of bergenin has been reported in six in vitro studies and in one in vivo study. Crude extracts/fractions of the aerial parts of M. erythrophylla have shown an inhibitory effect against Leishmania donovani, which is the etiological agent of visceral leishmaniasis, in a resazurin colorimetric test [37]. The extracts were incubated together with visceral leishmaniasis 1S (MHOM/SD/62/1S) promastigotes at concentrations of 0.16 to 100 µg/mL, in triplicate, followed by the addition of resazurin and amphotericin B (10-0.016 µg/mL), was used as the positive control. The results indicated that the crude extract had a moderate effect on antileishmanial activity (IC 50 of 61.6 µg/mL), and the hexane fraction showed good antileishmanial activity (IC 50 of 31.06 µg/mL) when compared with the reference drug, which was the positive control amphotericin B (IC 50 of 0.11 µM) [37]. Kaur and Kaur [103] reported antileishmanial activity of the ethanolic extract of B. ligulata root against L. donovani after finding satisfactory parasitic inhibition (IC 50 of 22.70 µg/mL), and phytochemical data revealed that the main compound in B. ligulata is bergenin, which suggests that the antileishmanial activity of these extracts is due to this compound [103]. Keshav et al. [62] reported that hydroxyethanolic extract of C. microphyllum showed good activity against sensitive and resistant strains of L. donovani, with an IC 50 of 14.40 µg/mL and 23.03 µg/mL, respectively. Kabran et al. [51] reported that bergenin isolated from leaves of M. oppositifolius had an effect against L. donovani (IC 50 of 73.3 µM).
Bergenin isolated from aerial parts of M. erythrophylla showed an inhibitory effect against L. donovani in resazurin colorimetric tests [37]. Bergenin was incubated together with protozoa, in triplicate, at concentrations of 0.08 to 50 µg/mL. Amphotericin B (10-0.016 µg/mL) was used as a positive control; the results showed the promising effects of bergenin against L. donovani (IC 50 of 53.7 µM) [37].
Antileishmanial activity was also evaluated in vivo. Kaur and Kaur [103] reported that ethanolic extract of B. ligulata root had an antileishmanial effect in inbred BALB/c mice infected intracardially with 10 7 promastigotes. In the study, 48 mice were divided into two groups, an uninfected group, and an infected group, and the infected groups had inflammation in the liver and spleen triggered by the recruitment of the inflammatory cytokines: interleukin 12 (IL-12), interleukin 4 (IL-4), interleukin 10 (IL-10) and interferon-gamma (IFN-γ). The infected group was treated with the ethanolic extract of B. ligulata orally at concentrations of 500 and 1000 mg/kg for 15 days, and the mice were euthanized at 1, 7, 14, and 21 days post-treatment. The spleen and liver were removed, then centrifuged, and the homogenate was used to quantify the parasitemia and determine the immune response by the parasite-specific enzyme-linked IgG1 and IgG2a isotopes, delayed-type hypersensitivity (DTH) responses, and the effect of recruited cytokines. The results indicated that the treatment with the extracts rich in bergenin at both concentrations tested significantly reduced the parasitic load, corresponding to 91.1% and 95.6%, respectively. The treatment with the extracts induced the IgG2a antibody response, which is considered an indicator of the Th1 type of immune response, thus contributing to parasitic reduction. The treatment also triggered inhibition of the Th2 activation pathway by reducing the production of inflammatory cytokines, especially IFN-γ at 5001.61 to 175.21 pg/mL, which contributed to the reduction of inflammation of the internal organs of infected mice [103].

Trypanocidal Activity
The trypanocidal activity was mainly evaluated using bergenin-rich extracts, with four in vitro studies and one in vivo study. Ethanolic extract of leaves of C. pluviosa rich in bergenin showed trypanocidal activity against Trypanosoma cruzi in vitro [72]. The extracts were incubated at concentrations of 10 to 500 µg/mL together with the parasitic suspension of T. cruzi in the infective trypomastigote form (2 × 10 5 /0.1 mL) in LIT (liver infusion tryptose) culture medium and, after 24 h, the suspension was microscopically quantified. The results indicated that the ethanolic extract of C. pluviosa showed good activity against the development of trypomastigotes of T. cruzi (IC 50 of 55 µg/mL). Nyasse et al. [109] reported that bergenin isolated from F. virosa leaves also exhibited inhibitory activity in the growth of T. brucei in the bloodstream (trypomastigotes), with an IC 50 value of 1 mM. Growth inhibition is achieved by inhibiting three glycolytic enzymes of the parasite: GAPDH (glyceraldehyde-3-phosphate dehydrogenase), PFK (phosphofructokinase), and PGK (phosphoglycerate kinase), which leads to the death of the parasite. Melos and Echearria [110] reported that trypanosomatids are highly dependent on glycolysis for ATP production, and, as many glycolytic enzymes have their own characteristics, they are considered to be potential targets for new chemotherapeutic agents.
Nyunt et al. [111] reported the trypanocidal activity of 11-O-acetylbergenin (isolated from the methanolic extract of V. repens) against Trypanosoma evansi trypomastigotes in vitro. 11-O-acetylbergenin was incubated in 96-well plates along with T. evansi in the trypomastigote form. Subsequently, the activity was determined by counting the number of parasites using a Neubauer hemocytometer. The findings indicated that 11-O-acetylbergenin inhibited (IC 50 of 0.17 mM) the growth of T. evansi, thus suggesting trypanocidal activity.
The trypanocidal activity of the ethanolic extract of the stem bark of S. gabonensis was investigated in rats infected with Trypanosoma congolense. Twenty albino rats in four groups (A, B, C, and D) of five rats were injected intraperitoneally with 0.5 mL of blood infected with T. congolese. A further ten rats (E and F) were not infected with the parasite. All the infected animals developed the following clinical manifestations: varying degrees of lethargy, mucosa, rough fur, reduced appetite, and depression. Groups A, B, and C were treated with the ethanolic extract of S. gabonensis from the stock solution orally via water intake. Group A received 0.5 mg/kg, Group B received 1 mg/kg, and Group C received 3.5 mg/kg; however, Group D did not receive any treatment, Group E received treatment with the stock solution (0.5 mg/kg) and Group F did not receive any treatment. The results show that the treated infected groups (A, B, and C) had a mortality rate of 40 to 80% with 13 to 32 days of survival, while the infected Group (D), which received no treatment, had a mortality rate of 100% with 12 to 23 days of survival. The other negative control groups did not suffer any impact. These results suggest that treatment with ethanolic extract of the stem bark of S. gabonensis, which is rich in bergenin, reduced the growth of T. congolense and prolonged the lifespan of the infected rats [69].

