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Article

Research article

J. Microbiol. Biotechnol. 2018; 28(5): 707-717

Published online May 28, 2018 https://doi.org/10.4014/jmb.1802.02027

Copyright © The Korean Society for Microbiology and Biotechnology.

Development and Characterization of an Anti-Acne Gel Containing Siamese Crocodile (Crocodylus siamensis) Leukocyte Extract

Weeraya Phupiewkham 1, 2, Qiumin Lu 3, Wisarut Payoungkiattikun 2, Threeranan Temsiripong 4, Nisachon Jangpromma 2, 5, Ren Lai 3 and Sompong Klaynongsruang 1, 2*

1Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand, 2Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand, 3Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences and Yunnan Provinces, Kunming Institute of Zoology, Kunming 650223, Yunnan, China, 4Sriracha Moda Co., Ltd., Sriracha, Chonburi 20110, Thailand, 5Office of the Dean, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand

Correspondence to:Sompong  Klaynongsruang
somklay.s@gmail.com

Received: February 19, 2018; Accepted: March 8, 2018

Abstract

Leukocytes are reportedly the first line of the innate immune defense and essential for the control of common bacterial infections. Therefore, in this work, the antibacterial activity of crocodile leukocyte extract against Propionibacterium acnes was evaluated, and we also characterized the related activity of skin infection. The leukocyte extract showed the minimum inhibitory concentration to be 100 μg/ml to P. acnes. SEM imaging demonstrated that the leukocyte extract adversely affected P. acnes cell permeability in a concentration-dependent manner. Furthermore, the crocodile leukocyte extract could significantly reduce proinflammatory markers and decrease inflammatory signs in infected mouse ears. The crude leukocyte extract was further purified using FPLC and RP-HPLC. The resulting fraction F5 was indicated as the anti-acne peptide-containing fraction. The molecular mass of the peptide contained in F5 was calculated to be 4,790.5 Da. N-Terminal sequencing revealed the amino acid sequence as GPEPVPAIYQ, which displays similarities to immunoglobulin A and leucine-rich repeat neuronal protein. This is the first reported amino acid sequence of a crocodile leukocyte extract that possesses anti-acne activity. To attempt to use it in a prototype cosmetic, an anti-acne gel containing crude crocodile leukocyte extract was formulated, resulting in seven gel formulations (G1, G2, G3, G4, G5, G6, and G7). The formulations G5, G6, and G7 exhibited 2-fold higher anti-acne activity than G1-G4. Investigation of accelerating stability studies of anti-acne gel formulations G5, G6, and G7 demonstrated that a low storage temperature (4ºC) is suitable for maintaining the physical properties and biological activity of the anti-acne gel products.

Keywords: Antimicrobial peptide, white blood cells extract, Propionibacterium acnes, skin bacterial infection, Crocodylus siamensis

Introduction

Acne vulgaris, known as acne, is a common chronic disease caused by abnormal sebaceous production within skin follicles. This disease often affects self-confidence. The pathological feature of acne starts when abnormal sebaceous production occurs. After that, abnormal keratinization in the follicle results in a comedo. Then, the inflammation process is triggered by Propionibacterium acnes, a skin-infecting bacterium [1]. Moreover, free radicals from lipid oxidation are demonstrated to be an indirect lesion of acne inflammation [2]. Although to date there are many therapeutics for acne treatment, they have a lot of side effects caused by chemical ingredients in the cosmetic products, which frequently result in skin irritation and bacterial resistance problems. Recently, many reports have demonstrated that natural active compounds such as proteins or peptides derived from plants and animals display anti-acne properties with low toxicity to humans [3]. Hence, many efforts have been made to use these compounds in the context of supplementary cosmetic products.

Tropical regions, particularly Southeast Asia, are a rich source of biodiversity, especially with high varieties of medicinal plant and animal extracts [4]. One medicinal derivative from animals is crocodile blood, a rich source of active proteins or peptides that demonstrate various biological properties [5]. Previous reports have shown that crocodile blood components, such as the serum of the American alligator (Alligator mississippiensis), exhibit antibacterial activity against Escherichia coli, and is anti-virus (HIV-1, WNV, and HSV-1) as well [6]. In addition, our reports demonstrate that Siamese crocodile (Crocodylus siamensis) blood had antibacterial activity, especially crocodile leukocytes. There are peptides that have been discovered from Siamese crocodile leukocyte extracts, including leucrocin I-IV [7]. These peptides exhibit broad-spectrum antimicrobial activity [7]. Moreover, crude crocodile leukocyte extract contains several biological properties, such as antioxidant activity [8] and anti-inflammatory activity [5]. Crocodile leukocyte extract is believed to represent a source of biologically active peptides, which may be suitable for developing a crocodile leukocyte-based cosmetics product as an anti-acne gel. Thus, in this study, the feasibility of preparing crocodile leukocyte extract anti-acne skin-care gel products was assessed.

Materials and Methods

Preparation and Extraction of Crocodile Leukocytes

Crocodile (C. siamensis) leukocytes were collected according to Theansungneon et al. [8]. Then, they were thawed on ice and resuspended with 10% (v/v) acetic acid solution. The suspension solution was homogenized by an ultrasonicator (Sonics Vibra Cell, VCX 750, USA) and the homogenate was centrifuged at 12,000 ×g for 30 min. The crude crocodile leukocyte extract was collected and concentrated by lyophilizers (LaboGene; Universal Analytical & Testing Instruments Ltd., China).

Antimicrobial Activity Assay

The antibacterial activity was determined by broth micro-microdilution antibacterial assay against P. acnes as modified according to Theansungneon et al. [8]. P. acnes ATCC 14916 was grown in supplemented BHI broth until the logarithmic phase and diluted to an absorbance at 600 nm (OD600) of 0.01. Then, the crude crocodile leukocyte extract was co-incubated with bacteria for 24 h at 37ºC under anaerobic conditions. The percentage of bacterial growth inhibition (BGI) was calculated as in Eq. (1), where ON and OS are the optical density of a negative control (ON) and sample (OS), respectively.

