Protective effects of arbutin against doxorubicin-induced cardiac damage.

BACKGROUND: Doxorubicin is an effective antineoplastic agent but has limited clinical application because of its cumulative toxicities, including cardiotoxicity. Cardiotoxicity causes lipid peroxidation, genetic impairment, oxidative stress, inhibition of autophagy, and disruption of calcium homeostasis. Doxorubicin-induced cardiotoxicity is frequently tried to be mitigated by phytochemicals, which are derived from plants and possess antioxidant, anti-inflammatory, and anti-apoptotic properties. Arbutin, a natural antioxidant found in the leaves of the bearberry plant, has numerous pharmacological benefits, including antioxidant, anti-bacterial, anti-hyperglycemic, anti-inflammatory, and anti-tumor activity. METHODS AND RESULTS: The study involved male Wistar rats divided into three groups: a control group, a group treated with doxorubicin (20 mg/kg) to induce cardiac toxicity, a group treated with arbutin (100 mg/kg) daily for two weeks before doxorubicin administration. After treatment, plasma and heart tissue samples were collected for analysis. The samples were evaluated for oxidative stress parameters, including superoxide dismutase, malondialdehyde, and catalase, as well as for cardiac biomarkers, including CK, CK-MB, and LDH. The heart tissues were also analyzed using molecular (TNF-α, IL-1ß and Caspase 3), histopathological and immunohistochemical methods (8-OHDG, 4 Hydroxynonenal, and dityrosine). The results showed that arbutin treatment was protective against doxorubicin-induced oxidative damage by increasing SOD and CAT activity and decreasing MDA level. Arbutin treatment was similarly able to reverse the inflammatory response caused by doxorubicin by reducing TNF-α and IL-1ß levels and also reverse the apoptosis by decreasing caspase-3 levels. It was able to prevent doxorubicin-induced cardiac damage by reducing cardiac biomarkers CK, CK-MB and LDH levels. In addition to all these results, histopathological analyzes also show that arbutin may be beneficial against the damage caused by doxorubicin on heart tissue. CONCLUSION: The study suggests that arbutin has the potential to be used to mitigate doxorubicin-induced cardiotoxicity in cancer patients.

Protective effect of arbutin against cyclophosphamide-induced oxidative stress, inflammation, and hepatotoxicity via Nrf2/HO-1 pathway in rats.

Cyclophosphamide (CP) is a potent anticancer drug widely employed in chemotherapy against various types of cancer. However, CP leads to toxicity to non-targeted organs, including the liver and this limits its clinical use. This study explored the role of arbutin (ARB) against CP-mediated oxidative and inflammatory reactions and hepatotoxicity. Rats were administered ARB (25 and 50 mg/kg) for 14 days and CP (150 mg/kg). CP triggered liver tissue injury with marked increase in serum AST, ALT, ALP, and bilirubin, and hepatic malondialdehyde (MDA) and nitric oxide (NO) coupled with diminution of GSH, SOD, catalase, and GPx. Liver NF-kB p65, NOS, IL-6, TNF-α, Bax and caspase-3 were upregulated by CP injection and IL-10 and Bcl-2 were decreased. ARB prevented liver injury, suppressed MDA, NO, NF-kB p65, inflammatory markers, Bax and caspase-3 in CP-treated rats. ARB restored antioxidants, IL-10 and Bcl-2, and enhanced Nrf2 and hemeoxygenase-1 (HO) both gene and protein in the liver of rats. In conclusion, these results pinpointed the protective role of ARB on oxidative and inflammatory reactions, apoptosis, and hepatotoxicity in rats. This hepatoprotective activity was linked to the ability of ARB to modulate Nrf2/HO-1 pathway.

Arbutin abrogates testicular ischemia/reperfusion injury in rats through repression of inflammation and ER stress.

