Purification and Partial Characterization of Bacillomycin L Produced by Bacillus amyloliquefaciens K103 from Lemon

Bacillus amyloliquefaciens K103 isolated from a lemon sample was used as a biocontrol agent to suppress Rhizoctonia solani Kühn and other fungal plant pathogens. Two antifungal compounds were purified from the culture broth using acid precipitation, gel permeation chromatography, and reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis indicated that the antifungal compounds were two isomers similar to bacillomycin L. One of the predominant active fractions was subjected to quadrupole time-of-flight mass spectrometry and amino acid analysis to determine its structural characteristics, revealing that the antifungal compound with a molecular mass of 1,034.5464 was identical to bacillomycin L. This is the second report of lemon microflora producing bacillomycin L or any antifungal compound, suppressing the growth of R. solani Kühn. Meanwhile, the study provided insights into the enormous potential of food microbial resources and bacillomycin L antibiotics in biological control and sustainable agriculture.     

Improved Production of (R)-1-phenyl-1,2-ethanediol by a Codon-optimized R-specific Carbonyl Reductase from Candida parapsilosis in Escherichia coli

An R-specific carbonyl reductase from Candida parapsilosis (CprCR) catalyzes the transformation of (R)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone. The gene rcr coding CprCR contains a few codons rarely used by Escherichia coli. In order to improve chiral alcohol production, three codon variants Δ24, aRCR, and mRCR of CprCR were designed through truncation of 4–27 bp disorder sequence at the 5′-terminus or/and adaption of nine rare codons. The effects of codon optimization on enzyme activity, protein production, and biotransformation were studied. Among these three types, the disorder sequence-truncated and rare codon-adapted variant mRCR presents the highest enzyme activity. When compared with CprCR, mRCR showed an increase of 35.6% in the total activity of cell-free extracts. The specific activity of mRCR presented similar increase in the cell-free extract with purified protein, which suggested that the codon optimization caused positive effect on protein productivity of variant enzyme. When microbial cells concentration was 30% (w/v), the molar conversion yield and enantiomeric excess of the mRCR variant reached 86.4% and 93.6%, which were increased 36.5% and 15.8% than those of wild-type at a high substrate concentration of 5 g/L. The work will supply a new method for improving chiral alcohol preparation with codon engineered microorganisms.     

MALDI-FTICR MS Imaging as a Powerful Tool to Identify Paenibacillus Antibiotics Involved in the Inhibition of Plant Pathogens

Nowadays, microorganisms are more and more often used as biocontrol agents for crop protection against diseases. Among them, bacteria of Bacillus and Paenibacillus genders are already used as commercial biocontrol agents. Their mode of action is supposed to be related to their production of antibiotics, such as cyclic lipopeptides, which exhibit great antimicrobial activities. We chose to work with a Paenibacillus polymyxa strain (Pp56) very resistant to various microorganisms. The bacteria were grown simultaneously with Fusarium oxysporum and we applied matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry to identify the antibiotics compounds present in the fungus growth inhibition area. We, therefore, identified fusaricidins A, B, and C and numerous members of the LI-F antibiotics family. MALDI-FTICR mass spectrometry imaging was then used to follow the diffusion of lipopeptides involved in the inhibitory activity over time. We analyzed the molecular content of the inhibitory area at different Pp56 and Fusarium incubation durations and concluded that some lipopeptides such as fusaricidin B and a mixture of LI-F05b/06b/08a were mainly involved in the defense mechanism of Pp56. Our study confirms that MALDI imaging may be a powerful tool to quickly determine which molecular species is involved in an antagonism with another microorganism, avoiding time-consuming steps of extraction, purification, and activity tests, which are still commonly used in microbiology.