BGI(%)=ON-OSON×100

Scanning Electron Microscopy (SEM)

The SEM was performed according to the method of Pakdeesuwan et al. [9] with slight modifications. First, P. acnes DMST 14916 was grown in Scheadler medium broth until it reached the logarithmic phase and was then harvested by centrifugation at 3,000 ×g for 5 min. The cell pellet was resuspended and diluted with fresh culture medium to a final concentration of 1 × 108 CFU/ml. The cell suspensions were treated with crude crocodile leukocyte extracts at concentrations of 0.5×, 1×, and 5× MIC, with incubation at 37ºC under anaerobic conditions for 24 h. After that, 100 μl aliquots of bacterial cell solutions were carefully pipetted and applied on a 0.2 μm cellulose acetate membrane filter (Sartorius AG, Germany) for 30 min, whereupon cells were fixed with 300 μl of 2 % (v/v) g lutaraldehyde (Sigma, USA) f or 1 h. T he f ixed material was dehydrated by rinsing repeatedly (for 15 min) with a series of ethanol fixing solutions containing 30%, 50%, 70%, 90%, and 100% ethanol, in order. Dry materials were coated by a sputter coater (SC7620; Polaron, UK) with gold palladium, and examined by SEM (LEO1450VP; LEO Electron Microscopy, UK) operating at 12-20 kV. Then, the cell morphological changes for all treatments of P. acnes DMST 14916 were visualized using SEM (model series 1450VP; Leo, UK).

In Vivo Anti-Inflammatory Activity of Crude Crocodile Leukocyte Extract

Animals. Male ICR mice (6-8 weeks old) were obtained from the National Laboratory Animal Center of Mahidol University (Salaya, Nakorn Pathom, Thailand). The animals were housed in an environmentally controlled room (temperature 22 ± 1ºC, 55 ± 5% relative humidity, and 12 h dark-light cycle) and given food and water ad libitum. All procedures in this study complied with the Guide for the Care and Use of Laboratory Animals and were approved by the ethics committee of the institutional animal care and use committee (IACUC) of Khon Kaen University, Khon Kaen, Thailand (AEKKU 32/2557).

In vivo anti-inflammatory activity. Anti-inflammatory activity of crude crocodile leukocyte extract was determined in the animal model according to the method of Huang et al. [10] with slight modifications. Male IRC mice (n = 42) were randomly divided into seven groups. All mice were given intradermal injections with P. acnes (1 × 107 CFU/20 μl) to the right ear, except for the non-infected group. Then, the injected ears were treated with crude crocodile leukocyte extracts (125, 250, 500, and 1,000 mg/kg) or clindamycin (RPC International Co., Ltd. Thailand) (300 mg/kg) as a positive control and compared with the (non-infected) group which was injected with phosphate-buffered saline (PBS, pH 7.4). Then, all male ICR mice were housed for 24 h before the histopathological examination, including toxicity test and signs of edema.

Histological examination. Triplicate inflammation analyses of the mouse ears were performed. The ears were cut and then fixed in 10% formaldehyde for 1 week. Then, the fixed tissues were embedded in paraffin blocks and sectioned into 3-4 μm wide slices. The cross-sections were stained with hematoxylin and eosin and then observed under a bright-field microscope (Nikon Eclipse 80i microscope; Nikon, Japan) [11].

Measurement of tumor necrosis factor alpha (TNF-α) levels in mice serum. The levels of TNF-α were quantified using the Mouse TNF-α ELISA Max standard (Biolegend, Inc., USA) according to the manufacturer’s instructions.

Peptide Purification

Crude crocodile leukocyte extract (0.05 g) was dissolved in double-distilled water (DDW) and centrifuged at 5,000 ×g for 10 min. The supernatant was collected and the protein concentration was measured by the Bradford method [12], using serum albumin as a protein standard. Subsequently, the crude crocodile leukocyte extract was subjected to the FPLC purification system (ÄKTA Explorer; GE Healthcare, Sweden) equipped with a Resource Q column (6 ml, 15 μm × 16 mm; GE Healthcare, Sweden) that had been previously equilibrated with 25 mM Tris-HCl buffer (pH 7.8)[13]. Then, the protein was eluted with a linear gradient of 1 M NaCl at a flow rate of 2 ml/min. The eluted peaks were assayed for antibacterial activity against P. acnes. Then, the highest antibacterial activity-containing peak was further subjected to an amicon ultra-15 centrifugal tube (Merk Millipore Ltd., Ireland) with 10 kDa molecular mass cut-off. The protein solution was centrifuged at 4,500 ×g for 40 min and then the retentate was collected and dried using a lyophilizer (Martin Christ freeze dryers, John Morris Group, Australia). After that, the dried material was dissolved in DDW before further purification using RP-HPLC (Water Corp., UK), as described by He et al. [13], equipped with a water XBridge C8 column (5 μm, 250 × 4.6 mm; Water Corp., UK). For RP-HPLC purification, the sample was pre-filtrated through a 0.2 μm filter membrane (Millex filters; Merck Millipore, Germany) before being injected into RP-HPLC, which was pre-equilibrated with 0.1% trifluoroacetic acid (0.1% TFA, solvent A). The peptide was eluted using a mobile phase system of 100% acetonitrile (solvent B). A linear gradient was performed with 0-60% solvent B for 60 min, and then 61-100% solvent B for 10 min at a flow rate of 1 ml/min. Fractions of purified peptides were collected, lyophilized, and resuspended in sterile DDW. Then, the purified peptides were rechecked for antimicrobial activity using the disc diffusion technique, and N-terminal amino acid sequencing was performed.