The aim of this study was to investigate the effects of arbutin (ARB) administration on oxidative stress, inflammation, endoplasmic reticulum (ER) stress and apoptosis in an experimental testicular torsion/detorsion (T/D)-induced testicular injury model for the first time. A total of 24 male Sprague-Dawley rats were divided into four groups with six rats in each group: sham control, T/D, T/D+ARB (50 mg/kg) and T/D+ARB (100 mg/kg). Torsion and detorsion times were applied as 4 h and 2 h, respectively. The levels of lipid peroxidation [malondialdehyde (MDA)] and oxidative stress [total oxidant status (TOS) and total antioxidant status (TAS)] in testicular tissues were determined using colorimetric methods. The levels of DNA damage [8-hydroxy-2'-deoxyguanosine (8-OHdG)], antioxidant system [superoxide dismutase (SOD) and catalase (CAT)], pro-inflammatory cytokines [high mobility group box 1 (HMGB1), nuclear factor kappa B protein 65 (NF-κB p65), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and myeloperoxidase (MPO)], ER stress [78-kDa glucose-regulated protein (GRP78), activating transcription factor 6 (ATF6) and CCAAT-enhancer-binding protein homologous protein (CHOP)] and apoptosis (caspase-3) markers in testicular tissues were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits. Johnsen's testicle scoring system was used for histological evaluation. In the T/D group, it was determined that statistically significant increasing in the levels of oxidative stress, inflammation, ER stress and apoptosis compared with sham control group (p < 0.05). ARB administrations statistically significantly restored testicular I/R damage in a dose dependent manner (p < 0.05). In addition, it was determined that the data of histological examinations supported the biochemical results. Our findings support the hypothesis that ARB may be used as a protective agent against T/D-induced testicular damage.

Identification of Sitogluside as a Potential Skin-Pigmentation-Reducing Agent through Network Pharmacology.

Many traditional Chinese medicines (TCMs) with skin-whitening properties have been recorded in the Ben-Cao-Gang-Mu and in folk prescriptions, and some literature confirms that their extracts do have the potential to inhibit pigmentation. However, no systematic studies have identified the specific regulatory mechanisms of the potential active ingredients. The aim of this study was to screen the ingredients in TCMs that inhibit skin pigmentation through a network pharmacology system and to explore underlying mechanisms. We identified 148 potential active ingredients from 14 TCMs, and based on the average "degree" of the topological parameters, the top five TCMs (Fructus Ligustri Lucidi, Hedysarum multijugum Maxim., Ampelopsis japonica, Pseudobulbus Cremastrae Seu Pleiones, and Paeoniae Radix Alba) that were most likely to cause skin-whitening through anti-inflammatory processes were selected. Sitogluside, the most common ingredient in the top five TCMs, inhibits melanogenesis in human melanoma cells (MNT1) and murine melanoma cells (B16F0) and decreases skin pigmentation in zebrafish. Furthermore, mechanistic research revealed that sitogluside is capable of downregulating tyrosinase (TYR) expression by inhibiting the ERK and p38 pathways and inhibiting TYR activity. These results demonstrate that network pharmacology is an effective tool for the discovery of natural compounds with skin-whitening properties and determination of their possible mechanisms. Sitogluside is a novel skin-whitening active ingredient with dual regulatory effects that inhibit TYR expression and activity.

Arbutin attenuates monosodium L-glutamate induced neurotoxicity and cognitive dysfunction in rats.