Antiviral Activity
Bergenin has shown promise in studies focused on antiviral activity since it was discovered that extracts of species of the genus Bergenia exert immunostimulating activity in response to viruses and pathogenic microorganisms after their invasion of biological systems [112]. In this context, it is reported that bergenin can induce the transmembrane glycoprotein CD64, which has an important role in the humoral immune response, using an Fc receptor that binds to monomeric antibodies such as IgG [113]. To date, about 8 in vitro studies have been performed, though no study evaluated in vivo conditions.
The methanolic extract of A. rivularis showed an antiviral effect against herpes simplex virus type 1 (HSV-1) and influenza A via a dye-uptake assay in HSV-1/Vero cell systems and influenza A virus/MDCK [85]. The cells were incubated together with the viruses in different concentrations of extracts (6.25 to 100 µg/mL), and the results revealed that the extracts exhibited good antiviral activity with consequent destruction of HSV-1 and influenza A viruses IC 50 of 6.25 µg/mL. These results suggest that extracts of A. rivularis exerted antiviral activity due to the presence of secondary metabolites since phytochemical investigations of the plant showed the presence of bergenin, flavonoids, and terpenoids [114]. Under the same conditions, Rajbhandar et al. [9] evaluated bergenin isolated from the rhizome of A. rivularis; however, they only tested the antiherpes activity, and the results were similar. Bergenin exhibited good activity against the herpes virus (IC 50 of 6.25 µg/mL), proving that bergenin has this potential and can be explored further.
Zuo et al. [88] evaluated bergenin isolated from the aqueous ethanolic extract of the aerial parts of S. melanocentra against the NS3 serine protease of the hepatitis C virus (HCV) in an enzyme-linked immunosorbent assay (ELISA). HCV is an enveloped virus with positive-stranded genomic RNA that encodes a polyprotein that is cleaved by the NS3 serine protease, thus allowing the development of HCV [115] and, therefore, has been targeted for the development of new therapies for HCV infection. In the study, bergenin was incubated along with NS3 serine protease, and the results indicated that bergenin significantly inhibited NS3 serine protease (IC 50 of 27.7 µg/mL).
Piacente et al. [116] evaluated the methanolic extract of aerial parts of A. japonica (the main constituent of bergenin) against the HIV virus in vitro and showed a moderate effect. However, when they tested bergenin and norbergenin isolated from the same extracts, both compounds showed weak antiHIV activity. The methanolic extract of the roots and bark of P. africanum was evaluated for its activity against HIV-1 reverse transcriptase (RT) [117]. Drugs that fight the HIV virus act as inhibitors of the reverse transcriptase, integrase, and protease enzymes. However, by inhibiting these enzymes, drugs prevent the multiplication of the virus in host cells. Reverse transcriptase is an enzyme that performs reverse transcription by producing DNA from RNA. It is also called RNA-dependent DNA polymerase. Therefore, the extracts incubated with this enzyme showed an inhibitory effect (IC 50 of 3.5 µg/mL) against the DNA-dependent RNA polymerase activity (RDDP) of RT; however, bergenin isolated from the same extract had no effect against the same enzyme at concentrations tested up to 100 µM [117].

Antibacterial Activity
The antibacterial activity of bergenin has been reported in 15 in vitro studies and 4 in vivo studies. Liu et al. [82] reported that the methanolic extract of leaves of B. purpurascens showed antibacterial activity against strains that commonly cause respiratory infections: Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Enterobacter cloacae. The bacteria were incubated in triplicate along with the extracts at concentrations of 0.8 to 512 µg/mL using the agar well diffusion method, with resazurin as the dye added to each well. The results showed that the extract of B. purpurascens inhibited the growth of all strains, with IC 50 ranging from 27 to 280 µg/mL. Other studies with the same approach using extracts that have bergenin had satisfactory results in vitro: methanol extracts of flowers of four angiosperm plant species Wendlandia thyrsoidea, Olea dioica, Lagerstroemia speciosa, and Bombax malabaricum against S. aureus, Bacillus cereus, Vibrio cholerae and E. coli, IC 50 ranging from 1.2 to 3.8 µg/mL [14]; ethanol extract of C. corean against resistant strains of S. aureus, IC 50 of 20 µg/mL [118]; methanolic extract of the rhizome of B. ligulata against E. coli and S. aureus, IC 50 of 250 µg/mg [119]; methanolic extract of the rhizome of A. rivularis against E. coli, IC 50 of 100 µg/mL [120].
Antibacterial activity was also determined in vivo. Liu et al. [82] evaluated the antibacterial activity of the methanolic extract of P. roxburghii (bergenin being the main constituent) against S. aureus in a neonatal Wister rat model. S. aureus is a bacterium of the Gram-positive cocci group that is part of the human microbiota, but that can cause diseases ranging from a simple infection, such as pimples and boils, to more serious ones, such as pneumonia and meningitis. Ten newborn rats were divided into four groups in which they received the treatments together with the S. aureus bacteria orally for four days in a row: Group I received saline, Group II received normal saline plus 2 mL of S. aureus, Group III received 50 mg of the extract plus 2 mL of S. aureus and Group IV received 100 mg of the extract plus 2 mL of S. aureus. The survival and weight of the rats were monitored for 9 days. The results showed that the survival rate of Group I was 80%. Infection with S. aureus led to mortality and reduced the survival % to 34.28% in Group II. However, in Groups III and IV, the percentage of survival was 48.57 and 60%, respectively, suggesting that extracts of B. purpurascens could reduce mortality caused by infection with S. aureus in newborn rats [82]. Kumar et al. [128] evaluated bergenin as an adjuvant immunotherapeutic agent for tuberculosis in mice. Mice were infected with 15 mL of M. tb H37Rv suspension using a Madison aerosol chamber, which targets the lungs and spleen. Days later, they were treated intraperitoneally with 4 mg of bergenin for 45 days. Then, the mice were euthanized, and the spleen and lung organs were removed. The results indicated that adjuvant therapy with bergenin protects mice against tuberculosis (IC 50 4 mg). It is suggested that this treatment induces the adaptive immune response by inducing memory T cells (CD8+ T, CD62L1o, and CD44hi), which can provide lasting protection against pathogens. This knowledge could be used to potentiate the BCG vaccine for TB [128]. Dias et al. [129] reported that the ethyl acetate leaf extract (EALE) of H. balsamifera (bergenin being the main constituent) had an effect on the inhibition of S. aureus in mealworms. The larvae were divided into three groups: Group I larvae infected with a lethal dose of S. aureus without treatment, Group II uninfected larvae plus treatment with 3 mg of EALE, and Group III larvae infected with a lethal dose of S. aureus plus treatment with 3 mg of EALE. The findings indicated that Group I had a mean lifespan of 1 day, Group II had no decrease in its lifespan and Group III had a prolonged lifespan (5 to 6 days), and, at the end of the evaluation, 50% of the larvae were still alive, indicating that EALE) inhibits the growth of S. aureus [130].