N-Terminal Amino Acid Sequencing Analysis

The purified antimicrobial peptides obtained by RP-HPLC were further analyzed for molecular mass by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF; Bruker, China) in positive-ion and linear mode according to the recommended operation parameters (ion acceleration voltage 20 kV, accumulation time of a single scan 50 sec). The polypeptide mass standard was used as an external standard. The accuracy of mass determinations was within 0.1%. Then, amino acid sequences of the purified peptides were determined by automated Edman degradation on an amino acid sequencer (Model PPSQ-31A Protein Sequencer; Shimadzu, UK).

Development of Crude Crocodile Leukocyte Extract Anti-Acne Gel

The anti-acne gel was formulated following the method of Vats and Sharma [14] with some modifications. Crude crocodile leukocyte extract was used as the main ingredient in the anti-acne gel preparations. Briefly, the gel was made in seven formulations according to the crude crocodile leukocyte extract concentration. Gel formulations G1 G4 had 0%, 0.01%, 0.02%, and 0.03% of crude crocodile leukocyte extract respectively. Hereafter, these are referred to as gel formula 1 (G1), gel formula 2 (G2), gel formula 3 (G3), and gel formula 4 (G4). Each of these contained deionized water, polysorbate 60, carbomer, PEG-40, hydrogenated castor oil, bisabolol, salicylic acid, Anthemis nobilis flower extract, Eugenia caryophylius (clove) flower oil, retinol (vitamin A), tocopheryl acetate (vitamin E), and ascorbic acid (vitamin C) as the gelling agents. The other formulations had an equal amount of crude crocodile leukocyte extract at a concentration of 2× MIC (20 mg/ 100 g gel weight), preservative (methylparaben), triethanolamine, glycerin and different types of gelling agents (sodium-carboxymethylcellulose (SCMC), carboxy-polymethyline (carbomer), and methylcellulose (MC)), and these were named formulas G5, G6, and G7, respectively.

Determination of the Anti-P. acnes Properties of Anti-Acne Gels

Each gel formulation (G1, G2, G3, G4, G5, G6, and G7) was used to test antibacterial activity. Of these formula gels, 0.5 g of each was dissolved in 0.5 ml of 0.5% dimethyl sulfoxide (DMSO). Then, bacterial growth inhibition assay was performed using the agar well diffusion technique as described by Valgas et al. [15]. Briefly, the logarithmic growth phase of P. acnes DMST 14916 was diluted to OD600 = 0.01 in BHI broth and swabbed on a BHI agar plate. Each 100 μl/well of dissolved gel solution was added into the well and incubated at 37ºC for 24 h under anaerobic conditions. The zone of inhibition was measured on the agar plate.

Accelerated Condition Determination

Physical properties of anti-acne gel. The best formula that contained high antibacterial activity was used for the study of accelerated storage condition.

Viscosity. Viscosities of the anti-acne gels (G5, G6, and G7) were determined using the Wells-Brookfield Cone/Plate Viscometer (DV1 Viscometer, Cone spindle: CPA 52Z; AMETEK Brookfield, USA). The sample was placed on the center of the plate and the viscosity was measured at the speed of 5 rpm at 37ºC.

pH. Twenty milligrams of anti-acne gels (G5, G6, and G7) was dissolved in 1 ml of 0.5% DMSO. Then, the pH of the samples was measured and recorded within 24 h.

Antibacterial activity determination. A 0.5 g sample of each gel formula (G5, G6, and G7) was dissolved in 0.5 ml of 0.5% DMSO. Then, antibacterial assay against P. acnes DMST 14916, Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 12228 was performed using the agar well diffusion technique.

Stability studies. The anti-acne gels (G5, G6, and G7) were kept at 4ºC and 45ºC for 3 months. Then, the samples were collected every month for determination of viscosity, pH, and antibacterial activity.

Statistical Analysis

Statistical values of all experimental results were calculated using analysis of variance, followed by Duncan’s multiple range test. These values are presented as the mean ± SD. A value of p < 0.05 was accepted as being significant.

Results

Anti-P. acnes Activity and Disruption of P. acnes Cell Morphology

After leukocyte extraction, the antibacterial activity against P. acnes ATCC 14916 was determined by liquid growth inhibition assay. The results demonstrated that the crude crocodile leukocyte extract showed anti-P. acnes activity in a concentration-dependent manner. An MIC of about 100 μg/ml was obtained (Fig. 1). In addition, the killing mechanism against P. acnes cells was visualized by SEM analysis (Fig. 2). Overall, the crude crocodile leukocyte extract affected bacterial cell membrane integrity. In detail, untreated bacterial cells illustrated a normal pleomorphic structure as regular rod-like structures with a smooth surface and fimbriae around the organism (Fig. 2A), whereas the negative control resulted in bacterial cell membrane shrinking. This might be an effect from responding to osmotic pressure (Fig. 2B). In addition, the clindamycin tested cells displayed cell shrinkage and blebbing (Fig. 2C). However, crude crocodile leukocyte extract-treated bacteria displayed irregularly deformed bacterial cell surfaces (Figs. 2D-2F). The abnormalities in cell morphology, including roughness, shrinking and cellular collapse, was found to be directly proportional to the crude crocodile leukocyte extract concentration (Figs. 2D-2F).

Figure 1. Antibacterial activity of crude leukocyte extracted from Crocodylus siamensis against Propionibacterium acnes DMST 14916 in liquid growth inhibition assay. Positive control (PC): Clindamycin (50 μg/ml); and negative control (NC): 0.01% acetic acid. Each bar represents the mean ± SD (n = 3). Different letters indicate significant differences between groups (p < 0.05).

