Cationic Detergent Research Articles

Cationic detergents enable the separation of membrane proteins of Plasmodium falciparum‐infected erythrocytes by 2D gel electrophoresis

The intraerythrocytic stage of Plasmodium falciparum alters the characteristics of its host cell by exporting selected plasmodial proteins. Although it is clear that the physicochemical and immunobiological properties of the host cell are modulated during parasite development, the involved plasmodial proteins and their mode of action are not completely known. Using cetyltrimethylammonium bromide (CTAB) or benzyldimethyl-n-hexadecylammonium chloride (16-BAC) for the first dimension and SDS for the second dimension, we separated proteins from membranes of human erythrocytes and of erythrocytes infected with the malaria parasite P. falciparum. Protein spots were analyzed by MALDI-TOF/TOF MS and annotated in respective 2D master gels. By using the alternative 2D approach, characteristic host cell membrane proteins and, more importantly, membrane-associated and exported plasmodial proteins were identified that might play a role in parasite-induced host cell modulation.

Purification and Characterization of Thermostable Alkaline Cellulase from Marine Bacterium Bacillus licheniformis AU01 by Utilizing Cellulosic Wastes

The present study was aimed to produce cellu- lase from Bacillus licheniformis AU01 by utilizing major cellulosic wastes. Among the cellulosic wastes tested, rice bran and peptone were found to be the best carbon and nitrogen source for production of cellulase (188 U ml -1 ). Through multi step purification an 8.66 fold purified protein with a specific activity of 620.8 U mg -1 was achieved with the molecular weight of 37 kDa. The optimum temperature and pH for CMCase activity was determined as 50 C and 9 respectively and more than 80 % activity retained at tem- perature 80 C and pH 12.0. The cellulase purified from present study was stable even in the presence of various cationic, anionic and commercial detergents. These remarkable qualities such as thermostability, pH stability and compatibility with detergents will make this cellulase as a potential candidate for industrial applications.

Aggregation and disaggregation of anionic aluminum phthalocyanines in cationic pre-micelle and micelle media: A fluorescence study

Abstract The interactions of two anionic aluminum phthalocyanines (Pcs), the tetrasulfonated (AlPcS 4 ) and the monosulfonated (AlPcS 1 ) species, with the cationic detergent n -dodecyltrimethylammonium chloride (DTAC) were investigated. These studies were performed by UV–visible absorption, steady-state and transient-state fluorescence, fluorescence lifetime imaging microscopy (FLIM), and fluorescence correlation spectroscopy (FCS). For both AlPcS 4 and AlPcS 1 , large complexes of Pc–DTAC were formed at low surfactant concentrations, in the pre-micelle range. These complexes were visualized by the FLIM technique, directly in a drop of the sample. As the DTAC concentration increased, the size of the complexes decreased significantly. Above the detergent critical micelle concentration ( CMC ≈ 22 mM), highly fluorescent monomers of both AlPcS 4 and AlPcS 1 were found to be strongly bound to the DTAC micelles surface: large equilibrium binding constants, K b = 3.5 × 10 5 and 6.2 × 10 4 , were obtained for AlPcS 4 and AlPcS 1 , respectively. The aggregation/disaggregation behavior of phthalocyanines likely results from a delicate balance in the system, between hydrophobic attractions (Pc–Pc) and electrostatic attractions (anionic Pc macrocycles – cationic DTAC headgroups) and repulsions (between anionic sulfonated groups of Pc macrocycles).

P-Glycoprotein-ATPase Modulation: The Molecular Mechanisms

P-glycoprotein-ATPase is an efflux transporter of broad specificity that counteracts passive allocrit influx. Understanding the rate of allocrit transport therefore matters. Generally, the rates of allocrit transport and ATP hydrolysis decrease exponentially with increasing allocrit affinity to the transporter. Here we report unexpectedly strong down-modulation of the P-glycoprotein-ATPase by certain detergents. To elucidate the underlying mechanism, we chose 34 electrically neutral and cationic detergents with different hydrophobic and hydrophilic characteristics. Measurement of the P-glycoprotein-ATPase activity as a function of concentration showed that seven detergents activated the ATPase as expected, whereas 27 closely related detergents reduced it significantly. Assessment of the free energy of detergent partitioning into the lipid membrane and the free energy of detergent binding from the membrane to the transporter revealed that the ratio, q, of the two free energies of binding determined the rate of ATP hydrolysis. Neutral (cationic) detergents with a ratio of q = 2.7 ± 0.2 (q > 3) followed the aforementioned exponential dependence. Small deviations from the optimal ratio strongly reduced the rates of ATP hydrolysis and flopping, respectively, whereas larger deviations led to an absence of interaction with the transporter. P-glycoprotein-ATPase inhibition due to membrane disordering by detergents could be fully excluded using 2H-NMR-spectroscopy. Similar principles apply to modulating drugs.