Antifungal Activity
The antifungal activity of bergenin has been reported in five in vitro studies. Bergenin isolated from the ethyl acetate fraction of E. uchi bark was tested against Candida albicans, Candida tropicalis, Candida guilliermondii, Aspergillus flavus, Aspergillus nidulans, and Aspergillus niger using the well agar diffusion method [67]. The fungi were coated in triplicate along with bergenin in different concentrations, and resazurin was added to each well as a dye. The results indicated that the presence of bergenin inhibits the growth of the yeasts C. albicans (IC 50 [125] reported that bergenin isolated from the crude methanolic extract of P. pterocarpum flowers had an inhibitory effect against Trichophyton mentagrophytes (IC 50 of 250 µg/mL), Epidermophyton floccosum (IC 50 of 500 µg/mL), Trichophyton rubrum (IC 50 of 500 µg/mL), Aspergillus niger (IC 50 of 500 µg/mL), and Botrytis cinerea (IC 50 of 250 µg/mL). Rolta et al. [119] reported that bergenin isolated from the methanolic extract of the rhizome of B. ligulata had an inhibitory effect against two Candida sp. resistant strains: C. albicans (MTCC277) and C. albicans (ATCC90028), both with IC 50 of 250 µg/mL. Pavithra et al. [14] reported that methanol extracts of flowers from four angiosperm plant species, W. thyrsoidea, O. dioica, L. speciosa, and B. malabaricum hadn't inhibitory effect against C. albicans. These results suggest that further studies should be carried out in order to determine a good inhibitory concentration, and this observation was also made by Nyemb et al. [123].

