Advanced Formulation Design for Topical Creams Assisted with Vibrational Spectroscopic Imaging.

Vibrational spectroscopic imaging has become useful analytical tools for quality control of drug products. In this study, we applied microscopic attenuated total reflection (ATR)-IR and confocal Raman microscopy to elucidate microscopic structure of creams and for the formulation design in the development of semi-solid drug products. The model creams were prepared with prednisolone (PRD) and fluconazole (FLC) as active pharmaceutical ingredients and oily solvents such as mineral oil (MO), isopropyl myristate (IPM), benzyl alcohol (BA) and diethyl sebacate (DES). As a result of microscopic ATR-IR imaging, several domains indicating oily internal phase were observed, which had absorption around 1732 and 1734 cm-1 derived from MO, IPM and DES. In addition, domains of BA around 1009 cm-1 were observed at the complemental or similar position in the formulation with MO or DES, respectively. These results suggested that the creams were oil-in-water type and the distribution of domains would reflect the compatibility of the solvents. The contents of PRD and BA were determined quantitatively in each layer after the intentional separation of the creams and the results agreed well with the imaging analysis. Whereas, confocal Raman imaging allowed to visualize the distribution of the components in depth direction as well as two-dimensional plane. In particular, the Raman imaging would ensure the coexistence of FLC and BA as oily phase in the cream. From these results, the feasibility of spectroscopic imaging techniques was successfully demonstrated for the formulation design of semi-solid dosage forms.

Effect of Chemical Penetration Enhancer-Adhesive Interaction on Drug Release from Transdermal Patch: Mechanism Study Based on FT-IR Spectroscopy, 13C NMR Spectroscopy, and Molecular Simulation.

Chemical penetration enhancers (CPEs) are commonly added into transdermal patches to impart improved skin permeation of drug. However, significant unexplained variability in drug release kinetics in transdermal patches is possible as a result of the addition of CPEs; investigations into the underlying mechanisms are still limited. In the present study, a diverse set of CPEs was employed to draw broad conclusions. Solubility parameters of CPEs and acrylate pressure-sensitive adhesive were calculated by molecular dynamics simulation and Fedors group contribution method to evaluate drug-adhesive miscibility. CPE-adhesive interaction was characterized by FT-IR study, 13C NMR spectroscopy, and molecular docking simulation. Results showed that release enhancement ratio (ERR) of CPEs for zolmitriptan was rank ordered as isopropyl myristate > azone > Plurol Oleique® CC497 > Span® 80 > N-methylpyrrolidone > Transcutol® P. It was found that solubility parameter difference (Δδ) between CPE and adhesive was negatively related with ERR. It was proved that hydrogen bonding between CPE and adhesive would increase drug release rate, but only if the CPE showed good miscibility with adhesive. CPE like isopropyl myristate, which had good miscibility with adhesive, could decrease drug-adhesive interaction leading to the release of drug from adhesive.

Isolation of new phytometabolites from Alpinia galanga willd rhizomes.

The current research was aimed to isolate newer phyto-metabolites from rhizomes of Alpinia galanga plant. Study involved preparation of Alpinia galanga rhizome methanolic extract, followed by normal phase column chromatography assisted isolation of new phytometabolites (using different combinations of chloroform and methanol), and characterization (by UV, FTIR, 13C-NMR, 1H-NMR, COSY, DEPT and Mass spectrometry). The isolation and characterization experiment offered two phytometabolites: an ester (Ag-1) and tetrahydronapthalene type lactone (Ag-2). Present study concludes and reports the two phytometabolites, benzyl myristate (Ag-1) and 3-Methyl-6α, 8ß-diol-7-carboxylic acid tetralin-11, 9ß-olide (Ag-2) for the first time in Alpinia galanga rhizome. The study recommends that these phytometabolites Ag-1 and Ag-2 can be utilized as effective analytical biomarkers for identification, purity and quality control of this plant in future.

Preparation and Characterization of Microemulsions Based on Antarctic Krill Oil.