Cell-based biosensor for measurement of phenol and nitrophenols toxicity

A cost-effective whole cell biosensor based on electrochemical technique to detect toxicities of phenol and nitrophenols has been developed. This method relied on the inhibition effect for respiratory chain activity of microorganism by toxicant, which was measured by chronoamperometry using mediator (ferricyanide). The current signals produced by suspended microorganisms and reoxidation of ferrocyanide were transformed to inhibiting efficiency directly, and 50% inhibiting concentration (IC₅₀) was chosen as the quantitative standard of toxicity. The test microorganisms used here consist of three bacilli (Escherichia coli, Enterobacter cloacae and Alcaligenes faecalis), two pseudomonas (Pseudomonas fluorescens and Pseucomonas putida) and one fungus (Trichosporon cutaneum). 3,5-Dichlorophenol (DCP) was taken as the reference toxicant. The results showed that the microorganisms which belong to the same bacterial family had similar trends of inhibitions on respiratory activity and similar IC50 values. By comparing the IC₅₀ values, P. fluorescens was the most sensitive one to DCP toxicity, its IC₅₀ was estimated to be 4.2 mg/L. pH 7.0 and together with the standard glucose-glutamic acid (GGA) as an exogenous material were taken for optimum conditions in this study. Here, P. fluorescens as model test microorganism was employed to assess toxicities of phenol and nitrophenols under the optimum conditions. IC₅₀ values of 291.4 mg/L for phenol, 64.1 mg/L for 2-NP, 71.4 mg/L for 3-NP and 14.0 mg/L for 4-NP were determined at 60 min, respectively. Comparison with the results of published data has confirmed that this cell biosensor is a sensitive and rapid alternative to toxicity screening of chemicals.     

In vitro assessment of N-(benzyl)chitosan derivatives against some plant pathogenic bacteria and fungi

Plant pathogenic bacteria and fungi negatively affect a large number of important fruit and vegetables during the growing season and throughout postharvest storage. Therefore, the current study focuses on the preparation of N-(benzyl)chitosan derivatives as antimicrobial agents to control these microorganisms. Chitosan was reacted with a set of aromatic aldehydes by reductive amination involving formation of the corresponding imines, followed by reduction with sodium borohydride to produce the N-(benzyl)chitosan derivatives. The end products were analyzed using ¹H NMR spectroscopy and the degrees of substitution ranged from 0.12 to 0.29. The antibacterial activity was evaluated in vitro against the crown gall disease Agrobacterium tumefaciens (Family: Rhizobiaceae; Class: Alpha Proteobacteria) and the soft mold disease Erwinia carotovora (Family: Enterobacteriaceae; Class: Gamma Proteobacteria) by the nutrient agar dilution method. A higher activity of chitosan and its derivatives was obtained with N-(o-ethylbenzyl)chitosan with a MIC of 500 mg/L against E. carotovora, while N-(o,p-diethoxybenzyl)chitosan was the most active one against A. tumefaciens with a MIC of 1050 mg/L. In addition, the in vitro antifungal assessment against root rot disease Fusarium oxysporum (Family: Tuberculariaceae; Class: Deuteromycetes) and the leaf spots and blights disease Pythium debaryanum (Family: Pythiaceae; Class: Oomycetes) was tested by a mycelial radial growth technique. The data showed that N-(o,p-diethoxybenzyl)chitosan was the most active one with an EC₅₀ of 400 and 468 mg/L for F. oxysporum and P. debaryanum, respectively. In addition, chitosan derivatives had a detrimental effect on spore germination for F. oxysporum. Most of these derivatives exhibited high inhibition percentage (>90%) of spore germination at 1000 mg/L.     

Utilization of sol–gel ceramics for the immobilization of living microorganisms

Two possible methods are described for using sol–gel technology to immobilize living microorganisms, either embedding the cells within thin silica layers, or using the technique of freeze-gelation to immobilize microorganisms within molded ceramic parts. The preparation and structure of both biocer variants are outlined, and examples are given for the activity and storage stability of embedded microorganisms. Silica layers were used to immobilize various bacteria and algae. Survival rates after storage are given for Pseudomonas fluorescence bacteria and for green algae such as H. pluvialis, C. vulgaris, B. braunii and N. limnetica embedded within thin transparent silica layers. The bioactivity of bacteria immobilized in freeze-gelation ceramics was investigated by monitoring glucose consumption for P. fluorescens NCIMB 11764, and phenol degradation for Rhodococcus ruber.     

Production of Enzymes by Paenibacillus chitinolyticus and Paenibacillus ehimensis to Obtain Chitooligosaccharides

Obtaining oligosaccharides from chitosan has been the focus of several studies in the pharmaceutical, chemical, food, and medical areas, due to their functional properties. Here, we evaluated the production potential of biologically functional chitooligosaccharides using enzymes extracts produced by Paenibacillus chitinolyticus and Paenibacillus ehimensis. After 48 h of fermentation, these microorganisms were able to produce chitosanases, which generated oligomers with a degree of polymerization between dimers and hexamers. The maximum conversion of chitosan to oligomers was 99.2 %, achieved after 12 h incubation of chitosan with enzymes produced by P. ehimensis. The chitooligosaccharides generated were capable of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical, reaching a maximum scavenging rate of 61 and 39 % when produced with P. ehimensis and P. chitinolyticus enzymes, respectively. The use of these enzymes in the crude form could facilitate their use in industrial applications.     