Electrochemical Functionalization of Single-Walled Carbon Nanotubes Films Obtained by Electrophoretic Deposition

In this paper, we report an electrode coated with single-walled carbon nanotubes (SWCNTs) thin-film prepared by the electrophoretic deposition (EPD) technique. SWCNTs electrodes are fabricated on indium tin oxide (ITO) glass substrates using a mixture of CNTs and a cationic detergent tetraoctylammonium bromide (TOAB) in tetrahydrofuran (THF) by applying a negative voltage to the ITO glass plate. The functionalization of these nanotubes is then achieved via electrochemical reduction of aryl diazonium salts, in a manner similar to that employed for functionalization of other carbon surfaces. A variety of diazonium salts have been used, including those that provide moieties conducive to further elaboration after attachment to the nanotubes.

A Cationic Phospholipid–Detergent Conjugate as a New Efficient Carrier for siRNA Delivery

Nucleic acids have a huge intrinsic therapeutic potential that, however, relies heavily on the development of methods for their delivery to their intracellular target site. Cationic lipids and polymers were introduced more than twenty years ago for the delivery of DNA molecules to cells. Since then, they have become useful tools for biomedical research and have also been adopted for siRNA delivery. Their mode of action has been roughly determined. It is based on the property of adherent cell lines to easily internalize large quantities of cationic complexes in endocytic compartments, and on the incorporation of membrane-perturbing elements within internalized complexes to trigger subsequent release of the nucleic acid payload into the cytosol. Cationic lipids forming hexagonal phase or formulations of cationic lipids with the fusogenic lipid 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE) enable both the formation of cationic nucleic acid complexes (lipoplexes) and rupture of the endosome membrane. Yet, the endosomolytic activity of the current effective synthetic nucleic acid delivery systems remains low and effective delivery is only achieved by using cell entry pathways with a high flow rate. The membrane-disruptive properties of detergents have been considered for improving nucleic acid delivery. However, the use of detergents remains delicate because direct plasma membrane permeation might have an irreversible impact on cell viability. Cationic detergents have been shown to provoke DNA condensation but the resulting lipoplexes have turned out to be inactive or only poorly active, in vitro, when particles are prepared from cationic detergent mixed with DOPE. This is explained by the rapid exchange of the detergent molecules between the complex and the aqueous environment, which results in an irreversible decondensation of the nucleic acid even before entry into the cells. On the other hand, preparation of DNA lipoplexes from cationic lipids in the presence of Tween-80, a non-ionic detergent, has led to systems with enhanced gene transfection properties both in vitro and in vivo. These results led us to explore the consequences on nucleic acid delivery of the conjugation of a detergent molecule to a cationic lipid so that the detergent cannot be depleted from the transfection particle. We selected Triton X-100 (TX100) for its well-known detergent properties and for synthetic convenience. Furthermore, TX100 does not solubilize cholesterol-enriched raft domains, which might be involved in cationic lipoplexes internalization. TX100 was covalently linked to the phosphate group of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a major component of biological membranes (Figure 1). Diacylglycerophosphocholines (PCs) are normal cellular metabolites and offer the opportunity to easily generate cationic lipids by O-alkylation of the phosphate group. Gorman et al. first demonstrated that a PC-derived phosphotriester (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, EDMPC) can mediate efficient gene transfer. Later on, derivatives of DOPC (i.e., ethyl-DOPC, EDOPC, Figure 1) and other PCs were developed by MacDonald et al. , the physical and transfection properties of which were extensively investigated. We hypothesized that the covalent attachment of a detergent molecule and a natural phospholipid to form a cationic phospholipid–detergent conjugate might improve cell uptake and transfection efficiency of lipoplexes prepared thereof. TX100 conjugation to DOPC was realized according to Scheme 1. Detergent activation with trifluoromethanesulfonyl anhydride provided the corresponding sulfonyl ester. This compound is unstable and decomposes in a few hours on standing at room temperature in chloroform. Analysis of degradation products is consistent with a deoligomerization process leading to the formation of dioxane as reported previously with other PEG derivatives. Consequently, the sulfonyl ester (1 equiv) was directly submitted to nucleophilic displacement by the anionic phosphate in DOPC (3 equiv). Conjugate 1 was obtained in 32% yield and unreacted DOPC (61%) was recovered after purification. The use of a larger excess of activated TX100 (7 equiv) improved the DOPC conversion but finally proved detrimental as the purification of the product became difficult (1: 22%; recovered DOPC: 24%). Control compound 2 was prepared similarly, by replacing Triton X-100 by polyethylene glycol mono[a] Dr. P. Pierrat, Dr. G. Creusat, Dr. G. Laverny, Prof. F. Pons, Dr. G. Zuber, Dr. L. Lebeau Laboratoire de Conception et Application de Mol!cules Bioactives CNRS, Universit! de Strasbourg, Facult! de Pharmacie 74 Route du Rhin, BP 60024, Illkirch (France) Fax: (+33)333-6885-4306 E-mail : llebeau@unistra.fr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201103645.