Figure 2. Observation of P. acnes DMST 14916 cell morphology using a scanning electron microscope. (A) Untreated P. acnes DMST 14916 cells. (B) Negative control (NC): sterilized double-distilled water-treated P. acnes DMST 14916 cells. (C) Positive control (PC): 50 μg/ml Clindamycin-treated P. acnes DMST 14916 cells. (D-F) Crude crocodile leukocyte extract at concentrations of 0.5× MIC, MIC, and 5× MIC were co-incubated with P. acnes DMST 14916.

Effect of Crocodile Leukocyte Extract on In Vivo P. acnes DMST 14916-Induced Inflammation

The histological analysis demonstrated that crude crocodile leukocyte extracts (125, 250, 500, and 1,000 mg/kg) could reduce the inflammation area in a concentration-dependent manner (Fig. 3). As a result, micro-abscesses were found in the dermis after 24 h of giving the P. acnes DMST 14916 injection. The inflammatory area in the mouse ear cross-sections predominantly consisted of neutrophil infiltration (Fig. 3B) when compared with the non-infected group (Fig. 3A). However, the 300 mg/kg clindamycin-treated group resulted in a dramatic decrease of the inflammation area with slight edema (Fig. 3C). In parallel, the crude crocodile leukocyte extract co-injected with P. acnes DMST 14916 demonstrated a reduction of ear swelling and infiltrated inflammatory cells in a dose-dependent manner (Figs. 3D-3G). In addition, the expression of proinflammatory cytokine TNF-α was investigated. From the results, the P. acnes DMST 14916-infected group showed a significant increase in TNF-α expression level. However, 250, 500, and 1,000 mg/kg crude crocodile leukocyte extract treatment groups showed significantly reduced TNF-α production (Fig. 4).

Figure 3. Histological analysis of mouse ears (A) Untreated: mouse ear injected with 20 μl vehicle (PBS, pH 7.4). (B) Negative control (NC): mouse ear infected with P. acnes DMST 14916 (1 × 107 CFU/20 μl). (C) Positive control (PC): mouse ear infected with P. acnes DMST 14916 (1 × 107 CFU/20 μl) and injected with 300 mg/kg clindamycin. (D-G) Mouse ear infected with P. acnes DMST 14916 (1 × 107 CFU/20 μl) and injected with crude crocodile leukocyte extracts (125, 250, 500, and 1,000 mg/kg). The arrows indicate areas of inflammation; scale bar = 100 μm.

Figure 4. Effect of crude crocodile leukocyte extracts (125, 250, 500, and 1,000 mg/kg) on inflammatory cytokine TNF-α production levels in serum of P. acnes DMST 14916 (1 × 107 CFU/20 μl)-induced mouse inflammation. Positive control (PC): mouse ear infected with P. acnes DMST 14916 (1 × 107 CFU/20 μl) and injected with 300 mg/kg clindamycin. Negative control (NC): mouse ear infected with P. acnes DMST 14916 (1 × 107 CFU/20 μl). All experiments were conducted in triplicate. Each bar represents the mean ± SD (n = 5). Different letters indicate significant differences between individual groups (p < 0.05).

Antimicrobial Peptide Purification

The results showed that crude crocodile leukocyte extract was purified into 10 peaks (viz., P1-P10) by anion-exchange chromatography (Fig. 5A). All peaks were then assayed for antimicrobial activity. Interestingly, P9 exhibited notable antibacterial activity with 30% P. acnes DMST 14916 inhibition (Fig. 5B). Then, P9 was further purified by C8 RP-HPLC, resulting in nine fractions (F1-F9) (Fig. 6). Only F5 exhibited the highest antibacterial activity against P. acnes (data not shown).

Figure 5. FPLC chromatogram of purified crude crocodile leukocyte extract using pre-packed columns (Resource Q) (A) Chromatogram obtained after gradient elution using buffer A containing 25 mM Tris-HCl and buffer B containing 50 mM NaCl in buffer A (pH 7.8) at a flow rate of 2 ml/min. (B) Antibacterial activity of FPLC fractions against P. acnes DMST 14916. Positive control (PC): Clindamycin (50 μg/ml); and negative control (NC): Solvent A. The data are expressed as the mean with standard deviation (n = 3). The different letters indicate significant differences between individual groups (p < 0.05).

Figure 6. RP-HPLC chromatogram of the active peak (P9). The sample was purified by C8 RP-HPLC using a linear elution gradient of 0-100% acetonitrile containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. The eluted peaks were monitored at a wavelength of 220 nm.

Molecular Weight Determination and N-Terminal Amino Acid Sequencing

The molecular mass of F5 was determined using MALDI-TOF/TOF mass spectrometry. The results show a single main molecular mass with the m/z ratio corresponding to 4,790.5 Da (Fig. 7). Then, the N-terminal amino acids of F5 were determined as Gly-Pro-Glu-Pro-Val-Pro-Ala-Ile-Tyr-Gln. BLAST search indicated that F5 belongs to integral membrane protein, which showed identity at 63% and 71%to immunoglobulin A (IgA) and leucine-rich repeat neuronal protein (LRRN) from C. siamensis, respectively.

Figure 7. Peptide mass spectrum of the anti-acne peptide (F5) as obtained from MALDI-TOF mass spectrometry and N-terminal sequences of anti-acne peptides. The molecular mass of the anti-acne peptides (F5) is represented by m/z.

Development of Crude Crocodile Leukocyte Extract Anti-Acne Gels

Biological property of anti-acne gel formulations. The above results demonstrated that crude crocodile leukocyte extract contains antibacterial peptides that affect acne vulgaris-causing bacteria. Thus, the agar-well diffusion assay was used to determine the antimicrobial activity of all anti-acne gel formulations. From the results, P. acnes DMST 14916 showed a higher sensitivity to G2, G3, and G4 gel formulas than S. aureus ATCC 25923, with the exception of S. epidermidis ATCC 12228. Overall, the results demonstrated that the developed anti-acne gel formulations G5, G6, and G7 displayed approximately 2-fold higher antibacterial activity than formulas G1-G4 (Table 1). Hence, anti-acne gel formulations G5, G6, and G7 were selected for study of the physical properties of the anti-acne gel before the accelerated condition assay.