Anti-inflammatory Activity
Several studies have evaluated the anti-inflammatory activity of bergenin, 16 in vitro and 12 in vivo. In all studies, bergenin showed that it has optimal cell viability ( Table 3). Nunumura et al. [131] reported the anti-inflammatory activity of bergenin (0.01-1000 µM isolated from the trunk bark of E. uchi) against three enzymes in vitro, namely, cyclooxygena se-1 (COX-1), cyclooxygenase-2 (COX-2) and phospholipase A2 (PLA2). The effects of bergenin against COX-1 and COX-2 were determined by measuring the levels of prostaglandin E2 (PGE2), and the effects against PLA2 were determined by measuring its concentration by means of HPGP (1 hexadecanoyl-2-10-pyrenyldecanoyl-sn-glycero-3phosphoglycerol). The results showed that bergenin selectively inhibited COX-2 (IC 50 of 1.2 µM), though it was poorly active against PLA2 (IC 50 of 156.6 µM) and was not able to inhibit COX-1 (IC 50 of 107.2 µM). These results corroborate the findings of Li et al. [129] (IC 50 of 100 µM) and De Oliveira et al. [132] (IC 50 of 1.2 µM). However, Jachak et al. [66] had a satisfactory result in COX-1 inhibition (IC 50 of 70.54 µM) and demonstrated that bergenin inhibits both COX-1 and COX-2. COX-1 and COX-2 enzymes convert arachidonic acid to prostaglandin E2; thus, its inhibition can cause relief in inflammatory symptoms, as occurs with ibuprofen and celecoxib [133]. Bergenin showed an anti-inflammatory effect in human immortalized keratinocytes (HaCaT) [134] induced by interferon (TNF-α). These were treated with bergenin at concentrations of 0.01 to 200 µM, and the results show that bergenin (IC 50 of 50 µM) triggers activation of the Nrf2 (erythroid-derived nuclear factor 2) pathway and inactivation of the NF-κB (nuclear factor k-b) pathway, inducing upregulation of IL-6 and IL-8 in HaCa. In other words, it reduces the levels of IL-6 and IL-8 and demonstrates an anti-inflammatory effect since blocking the expression levels of these two cytotoxins reduces inflammation. These results are similar to the findings of Chen et al. [135] and suggest that bergenin (IC 50 of 100 µM) inhibits the pro-inflammatory response induced by TNF-α by blocking the NF-κB signaling pathway.
Shah et al. [126] isolated bergenin from the crude extract of M. philippenensis and then subjected it to chemical derivatization, obtaining 16 different synthetic derivatives. They subsequently tested its anti-inflammatory activity in vitro. Bergenin and its derivatives were incubated with macrophages treated with LPs (lipopolysaccharides) to determine whether bergenin could activate the iNO pathway for nitric oxide (NO) production and inhibit TNF-α production to normalize the cells. The results showed that only compounds 1g (IC 50 of 322.1 µM) and 1h (IC 50 of 253.2 µM) showed significant activity for NO production ( Figure 2). Jungo et al. [136] also obtained good results, which suggests that the suppression of inflammatory cytokines may be associated with excessive production of NO (IC 50 of 30 µM).
In vivo studies have determined the anti-inflammatory activity of bergenin in edema of the paw, ear, intestine, lungs, and mammary glands of experimentally induced rats and mice. Male BALB/c mice were experimentally induced into acute pulmonary injury/edema by intranasal inhalation of LPs. The mice showed histological changes, with increases in the activity of myeloperoxidases (MPO) in lung tissues and inflammatory cells (BALF) and decreases in cytokines (in BALF and serum) and, after 12 h, were treated with bergenin at concentrations of 50, 100 and 200 mg/kg. The results indicated that bergenin (IC 50 of 50 mg/kg) repaired the injured tissue and normalized the edema at all concentrations tested: decreased MPO activity (decumulated neutrophils in lung tissues), decreased inflammatory cells (neutrophils and macrophages in BALF), increased production of inflammatory cytokines (IL-1β and IL-6 in BALF, IL-1β, TNF-α and IL-6 in LPA serum), markedly inhibited the phosphorylation of NF-kB p65. However, it inhibited the expression of MyD88 (myeloid differentiation factor 88) though not the expression of NF-κB p65 in lung tissues, which indicates that bergenin partially suppresses its production. In addition, bergenin also inhibited nuclear translocation and phosphorylation of NF-κB p65 stimulated by LPs in Raw264 cells, indicating that bergenin has anti-inflammatory effects in LPS-induced pulmonary edema [109].
Wistar rats were induced to acute ulcerative colitis by TNBS (2,4,6-trinitrobenzenesulfonic acid), causing an intestinal inflammation with tissue damage, and were subsequently treated with bergenin at concentrations of 12 to 100 mg/kg/day [137]. The results indicated that bergenin (IC 50 of 25mg/kg) decreased the signs of macroscopic and microscopic colitis damage and reduced the degree of neutrophilic infiltration in the colon tissue. In addition, it was able to negatively regulate the expression of COX-2, iNOS, IkB-α, and pSTAT3 proteins (phosphorylated transducer and activator of immunohistochemical expression of expression-3) as well as activating inflammasome signaling pathways [137]. This pathway consists of an intracellular multiprotein complex that acts in the activation of enzymes of the cysteine-aspartate proteases (CASPASES) family as an essential structure for the regulation of immunity under physiological conditions and in recognition of danger signals with subsequent recruitment of cytokines that will normalize inflammation [138] The findings did not differ from those of Wang et al. [139] who tested bergenin isolated from the herb S. stolonifera. Gao et al. [10] reported that bergenin plays an anti-inflammatory role through the modulation of MARPK and NF-kB signaling pathways in an LP-induced mammary gland mastitis mouse model. Mice with mastitis induced in the mammary gland showed an excessive concentration of the pro-inflammatory cytokines NO, TNF-α, IL-1β, and IL-6, but only after treatment with bergenin. The results suggest that bergenin reduced the expression of pro-inflammatory cytokines, NO, TNF-α, IL-1β, and IL-6 by inhibiting the activation of the signaling pathways NF-kB and MAPKs, resulting in tissue normalization. These two pathways are responsible for the expression of inflammatory processes [140]. Inhibition of these two pathways was also observed in BALB/c rats (IC 50 of 10 µM) with inflammation caused by Klebsiella pneumonia [141]. Souza et al. [142] reported that the phenolic extract of the stem bark of E. uchi (the main constituent being bergenin) had an anti-inflammatory effect in the edema of mice paws induced by intraplantar injection of carrageenan. Carrageenan is an inflammatory agent and produces inflammation by releasing prostaglandins, causing the formation of edema [143]. The results suggested that the phenolic extracts normalized edema by inhibiting COX-2, the main COX isoform induced during inflammation, to regulate the production of prostaglandins at the site of inflammation [128]. These results are similar to the findings of Borges [142], who tested acetylbergenin isolated from the stem bark of E. uchi in a paw edema model in rats induced experimentally by intraplantar injection of 100 µL of 1% carrageenan in the right paw of rats of the MacCoy lineage. Bergenin had an anti-inflammatory effect in a model of inflammation induced by Freund's complete adjuvant (FCA) [73,144]. Mice were experimentally induced into edema by injection of FCA in the plantar region of the right paw. After this procedure, the recruitment of pro-inflammatory cytokines in the site was observed. The treatment with bergenin determined the suppression (IC 50 of 12.5 mg/kg) of IL-1β, TNF-α, and IL-10 levels and normalized the tissues, suggesting that the anti-inflammatory effect of bergenin may be closely linked to the inhibition of these inflammatory cytokines [145]. It is known that they precede the release of the final mediators of hyperalgesia, i.e., prostaglandins and sympathetic amines [146]. These results are similar to the findings of Bharate et al. [22], who tested bergenin-rich extracts of B. ciliata.
Male Sprague-Dawley rats were experimentally exposed to tobacco smoke and developed chronic bronchitis. Later, they were treated with bergenin (87 mg/kg) and dexamethasone (0.2 mg/kg). The results indicated that both compounds suppressed inflammatory cell infiltration and inhibited mucus secretion, in addition to reducing white blood cells in BALF [147]. The authors suggest that the anti-inflammatory mechanism of action of bergenin may be associated with the alteration of branched-chain amino acid (BCAA) metabolism, glycine, serine, and threonine metabolism, and glycolysis to treat chronic bronchitis. It has been proven that these metabolic changes have an influence on the inflammatory response [148].
Bergenin exhibited antioxidant activity in HepG2 cells that were induced to oxidative damage by sodium selenite in vitro [161]. The cells treated with sodium selenium caused an oxidative-antioxidant imbalance that damaged them as it decreased antioxidant enzymes, which allowed the proliferation of free radicals that, in turn, triggered pro-inflammatory cascades and induced the damage. HepG2 was co-incubated with sodium selenite (10 µM) and bergenin (75,150, and 300 µM) for 24 h. The results indicated that bergenin (IC 50 of 75 µM) exerted protective effects against oxidative stress induced by sodium selenite in HepG2 cells since it sequesters free radicals and normalizes the oxidative stress balance. Similar protection was observed in PC12 cells, a cell line derived from a pheochromocytoma of the adrenal medulla of rats, with norbergenin derivatives isolated from D. crassiflora stems (IC 50 of 0.1 and 10 µM) [162].
BALB/c mice immunosuppressed by cyclophosphamide (Cy) experimentally induced a decrease in the action of enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)], which are important in response to oxidative stress and accelerate the formation of free radicals and, consequently, the spleen and thymus suffered lesions [163]. Manente et al. [164] reported that oxidative cell damage is a major side effect of chemotherapy drugs, including cyclophosphamide. Cyclophosphamide disrupts the redox balance and causes tissue damage; however, after treatment with bergenin, the results suggested that bergenin (IC 50 of 20 mg/kg) reversed the Cy-induced decrease in total antioxidant capacity, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities. This improved humoral and cellular immune functions and increased antioxidant activity. This effect is justified by the fact that bergenin acts as a free radical scavenger or a redox-regulating agent, effectively avoiding Cy-induced oxidative stress injury by increasing antioxidant enzymes and oxidative-reducing enzymes [163]. These enzymes, when increased at the site of injury, convert harmful oxygen into lessreactive hydrogen peroxide, catalyzing the dismutation of superoxide into oxygen and hydrogen peroxide [164]. Lee et al. [165] reported that bergenin had an antioxidant effect in mice treated with morphine. Morphine treatment induced oxidative stress by decreasing antioxidant enzymes and causing the proliferation of free radicals, and this allowed inflammation of the brain since oxidative stress and inflammation are interdependent. Free radicals activate pro-inflammatory genes that trigger a cascade of progressive inflammation; however, treatment with bergenin (IC 50 of 20 mg/kg) had an antioxidant effect and played a role in antinarcotic effects through adaptation to morphine-induced oxidative stress in the brain. Sriset et al. [162 reported that bergenin attenuates sodium selenite-induced hepatotoxicity by improving hepatic oxidant-antioxidant balance in ICR mice. Mice were orally administered sodium selenite (4 m/kg), which caused liver damage through oxidativeantioxidant imbalance. This is because there is an increase in plasma levels of enzymes aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, and there is an increased proliferation of reactive oxygen species by decreasing antioxidant enzymes, resulting in lipid peroxidation in plasma. After treatment with bergenin, the results indicated that bergenin (IC 50 of 10 mg) restored normal tissue damaged by the antagonistic effect exhibited by sodium selenite. The effect of bergenin on membrane lipid peroxidation and ascorbic acid level in tissues was studied using pathogen-free weaned rats as the experimental animal and 2,4-dinitrophenyl hydrazine (2,4-DNPH) as the experimental oxidant [166,167]. Lipid peroxidation was experimentally induced with 2,4-DNPH via the intraperitoneal route, which allowed a greater proliferation of reactive oxygen species and triggered processes that led to tissue damage by oxidative-antioxidant imbalance. In this context, three primary antioxidant enzymes were analyzed, namely catalase, superoxide dismutase (SOD), and glutathione peroxidase, and two non-enzymatic antioxidants, namely vitamin E (α-tocopherol) and vitamin C (ascorbic acid). After treatment with bergenin isolated from S. gabonensis stem bark extract (main constituent being bergenin), the results indicated that the bark extract (IC 50 of 2.8 mg/100 g) exhibited divergent effects on antioxidant enzymes: impaired the enzyme-inducing action of 2,4-DNPH on liver and red blood cell catalase, reduced the depressant effect of SOD, and neither 2,4-DNPH nor the extract had any measurable effect on glutathione peroxidase. The bark extract also exerted a sparing effect on tissue antioxidant vitamins, ascorbic acid, and vitamin E, effectively inhibiting their depletion by 2,4-DNPH in the liver, red blood cells, and brain. These results suggest that the antioxidant mechanism of action of the bark extract against membrane peroxidation is multifactorial/multisystem, involving catalase inhibition, enhancing the SOD capacity of the liver and red blood cells, and sparing tissue depletion/use of vitamins C (ascorbic acid) and E (α-tocopherol) [166].