Electrostatic Protein Immobilization Using Charged Polyacrylamide Gels and Cationic Detergent Microfluidic Western Blotting

We report a novel protein immobilization matrix for fully integrated microfluidic Western blotting (WB). The electrostatic immobilization gel (EIG) enables immobilization of all proteins sized using cetyl trimethylammonium bromide polyacrylamide gel electrophoresis (CTAB-PAGE), for subsequent electrophoretic probing with detection affinity reagents (e.g., labeled antibodies). The "pan-analyte" capture strategy introduced here uses polyacrylamide gel grafted with concentrated point charges (zwitterionic macromolecules), in contrast to existing microfluidic WB strategies that rely on a sandwich immunoassay format for analyte immobilization and detection. Sandwich approaches limit analyte immobilization to capture of only a priori known targets. A charge interaction mechanism study supports the hypothesis that electrostatic interaction plays a major role in analyte immobilization on the EIG. We note that protein capture efficiency depends on both the concentration of copolymerized charges and ionic strength of the gel buffer. We demonstrate pan-analyte immobilization of sized CTAB-laden model proteins (protein G, ovalbumin, bovine serum albumin, β-galactosidase, lactoferrin) on the EIG with initial capture efficiencies ranging from 21 to 100%. Target proteins fixed on the EIG (protein G, lactoferrin) are detected using antibody probes with signal-to-noise ratios of 34 to 275. The approach advances protein immunoblotting performance through 200× reduction on sample consumption, 12× reduction in assay duration, and automated assay operation, compared to slab-gel WB. Using the microfluidic WB assay, assessment of lactoferrin in human tear fluid is demonstrated with a goal of advancing toward nonbiopsy-based diagnosis of Sjögren's Syndrome, an autoimmune disease.

Denaturation of human serum albumin under the action of cetyltrimethylammonium bromide according to fluorescence polarization data of protein

Denaturation of human serum albumin (HSA) under the action of cationic detergent cetyltrimethylammonium bromide (CTAB) is studied at different pH values by estimating the rotational diffusion of protein via fluorescence polarization. The degree of polarization of HSA tryptophan fluorescence, the rotational relaxation time, the rotational diffusion coefficient and the effective Einstein radius of the HSA molecules in solutions with different CTAB concentrations at different pH values are determined. The obtained rotational diffusion parameters of the HSA molecules show that under the action of CTAB, HSA denaturation has a one-stage character and proceeds more intensely and effectively at pH values higher than the pI value of protein (4.7).