Table 1 . Antimicrobial activity of anti-acne gels..

SampleInhibition zone (mm)a

Staphylococcus aureus ATCC 25923Staphylococcus epidermidis ATCC 12228Propionibacterium acnes DMST 14916
G1 (without crude leukocyte extract)---
G2 (0.01% of crude leukocyte extract)9.00±2.00-13.00±0.00
G3 (0.02% of crude leukocyte extract)9.33±0.00-14.34±1.15
G4 (0.03% crude leukocyte extract)7.00±2.89-15.67±8.08
G5 (SCMC+2× MIC crude leukocyte extract)14.33±1.15-18.67±3.21
G6 (Carbomer 934+2× MIC crude leukocyte extract)17.00±7.21-10.67±0.58
G7 (MC+2× MIC crude leukocyte extract)18.33±5.77-14.67±4.73
Clindalin gel (1% Clindamycin)26.67±2.89-28.34±2.89
Positive control (30 µg Clindamycin)34.00±2.8932.00±0.0040.00±0.00
Negative control (10% DMSO)---

aAll experiments were performed in triplicate. Carbomer, carboxy-polymethyline; SCMC, sodium-carboxymethylcellulose; MC, methylcellulose.



Determination of Physical Properties of Anti-Acne Gel

The characteristics of freshly prepared anti-acne gel via analysis of the physical properties of anti-acne gel formulas G5, G6, and G7 were determined. The results of the physical gel properties of color, appearance, pH, and viscosity are shown in Table 2. Formula G5 exhibited a light yellow color and was translucent. Formulas G6 and G7 were colorless and also translucent. In addition, the pH value of all formulations (G5, G6, and G7) was seen to be within 4.38-7.84. The viscosity ranged from 1,463.67 to 14,693.33 cP.

Table 2 . Evaluation of physical properties of anti-acne gel formulations..

aAll experiments were performed in triplicate; ± shows a standard deviation; cP, centipoise..



Study of the Storage Stability of the Anti-Acne Gels

The viscosity and pH of anti-acne gels (gel formulas G5, G6, and G7) with different storage conditions are shown in Table 3. No significant difference in viscosity was detected for gel formulation G5 during an accelerated storage period at 4ºC for 2 months. However, the viscosity of gel formulation G6 changed slightly after 2 months of storage. On the other hand, low stability of viscosity was detected in gel formulation G7. Low stability of viscosity was detected in gel formulas G5 and G6 during accelerated storage conditions at 45ºC. However, gel formulation G7 displayed high stability of viscosity after 2 months storage. The pH of gel formulations G5, G6, and G7 displayed high stability characteristics during 3 months of storage. In addition, the residual anti-skin bacteria activity during 3 months of storage at 4ºC was also investigated. The results demonstrated that the three anti-acne gel formulations (G5, G6, and G7) contained antibacterial activity against P. acnes DMST 14916 and also against S. aureus ATCC 25923 after storage for 3 months. Among these three gel formulations, G5 seemed to express the highest anti-skin bacteria activity against P. acnes DMST 14916 and S. aureus ATCC 25923 (Table 4). Months 1, 2, and 3 had anti-P. acnes DMST 14916 activity at 15.67 ± 1.15, 19.00 ± 1.73, and 14.67 ± 3.51 mm, respectively. Anti-S. aureus ATCC 25923 activity was 15.00± 0.70, 14.50 ± 0.71 and 11.50 ± 0.71 mm at 1, 2, and 3 months of storage, respectively. On the other hand, P. acnes DMST 14916 displayed the highest sensitivity to these three anti-acne gel formulations at all accelerated conditions at 45ºC for 3 months (Table 5).

Table 3 . Stability studies of anti-acne gel formulations..

ParameterRefrigerated storage period (Month)

Month 1Month 2Month 3
Viscosity
4 °C
G51747.33±5.66a1714.67±8.49a1440.00±31.13b
G6435.70±51.75b569.30±21.95a599.95±38.14a
G71388.33±91.92c1742.67±12.73a1680.33±8.15b
45 °C
G51613.00±35.68a1527.00±48.82b1344.33±39.07c
G6471.90±47.38c1696.00±10.36a582.30±27.65b
G71806.33±38.28a1605.50±18.95b1640.33±18.39b
pH
4 °C
G55.22±0.05a5.26±0.03a5.40±0.02a
G66.11±0.05a6.15±0.06a6.31±0.06a
G74.05±0.02a4.13±0.02a4.43±0.01a
45 °C
G55.36±0.01a5.54±0.05a5.35±0.03a
G66.49±0.13a6.07±0.01a6.25±0.03a
G74.15±0.02a4.03±0.12a4.35±0.06a

*All experiments were performed in triplicate; ± shows a standard deviation..

A different lowercase letter in the same row within the same formula with previous storage at 4°C denotes significant difference (p < 0.05) compared with the positive control (50 μg of clindamycin) of antibacterial activity..



Table 4 . Zone of inhibition of anti-acne gels after storage for 3 months at 4ºC..