Antinociceptive Activity
The antinociceptive activity is linked to the analgesic activity of bergenin, blocks the sensory neurons, and gives the sensation of pain relief in vivo. Obviously, all the studies presented in the previous sections, especially the anti-inflammatory and antioxidant activities, show the ability of bergenin to restore the normality of inflamed tissue or an organ or to restore the antioxidant oxidant balance and cause the mice or rats to feel pain relief. Ethanolic extract of D. dentatus leaves, which are rich in bergenin, showed antinociceptive activity in male Swiss mice. The mice were induced by formalin intraperitoneally, causing joint inflammation, knee edema, leukocyte infiltration, hyperalgesia, and sensation of excessive cold; however, oral treatment with the extract (100 and 300 mg/kg) reduced leukocyte infiltration and normalized the injured tissues, which consequently significantly inhibited nociceptive sensitivity and sensitivity to cold (IC 50 of 100 mg/kg). The results suggest that the ethanolic extract could interfere with the peripheral and central pain mechanisms of nociception induced by formalin [171]. Bharate et al. [22] reached the same conclusion using the ethanolic extract of the rhizome of bergenin-rich B. ciliate (IC 50 of 2 g/kg) in rats induced to nociception by acetic acid and the formalin-induced paw-licking method and suggested that upregulation of inflammation is inversely proportional to antinociceptive activity, i.e., the bergenin contained in the extract enables inhibition of both nociceptive response phases as anti-inflammatory processes are activated. Souza et al. [143] reported that the extract of the bark of E. uchi presented good antinociceptive activity (IC 50 of 100 mg/kg) in the test of abdominal contortions induced by acetic acid. They suggested that the mechanism of action is associated with the inhibition of the formation of pro-inflammatory mediators. Paulino et al. [62] claim that bergenin has the potential to exert antinociceptive activity and may affect the opioid system. Opioids act by promoting the opening of potassium channels and inhibiting the opening of voltage-gated calcium channels, thus causing hyperpolarization and reduced neuronal excitability. In addition, there is a reduction in the release of transmitters (by inhibiting the entry of Ca 2+ ), blocking the sensation of pain.
Bergenin isolated from the rhizome of B. ciliata showed antinociceptive activity (IC 50 of 10 mg/kg) by normalizing the antioxidant oxidative balance and de-accumulation of IL-2 in an induced hyperoxaluric rat model [172]. Semisynthetic acetylbergenin, the product of acetylation of bergenin isolated from the cortex of E. uchi, also showed good antinociceptive activity in Swiss albino rats with abdominal contraction [173]. The abdominal contraction was induced in rats by intraperitoneal injection with 0.6% acetic acid triggering contractions, thereby raising levels of prostaglandin E2, which triggers local inflammatory processes by raising COX-2, in addition to interfering with nociceptive mechanisms causing extreme pain in rats; however, treatment with bergenin (IC 50 of 6.8 mg/kg) normalized inflammation and inhibited nociception induced by acetic acid.

AntiArthritic Activity
Bergenin has been studied in order to verify its antiarthritic potential. In rat or mouse models that experimentally induced inflammation in the joints, seven studies were able to discriminate antiarthritic activity in vivo. Branquinho et al. [171] reported the activity of the ethanolic extract of leaves of D. dentatus, which are rich in bergenin, in male Swiss mice against rheumatoid arthritis. In the study, mice were experimentally induced to joint inflammation (including knee edema, leukocyte infiltration, and hyperalgesia) by zymosan through a single intraperitoneal dose of 200 µL. After treatment with the extract (0.3 to 30 mg/kg), the results suggested that the extract showed therapeutic action in rheumatoid arthritis and decreased the amount of inflammation through the de-accumulation of proinflammatory cytokines and by modulating the immune response in the cells. Bharat et al. [122] came to the same conclusion using the ethanolic extract of the rhizome of bergenin-rich B. ciliata (IC 50 of 2 g/kg) in rats induced to arthritis using Mycobacterium.
Bergenin isolated from the rhizome of B. ciliata was shown to be a potent antiarthritic agent in an induced hyperoxaluric rat model [172]. Hyperoxaluria was induced using ethylene glycol, and this induction caused oxidative stress, especially in the joints, which caused a proliferation of pro-inflammatory cytokines, causing edema. However, treatment with bergenin (IC 50 of 10 mg/kg) inhibited the proliferation of IL-β and sequestered reactive oxygen species, thus normalizing the joints. In the study by Jain et al. [174], bergenin and its derivatives exerted antiarthritic activity, possibly by inhibiting pro-inflammatory cytokines and producing TNF-α. Nazir et al. [175] reported that the effect of bergenin and norbergenin against adjuvant-induced arthritis, both at doses above 2 mg/kg, is achieved by possible modulation of the Th1/Th2 cytokine balance.
Methanolic extract of C. capitella and its metabolite 11-O-(4 -O-methyl galloyl)-bergenin, isolated from aerial parts, showed an antiarthritic effect on rat hind paws. Arthritis was induced by intraplantar injection of 0.1 mL of complete Freund's adjuvant (CFA) into the subplantar tissue of the hind paw. CFA produced definitive edema within 24 h, with progressive arthritis on day 9 after inoculation according to the elevated levels of TNF-α, IL-1β, IL-6, and myeloperoxidase activity. There was also an increase in rheumatoid factor (RF) and anticyclic citrullinated peptide antibody (antiCCP), which are considered predictors of severe arthritis. Subsequently, they were treated with the extract (250 mg/kg) and 11-O-(4 -O-methyl galloyl)-bergenin (5, 10, and 20 mg/kg). The findings indicated that both treatments inhibited the production of pro-inflammatory cytokines, and serum levels of RF and antiCCP were normalized, with an IC 50 of 250 mg/kg for the extract and 20 mg/kg for 11-O-(4 -O-methyl galloyl)-bergenin [176].