Application of Green Algal Planktochlorella nurekis Biomasses to Modulate Growth of Selected Microbial Species

As microalgae are producers of proteins, lipids, polysaccharides, pigments, vitamins and unique secondary metabolites, microalgal biotechnology has gained attention in recent decades. Microalgae can be used for biomass production and to obtain biotechnologically important products. Here, we present the application of a method of producing a natural, biologically active composite obtained from unicellular microalgae of the genus Planktochlorella sp. as a modulator of the growth of microorganisms that can be used in the cosmetics and pharmaceutical industries by exploiting the phenomenon of photo-reprogramming of metabolism. The combination of red and blue light allows the collection of biomass with unique biochemical profiles, especially fatty acid composition (Patent Application P.429620). The ethanolic and water extracts of algae biomass inhibited the growth of a number of pathogenic bacteria, namely Enterococcus faecalis, Staphylococcus aureus PCM 458, Streptococcus pyogenes PCM 2318, Pseudomonas aeruginosa, Escherichia coli PCM 2209 and Candida albicans ATCC 14053. The algal biocomposite obtained according to our procedure can be used also as a prebiotic supplement. The presented technology may allow the limitation of the use of antibiotics and environmentally harmful chemicals commonly used in preparations against Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli or Candida spp.     

Identification and in silico molecular modelling study of newly isolated Bacillus subtilis SI-18 strain against S9 protein of Rhizoctonia solani

The numerous bioactive components from Bacillus subtilis are commonly used as antimicrobial agents for reducing plant diseases caused by fungal pathovars. In this study, we isolated and identified B. subtilis SI-18 strain from twenty isolates of rhizosphere soil through morphological and molecular approaches, and explored its inhibitory activities against Rhizoctonia solani. According to morphological features and 16S rRNA and gyrB gene sequence analysis, B. subtilis SI-18 strain was identified. Additionally, the culture filtrate of B. subtilis SI-18 resulted in the suppression of R. solani mycelium growth and material leakage from the cells. Then, we have performed homology modelling and molecular docking study of S9 protein from R. solani where three potential compounds (D1, D2, and D3) were identified among 134 antimicrobial compounds derived from B. subtilis group based on higher binding energy and interaction at the active grove of the target protein. The D1 compound creates alkyl bond at Val48 whereas D2 also binds with Val48 by creating hydrogen bond. On the other hand, two hydrogen bonds were observed at Val48 and Ile52 by D3, which might be responsible for possible blocking of the target S9 protein of R. solani. To validate the docking study and understand the change in drug-ligand conformation, molecular dynamics simulation was assessed where rigid conformation was found for D1, D2 and D3 complexes. Moreover, ADMET study confirms that no toxicity and carcinogenicity were found for screened compounds. Based on our studies, we demonstrated that compounds D1, D2, and D3 derived from B. subtilis can be a potential inhibitor of S9 protein of R. solani that might be a possible strategy for fungal disease prevention.     

Chitosan matrices modified with carbon nanotubes for use in mediated microbial biosensing

We describe a microbial sensor based on Pseudomonas fluorescens cells that was prepared by modifying graphite electrodes with chitosan and carbon nanotubes. Chronoamperometry was performed at +0.3 V in the presence of hexacyanoferrate as a mediator and revealed a good response to glucose which is linear in the 1.0 to 5.0 mM concentration range. Linearity was defined by the equation of y?=?102.120x?13.279 (R ²?=?0.998) (y shows current density as nA.cm^?2 and x shows glucose concentration in mM). The effect of the CNTs on the response was compared to that of electrodes made without CNTs.

Genomic and Metabolomic Investigation of a Rhizosphere Isolate Streptomyces netropsis WLXQSS-4 Associated with a Traditional Chinese Medicine

Rhizosphere microorganisms play important ecological roles in promoting herb growth and producing abundant secondary metabolites. Studies on the rhizosphere microbes of traditional Chinese medicines (TCMs) are limited, especially on the genomic and metabolic levels. In this study, we reported the isolation and characterization of a Steptomyces netropsis WLXQSS-4 strain from the rhizospheric soil of Clematis manshurica Rupr. Genomic sequencing revealed an impressive total of 40 predicted biosynthetic gene clusters (BGCs), whereas metabolomic profiling revealed 13 secondary metabolites under current laboratory conditions. Particularly, medium screening activated the production of alloaureothin, whereas brominated and chlorinated pimprinine derivatives were identified through precursor-directed feeding. Moreover, antiproliferative activities against Hela and A549 cancer cell lines were observed for five compounds, of which two also elicited potent growth inhibition in Enterococcus faecalis and Staphylococcus aureus, respectively. Our results demonstrated the robust secondary metabolism of S. netropsis WLXQSS-4, which may serve as a biocontrol agent upon further investigation.     