SampleInhibition zone (mm)

P. acnes DMST 14916S. aureus ATCC 25923S. epidermidis ATCC 12228
Month 1
Positive control, clindamycin (50 µg)31.33±1.15a29.00±1.14aND
Negative control, 5% DMSO0i0gND
Crude leukocyte extract21.00±1.00c14.00±1.41cND
Anti-acne gel formula G515.67±1.15d15.00±0.70bND
Anti-acne gel formula G610.67±0.58h10.00±0.00fND
Anti-acne gel formula G713.67±0.58f12.50±3.54dND
Month 2
Positive control, clindamycin (50 µg)21.67±1.15c29.00±1.41aND
Negative control, 5% DMSO0i0gND
Crude leukocyte extract15.33±1.53d14.00±1.41cND
Anti-acne gel formula G519.00±1.73c14.50±0.71cND
Anti-acne gel formula G611.67±1.15g13.00±1.41dND
Anti-acne gel formula G712.00±0.00g12.50±0.71dND
Month 3
Positive control, clindamycin (50 µg)24.67±0.58b29.00±1.14a28.33±0.58a
Negative control, 5% DMSO0i0g0e
Crude leukocyte extract13.33±2.08f12.00±0.00e0e
Anti-acne gel formula G514.67±3.51e11.50±0.71f10.33±0.58b
Anti-acne gel formula G611.00±1.00h11.50±0.71f10.33±0.58b
Anti-acne gel formula G714.67±0.58e11.00±1.14f11.00±1.00b

*All experiments were performed in triplicate..

A different lowercase letter in the same column within the same formula with previous storage at 4°C denotes significant difference (p < 0.05) compared with the positive control (50 g of clindamycin) of antibacterial activity..



Table 5 . Zone of inhibition of anti-acne gel after storage for 3 months at 45ºC..

SampleInhibition zone (mm)

P. acnes DMST 14916S. aureus ATCC 25923S. epidermidis ATCC 12228
Month 1
Positive control, clindamycin (50 µg)32±2.89a29±1.14aND
Negative control, 5% DMSO0h0eND
Crude leukocyte extract21±0.71c13.5±2.12bND
Anti-acne gel formula G515±0.00e12.5±3.54cND
Anti-acne gel formula G610±0.00g10±0.00dND
Anti-acne gel formula G720±0.00d10±0.00dND
Month 2
Positive control, clindamycin (50 µg)25±0.58bNDND
Negative control, 5% DMSO0hNDND
Crude leukocyte extract16±0.00eNDND
Anti-acne gel formula G521±1.15cNDND
Anti-acne gel formula G611±1.15gNDND
Anti-acne gel formula G711±1.15gNDND
Month 3
Positive control, clindamycin (50 µg)24±1.15b30±0.00a31±1.41a
Negative control, 5% DMSO0h0e0e
Crude leukocyte extract15±0.00e0e0e
Anti-acne gel formula G520±0.00d12±0.00c20±0.00b
Anti-acne gel formula G611±1.41g12±0.00c15±0.00c
Anti-acne gel formula G713±1.73f12±0.00c11±1.41d

*All experiments were performed in triplicate..

A different lowercase letter in the same column within the same formula with previous storage at 45°C denotes significant difference (p < 0.05) compared with the positive control (50 g clindamycin) of antibacterial activity..


Discussion

The effect of crude crocodile leukocyte extract on antibacterial activity against P. acnes and on P. acnes cell morphology changes were investigated using broth dilution assay and SEM. The results correlated with Pata et al. [7], who reported the broad-spectrum antimicrobial properties of Siamese crocodile white blood cell extracts. In addition, they also discovered novel antimicrobial peptides (leucrocins I-IV) that were derived from crocodile leukocyte extract. Here, we found that crocodile leukocyte extract could inhibit the growth of P. acnes by the disruption and permeabilization of bacterial cell membranes. These properties might result from active proteins or peptides contained in Siamese crocodile white blood cells. The research evidence demonstrates that this mechanism is an essential step for the bacterial killing property of peptide substances [16]. Based on this recent discovery, crocodile white blood cell extract might be suitable for use as an active ingredient of cosmetic products for treating acne vulgaris infection or related skin diseases. Thus, the effect of crocodile white blood cell extract on an in vivo model of inflammation should be pursued. From the obtained results, crude crocodile leukocyte extract decreased the inflammation caused by P. acnes infection through the downregulation of TNF-α production levels. Hence, crude crocodile leukocyte extract could decrease the inflammatory signs [10, 18]. Crude crocodile leukocyte extract not only exhibits significant anti-inflammatory activity by reducing TNF-α production, but might also promote the wound-healing process [19].

Encouraged by the results of the inhibitory mechanism study against P. acnes on the in vitro and in vivo models, we believe that the promising biological activities might result from the active peptides or proteins contained in the crocodile leukocyte extract. Hence, an effort to purify active peptides was performed using anion-exchange and reverse-phase high-performance liquid column chromato-graphy. Moreover, protein purification and N-terminal amino acid sequencing were performed. The results characterized the obtained peptide as having a strong hydrophobicity, with a molecular mass of 4,790.5 Da. The N-terminal sequencing indicates that this peptide is a novel peptide derived from crocodile leukocyte extract, and displays similarities to IgA and LRRN. This protein is involved in the transmembrane peptides/proteins (TMPs) family. TMPs have been previously characterized as having highly hydrophobic properties that enabled them to penetrate through the membrane and lipid bilayer, and interact with the hydrophobic tails of the lipid molecules in the interior of the bilayer of bacteria [20]. These results suggest a possible anti-P. acnes mechanism for the novel peptide derived from crocodile leukocyte extract. In addition this is the first report of anti-acne peptides derived from animals [21].

According to the collected results, the promising anti-P. acnes and anti-inflammation activities of crocodile leukocyte extract might make it a good candidate for use as an active ingredient of cosmetic products. Cosmetic formulations in the form of emulsions, such as gel or emulsion, are the most often used for skin-care products [22]. Gels are also absorbed faster than creams or other skin-care products, and then can release an active ingredient as well because they are mostly water-based [23]. Moreover, the accelerated storage condition results reflected the stability of the cosmetic products during shelf life [24]. The results demonstrate significantly that a low storage temperature (4ºC) is suited for maintaining the physical properties and biological activity of the anti-acne gel products.

