Antibacterial and antibiofilm effects of Camellia oleifera seed dreg extract and its application in cosmetics

Cosmetic care products contain a high proportion of water and nutrients. Therefore, preventing bacterial growth is an important issue to ensure product quality and safety. The application of antibacterial natural ingredients derived from plants is considered to have the potential to maintain product quality and reduce the use of chemicals in formulations. Additionally, chemically synthesized antiseptic and antibacterial agents are widely used in the industry at present. However, some preservative ingredients have been reported that may cause skin irritation, redness, allergies, and even dermatitis.


| INTRODUC TI ON
The genus Camellia comprises a group of more than 100 species native to Southern Asia.The oil content in seeds of camellia is high, averaging approximately 30%, 1 and the content may be as high as greater than 50% in certain Camellia oleifera cultivars. 2Camellia oil has a high monounsaturated fatty acid content, 3,4 and is considered a healthy type of oil.It also contains other functional components such as saponins, polyphenol, and squalene. 5In addition to the high economic value of the oil, defatted camellia seed residue possesses insecticidal effects, and has been used in agriculture and aquaculture. 6,7Saponins in defatted camellia seed residue are considered the key and major compounds with bioactivities, and can be extracted with water or solvents. 8ponins are plant-based natural surfactants that constitute a large group of glycosides, including a steroid or triterpenoid sapogenin (non-saccharide) linked to one or more oligosaccharide moieties. 9They are classified into two main subclasses, steroidal, and terpenoid saponins, based on their different non-saccharide structures. 10They are detergent-like natural surfactants showing biological activities against bacterial and fungal strains.Previous studies have reported the use of saponins as biodegradable and renewable alternatives to synthetic surfactants, 11 and recent research has demonstrated that saponins have a toxic effect against pathogenic bacteria. 12With increasing environmental concern, there is a trend toward replacing synthetic products with natural equivalents by the pharmaceutical industry in food and cosmetics.
Microorganism control is an extremely important link in the cosmetics industry.Every country has specific laws and regulations.However, it is not uncommon for cosmetics to be contaminated or fail to pass the test, which will have a great impact on the quality, efficacy, and safety of products.Pseudomonas aeruginosa and Staphylococcus aureus are important pathogens that cause food poisoning, infections of skin and soft tissue, pneumonia, and sepsis. 13,14Some pathogenic bacteria strains can become lethal, and resistant bacteria can cause critical infections in immunocompromised patients, leading to healthcare-associated infections, septic shock, pneumonia, and wound infections.Conventional studies on microbiology focus on cultured planktonic bacteria.However, most bacteria exist in natural and clinical environments, and are known to be associated with living and nonliving surfaces. 15When bacteria form a biofilm, it is highly difficult to conquer the infection.This is because biofilms are complex and aggregated communities of microorganisms, embedded in a self-producing matrix of extracellular polymeric substances, that become more difficult for the host immune system to overcome. 16[19][20] Combined with their safety, naturally-occurring sources, and antimicrobial activities, saponins have the potential to be developed into decontamination products for use in the food industry and drugs for clinical use.Therefore, the primary objective of this study was to investigate the antibacterial effects of C. oleifera seed dregs (CTSD) extract on planktonic bacteria and biofilms.The results of this study will provide a reference for the development of CTSD as new antibacterial agents to improve the efficiency of infection control.Further add the CTSD to the product formulation to evaluate its stability, preservative, and other properties as a reference for future commercialization.

| Camellia oleifera tea seed dregs extract preparation and analysis
C. oleifera seed dregs were purchased from Chenxing Oil Company (Pingtung, Taiwan).Fine powder of seed dregs (200 g) was soaked in 800 mL 50% ethanol for 24 h, then subjected to heat reflux extraction for 6 h at 90°C.The supernatant was collected and filtered using Whatman No. 1 filter paper, and concentrated under reduced pressure for 2 h at 50°C, followed by freeze drying.Yield of extract = quantity of extract/quantity of raw material × 100%.Furthermore, The HPLC (Hitachi, Inc., Tokyo, Japan) with Phenomenex Luna C-18 column was used for analyzing the components of CTSD. 10 μL of sample was injected and eluted with water-acetonitrile (9.5:0.5 to 6:4) at the flow rate 1 mL/min, and detected under 215-nm wavelength of UV light.