Antarctic krill oil is high in nutritional value and has biological functions like anti-inflammation and hypolipidemic effects. But it has and unpleasant smell, and unsaturated fatty acids are prone to oxidative deterioration. Its high viscosity and low solubility in water make it difficult for processing. Microemulsion can be a new promising route for development of krill oil product. We determined a formula of krill oil-in-water microemulsion with krill oil: isopropyl myristate = 1:3 as oil phase, Tween 80:Span 80 = 8:2 as surfactant, ethanol as co-surfactant and the mass ratio of surfactant to co-surfactant of 3:1. After screening the formula, we researched several characteristics of the prepared oil-in-water microemulsion, including electrical conductivity, microstructure by transmission electron microscope and cryogenic transmission electron microscope, droplet size analysis, rheological properties, thermal behavior by differential scanning calorimeter and stability against pH, salinity, and storage time.

Role of sodium l-cysteine alginate conjugate and isopropyl myristate to enhance the permeation enhancing activity of BCS class III drug from TDDS; optimization by central composite design and in vivo pharmacokinetics study.

OBJECTIVE: The objective of the present research was to study the effect and optimization of sodium alginate l-cysteine conjugate and permeation enhancer on permeation of high soluble low permeable ropinirole hydrochloride from the transdermal formulation. METHODS: Sodium alginate l-cysteine conjugate was prepared and characterized and the same was added into a transdermal formulation along with IPM as a permeation enhancer. Twelve primary formulations were prepared by solvent casting method and evaluated. The results were fed into Design Expert® Software to obtain optimized formulation. The optimized formulation was evaluated for physicochemical, ex vivo permeation, stability, skin irritation, and pharmacokinetic studies. RESULTS: The results of the characterization of prepared sodium alginate l-cysteine conjugate confirmed the thiolation process. Stability studies suggested that the drug was compatible with all the excipients. SEM images of the transdermal patch revealed that the amorphous drug was uniformly distributed. From the design space, the optimized formulation from the polymer's ratio (SA: SACC; 4:6) and IPM 9.5%w/w of polymers weight showed target steady state flux 9.004 µg/cm2/h with maximum drug permeation. The increased target flux and maximum drug permeation from an optimized patch suggested that there was an effect of SACC on ropinirole hydrochloride permeation in the presence of IPM as a permeation enhancer. Pharmacokinetic studies in rabbits showed that the optimized patch improved bioavailability as compared to marketed oral tablets. CONCLUSIONS: The study was concluded that there was a positive effect of sodium alginate l-cysteine conjugate and IPM on ropinirole hydrochloride permeation from the transdermal formulation.

Formulation and Structural Study of a Biocompatible Water-in-Oil Microemulsion as an Appropriate Enzyme Carrier: The Model Case of Horseradish Peroxidase.

A novel biocompatible water-in-oil microemulsion was developed using nonionic surfactants and was investigated as a potential enzyme delivery system for pharmaceutical applications. The system was composed of isopropyl myristate/polysorbate 80 (Tween 80)/distilled monoglycerides/water/propylene glycol (PG), had a low total surfactant concentration (8.3% w/w), and was able to incorporate approximately 3% w/w aqueous phase containing horseradish peroxidase (HRP). Structural and activity aspects of the system were studied using a variety of techniques such as dynamic light scattering (DLS), electron paramagnetic resonance (EPR), and dynamic interfacial tension. The apparent hydrodynamic diameter of the empty droplets was calculated at about 37 nm. Different enzyme concentrations, ranging from 0.01 to 1.39 µM, were used for both DLS and EPR studies to effectively determine the localization of the macromolecule in the microemulsion. According to the results, for high enzyme concentrations, a participation of HRP in the surfactant monolayer of the microemulsion is evident. The number of reverse micelles in the microemulsion was defined by a theoretical model and was used to clarify how the enzyme concentration affects the number of empty and loaded reverse micelles. To assure that the system allows the enzyme to retain its catalytic activity, an oxidative reaction catalyzed by HRP was successfully carried out with the use of the model substrate 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid]. The influence of several parameters such as temperature, pH, and PG concentration was examined to optimize the reaction conditions, and a kinetic study was conducted revealing an ordered-Bi-Bi mechanism. Values of all kinetic parameters were determined. The release of the encapsulated enzyme was studied using an adequate receiver phase, revealing the effectiveness of the proposed microemulsion not only as a microreactor but also as a carrier for therapeutic biomolecules.

Investigation of the Compatibility of the Skin PAMPA Model with Topical Formulation and Acceptor Media Additives Using Different Assay Setups.