Cytotoxic Plant Extracts towards Insect Cells: Bioactivity and Nanoencapsulation Studies for Application as Biopesticides

The potential of plant extracts as bioinsecticides has been described as a promising field of agricultural development. In this work, the extracts of Punica granatum (pomegranate), Phytolacca americana (American pokeweed), Glandora prostrata (shrubby gromwell), Ulex europaeus (gorce), Tagetes patula (French marigold), Camellia japonica red (camellia), Ruta graveolens (rue or herb-of-grace) were obtained, purified, and their activity against Spodoptera frugiperda (Sf9) insect cells was investigated. From the pool of over twenty extracts obtained, comprising different polarities and vegetable materials, less polar samples were shown to be more toxic towards the insect cell line Sf9. Among these, a dichloromethane extract of R. graveolens was capable of causing a loss of viability of over 50%, exceeding the effect of the commercial insecticide chlorpyrifos. This extract elicited chromatin condensation and the fragmentation in treated cells. Nanoencapsulation assays of the cytotoxic plant extracts in soybean liposomes and chitosan nanostructures were carried out. The nanosystems exhibited sizes lower or around 200 nm, low polydispersity, and generally high encapsulation efficiencies. Release assays showed that chitosan nanoemulsions provide a fast and total extract release, while liposome-based systems are suitable for a more delayed release. These results represent a proof-of-concept for the future development of bioinsecticide nanoformulations based on the cytotoxic plant extracts.     

Keratinolytic enzyme-mediated biodegradation of recalcitrant feather by a newly isolated Xanthomonas sp. P5

We isolated and characterized a novel feather-degrading Xanthomonas sp. P5 with keratinolytic activity. In improved medium containing 0.1% (w/v) feather, maximal keratinolytic activity was observed at 5 days (69.0 ± 0.6 U/mL). This value was 7.1-fold higher than the yield in basal feather medium. The strain P5 degraded feather completely after 7 days. Feather degradation resulted in free thiol group, soluble protein and amino acids formation, indicating that sulfitolysis and proteolysis may be responsible for feather degradation by the strain P5. Total free amino acid concentration in the cell-free supernatant was around 188.6 μM. Asparagine, methionine, histidine and threonine were the major amino acids released in the culture. Xanthomonas sp. P5 exhibited multiple plant growth-promoting attributes such as siderophore, indoleacetic acid, ammonia, hydrolytic enzyme and antifungal activity. Our results indicate that Xanthomonas sp. P5 could be not only used to upgrade the nutritional value of recalcitrant feather waste but is also a potential candidate for the development of biofertilizer or biocontrol agent applicable to crop plant soil.     

Cloning and characterization of Pfl_1841, a 2-methylenebornane synthase in Pseudomonas fluorescens PfO-1

The pfl_1841 gene from Pseudomonas fluorescens PfO-1 is the only gene in any of the three sequenced genomes of the Gram-negative bacterium P. fluorescens, that is, annotated as a putative terpene synthase. The predicted Pfl_1841 protein, which harbors the two strictly conserved divalent metal binding domains found in all terpene cyclases, is closely related to several known or presumed 2-methylisoborneol synthases, with the closest match being to the MOL protein of Micromonaspora olivasterospora KY11048 that has been implicated as a 2-methylenebornane synthase. A synthetic gene encoding P. fluorescens Pfl_1841 and optimized for expression in Escherichia coli was expressed and purified as an N-terminal His₆-tagged protein. Incubation of recombinant Pfl_1841 with 2-methylgeranyl diphosphate produced 2-methylenebornane as the major product accompanied by 1-methylcamphene as well as other minor, monomethyl-homomonoterpene hydrocarbons and alcohols. The steady-state kinetic parameters for the Pfl_1841-catalyzed reaction were K_M=110±13 nM and k_cat=2.4±0.1×10⁻² s⁻¹. Attempts to identify the P. fluorescens SAM-dependent 2-methylgeranyl diphosphate synthase have so far been unsuccessful.     