Neomycin–phenolic conjugates: Polycationic amphiphiles with broad-spectrum antibacterial activity, low hemolytic activity and weak serum protein binding

Here we present a proof-of-concept study, combining two known antimicrobial agents into a hybrid structure in order to develop an emergent cationic detergent-like interaction with the bacterial membrane. Six amphiphilic conjugates were prepared by copper (I)-catalyzed 1,3-dipolar cycloaddition between a neomycin B-derived azide and three alkyne-modified phenolic disinfectants. Three conjugates displayed good activity against a variety of clinically relevant Gram positive and Gram negative bacteria, including MRSA, without the high level of hemolysis or strong binding to serum proteins commonly observed with other cationic antimicrobial peptides and detergents.

Two-Phase Titration Method for Cationic Surface Active Agents Determination with Use of 5-(3-Bromo-4-hydroxy-5-methoxyphenyl)-7,7-dimethyl-7H-indolo[1,2-a]quinolinium Perchlorate Dye

Abstract The direct two-phase titration method with use of solvatochromic dye, i. e. 5-(3-bromo-4-hydroxy-5-methoxyphenyl)-7,7-dimethyl-7H-indolo[1,2-a]quinolinium perchlorate (1) for cationic surfactants determination is presented which is the modification of an earlier described procedure for anionic surface active substances detection. The method was tested on standard solution of benzethonium chloride (cationic detergent) which enables to estimate its accuracy as well as on few commercially available products to verify its utility. The influence of pH value of the probe on the titration endpoint determination was discussed and a simple solution of this problem has been proposed.

The Amphiphilicity of Allorcits and the Rate of ATP Hydrolysis and Flopping by P-Glycoprotein

We investigated how the amphiphilicity of detergents influences the rate of ATP hydrolysis by P-glycoprotein. To this purpose we used 34 electrically neutral and cationic detergents with different head groups and alkyl chain lengths and thus varied amphiphilicity. The free energy of detergent partitioning into the lipid membrane (dominated by the alkyl chain) and the free energy of detergent binding from the membrane to the transporter (due to the polar head group) were determined. For seven of these detergents the rate of ATP hydrolysis (Log scale) decreased linearly with increasing affinity from water to the transporter as observed previously for typical P-glycoprotein allocrits. Although, structurally closely related the remaining 27 detergents significantly reduced the rate of ATP hydrolysis. Inhibition of the P-glycoprotein-ATPase due to membrane disordering could be excluded based on order parameter measurements of deuterated lipid bilayers in the presence of relevant detergent concentrations using D-NMR spectroscopy. An optimal rate of ATP hydrolysis was observed only if the two free energies of binding were in appropriate proportion to each other, i.e., if the ratio of the two free energies of binding was slightly above three for cationic, and somewhat below three for neutral detergents. Lower or higher ratios inhibited the P-glycoprotein-ATPase activity and effective allocrit flopping.

Effect of solid-phase chemical modification on the features of the lipase from Thermomyces lanuginosus

The lipase from Thermomyces lanuginosus (TLL) was immobilized on octyl Sepharose and further modified with ethylenediamine (EDA) after activation of the carboxylic groups with carbodiimide. Different degrees of modification of the carboxyl groups were carried out by controlling the concentration of carbodiimide (10%, 50% or 100%). Subsequently, the effect of incubation of the modified preparations on hydroxylamine to recover the modified tyrosine was also studied. The modified enzymes exhibited a mobility in native electrophoresis quite different from that of the unmodified lipase (as expected by the changes in charge), and required higher concentrations of cationic detergent to become desorbed from the support. Interestingly, the chemical modification of the immobilized TLL produced an improvement in its activity, proportional to the amination degree. This increase in activity was much more significant at pH 10, where the fully modified preparation increased the activity by a factor of 10 as compared to the unmodified preparation. Moreover, the incubation of the chemically aminated preparations in a hydroxylamine solution improved the activity by an additional factor of 1.2. The fully aminated and incubated in hydroxylamine preparation exhibited a thermostability higher than that of the unmodified preparation, mainly at pH 5 (almost a 30 fold factor). In the presence of tetrahydrofurane, some stabilization was observed at pH 7, while at pH 9 the stability of the modified enzyme decreased (under all the assayed amination degrees) when compared to that of the unmodified enzyme. Thus, this simple protocol may be a rapid and efficient way of preparing a TLL biocatalyst with higher activity and stability, although this will depend on the inactivation conditions.