The Skin Parallel Artificial Membrane Permeability Assay (PAMPA) is a 96-well plate-based skin model with an artificial membrane containing free fatty acid, cholesterol, and synthetic ceramide analogs to mimic the stratum corneum (SC) barrier. The current study evaluates the compatibility of lipophilic solvents/penetration enhancer, topical emulsions containing different emulsifier systems, and organic acceptor media additives with the artificial membrane of the assay. Additionally, different assay setups (standard setup: donor in bottom plate versus modified setup: donor in top plate) were compared. Methylparaben (MP), ethylparaben (EP), and propylparaben (PP) were used as model permeants and internal standards for proper assay execution. The permeation order of the parabens (MP > EP > PP) remained the same with different lipophilic solvents, and the ranking of lipophilic solvents was comparable under standard and modified conditions (isopropyl myristate, IPM > dimethyl isosorbide, DMI ≥ propylene glycol, PG > diisopropyl adipate, DIPA). Pre-incubation of the Skin PAMPA plates with IPM, DIPA, and DMI, as well as with formulations that contain non-ionic emulsifiers, and acceptor solutions containing DMSO or EtOH (≤ 50%) for 4 h did not increase the percentage of permeated parabens in the main experiment, suggesting that those compounds do not make the artificial membrane more permeable. High-resolution mass spectrometry confirmed that acceptor solutions with ≤ 50% DMSO or EtOH do not extract stearic acid, cholesterol, and certramides at standard assay conditions. Hence, if certain constraints are considered, the Skin PAMPA model can be used as a pre-screening tool for topical formulation selection.

Preparation of microemulsion containing Lycopersicon esculentum extract: In vitro characterization and stability studies.

Lycopene, the active component of Lycopersicon esculentum species, has been reported for the protecting capabilities against ultra-violet induced skin pigmentation, antioxygen and antityrosinase activities. In the present study, extract of tomato fruit was obtained from the Lycopersicon esculentum plant using solvent system comprised of hexaneethanol-acetone. The phyto chemical active constituent lycopene was then identified by spectrophotometric technique at 470nm. Micro emulsions were developed containing different ratio of water, isopropyl myristate (oil), tween 80 and propylene glycol as surfactant and co-surfactant respectively via pseudoternary phase diagram. Various physicochemical tests were performed including globular size, conductivity, viscosity, scanning electron microscopy (SEM), refractive index (RI) and pH measurement for the formulation characterization. Results of physical and chemical stability studies showed that the micro emulsion with proportion of surfactant: co-surfactant of 2:1 (Smix) was found to be optimized formulation and with enhanced stability. Therefore, concluded that the stability of the micro emulsion was dependent on the proportions of surfactant co-surfactant, water and oil in the preparation.

Development, characterization and evaluation of ginger extract loaded microemulsion: In vitro and Ex vivo release studies.

Zingeber officinale (ginger) has been used for a long time in conventional medicine for the management of many diseases most important of which is inflammatory diseases. The aim of this study was formulation of topical microemulsion system to enhance the solubility, stability and release profile of ginger extract, as it is unstable in the presence of light, air, heat and long term storage. The solubility of ginger extract in different oils, surfactants, and cosurfactants was determined in order to find the optimal components for microemulsion. Isopropyl myristate (IPM) was selected as oil phase, tween 80 and PEG 400 were selected as surfactant and co-surfactant respectively based on highest solubility values. Pseudo-ternary phase diagram was constructed in order to find out the microemulsion region. The prepared microemulsions were evaluated for pH, viscosity, conductivity, refractive index, globular size, zeta potential, polydispersity index, ginger extract content, in-vitro and ex-vivo release profiles. The formulation GE1 showed best physicochemical properties with smallest globular size (19.75nm), highest release rate and flux value. It also showed significant (p<0.05) anti-inflammatory effect as compared to reference piroxicam drug solution. It is concluded that ginger extract can be used to develop stable microemulsion system with better skin permeation and promising antiinflammatory activity.

Electron Paramagnetic Resonance and Small-Angle X-ray Scattering Characterization of Solid Lipid Nanoparticles and Nanostructured Lipid Carriers for Dibucaine Encapsulation.