Epigenetic Modifiers Affect the Bioactive Compounds Secreted by an Endophyte of the Tropical Plant Piper longum

Seven endophytic fungi were isolated from the tropical medicinal plant Piper longum L. After preliminary screening, Phomopsis heveicola was selected for the epigenetic activation treatments. The antibacterial, antifungal, and antioxidant potentials of crude extracts obtained from the treatments (with and without epigenetic modifiers) were analyzed in vitro. The extracts inhibited growth of the human pathogens Pseudomonas aeruginosa, Shigella sonnei, Streptococcus pyogenes, and Salmonella typhi, as well as the phytopathogens Puccinia recondita, Rhizoctonia solani, Phytophthora infestans, and Botrytis cinerea. Furthermore, DPPH-scavenging activity was higher in valproic acid treated extracts. Volatile chemicals with known biological activities (measured with GC-MS/MS), were released in the valproic acid treatment. The antimicrobial potentials of the extracts were confirmed using MRM/MS analysis. The experiments revealed a new promising endophytic fungus, P. heveicola, to be utilized in biological plant protection and in biomedical applications.     

Design,synthesis, biological evaluation and in silico studies of N-(pyridin-2-yl)-benzamides derivatives as quorum sensing inhibitors

The emergence of bacterial resistance against chemical treatment is a big threat to the efficacy of bacterial infection treatment. One of the major reasons for resistance to antimicrobial agents is growth of microorganisms in biofilm. An alternative treatment by developing novel anti-biofilm agents had led to the concept of quorum sensing (QS) inhibition, which primarily targets QS signaling system by disrupting cell-cell communication. Therefore, this study focuses to develop novel antimicrobial agents which work by QS inhibition and act as anti-biofilm agents against Bacillus Subtilis and Pseudomonas Aeruginosa. In this work, a natural product-like scaffolds from Asinex library were screened and N-pyridin-2-yl-benzamide moiety was chosen to design and synthesize. Synthesized compounds were evaluated for potential anti-biofilm activity for the aforesaid microorganisms and also checked for cell viability assay, where two potent compounds 3a and 3c showed their static biofilm activity to ∼59% and ∼58% at 100 μM, respectively against Bacillus subtilis. These synthesized compounds were investigated for physicochemical parameters and binding mode prediction through molecular modelling tools. The interactions and stability of these compounds showed better affinity towards TasA and LasR proteins from Bacillus subtilis and Pseudomonas aeruginosa, respectively. Furthermore, molecular dynamic simulation for 100 ns was executed in order to appreciate the stability of the protein and ligand complex. The overall results promised that N-pyridin-2-yl-benzamide derivatives can be discovered as a lead in developing potent anti-quorum sensing agents against various bacteria.     

Slow-Release Nutrient Capsules for Microorganism Stimulation in Oil Remediation

As the concern towards environmental deterioration grows worldwide, new technological achievements become essential for all countries. Among the technologies with great potential of bioremediation is microencapsulation of active material. Several studies have investigated the use of controlled release of active materials as a way of biostimulation and supplying the nutrients necessary for the bioremediation process. In fact, as the use of microorganisms has a great potential in degrading crude oils, this work aims to use that technology and to associate it to produce controlled-release capsules of nitrogen, phosphorus, and potassium (N, P, and K) for bioremediation purposes. For the capsule formulation, polymers of sodium alginate, Capsul®, and the commercial fertilizer NPK from Sempre Verde Inc. were used. Crude oil was the only carbon source and mineral medium for microorganism growth. Controlled-release nutrient capsules, with 4 mm in diameter, made of 3.0 % alginate (w/v) and 4.0 % Capsul® (w/v) were produced. Those capsules were used in association with a microbial consortium, in a liquid phase bioremediation process, having degraded 43.6 % of the total hydrocarbon within 240 h, evidencing thus as a promising tool for hydrocarbon bioremediation.     