| Determination of total triterpenoid content
The total triterpenoid content was estimated by the vanillin and perchloric acid method using the standard ursolic acid calibration curve.Briefly, after a 200 μL sample solution in a 10 mL test tube was heated to evaporation in a water-bath, 1 mL new mixed 5% (w/v) vanillin-acetic acid solution and 1.8 mL sulfuric acid were added.
The mixed solution was incubated at 70°C for 20 min, then cooled in running water for 2 min.The absorbance was measured at 573 nm against the blank using a spectrophotometer (BioRad Model 680 Microplate Reader).The blank consisted of all reagents and solvents without sample solution.

| Cellular toxicity toward on CCD-966SK cells
Safety of the CTSD was evaluated by MTT analysis according to ISO10993-5 and our previous study. 21 Briefly, 5 × 10 3 cells seeded and grown onto a 96-well plate.Cells treated with various concentrations of CTSD and incubated for 24 h.

| DPPH and ROS assay
Assessing free radical scavenging activity of sample in vitro and in cultured CCD-966SK cells.The protocol of DPPH and ROS analysis was carried out according to the methods previously published by our laboratory. 22,23Briefly, for DPPH test, the stock DPPH solution was prepared in 95% methanol, mixed evenly with sample at indicated concentrations, and kept them away from light for 30 min.
The resting DPPH level was calculated as the percentage of absorbance using the following formula: % DPPH level = 1 − (OD control − O D sample )/OD control × 100%.For the intracellular ROS determination, the DCF-DA assay was utilized.The normal human skin fibroblast CCD-966SK cells were incubated for 30 min with 10 μM DCF-DA and washed with PBS, followed by treatment with H 2 O 2 alone or in combination with nontoxic various concentrations of tested sample for 2 h.The fluorescence intensities of DCF were quantified using a microplate reader (Turner Biosystems), with an excitation wavelength of 485 nm, and an emission wavelength of 525 nm.

| Determination of the time-kill curve
Two bacterial strains, P. aeruginosa (BCRC10733) and S. aureus (BCRC 15201) were used in the current study.First, the rates of kill of the test microorganisms upon treatment with CTSD were evaluated.Overnight broth cultures were adjusted to a concentration of 10 7 CFU/mL, treated with CTSD ranging from 0 to 250 μg/mL, and incubated for different time periods.Then, appropriate amounts of bacterial liquid were taken from each tube and spread on a plate for an additional 24 h of incubation in order to calculate the numbers of colonies.

| Determination of minimum inhibitory and minimum bactericidal concentrations
CTSD was diluted with Muller Hinton broth (MHB) to achieve concentrations ranging from 0 to 1 mg/mL using the two-fold serial dilution method.Inocula were added into a 20 mL tube containing 10 mL MHB to achieve an initial inoculum of approximately 10 7 CFU/mL, then incubated at 37°C with shaking for 24 h.The lowest concentration of CTSD that did not result in visible growth by macroscopic observation was determined as the minimum inhibitory concentration (MIC).Further, appropriate amounts of bacterial liquid were taken from each tube, spread on a plate, and incubated for 18 h for colony-counting.The lowest concentration of CTSD that resulted in the complete absence of colonies was defined as the minimum bactericidal concentration (MBC).

| Potassium ion efflux and release of 260-nm absorbing materials analysis
The 10 8 CFU/mL microorganism suspension was exposed to CTSD at the half of MIC, MIC and double the MIC, and the extracellular potassium concentration was measured at each pre-established interval using a potassium ion analyzer (Horiba, Kyoto, Japan).For the measurement of 260-nm absorbing intracellular material, the bacterial suspension was centrifuged and the OD 260 value of the supernatant was monitored using a spectrophotometer and the 0.5 × MIC, MIC, and 2 × MIC of CTSD containing distilled H 2 O instead of cultured medium were used as the blank, respectively.

| Eradication of established biofilms
Eradication of biofilms was performed using a Calgary Biofilm Device (CBD) and an MBEC assay system (Innovotech Inc., Edmonton, AB, Canada).Bacterial suspension (200 μL) at 10 7 CFU/ mL was added to a 96-well microtiter plate.The CBD peg lid was then fitted inside the plate, and the assembled device was placed on a gyrorotatory shaker at 110 rpm in a humidified incubator for 24 h.Following incubation, the biofilms were rinsed to remove loosely adherent cells, then placed into the challenge plates containing CTSD at various concentrations for overnight culture.The challenged biofilms were then transferred to a recovery plate containing medium.The biofilms were removed from all pegs by sonication and the recovery medium containing the sonicated biofilms was serially-diluted, spread on plates, and subjected to 24 h of incubation for colony-counting.The minimum biofilm eradication concentration (MBEC) represents the lowest dilution at which no bacterial regrowth was observed.