Excitotoxicity, oxidative stress, and neuro-inflammation underlie the pathogenesis of neurodegenerative brain disorders. Although L-glutamate is the prime excitatory neurotransmitter involved in diverse brain functions, however, overabundance at synapse can activate cell death mechanisms. Previous studies indicate that arbutin affords relief in metabolic, cardiovascular, and gastrointestinal disorders. Recently, arbutin showed benefits in animal models of epilepsy, Parkinson's disease, and Alzheimer's disease that further expanded its therapeutic potential against brain disorders. In the present study, we aimed to evaluate the potential of arbutin against monosodium L-glutamate (MSG) neurotoxicity in rats. Wistar rats (male, 180-200 g) were administered MSG (4 mg/kg) and arbutin (50 and 100 mg/kg) intraperitoneally for 21 days. Cognitive functions were assessed using elevated plus maze and novel object recognition task. Biochemical parameters of oxidative stress, tumour necrosis factor-α (TNF-α), γ-amino butyric acid (GABA), acetylcholinesterase (AChE) activity, lactate dehydrogenase (LDH), and intracellular cation-levels (Na+, Ca2+, K+) were determined using whole brain. Administration of MSG augmented cation-levels, oxidative stress, inflammation, AChE, and LDH activities, and decreased GABA levels in the brain. Arbutin (50 and 100 mg/kg, i.p.) significantly decreased these biochemical disturbances in the brain of MSG administered rats. Behavioural results showed that MSG triggered cognitive deficits in rats that were significantly attenuated by arbutin. Histopathological findings in hippocampus and cortex revealed neuroprotective outcome of arbutin treatments against MSG. MK-801 and N(G)-nitro-L-arginine methyl ester (L-NAME) enhanced memory and neuroprotective effects in rats treated with arbutin and MSG. Arbutin may afford therapeutic advantages in neurodegenerative brain disorders by suppressing the excitotoxic pathways.

Regulation of gene expression: Cryptic β-glucoside (bgl) operon of Escherichia coli as a paradigm

Bacteria have evolved various mechanisms to extract utilizable substrates from available resources and consequently acquire fitness advantage over competitors. One of the strategies is the exploitation of cryptic cellular functions encoded by genetic systems that are silent under laboratory conditions, such as the bgl (β-glucoside) operon of E. coli. The bgl operon of Escherichia coli, involved in the uptake and utilization of aromatic β-glucosides salicin and arbutin, is maintained in a silent state in the wild type organism by the presence of structural elements in the regulatory region. This operon can be activated by mutations that disrupt these negative elements. The fact that the silent bgl operon is retained without accumulating deleterious mutations seems paradoxical from an evolutionary view point. Although this operon appears to be silent, specific physiological conditions might be able to regulate its expression and/or the operon might be carrying out function(s) apart from the utilization of aromatic β-glucosides. This is consistent with the observations that the activated operon confers a Growth Advantage in Stationary Phase (GASP) phenotype to Bgl+ cells and exerts its regulation on at least twelve downstream target genes.

Arbutin, an intracellular hydroxyl radical scavenger, protects radiation-induced apoptosis in human lymphoma U937 cells.

Ionizing radiation (IR) can generate reactive oxygen species (ROS). Excessive ROS have the potential to damage cellular macromolecules including DNA, proteins, and lipids and eventually lead to cell death. In this study, we evaluated the potential of arbutin, a drug chosen from a series of traditional herbal medicine by measuring intracellular hydroxyl radical scavenging ability in X-irradiated U937 cells. Arbutin (hydroquinone-ß-D-glucopyranoside), a naturally occurring glucoside of hydroquinone, has been traditionally used to treat pigmentary disorders. However, there are no reports describing the effect of arbutin on IR-induced apoptosis. We confirmed that arbutin can protect cells from apoptosis induced by X-irradiation. The combination of arbutin and X-irradiation could reduce intracellular hydroxyl radical production and prevent mitochondrial membrane potential loss. It also could down-regulate the expression of phospho-JNK, phospho-p38 in whole cell lysate and activate Bax in mitochondria. Arbutin also inhibits cytochrome C release from mitochondria to cytosol. To verify the role of JNK in X-irradiation-induced apoptosis, the cells were pretreated with a JNK inhibitor, and found that JNK inhibitor could reduce apoptosis induced by X-irradiation. Taken together, our data indicate that arbutin plays an anti-apoptotic role via decreasing intracellular hydroxyl radical production, inhibition of Bax-mitochondria pathway and activation of the JNK/p38 MAPK pathway.

Role of metabolism by the human intestinal microflora in arbutin-induced cytotoxicity in HepG2 cell cultures.