Dibucaine (DBC) is one of the most potent long-acting local anesthetics, but it also has significant toxic side effects and low water solubility. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have been proposed as drug-delivery systems to increase the bioavailability of local anesthetics. The purpose of the present study was to characterize SLNs and NLCs composed of cetyl palmitate or myristyl myristate, a mixture of capric and caprylic acids (for NLCs only) plus Pluronic F68 prepared for the encapsulation of DBC. We intended to provide a careful structural characterization of the nanoparticles to identify the relevant architectural parameters that lead to the desirable biological response. Initially, SLNs and NLCs were assessed in terms of their size distribution, morphology, surface charge, and drug loading. Spectroscopic techniques (infrared spectroscopy and electron paramagnetic resonance, EPR) plus small-angle X-ray scattering (SAXS) provided information on the interactions between nanoparticle components and their structural organization. The sizes of nanoparticles were in the 180 nm range with low polydispersity and negative zeta values (-25 to -46 mV). The partition coefficient of DBC between nanoparticles and water at pH 8.2 was very high (>104). EPR (with doxyl-stearate spin labels) data revealed the existence of lamellar arrangements inside the lipid nanoparticles, which was also confirmed by SAXS experiments. Moreover, the addition of DBC increased the molecular packing of both SLN and NLC lipids, indicative of DBC insertion between the lipids, in the milieu assessed by spin labels. Such structural information brings insights into understanding the molecular organization of these versatile drug-delivery systems which have already demonstrated their potential for therapeutic applications in pain control.

Predicting the Surface Tension of Liquids: Comparison of Four Modeling Approaches and Application to Cosmetic Oils.

The efficiency of four modeling approaches, namely, group contributions, corresponding-states principle, σ-moment-based neural networks, and graph machines, are compared for the estimation of the surface tension (ST) of 269 pure liquid compounds at 25 °C from their molecular structure. This study focuses on liquids containing only carbon, oxygen, hydrogen, or silicon atoms since our purpose is to predict the surface tension of cosmetic oils. Neural network estimations are performed from σ-moment descriptors as defined in the COSMO-RS model, while methods based on group contributions, corresponding-states principle, and graph machines use 2D molecular information (SMILES codes). The graph machine approach provides the best results, estimating the surface tensions of 23 cosmetic oils, such as hemisqualane, isopropyl myristate, or decamethylcyclopentasiloxane (D5), with accuracy better than 1 mN·m-1. A demonstration of the graph machine model using the recent Docker technology is available for download in the Supporting Information.

Preparation and evaluation of novel microemulsion-based hydrogels for dermal delivery of benzocaine.

The purpose of the current research was to prepare and evaluate the potential use of microemulsion-based hydrogel (MBH) formulations for dermal delivery of benzocaine (BZN). The pseudoternary-phase diagrams were constructed for various microemulsions composed of isopropyl myristate (IPM) as oil phase, Span 20, Tween 20, Tween 80, cremophor EL and cremophor RH40 as surfactants, ethanol as cosurfactant and distilled water as aqueous phase. Finally, concentration of BZN in microemulsions was 2% (w/w). The physicochemical properties, such as conductivity, viscosity, pH, droplet size, polydispersity index and zeta potential of microemulsions, were measured. Carbopol 940 was used to convert BZN-loaded microemulsions into gel form without affecting their structure. Furthermore, excised rat abdominal skin was used to compare permeation and penetration properties of BZN loaded M3 and M3BHs with BZN solution. According to ex vivo study results, BZN-loaded M3BH1 showed highest flux values and high release rate values, and furthermore, this gel formulation had low surfactant content. Finally, in order to learn the localization of formulations within the dermal penetration, formulations and BZN solution were labeled with red oil O and subjected to fluorescence observation. In conclusion, BZN-loaded MBHs could be offered as a promising strategy for dermal drug delivery.

Carbon nanotube membranes to predict skin permeability of compounds.

In the present study, carbon nanotube (CNT) membranes were prepared to predict skin penetration properties of compounds. A series of penetration experiments using Franz diffusion cells were performed with 16 different membrane compositions for model chemicals. Similar experiments were also carried out with same model molecules using five different commercially available synthetic membranes and human skins for the comparison. Model chemicals were selected as diclofenac, dexketoprofen and salicylic acid. Their permeability coefficients and flux values were calculated. Correlations between permeability values of model compounds for human skins and developed model membranes were investigated. Good correlations were obtained for CNT membrane, isopropyl myristate-treated CNT membrane (IM-CNT membrane) and bovine serum albumin-cholesterol, dipalmitoyl phosphatidyl choline-treated membrane (BSA-Cholesterol-DPPC-IM-CNT membrane). An artificial neural network (ANN) model was developed using some molecular properties and penetration coefficients from pristine CNT membranes to predict skin permeability values and quite good predictions were made.