In summary, the crude crocodile leukocyte extract displayed notable antibacterial activity against skin-infecting bacteria. In addition, the extract could reduce inflammatory signs in P. acnes-infected mice. We speculate that this activity is derived from active peptides or proteins contained in the crude crocodile leukocyte extract. Hence, the collective results indicate that crude crocodile leukocyte extract may be a good candidate for development as an active ingredient of anti-acne gels. Thus, various anti-acne gel formulations have been developed. The results indicate that anti-acne gel formulations G5-G7 displayed more suitable anti-P. acnes activity than G1-G4. In addition, anti-acne gel formulations G5, G6, and G7 displayed high stability during 3 months of accelerated conditions test.

Acknowledgments

This research was supported by the Royal Golden Jubilee (RGJ) Ph.D. Program of Thailand Research Fund (grant number PHD/0141/2553), and we would like to thank the Protein and Proteomics Research Center for Commercial and Industrial Purposes (Pr°CCI), Faculty of Science, Khon Kaen University, Thailand for access to their laboratory equipment.

Conflict of Interest


The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Antibacterial activity of crude leukocyte extracted from Crocodylus siamensis against Propionibacterium acnes DMST 14916 in liquid growth inhibition assay. Positive control (PC): Clindamycin (50 μg/ml); and negative control (NC): 0.01% acetic acid. Each bar represents the mean ± SD (n = 3). Different letters indicate significant differences between groups (p < 0.05).
Journal of Microbiology and Biotechnology 2018; 28: 707-717https://doi.org/10.4014/jmb.1802.02027

Fig 2.

Figure 2.Observation of P. acnes DMST 14916 cell morphology using a scanning electron microscope. (A) Untreated P. acnes DMST 14916 cells. (B) Negative control (NC): sterilized double-distilled water-treated P. acnes DMST 14916 cells. (C) Positive control (PC): 50 μg/ml Clindamycin-treated P. acnes DMST 14916 cells. (D-F) Crude crocodile leukocyte extract at concentrations of 0.5× MIC, MIC, and 5× MIC were co-incubated with P. acnes DMST 14916.
Journal of Microbiology and Biotechnology 2018; 28: 707-717https://doi.org/10.4014/jmb.1802.02027

Fig 3.

Figure 3.Histological analysis of mouse ears (A) Untreated: mouse ear injected with 20 μl vehicle (PBS, pH 7.4). (B) Negative control (NC): mouse ear infected with P. acnes DMST 14916 (1 × 107 CFU/20 μl). (C) Positive control (PC): mouse ear infected with P. acnes DMST 14916 (1 × 107 CFU/20 μl) and injected with 300 mg/kg clindamycin. (D-G) Mouse ear infected with P. acnes DMST 14916 (1 × 107 CFU/20 μl) and injected with crude crocodile leukocyte extracts (125, 250, 500, and 1,000 mg/kg). The arrows indicate areas of inflammation; scale bar = 100 μm.
Journal of Microbiology and Biotechnology 2018; 28: 707-717https://doi.org/10.4014/jmb.1802.02027

Fig 4.

Figure 4.Effect of crude crocodile leukocyte extracts (125, 250, 500, and 1,000 mg/kg) on inflammatory cytokine TNF-α production levels in serum of P. acnes DMST 14916 (1 × 107 CFU/20 μl)-induced mouse inflammation. Positive control (PC): mouse ear infected with P. acnes DMST 14916 (1 × 107 CFU/20 μl) and injected with 300 mg/kg clindamycin. Negative control (NC): mouse ear infected with P. acnes DMST 14916 (1 × 107 CFU/20 μl). All experiments were conducted in triplicate. Each bar represents the mean ± SD (n = 5). Different letters indicate significant differences between individual groups (p < 0.05).
Journal of Microbiology and Biotechnology 2018; 28: 707-717https://doi.org/10.4014/jmb.1802.02027

Fig 5.

Figure 5.FPLC chromatogram of purified crude crocodile leukocyte extract using pre-packed columns (Resource Q) (A) Chromatogram obtained after gradient elution using buffer A containing 25 mM Tris-HCl and buffer B containing 50 mM NaCl in buffer A (pH 7.8) at a flow rate of 2 ml/min. (B) Antibacterial activity of FPLC fractions against P. acnes DMST 14916. Positive control (PC): Clindamycin (50 μg/ml); and negative control (NC): Solvent A. The data are expressed as the mean with standard deviation (n = 3). The different letters indicate significant differences between individual groups (p < 0.05).
Journal of Microbiology and Biotechnology 2018; 28: 707-717https://doi.org/10.4014/jmb.1802.02027

Fig 6.

Figure 6.RP-HPLC chromatogram of the active peak (P9). The sample was purified by C8 RP-HPLC using a linear elution gradient of 0-100% acetonitrile containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. The eluted peaks were monitored at a wavelength of 220 nm.
Journal of Microbiology and Biotechnology 2018; 28: 707-717https://doi.org/10.4014/jmb.1802.02027

Fig 7.

Figure 7.Peptide mass spectrum of the anti-acne peptide (F5) as obtained from MALDI-TOF mass spectrometry and N-terminal sequences of anti-acne peptides. The molecular mass of the anti-acne peptides (F5) is represented by m/z.
Journal of Microbiology and Biotechnology 2018; 28: 707-717https://doi.org/10.4014/jmb.1802.02027

Table 1 . Antimicrobial activity of anti-acne gels..