| Long-term stability of cosmetics formula evaluation
Four different cosmetic formulations were designed.The composition of formulations is showed in Table 2. Ingredients other than methyl paraben and CTSD are homogenized using vacuum homogenizer (Prema, Taiwan) at 13000 rpm for 5 min, equipped with a water circulation at 25°C.Then add methyl paraben and CTSD to the emulsion and continue stirring with IKA blender RW20 (IKA, Staufen, Germany) for additional 30 min.The products were stored at 25°C.Stability parameters including appearance by visual observation, and physical-chemical properties were also determined.The stability was evaluated using high-speed centrifugation at 10000×g for 15 min.Both pH and viscosity were also determined.These assessments carried out at baseline upon product completion, following 25°C storage for 24 days and acceleration testing by six cycles heating and cooling, each condition at 4 and 45°C for 48 h.Viscosity was measured using viscometer (Brookfield viscometer, Model DEVLV), spindle 64 and rotated at 20 rpm.Measurement of the pH and viscosity were made in two independent experiments, each performed in duplicate.

| Antimicrobial activity of CTSD in O/W emulsion by challenge tests
The preservative efficacy in cosmetics emulsion was performed according to the Cosmetic, Toiletry, and Fragrance Association (CTFA) guidelines.Inoculation of P. aeruginosa or S. aureus 10 6 CFU/g to each cosmetic formulation and store at 25°C.The viable microorganism number was determined after contact time of 3, 7, 14, 21, and 28 days.Cell viability was determined by direct colony counting after appropriate dilution.

| Statistical analysis
All determinations were performed with three independent experiments, each performed in triplicate.Data are presented as the mean ± SD (standard deviation).The statistical analysis was performed using the unpaired t-test, and the p value less than 0.05 was taken to indicate significant difference.

| Component analysis, yield, and total triterpenoid content
The HPLC analysis for CTSD showed one sharp major peak (84.0% of total area, retention time: 13.7 min) accompanied by several minor peaks (Figure 1).The major peak was presumed to indicate saponin which is the major component in defatted camellia seed residue.Three triplicate preparations were performed.The extraction yield of CTSD was 11.75 ± 0.22%.In the triterpenoid quantitation experiment, the calibration equation for ursolic acid was Y = 0.0668X + 0.3127 (R 2 = 0.9757), in which Y was the absorbance value and X was the concentration of ursolic acid.The total triterpenoid content in the CTSD was 42.58 ± 1.39 mg ursolic acid equivalent (UAE)/g extract.

| Cytotoxicity
To apply CTSD to skin health care product, cytotoxicity in human skin normal cells, CCD-966SK was performed according the international regulation ISO 10993-5 protocol.As shown in Figure 2A, CTSD incubation for 24 h at a concentration less than 62.5 μg/mL, cell growth is the same as medium only control.Although the cell viability was significantly lower at the concentration of 125 μg/mL compared with the medium control group, it retained more than 80% of the cell viability.The positive control 10% DMSO caused severe cell death (data not shown).

| Antioxidant ability of CTSD
First of all, in vitro DPPH assay was employed to estimate the potential antioxidant function.DPPH is based on the spectrophotometric measurement of the DPPH concentration change resulting from the reaction with an antioxidant.The CTSD showed significant free radical scavenging ability at nontoxic concentrations.The scavenging effect is comparable to the strong antioxidant BHT at the same concentration (Figure 2B).Further, we analyzed the oxidative response induced by hydrogen peroxide (H 2 O 2 ) in cultured CCD-966SK system and characterized the protective potential of CTSD.H 2 O 2 induced significantly higher levels of ROS compared to vehicle treated cells.Critically, CTSD efficiently blocked the H 2 O 2generated ROS at noncytotoxic concentrations; lower dosages of CTSD also exerted significant activity in terms of reducing the ROS level in a dose-dependent manner (Figure 2B).Thus, the two experiments show the same dose-dependent trend, CTSD has a significant effect of scavenging free radicals and enough safety.This suggests that the current prepared extract may reduce environmental, such as UV-induced free radicals, for use in antiaging and anti-wrinkle care products.