Ultrasound- assisted emulsification microextraction for separation of trace amounts of antimony prior to FAAS determination

We describe a simple and rapid method for the ultrasound-assisted microextraction of antimony using the solidified floating organic drop method. The effects of pH, type and volume of the extractant, time of sonication, amount of chelating agent, type and amount of surfactant were investigated and optimized. Bromopyrogollol red is acting as the chelating agent. Antimony(III) ion was extracted into finely dispersed droplets of undecanol after ion-pair formation with the water soluble chelator and the cationic detergent benzyldimethyltetradecylammonium chloride. Flame atomic absorption spectrometry was used for the detection. The resulting calibration is linear in the concentration range from 4.0 to 900 ng mL-1 of Sb(III) with a correlation coefficient of 0.9981. The enrichment factor is 67, the detection limit is 0.62 ng mL-1, and the relative standard deviation is ± 3.6% (at 100 ng mL-1; for n = 10). The method was successfully applied to the determination of antimony in water samples.

Structural studies on interaction of lysozyme with cationic detergents, cetyl- and dodecyl-trimethylammonium bromides

Structural studies on interaction of lysozyme with cationic detergents, cetyl- and dodecyl-trimethylammonium bromides

Benzalkonium chloride (BAK) induces apoptosis or necrosis, but has no major influence on the cell cycle of Jurkat cells

Benzalkonium chloride (BAK) is a cationic detergent with a very slow turnover. Because of its strong antibacterial activities, BAK is widely used especially in dentistry and ophthalmology. It is the most commonly used preservative in topical ophthalmic medications. Due to chronicity and widespread use of such treatments, BAK's side effects are of great importance. BAK toxicity for adherent cells, probably related to its pro-oxidative activities, is time- and dose-dependent. Although lymphocytes often infiltrate superficial eye tissues, the BAK influence on them is yet to be established. The aim of this study was to check BAK cytotoxicity on T lymphocytic Jurkat line cells and to verify the suggestion that BAK can induce G2M cell blocks. A dose- and time-dependent cytotoxic effect of BAK on lymphoid cells in relatively low concentrations was shown in this study. In lower concentrations, it shows a moderate apoptotic and minimal antiproliferative effect on Jurkat cells, while in higher concentrations it shows a rapid necrotic effect. No G2M cell blocks were observed. Our findings could suggest lymphoid dysfunction during intensive, prolonged topical BAK treatment, even at dosages relatively non-toxic to epithelial eye cells.

Denaturation of human serum albumin initiated by cetyltrimethylammonium bromide as monitored via the intrinsic fluorescence of the protein and fluorescence of an eosin probe

An analysis of the intrinsic fluorescence of the protein and the fluorescence of the eosin molecular probe in solutions of the protein was used to study the denaturation of human serum albumin (HSA) under the action of cetyltrimethylammonium bromide (CTAB), a cationic detergent, at various pH values. The denaturation of HSA under the influence of CTAB is a single-stage process over the entire pH range covered, pH (3.5–8.0). The maximum possible loosening of HSA globules in the presence of CTAB is reached at [CTAB] = 4 mmol/l. This detergent effectively denatures HSA at pH values higher than the pI of the protein (4.7).

Strong Response of Multilayer Polyelectrolyte Films to Cationic Surfactants

The influence of common cationic surfactants on the physical properties of differently composed polyelectrolyte films prepared by the layer-by-layer (LbL) technology was investigated. Free-standing polyelectrolyte films as microcapsules showed a fast, strong response to the addition of less than 1 mM cationic surfactant cetyltrimethylammonium bromide (CeTAB). As a function of the polyelectrolyte composition, the behavior of the capsules varied from negligible changes to complete disintegration via strong swelling. The response of microcapsules consisting of (poly(allylamine hydrochloride)(PAH)/poly(styrene sulfonate)(PSS))(4) was associated with a 5-fold volume increase, a fast switch of permeability, and in the case of fluorescently labeled films a 4-fold increase in fluorescence intensity. The kinetics and strengths of the interaction process were investigated by confocal laser scanning microscopy (CLSM) and fluorescence spectroscopy. Also, the relative stabilities of the polycation/polyanion and surfactant/polyanion complexes were determined. A mechanism was suggested to explain the interactions between the cationic surfactants and polyelectrolyte capsules. The strong response can be exploited in potential applications such as the triggered release of drugs or other encapsulated materials, the fluorescence-based detection of cationic detergents, and a switchable stopper in microchannels. However, the high sensitivity of LbL films to traces of cationic surfactants can also limit their applicability to the encapsulation of drugs or other materials because pharmaceutical or technical formulations often contain cationic surfactants as preservatives such as benzalkonium salts (BAC). It was demonstrated that undesired capsule opening can be effectively prevented by cross-linking the polyelectrolyte multilayers.