Antiulcerogenic Activity
The antiulcerogenic activity of bergenin has been reported in several experimental ulcers. In this context, about six studies were carried out, one in vitro and five in vivo. Bergenin and norbergenin (isolated from the aqueous extract of M. japonicus) showed an inhibitory effect on the bovine adrenal tyrosine hydroxylase (TH) enzyme in vitro [177]. The increased activity of this enzyme is suggested to be related to the occurrence of gastric ulcers in patients stressed by the cold. The findings showed that bergenin and norbergenin inhibited TH activity by 12.2% and 51.0% at the concentration of 5 µg/mL, 16.1% and 51.6% at the concentration of 10 µg/mL, and 29.0% and 53.4% at the concentration of 20 µg/mL, respectively [177].
Male Donryu rats received treatment with bergenin (30 to 1000 mg/kg) orally and were subsequently placed in a cage and immersed up to the level of the xiphoid process in a water bath (23 • C) for 7 h, then euthanized. The stomach was removed and submerged in a formalin solution inducing inflammation, allowing the marking of the inner and outer layers of the gastric wall. Finally, the stomach was incised along the curvature, and the lesions were examined [178]. The results indicated that bergenin (IC 50 30 mg/kg) inhibited the development of stress-induced gastric ulcers in rats. One of the mechanisms attributed to its effectiveness may be the inhibition of acetylcholine release, which induces acid secretion and improves gastric motility [178]. Oral administration of bergenin and norbergenin isolated from the leaves and roots of F. microcarpa and luvangetin showed significant protection against pyloric gastric ulcers in rats induced by aspirin and colds, suggesting that the gastroprotective effects of bergenin and norbergenin may be due to increased prostaglandin production [41].
Semisynthetic acetylbergenin (1i) isolated from E. uchi was effective in preventing gastric ulcers [152]. Acute gastric ulcer was induced by stress, according to Basile et al. [41]. Wistar rats were fasted with free access to water for 24 h, then treated with distilled water (0.5 mL), acetylbergenin (6.8 mg/kg), and indomethacin (10 mg/kg). Each animal was kept for 17 h in a container tube, which was immersed vertically until the water reached the neck region of the animal in a tank with running water at 25 • C. In addition, the rats were euthanized by CO 2 inhalation. Their stomachs were immediately excised and opened by cutting along the largest curvature for examination of the inner wall, and the lesions were counted. The results indicated that treatment with indomethacin (10 mg/kg) produced more lesions when compared with acetylbergenin at a dose of 6.8 mg/kg, revealing that acetylbergenin has a protective effect [152].

Antidiabetic/Antiobesity Activity
Antidiabetic/antiobesity activity was investigated in eight in vivo studies. The alcoholic extract of C. digyna root rich in bergenin showed an antidiabetic effect in diabetic rats induced by streptozotocin-nicotinamide. The rats received a single dose of streptozotocinnicotinamide (65 mg/kg) intraperitoneally, which induced fatal hypoglycemia due to the massive release of pancreatic insulin. After 6 h, the rats received 10% glucose solution and continued receiving more glucose by injection until reaching a glucose level of 200 mg/dL in fasting. Treatment with the extract (250 to 750 mg/kg) for 14 days reduced dose-dependent blood glucose levels from 200 mg/dL to 146.33 mg/dL (IC 50 of 250 mg/kg), and it was also noted that it was able to reduce the body weight of the rats [179]. Hyperglycemia is recognized as a common complication of diabetes mellitus. Reduced insulin secretion causes a variety of disruptions in metabolic and regulatory mechanisms that lead to lipid accumulation. Bergenin significantly reduces triacylglycerols and total cholesterol in diabetic rats. The lipid-lowering effect of bergenin and other antidiabetic drugs reduces the risk of vascular complications [179]. San et al. [91] reported that the methanolic extract of the root of C. javana affected glucose uptake (100 µg/mL) in L6 skeletal muscle cells according to the reported methods [91,180].
Rats fed a hyperglycemic diet increased blood glucose to 220 mg/dL and developed type 2 diabetes, then received intragastric treatment with bergenin (10, 20, 40 mg/kg) or metformin (25 mg/kg) as the positive control from 8 to 16 weeks [181]. Treatment with bergenin (10, 20, 40 mg/kg) significantly reduced the concentration of glucose in the blood from 220 to 86.19 mg/dL against 80.45 mg/dL of metformin. Bergenin significantly improved the insulin sensitivity index, reduced liver damage and oxidative changes, and brought antioxidants and lipids back to normal, suggesting that bergenin can be used as a functional drug or as an adjunct in the management of insulin resistance and associated fatty liver disease [181]. Other studies have also determined the IC 50 for the inhibition of the development of type 2 diabetes with antiobesity consequences; namely, bergenin isolated from the methanolic extract of F. racemosa (IC 50 of 200 mg/kg) [182] and bergenin isolated from the rhizomes of B. crassifolia (IC 50 of 100 mg/kg) [183].
Methoxybergenin, a natural derivative isolated from the stem bark of V. pauciflora, showed an antidiabetic effect in diabetic Wistar rats induced via alloxan [184]. Twenty-five Wistar rats with a body weight of 100 g, presenting a normal glycemia (50-125 mg/dl), were induced via alloxan at a dose of 150 mg/kg intraperitoneally, which subsequently presented an elevation of the blood glucose level (440 mg/dL), thus damaging the production of insulin by the cells of the pancreas. Treatment with methoxybergenin after 21 days of treatment exhibited good antidiabetic activity (IC 50 of 191 mg/200 g), and this inhibition allowed the reduction of the body weight of the rats, which suggests antiobesity activity [184].
Kumar et al. [185] reported that bergenin isolated from the methanolic extract of M. philippinensis showed moderate antiglycation activity (IC 50 of 186.73 µg/mL), which suggests participation in glycemic regulation. This effect allows sugar molecules not to be fixed in large quantities in proteins, especially hemoglobin, in addition to avoiding increased oxidative stress. The same was observed with respect to 11-O-galloylbergenin isolated from the ethyl acetate fraction of P. peltatum (IC 50 of 0.1 µg/mL) [186].

AntiArrhythmic Activity
Studies on the antiarrhythmic activity of bergenin are scarce; however, only one study undertook this assessment [7]. Bergenin isolated from the aerial parts of F. virosa was investigated for its antiarrhythmic effects at concentrations of 0.2 mg/kg, 0.4 mg/kg, and 0.8 mg/kg, and showed distinct therapeutic effects on arrhythmias induced by barium chloride (BaCl2) in rats. At concentrations of 0.4 mg/kg and 0.8 mg/kg, bergenin significantly countered arrhythmias induced by coronary artery ligation and reperfusion. At a concentration of 0.8 mg/kg, bergenin raised the atrial fibrillation threshold in rabbits from 1.34 mV to 1.92 mV, suggesting that bergenin has the potential to treat cardiac arrhyth-mias [7]. According to Filho et al. [187], the elevation of the atrial fibrillation threshold allows the electric current to travel with sufficient intensity to contract the muscles of the ventricles and cause involuntary systole.