A cascade screening approach for the identification of Bcr-Abl myristate pocket binders active against wild type and T315I mutant.

The major clinical challenge in drug-resistant chronic myelogenous leukemia (CML) is currently represented by the Bcr-Abl T315I mutant, which is unresponsive to treatment with common first and second generation ATP-competitive tyrosine kinase inhibitors (TKIs). Allosteric inhibition of Bcr-Abl represent a new frontier in the fight against resistant leukemia and few candidates have been identified in the last few years. Among these, myristate pocket (MP) binders discovered by Novartis (e.g. GNF2/5) showed promising results, although they proved to be active against the T315I mutant only in combination with first and second generation ATP-competitive inhibitors. Here we used a cascade screening approach based on sequential fluorescence polarization (FP) screening, in silico docking/dynamics studies and kinetic-enzymatic studies to identify novel MP binders. A pyrazolo[3,4-d]pyrimidine derivative (6) has been identified as a promising allosteric inhibitor active on 32D leukemia cell lines (expressing Bcr-Abl WT and T315I) with no need of combination with any ATP-competitive inhibitor.

Skin Permeation of Testosterone from Viscoelastic Lecithin Reverse Wormlike Micellar Solution.

We evaluated testosterone-containing lecithin reverse wormlike micelles (reverse worms) composed of a polar substance/lecithin/isopropyl myristate for transdermal application. Water, D-ribose, or tetraglycerol were used as the polar substance and were key ingredients for forming the reverse worms. Using the reverse worms, 1 wt% of testosterone could be stably solubilized. When using D-ribose as polar substance, the maximum zero-shear viscosity of the reverse worms solution was higher than that of systems using water or tetraglycerol as the polar substance. The mechanism of skin permeation of testosterone from reverse worms solution was elucidated using skin permeation experiments with hairless mouse skin. When the structure of the reverse worms transitioned to lamellar liquid crystals at the skin/formulation interface, testosterone became supersaturated in the formulations. The structural transition occurred in systems using water or D-ribose as the polar substance, increasing the flux of testosterone. The flux of testosterone from reverse worms solution thus depends on the type of polar substance used.

Development, Characterization and Skin Interaction of Capsaicin-Loaded Microemulsion-Based Nonionic Surfactant.

The aim of this study was to develop novel microemulsions (MEs) for the transdermal delivery of capsaicin. Microemulsion-based nonionic surfactants consisting of isopropyl myristate as the oil phase, various nonionic surfactants as the surfactant (S), various glycols or alcohol as the co-surfactant (CoS), and reverse osmosis water as the aqueous phase were formulated. Based on the optimal ME obtained from Design Expert, MEs containing a fixed concentration of oil, water or surfactant were prepared while varying the amounts of the other two fractions. The results indicated that the skin permeation flux of low dose capsaicin (0.15% (w/w)) was significantly higher for the selected ME than the commercial product and capsaicin in ethanol (control) by approximately two- and four-fold, respectively. We successfully demonstrated the feasibility of the transdermal delivery of capsaicin-loaded ME using a low concentration of nonionic surfactant and ethanol. Moreover, the optimization using computer program helped to simplify the development of a pharmaceutical product.

A new TLC bioautographic assay for qualitative and quantitative estimation of lipase inhibitors.

INTRODUCTION: Lipase inhibitory assays based on TLC bioautography have made recent progress; however, an assay with greater substrate specificity and quantitative capabilities would advance the efficacy of this particular bioassay. OBJECTIVE: To address these limitations, a new TLC bioautographic assay for detecting lipase inhibitors was developed and validated in this study. METHODS: The new TLC bioautographic assay was based on reaction of lipase with ß-naphthyl myristate and the subsequent formation of the purple dye between ß-naphthol and Fast Blue B salt (FBB). The relative lipase inhibitory capacity (RLIC) was determined by a TLC densitometry with fluorescence detection, expressed as orlistat equivalents in millimoles on a per sample weight basis. Six pure compounds and three natural extracts were evaluated for their potential lipase inhibitory activities by this TLC bioautographic assay. RESULTS: The ß-naphthyl myristate as the substrate improved the detection sensitivity and specificity significantly. The limit of detection (LOD) of this assay was 0.01 ng for orlistat, the current treatment for obesity. This assay has acceptable accuracy (92.07-105.39%), intra-day and inter-day precisions [relative standard deviation (RSD), 2.64-4.40%], as well as intra-plate and inter-plate precisions (RSD, 1.8-4.9%). CONCLUSION: The developed method is rapid, simple, stable, and specific for screening and estimation of the potential lipase inhibitors.