In Vivo 2H-NMR Study of the Action of Antibacterial Agents on Escherichia Coli Membranes

Contrary to eukaryote membranes, bacterial membranes are negatively charged owing to the presence of phosphatidylglycerol (PG) or cardiolipin in their inner membranes. One of the action mechanisms of antibiotics such as antimicrobial peptides is to selectively interact with negatively charged lipids to create a breach in the bacterial membrane. The characterization of the cellular membrane integrity is, thus, a key element in understanding the mechanism of action of antimicrobial agents. Solid-state nuclear magnetic resonance spectroscopy (SS-NMR) is a useful tool that allows probing the organization and dynamics of phospholipids in a bilayer. In this work, we have performed an in vivo 2H-NMRstudy of Escherichia coli membranes labeled with deuterated palmitic acid. More specifically, we have studied the effect of nanoparticles (NPs) and antibacterial molecules on the bacteria membrane. The 2H-NMR spectra obtained on intact cells show that the E. coli membranes were successfully labeled according to previous work. A 10-hour exposure to the cationic detergent cetyltrimetylammonium chloride (CTAC) shows increased membrane fluidity at high detergent concentration (320µM). The study of the effect of fullerenol reveals that these NPs increase the lipid mobility in the membrane especially after 8 hours of exposure. The 2H-NMR spectra obtained in the presence of polymyxin B show no fast-tumbling membrane fragments, although UV analysis indicates the leakage of the cell content. These results suggest that this antibiotic would perforate E. coli membranes without their complete disruption. Our work illustrates the use of in vivo2H NMR studies to understand the specific action of different substances on labeled biological membranes.

Adsorption of Tetramethylthiourea in the Presence of Cationic Detergent at Interface Electrode / Aqueous Perchlorate Solutions

The mixed adsorption layers of tetramethylthiourea and a cationic detergent formed at electrode surface in NaClO4 (1 mol dm -3 ) are described. The systems were characterized by the measurement of dif- ferential capacity, zero charge potential, and surface tension at this potential. The data were analyzed to obtain the surface pressure and relative surface excess of tetramethylthoiurea as a function of electrode charge at bulk concentration of studied compounds. It was stated that the adsorbing tetramethylthoiurea molecule pushed out the water molecule instead of the detergent molecule from the electrode surface. The constants of the Frumkin and corrected Flory-Huggins isotherm point to a decreased TMTU adsorp- tion energy in the cationic detergents presence and stronger repulsive interaction. Electrostatic para- meters of the inner layer were assigned. In the range of more negative potentials, mixed adsorption layers were described by investigating the kinetics of the Zn 2+ ions electroreduction as a piloting ion. (doi: 10.5562/cca1791)

Antiviral and Virucidal Activities of N-Cocoyl-L-Arginine Ethyl Ester

Various amino acid-derived compounds, for example, Nα-Cocoyl-L-arginine ethyl ester (CAE), alkyloxyhydroxylpropylarginine, arginine cocoate, and cocoyl glycine potassium salt (Amilite), were examined for their virucidal activities against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), influenza A virus (IAV), and poliovirus type 1 (PV-1) in comparison to benzalkonium chloride (BKC) and sodium dodecylsulfate (SDS) as a cationic and anionic control detergent and also to other commercially available disinfectants. While these amino acid-derived compounds were all effective against HSV-1 and HSV-2, CAE and Amilite were the most effective. These two compounds were, however, not as effective against IAV, another enveloped virus, as against HSV. Cytotoxicity of CAE was weak; at 0.012%, only 5% of the cells were killed under the conditions, in which 100% cells were killed by either SDS or BKC. In addition to these direct virucidal effects, CAE inhibited the virus growth in the HSV-1- or PV-1-infected cells even at 0.01%. These results suggest a potential application of CAE as a therapeutic or preventive medicine against HSV superficial infection at body surface.