A possible role for metabolism by the human intestinal microflora in arbutin-induced cytotoxicity was investigated using human hepatoma HepG2 cells. When the cytotoxic effects of arbutin and hydroquinone (HQ), a deglycosylated metabolite of arbutin, were compared, HQ was more toxic than arbutin. Incubation of arbutin with a human fecal preparation could produce HQ. Following incubation of arbutin with a human fecal preparation for metabolic activation, the reaction mixture was filter-sterilized to test its toxic effects on HepG2 cells. The mixture induced cytotoxicity in HepG2 cells in a concentration-dependent manner. In addition, the mixture considerably inhibited expression of Bcl-2 together with an increase in Bax expression. Likewise, activation stimulated cleavage of caspase-3 and production of reactive oxygen species in HepG2 cell cultures. Furthermore, induction of apoptosis by the intestinal microflora reaction mixture was confirmed by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling assay. Taken together, these findings suggest that the human intestinal microflora is capable of metabolizing arbutin to HQ, which can induce apoptosis in mammalian cells.

Role of metabolism by intestinal bacteria in arbutin-induced toxicity in vitro.

A possible role of metabolism by intestinal bacteria in arbutin-induced toxicity was investigated in mammalian cell cultures. Following an incubation of arbutin with intestinal bacteria, either Bifidobacterium longum HY81 or Bifidobacterium adolescentis, for 24 h, its aglycone hydroquinone could be produced and detected in the bacterial culture media. The bacterial growth was not affected up to 10 mM arbutin in the culture medium. When the toxicity of bacteria cultured medium with arbutin was tested in the HepG2 cell lines, the medium with arbutin was more toxic than either parent arbutin only or bacteria cultured medium without arbutin, indicating that metabolic activation might be required in arbutin-induced toxicity. In addition, bacteria cultured medium with arbutin could suppress LPS and ConA mitogenicity in splenocyte cultures prepared from normal mice. The results indicate that the present toxicity testing system might be applied for assessing the possible role of metabolism by intestinal bacteria in certain chemical-induced toxicity in mammalian cell cultures.

Inhibitory effects of arbutin-beta-glycosides synthesized from enzymatic transglycosylation for melanogenesis.

To develop a new skin whitening agent, arbutin-beta-glycosides were synthesized and evaluated for their melanogenesis inhibitory activities. Three active compounds were synthesized via the transglycosylation reaction of Thermotoga neapolitana beta-glucosidase and purified by recycling preparative HPLC. As compared with arbutin (IC(50 )= 6 mM), the IC(50 )values of these compounds were 8, 10, and 5 mM for beta-D -glucopyranosyl-(1-->6)-arbutin, beta-D: -glucopyranosyl-(1-->4)-arbutin, and beta-D -glucopyranosyl-(1-->3)-arbutin, respectively. beta-D: -Glucosyl-(1-->3)-arbutin also exerted the most profound inhibitory effects on melanin synthesis in B16F10 melanoma cells. Melanin synthesis was inhibited to a significant degree at 5 mM, at which concentration the melanin content was reduced to below 70% of that observed in the untreated cells. Consequently, beta-D: -glucopyranosyl-(1-->3)-arbutin is a more effective depigmentation agent and is also less cytotoxic than the known melanogenesis inhibitor, arbutin.

Mutagenicity of arbutin in mammalian cells after activation by human intestinal bacteria.