Transfollicular enhancement of methyl myristate loaded in cationic niosomes incorporated in hair lotion.

Hair lotion containing methyl myristate loaded in cationic niosomes (HL-MMnio) composed of Brij72/cholesterol/DDAB at 7:3:0.65 molar ratio was developed. The remaining percentages of MM loaded in cationic niosomes in hair lotion were higher than free MM in hair lotion of about 1.2 times. The cumulative amounts in porcine skin and the receiver compartment of MM loaded in cationic niosomes incorporated in hair lotion were higher than those of free MM in hair lotion of 1.45 and 1.32 times, respectively. HL-MMnio showed very slightly irritation on rabbit skin, which was disappeared after 4 d. For melanogenesis induction in C57BL/6 mice with aged-induced grey body coat hairs, the highest pigmentation scores of HL-MMnio applied on the dorsal area were observed after 21 days, while hair lotion containing the free MM indicated after 35 days. This study has suggested that HL-MMnio was the high potential formulation for canities treatment.

Influence of sonophoresis and chemical penetration enhancers on percutaneous transport of penbutolol sulfate.

The effect of ultrasound and chemical penetration enhancers on transcutaneous flux of penbutolol sulfate across split-thickness porcine skin was investigated. Penbutolol sulfate is a potent, noncardioselective beta-blocker, which is used for the management of hypertension. The drug is one of the most lipid soluble of the ß-adrenoceptor antagonists used clinically. It has an n-octanol/pH 7.4 buffer partition coefficient of 179 compared to a value of 22 for propranolol. The amount of penbutolol sulfate transported across the skin is low. In this project, we studied the effect of sonophoresis and chemical penetration enhancers on transdermal delivery of penbutolol sulfate. Low-frequency sonophoresis at a frequency of 20 kHz increased transcutaneous flux of penbutolol sulfate by 3.5-fold (27.37 ± µg cm-2 h-1) compared to passive delivery (7.82 ± 1.72 µg cm-2 h-1). We also investigated the effect of 50% ethanol, 1% limonene and 2% isopropyl myristate (IPM) on transcutaneous permeation of penbutolol sulfate. IPM, ethanol and limonene at the concentration of 1%, 50% and 2%, respectively, increased the steady-state flux values of penbutolol sulfate 2.2- (17.07 ± 3.24 µg cm-2 h-1), 2.6 - (19.40 ± 6.40 µg cm-2 h-1) and 3.4-times (26.38 ± 5.01 µg cm-2 h-1) compared to passive delivery (7.76 ± 2.9 µg cm-2 h-1). The results demonstrate that although there were slight increases in flux values, ultrasound, ethanol, limonene and IPM did not significantly enhance the transdermal delivery of penbutolol sulfate. Future studies will examine ways of optimizing sonophoretic and chemical enhancer parameters to achieve flux enhancement.

Methyl 3-Hydroxymyristate, a Diffusible Signal Mediating phc Quorum Sensing in Ralstonia solanacearum.

Ralstonia solanacearum, a plant pathogenic bacterium causing "bacterial wilt" on crops, uses a quorum sensing (QS) system consisting of phc regulatory elements to control its virulence. Methyl 3-hydroxypalmitate (3-OH PAME) was previously identified as the QS signal in strain AW1. However, 3-OH PAME has not been reportedly detected from any other strains, and this suggests that they produce another unknown QS signal. Here we identify (R)-methyl 3-hydroxymyristate [(R)-3-OH MAME] as a new QS signal that regulates the production of virulence factors and secondary metabolites. (R)-3-OH MAME was synthesized by the methyltransferase PhcB and sensed by the histidine kinase PhcS. The phylogenetic trees of these proteins from R. solanacearum strains were divided into two groups, according to their QS signal types--(R)-3-OH MAME or (R)-3-OH PAME. These results demonstrate that (R)-3-OH MAME is another crucial QS signal and highlight the unique evolution of QS systems in R. solanacearum.