SampleInhibition zone (mm)a

Staphylococcus aureus ATCC 25923Staphylococcus epidermidis ATCC 12228Propionibacterium acnes DMST 14916
G1 (without crude leukocyte extract)---
G2 (0.01% of crude leukocyte extract)9.00±2.00-13.00±0.00
G3 (0.02% of crude leukocyte extract)9.33±0.00-14.34±1.15
G4 (0.03% crude leukocyte extract)7.00±2.89-15.67±8.08
G5 (SCMC+2× MIC crude leukocyte extract)14.33±1.15-18.67±3.21
G6 (Carbomer 934+2× MIC crude leukocyte extract)17.00±7.21-10.67±0.58
G7 (MC+2× MIC crude leukocyte extract)18.33±5.77-14.67±4.73
Clindalin gel (1% Clindamycin)26.67±2.89-28.34±2.89
Positive control (30 µg Clindamycin)34.00±2.8932.00±0.0040.00±0.00
Negative control (10% DMSO)---

aAll experiments were performed in triplicate. Carbomer, carboxy-polymethyline; SCMC, sodium-carboxymethylcellulose; MC, methylcellulose.


Table 2 . Evaluation of physical properties of anti-acne gel formulations..

aAll experiments were performed in triplicate; ± shows a standard deviation; cP, centipoise..


Table 3 . Stability studies of anti-acne gel formulations..

ParameterRefrigerated storage period (Month)

Month 1Month 2Month 3
Viscosity
4 °C
G51747.33±5.66a1714.67±8.49a1440.00±31.13b
G6435.70±51.75b569.30±21.95a599.95±38.14a
G71388.33±91.92c1742.67±12.73a1680.33±8.15b
45 °C
G51613.00±35.68a1527.00±48.82b1344.33±39.07c
G6471.90±47.38c1696.00±10.36a582.30±27.65b
G71806.33±38.28a1605.50±18.95b1640.33±18.39b
pH
4 °C
G55.22±0.05a5.26±0.03a5.40±0.02a
G66.11±0.05a6.15±0.06a6.31±0.06a
G74.05±0.02a4.13±0.02a4.43±0.01a
45 °C
G55.36±0.01a5.54±0.05a5.35±0.03a
G66.49±0.13a6.07±0.01a6.25±0.03a
G74.15±0.02a4.03±0.12a4.35±0.06a

*All experiments were performed in triplicate; ± shows a standard deviation..

A different lowercase letter in the same row within the same formula with previous storage at 4°C denotes significant difference (p < 0.05) compared with the positive control (50 μg of clindamycin) of antibacterial activity..


Table 4 . Zone of inhibition of anti-acne gels after storage for 3 months at 4ºC..

SampleInhibition zone (mm)

P. acnes DMST 14916S. aureus ATCC 25923S. epidermidis ATCC 12228
Month 1
Positive control, clindamycin (50 µg)31.33±1.15a29.00±1.14aND
Negative control, 5% DMSO0i0gND
Crude leukocyte extract21.00±1.00c14.00±1.41cND
Anti-acne gel formula G515.67±1.15d15.00±0.70bND
Anti-acne gel formula G610.67±0.58h10.00±0.00fND
Anti-acne gel formula G713.67±0.58f12.50±3.54dND
Month 2
Positive control, clindamycin (50 µg)21.67±1.15c29.00±1.41aND
Negative control, 5% DMSO0i0gND
Crude leukocyte extract15.33±1.53d14.00±1.41cND
Anti-acne gel formula G519.00±1.73c14.50±0.71cND
Anti-acne gel formula G611.67±1.15g13.00±1.41dND
Anti-acne gel formula G712.00±0.00g12.50±0.71dND
Month 3
Positive control, clindamycin (50 µg)24.67±0.58b29.00±1.14a28.33±0.58a
Negative control, 5% DMSO0i0g0e
Crude leukocyte extract13.33±2.08f12.00±0.00e0e
Anti-acne gel formula G514.67±3.51e11.50±0.71f10.33±0.58b
Anti-acne gel formula G611.00±1.00h11.50±0.71f10.33±0.58b
Anti-acne gel formula G714.67±0.58e11.00±1.14f11.00±1.00b

*All experiments were performed in triplicate..

A different lowercase letter in the same column within the same formula with previous storage at 4°C denotes significant difference (p < 0.05) compared with the positive control (50 g of clindamycin) of antibacterial activity..


Table 5 . Zone of inhibition of anti-acne gel after storage for 3 months at 45ºC..

SampleInhibition zone (mm)

P. acnes DMST 14916S. aureus ATCC 25923S. epidermidis ATCC 12228
Month 1
Positive control, clindamycin (50 µg)32±2.89a29±1.14aND
Negative control, 5% DMSO0h0eND
Crude leukocyte extract21±0.71c13.5±2.12bND
Anti-acne gel formula G515±0.00e12.5±3.54cND
Anti-acne gel formula G610±0.00g10±0.00dND
Anti-acne gel formula G720±0.00d10±0.00dND
Month 2
Positive control, clindamycin (50 µg)25±0.58bNDND
Negative control, 5% DMSO0hNDND
Crude leukocyte extract16±0.00eNDND
Anti-acne gel formula G521±1.15cNDND
Anti-acne gel formula G611±1.15gNDND
Anti-acne gel formula G711±1.15gNDND
Month 3
Positive control, clindamycin (50 µg)24±1.15b30±0.00a31±1.41a
Negative control, 5% DMSO0h0e0e
Crude leukocyte extract15±0.00e0e0e
Anti-acne gel formula G520±0.00d12±0.00c20±0.00b
Anti-acne gel formula G611±1.41g12±0.00c15±0.00c
Anti-acne gel formula G713±1.73f12±0.00c11±1.41d

*All experiments were performed in triplicate..

A different lowercase letter in the same column within the same formula with previous storage at 45°C denotes significant difference (p < 0.05) compared with the positive control (50 g clindamycin) of antibacterial activity..


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