Arbutin (hydroquinone-beta-D-glucopyranoside) is present in various food plants. Its aglycone, hydroquinone, is mutagenic and carcinogenic. We investigated whether hydroquinone may be released under conditions encountered in the human gastrointestinal tract. Arbutin was stable in artificial gastric juice. Fecal slurries from nine human subjects completely converted arbutin (2 mM) into hydroquinone. Four of nine representative human intestinal species investigated, namely Eubacterium ramulus, Enterococcus casseliflavus, Bacteroides distasonis, and Bifidobacterium adolescentis, deglycosylated arbutin at rates of 21.08, 16.62, 8.43 and 3.59 nmol x min(-1) x (mg protein)(-1), respectively. In contrast, homogenates from small intestinal mucosa and cytosolic fractions from colon mucosa deglycosylated arbutin at substantially lower rates: 0.50 and 0.09 nmol x min(-1) x (mg protein)(-1), respectively. Arbutin, unlike hydroquinone, did not induce gene mutations in Chinese hamster V79 cells in the absence of an activating system. However, in the presence of cytosolic fractions from E. ramulus or B. distasonis, arbutin was strongly mutagenic. Cytosolic fraction from Escherichia coli, showing no arbutin glycosidase activity, was not able to activate arbutin in this model system. The release of the proximate mutagen hydroquinone from arbutin by intestinal bacteria in the immediate vicinity of the colon mucosa may pose a potential risk.

Effects of alpha- and beta-arbutin on activity of tyrosinases from mushroom and mouse melanoma.

The effects of alpha- and beta-arbutin on the activity of tyrosinases from mushroom and mouse melanoma were examined. alpha-Arbutin was synthesized from hydroquinone and starch using glucoside synthetase (GSase). beta-Arbutin inhibited both tyrosinase activities from mushroom and mouse melanoma. alpha-Arbutin inhibited only the tyrosinase from mouse melanoma, 10 times as strongly as beta-arbutin. The IC50 of alpha-arbutin was 0.48 mM and its inhibitory mechanism was speculated to be mixed type inhibition, while that of beta-arbutin was noncompetitive.

Identification of catalytic residues in the beta-glucoside permease of Escherichia coli by site-specific mutagenesis and demonstration of interdomain cross-reactivity between the beta-glucoside and glucose systems.

beta-Glucoside Enzyme II (IIBgl) of the Escherichia coli phosphotransferase system transports and phosphorylates beta-glucosides, whereas the glucose Enzyme II-III pair (IIGlc-IIIGlc) transports and phosphorylates glucose as well as certain aliphatic alpha- and beta-glucosides. Comparisons of their respective amino acid sequences previously revealed that both systems are homologous and must be evolutionarily related. To gain more insight into the details of the transport mechanism, we made use of the observed homologies among phosphotransferase system permeases to design a suitable set of site-specific mutants within the gene encoding IIBgl. This set was used to study in vivo fermentation and to analyze in vitro P-enolpyruvate-dependent sugar phosphorylation as well as sugar phosphate-dependent sugar transphosphorylation. The following results were obtained. (i) IIBgl transports and phosphorylates glucose as well as aryl- and alkyl-beta-glucosides; (ii) histidyl 547 is essential for the phosphorylation of IIBgl by the histidine-containing phosphoryl carrier protein of the phosphotransferase system (HPr) (first phosphorylation site); (iii) both cysteyl 24 and histidyl 306 are essential for the transfer of the phosphoryl group to the sugar; (iv) replacement of Cys-24 by serine leads to uncoupling of sugar transport from phosphorylation; and (v) histidyl 183 is important for substrate specificity. Our studies also revealed heterologous phosphoryl transfer between the beta-glucoside and glucose permease components which probably occurs as follows: 1) HPr-P----IIBgl (His-547)----IIGlc----alkyl-alpha- or -beta-glucosides or glucose (but not aryl-beta-glucosides) and 2) HPr-P----IIIGlc----IIBgl (Cys-24 or His-306)----alkyl- or aryl-beta-glucosides or glucose (but not methyl-alpha-glucoside). In addition to the essential residues noted above, several residues in IIBgl were identified which when mutated reduced the in vitro catalytic efficiency of the enzyme more than 10-fold. Thus, aspartyl 551 and arginyl 625 appeared to function together with histidyl 547 in phosphoryl transfer involving the first phosphorylation site in the permease, whereas histidyl 183 appeared to function together with cysteyl 24 and histidyl 306 in phosphoryl transfer involving the second phosphorylation site in the permease.