Anticancer Activity
The anticancer activity of bergenin has been reported in 13 in vitro studies and 2 in vivo studies. Bergenin-rich E. agallocha leaf extract showed anticancer activity in a cervical cancer cell line (SiHa HPV 16+) [45]. The extract was matched with a cancer cell line and peripheral healthy blood mononuclear cells (PBMC) using an MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to assess its cytotoxicity. The findings indicated that bergenin-rich extracts exhibited remarkable activity against the SiHa cell line (IC 50 of 15.538 µg/mL), whereas for healthy cells (PBMC), the extracts induced cell proliferation, suggesting that they do not produce cytotoxicity in normal healthy cells. The mechanism of action of this effect may be associated with blocking the action of the GLI-related protein (glioma-associated oncogene homolog). GLI is a transcriptional effector involved in developing tumors; thus, blocking its action leads to the inhibition of its translocation to the nucleus [188]. Konoshima et al. [160] describe GLI as an effective inhibitor of the Hedgehog, a signaling pathway in cancer therapy. Bergenin has shown effects on human colorectal adenocarcinoma cell line HCT116 [189]. Bergenin markedly inhibited (IC 50 of 30 µM) the growth of HCT116 cells with cellular apoptosis of up to 5.21%. When the influence of bergenin on the cell cycle was evaluated, it was found that it increased the number of cells in the Gap 1 (G1) phase with a decrease in the percentage of cells in the synthesis phase (S) in 24 h, showing that bergenin can lead to DNA damage in HCT116 cells by increasing the phosphorylation of the histone variant H2AX in Ser139 and generation of reactive oxygen species (ROS). The canonical PI3K/AKT/mTOR signaling pathway emerges as a critical regulator of the cell proliferation pathway [190]. This pathway is very important and is involved in the regulation of cell proliferation, cell cycle progression, apoptosis, and morphogenesis in different organs [191,192]. Bergenin, isolated from the bark of F. religiosa, F. virens, F. glomerata, F. benghalensis, and T. pulpunea, showed effects on cervical cancer lines of Hela and SiHa cancer cells (IC 50 of 125.8 and 96.0 µg/mL respectively) using an MTT assay [42]. Newell et al. [77] reported anticancer activity of bergenin isolated from the seed of five Ardisia species, namely A. japonica, A. escallonioides, A. mamillata, A. crenata, and A. compressa, with the inhibition of human topoisomerase II enzyme and cytotoxicity in human liver cancer cells (HepG2) in vitro. The findings indicated that bergenin showed catalytic inhibition of topoisomerase II (IC 50 of 18 µM) and cytotoxicity (IC 50 of 18 µM) against HepG2 cells, and it was found that bergenin showed a tendency to accumulate cells in the G1 phase and reduction of G2/M leading to apoptosis of malignant cells.
Bergenin (isolated from the herb B. purpurascens) inhibited the activity of human hepatic cytochrome P450 enzymes (CYP), CYP3A4, 2E1, and 2C9, with IC 50 values of 14.39, 22.83, and 15.11 µM, respectively [193]. Enzyme kinetics studies showed that bergenin was not only a non-competitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP2E1 and CYP2C9. These enzymes at high levels may favor extrinsic factors (e.g., alcohol and tobacco consumption), triggering the development of oral cancer [193].
Bulugahapitiya et al. [40] reported anticancer activity of ethyl acetate extract (EtOAc) from F. leucopyrus leaves, bergenin and bergenin diastereomer (at C-9 and C-14) both isolated from EtOAc, against human ovarian carcinoma. Anticancer activity was evaluated using cell proliferation assays (MTS) and human telomerase reverse transcriptase (hTERT) in human ovarian carcinoma (A 2780). The MTS assay showed significant antiproliferation activity with an IC 50 of 36.35, 12.36, and 48.53 µg/mL for EtOAc, bergenin, and bergenin diastereoisomer, respectively. A rapid depletion of the hTERT content in human ovarian cancer cells was observed for the bergenin diastereomer in the concentration range of 50-200 µg/mL. The findings suggested the anticancer activity of F. leucopyrus leaves against human ovarian cancer, and bergenin and its isomer were identified as the compounds responsible for the anticancer activity of this plant. It can effectively inhibit the proliferation of cancer cells and inhibit the human telomerase reverse transcriptase enzyme, which is directly responsible for the activation of cancer cell telomerase [194]. These results resemble the findings of Mettihewa et al. [195]. Bergenin, isolated from S. stolonifera, was shown to induce apoptosis (IC 50 of 6.1 µM) in BGC-823 carcinoma cells in an MTT assay [196].
Esterified bergenin 5c (11-hydroxyl-modified bergenin), a semisynthetic derivative of bergenin, was evaluated for its antitumor properties in vitro and in vivo [197]. The findings showed that it trapped HepG2 cells (IC 50 of 4.23 ± 0.79 mM) in the G2/M phase and induced cell apoptosis. In addition, compound 5c suppresses (IC 50 30 mg/kg) tumor growth in Heps xenograft-bearing mice with low toxicity. Liu et al. [198] reported that the methanolic extract, which is rich in bergenin and norbergenin, from S. stolonifera showed an antitumor effect, and its lung tumor inhibition rate can reach 49.2% (IC 50 of 5.150 mg/g). The extract showed some capacity in the recovery of the immune system and hematological system of Lewis mice, in addition to causing necrosis in tumor cells and decreased macrophage density to inhibit the growth of lung tumors [198]. De-Biao et al. [199] reported anticancer activity of 3, 4, 11-trihydroxyl modified bergenin derivatives in an MTT assay in the inhibition of DU-145 and BGC-823 cells, with significant inhibitions of compounds 3a to 7a (3, 4, 11-trihydroxyl modified derivatives of bergenin). IC 50 ranged from 20.89-100 µM for DU-145 and 23.99-100 µM.
11-O-Galloylbergenin, a natural bergenin derivative isolated from leaves of C. coreana, was shown to be a potent antitumor agent in human osteosarcoma cells (MG63 cells) in an MTT assay. The findings showed that it inhibited the proliferation of MG63 cells and induced cellular apoptosis. This phenomenon was accompanied by the upregulation of the p53 and p21 genes [200]. The p53 gene encodes the tumor suppressor protein p53, which plays a significant role in tumor development and regulates the cell cycle and apoptosis, especially in the early events of osteosarcoma tumorigenesis [200].