Detection of Fungi and Oomycetes by Volatiles Using E-Nose and SPME-GC/MS Platforms

Fungi and oomycetes release volatiles into their environment which could be used for olfactory detection and identification of these organisms by electronic-nose (e-nose). The aim of this study was to survey volatile compound emission using an e-nose device and to identify released molecules through solid phase microextraction–gas chromatography/mass spectrometry (SPME–GC/MS) analysis to ultimately develop a detection system for fungi and fungi-like organisms. To this end, cultures of eight fungi (Armillaria gallica, Armillaria ostoyae, Fusarium avenaceum, Fusarium culmorum, Fusarium oxysporum, Fusarium poae, Rhizoctonia solani, Trichoderma asperellum) and four oomycetes (Phytophthora cactorum, P. cinnamomi, P. plurivora, P. ramorum) were tested with the e-nose system and investigated by means of SPME-GC/MS. Strains of F. poae, R. solani and T. asperellum appeared to be the most odoriferous. All investigated fungal species (except R. solani) produced sesquiterpenes in variable amounts, in contrast to the tested oomycetes strains. Other molecules such as aliphatic hydrocarbons, alcohols, aldehydes, esters and benzene derivatives were found in all samples. The results suggested that the major differences between respective VOC emission ranges of the tested species lie in sesquiterpene production, with fungi emitting some while oomycetes released none or smaller amounts of such molecules. Our e-nose system could discriminate between the odors emitted by P. ramorum, F. poae, T. asperellum and R. solani, which accounted for over 88% of the PCA variance. These preliminary results of fungal and oomycete detection make the e-nose device suitable for further sensor design as a potential tool for forest managers, other plant managers, as well as regulatory agencies such as quarantine services.     

Ocimum campechianum Mill. from Amazonian Ecuador: Chemical Composition and Biological Activities of Extracts and Their Main Constituents (Eugenol and Rosmarinic Acid)

The essential oil (EO), the methanolic (MeOH), and the 70% ethanolic (70% EtOH) extracts obtained from the aerial parts of Ocimum campechianum Mill. (Ecuador) were chemically characterized through gas-chromatography coupled to mass spectrometry detector (GC-MS), high-performance liquid chromatography coupled to diode array-mass spectrometry detectors (HPLC-DAD-MS) and studied for their in vitro biological activity. The radical scavenger activity, performed by spectrophotometric 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, highlighted significant IC₅₀ values for the EO, extracts and their main constituents (eugenol and rosmarinic acid). EO (and eugenol) showed noteworthy activity against Pseudomonas syringae pv. syringae and a moderate effect against clinical Candida strains, with possible synergism in association to fluconazole against the latter microorganisms. The extracts and pure molecules exhibited weak cytotoxic activity against the HaCat cell line and no mutagenicity against Salmonella typhimurium TA98 and TA100 strains, giving indication of safety. Instead, EO showed a weak activity against adenocarcinomic human alveolar basal epithelial cells (A549). The above-mentioned evidence leads us to suggest a potential use of the crude drug, extracts, and EO in cosmetic formulation and food supplements as antioxidant agents. In addition, EO may also have a possible application in plant protection and anti-Candida formulations.     

Palmatine,a Bioactive Protoberberine Alkaloid Isolated from Berberis cretica,Inhibits the Growth of Human Estrogen Receptor-Positive Breast Cancer Cells and Acts Synergistically and Additively with Doxorubicin

Palmatine (PLT) is a natural isoquinoline alkaloid that belongs to the class of protoberberines and exhibits a wide spectrum of pharmacological and biological properties, including anti-cancer activity. The aim of our study was to isolate PLT from the roots of Berberis cretica and investigate its cytotoxic and anti-proliferative effects in vitro alone and in combination with doxorubicine (DOX) using human ER⁺/HER2⁻ breast cancer cell lines. The alkaloid was purified by column chromatography filled with silica gel NP and Sephadex LH-20 resin developed in the mixture of methanol: water (50:50 v/v) that provided high-purity alkaloid for bioactivity studies. The purity of the alkaloid was confirmed by high resolution mass measurement and MS/MS fragmentation analysis in the HPLC-ESI-QTOF-MS/MS-based analysis. It was found that PLT treatment inhibited the viability and proliferation of breast cancer cells in a dose-dependent manner as demonstrated by MTT and BrdU assays. PLT showed a quite similar growth inhibition on breast cancer cells with IC₅₀ values ranging from 5.126 to 5.805 µg/mL. In contrast, growth of normal human breast epithelial cells was not affected by PLT. The growth inhibitory activity of PLT was related to the induction of apoptosis, as determined by Annexin V/PI staining. Moreover, PLT sensitized breast cancer cells to DOX. Isobolographic analysis revealed synergistic and additive interactions between studied agents. Our studies suggest that PLT can be a potential candidate agent for preventing and treating breast cancer.