Hepatoprotective Activity
In three in vitro studies and seven in vivo studies, bergenin showed hepatoprotective activity. Bergenin isolated from the cortex of M. japonicus exhibited good hepatoprotective activity by removing hepatotoxicity in a rat liver-cell assay induced by carbon tetrachloride (CCl 4 ) [201]. Hepatocytes were isolated from rats using the method of Berry and Friend [202] and were then cultured [198]. After one day of plating, they were exposed to CCl 4 (10 mM), which induced hepatotoxicity by metabolic activation. In other words, CCl 4 is metabolically activated by cytochrome P-450-dependent mixed oxidase in the endoplasmic reticulum to form a trichloromethyl free radical (CCl 3 ), which, combined with lipids and proteins in the presence of oxygen to induce lipid peroxidation [203], results in changes in the structures of the endoplasmic reticulum system and other membranes, loss of metabolic enzyme activation, reduced protein synthesis, and loss of glucose-6-phosphatase activation, thus leading to liver damage [204]. However, treatment with bergenin (IC 50 of 300 µM) lowered the activity of enzymes whose high levels indicate liver damage, namely pyruvic transaminase (GPT) sorbitol dehydrogenase (SDH) [201]; both enzymes are also associated with lipid peroxidation [205]. In vivo studies positively confirmed the hepatoprotective activity in rats with experimentally induced hepatotoxicity by CCl 4 , and the IC 50 was determined. Bergenin isolated from the cortex of M. japonicus exhibited a hepatoprotective effect in rats (IC 50 of 50 mg/kg) and normalized the decreased activities of glutathione S-transferase and glutathione reductase, in addition to significantly preventing the elevation of hepatic malondialdehyde formation and depletion of reduced glutathione content in the liver [206]. Bergenin isolated from the herb S. stolonifera exhibited (IC 50 of 100 mg/kg) a hepatoprotective effect and detoxified liver cells [206]; bergenin isolated from the extract of P. pterocarpum showed a hepatoprotective effect in albino rats (IC 50 of 100 mg/kg) reducing excessive levels of the enzymes alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyltransferase (γ-GT), direct bilirubin (DB) and total bilirubin (TB) [207,208]; bergenin and 11-O-galloylbergenin, isolated from the leaves of A. edulis, have remarkable antihepatotoxic activity against CCl 4 and galactosamine cytotoxicity in cultured primary rat hepatocytes (IC 50 of 100 mg/kg) [122]. Mondal et al. [209] reported that the hepatoprotective capacity of bergenin could also be related to the fact that it can sequester free radicals and restore the antioxidant oxidant balance.
The hepatoprotective effects of acetylbergenin have been examined against D-galactos amine (GalN). Ga1N induced liver damage in rats, compared to previously reported bergenin [137]. Acetylbergenin was synthesized from the acetylation of bergenin, isolated from M. japonicus, to increase lipophilic and physiological activities. Acetylbergenin was administered orally once daily for 7 days, and then GalN (400 mg/kg) was injected at 24 h and 96 h after the final acetylbergenin administration. Acetylbergenin reduced elevated serum enzymatic activities of alanine/aspartate aminotransferase, sorbitol dehydrogenase, glutamyltransferase, and GalN-induced hepatic malondialdehyde formation. Acetylbergenin also significantly restored GalN-induced decreased glutathione levels and decreased glutathione S-transferase and glutathione reductase activities to normalization. Therefore, these results suggest that acetylbergenin has hepatoprotective effects against GalN-induced hepatotoxicity by inhibiting lipid peroxidation and maintaining an adequate level of GSH for xenobiotic detoxification as its underlying hepatoprotective mechanisms. In addition, lipophilic acetylbergenin showed more activity in hepatoprotection than the much less lipophilic bergenin that was previously reported [210].

Neuroprotective Activity
The neuroprotective activity of bergenin has been reported in two in vitro studies. Bergenin showed neuroprotective activity against Alzheimer's disease (AD) [211], a chronic progressive neurodegenerative disease, which often occurs in the elderly and negatively affected intellectual abilities and cognitive processes. Human neuroblastoma cell lines (SH-SY5Y) were treated in an MTT assay with N-methyl D-aspartate (NMDA) at the concentration of 2.5 mM per 24 h, which led to reduced cell viability (49.33%). In this context, this concentration was selected to induce cytotoxicity in SH-SY5Y, along with pretreatment with bergenin (5 to 50,000 nM) in an MTT assay. The findings revealed that pretreatment with bergenin led to NMDA concentration-dependent reversal in the concentration range of 5-500 nM. Bergenin at 500 nM led to the greatest increase in cell survival-up to 81.754% [211]. Takahashi et al. [46] reported the neuroprotective activity of norbergenin derivatives isolated from the methanolic extract of the bark of M. japonicus, and this suggests that it is closely related to their ability to sequester reactive oxygen species and thus restore antioxidant oxidative balance.
Suzuki et al. [212] reported the protective effect of norbergenin-11-caproate (a semisynthetic derivative of norbergenin isolated from M. japonicus) against cell damage in human neuroblastoma IMR-32 cells treated with tunicamycin. When IMR-32 cells were treated with tunicamycin, their viability in an MTT assay was decreased in a dose-dependent manner (0.01-1 µM). Treatment with norbergenin-11-caproate (10 mM) showed complete protection against the cell growth inhibitory effect of tunicamycin but did not inhibit the induction of Bip/GRP78 mRNA, suggesting the therapeutic potential of this derivative.

Cardioprotective Activity
Studies on the cardioprotective activity of bergenin are scarce, with only one study investigating this effect. Thirty rats were experimentally induced to myocardial infarction by isoproterenol (ISO), which sharply increased the ST and deep Q wave, in addition to causing leakage of cardiac marker enzymes, such as cTnI (cardiac troponin I), CPK (creatine phosphokinase), CK-MB (creatine kinase MB isoenzyme), LDH (lactate dehydrogenase), ALT (alanine aminotransferase) and AST (aspartate aminotransferase), from cardiac tissue to circulation, cell membrane rupture, hypoxia, and cardiac hypertrophy. Subsequently, they were treated twice within 24 h with bergenin at doses of 1 and 3 mg/kg via injection for 5 days to determine the cardioprotective response. The results indicated that bergenin (IC 50 of 1 mg/kg) sharply restricted ST segment elevation induced by isoproterenol, Q wave, and ECG pattern, indicating that it has protective effects on the cell membrane since it prevents the extension of myocardial damage (induced by ISO) by strengthening the myocardial cell membrane and tissue architecture, and also normalizes marker enzymes [211].

Conclusions and Future Perspective
Because it is a secondary metabolite with immeasurable pharmacological potential and because it is distributed in many plant species (at least 112 species belonging to 34 families), bergenin has aroused the interest of researchers in the medical and biotechnological fields. Both its derivatives (natural and semisynthetic) and its extracts are well studied, with phytochemical confirmation of its highest concentration, and in none of the studies has it been observed to be cytotoxic to healthy cells. At least 17 activities were well studied, of which we can highlight the following: antimalarial, antileishmanial, trypanocidal, antiviral, antibacterial, anti-inflammatory, antioxidant, antinociceptive, antiarthritic, antiulcerogenic, antidiabetic, anticancer and hepatoprotective activities. However, it is emphasized that more studies should be carried out in order to further explore its pharmacological potential, especially the unraveling of the different mechanisms of action against infectious-contagious pathogens that are part of tropical and neglected infectious diseases and which have wide worldwide distribution such as malaria and leishmaniasis. For activities whose mechanisms of action are already well known, such as anti-inflammatory and antioxidant activities, there may be interest in the production of synthetic derivatives, with conducting of preclinical and clinical studies, since in preclinical studies in vitro and in vivo (in mice) with natural bergenin as well as its semisynthetic derivatives, it has been shown to be safe.