| Time-and concentration-associated bactericidal kinetics
The kinetics of bacteria-killing of CTSD are displayed in Figure 3.At lower concentrations, the bactericidal effect was less significant, the number of viable cells decreasing gradually with an increasing concentration of CTSD.Comparing the number of colonies, the CTSD had a greater bactericidal power against S. aureus.Importantly, the extract exhibits superior antimicrobial efficacy at noncytotoxic concentrations.

| Antimicrobial activity
To investigate the capacity of CTSD for preventing planktonic bacteria growth, the MIC, and MBC values were determined using the two-fold serial dilution method.The results are presented in Table 1.CTSD displayed antibacterial activity against both tested Gram-positive (S. aureus) and Gram-negative (P.aeruginosa) bacteria, with recorded MIC of 15.7 and 31.3 μg/mL against S. aureus and P. aeruginosa, respectively.In addition, the MBC were 31.3 and 62.5 μg/mL against S. aureus and P. aeruginosa, respectively.
Although both bacteria were found to be susceptible to CTSD in this study, Gram-positive S. aureus showed greater susceptibility to the treatment.

| Eradication of biofilm activity
In natural environments, such as within host tissue or on a surface exposed to unfavorable growth conditions (UV, shear force, and dryness), bacteria have adapted, and exist as adherent populations called biofilms.Due to poor growth conditions, biofilms may grow more slowly and have poor motility; however, planktonic free bacteria in laboratory cultures provide sufficient nutrients, and therefore replicate rapidly.Usually, planktonic bacteria are more sensitive to antibiotics.Thus, subsequent experiments were conducted to evaluate the biofilm eradication ability of CTSD using the well-established CBD.The MBEC values are presented in Table 1; the MBEC against S. aureus and P. aeruginosa were 62.5 and 125 μg/mL, respectively.

| Leakage of potassium ions
An antibacterial mode of CTSD against pathogenic bacteria was determined by monitoring the release of intracellular potassium ions.
As shown in Figure 4, release of potassium from the two tested microorganisms occurred at 30 min after CTSD addition.Treatment with 1× MIC caused significant potassium ion loss, and treatment with double the MIC induced rapid and massive release of potassium ions by the two pathogenic bacteria.S. aureus was more sensitive to CTSD than P. aeruginosa, which was in agreement with the MIC and MBC shown in Table 1.

| Release of 260-nm absorbing materials
Another strategy for investigating the action mode of CTSD against bacteria is examining the release of 260-nm absorbing materials, which represents DNA release from bacteria damaged by CTSD treatment.As shown in Figure 5, the absorbance at 260 nm of the culture filtrates of the tested bacteria treated with CTSD at 0.5 × MIC, MIC and 2 × MIC revealed a gradually increasing release of 260-nm absorbing materials with respect to exposure time.The results provided direct evidence that CTSD has antibacterial effects on both S. aureus and P. aeruginosa.

| Preservative efficacy evaluation by challenge test
Four formulations were designed to evaluate the preservative effect of actual products, including no preservatives, mainstream methyl paraben preservatives, and extracts as preservatives (Table 2).
The results of challenge test were demonstrated in Figure 6.Lack of preservatives allows bacteria to grow, importantly, emulsion formulations with extract or methyl paraben as preservatives significantly reduce bacterial counts from days 7 to 28 either inoculated with S. aureus or P. aeruginosa.CTSD concentration at 0.2% has a slightly insufficient effect, while 0.5% of CTSD has almost the same preservative effect as methyl paraben.Based upon the ISO 11930 Acceptance Criteria, for bacteria, the product must reveal no less than a 3-log reduction from the initial count at 7th day, and no increase from the Day 7 to Day 28.Thus, CTSD with a concentration of 0.5% can meet this standard.

| Physicochemical and rheological properties
The various characteristics of fresh prepared emulsion, 28-day storage at 25°C, and six cooling-heating storage were determined.As shown in Table 3, the pH value is between 5.4 and 5.9.There is no significant difference between the different storage conditions of each formula.Recipe 1 (methyl paraben as preservative) after storage by the acceleration test, the viscosity was significantly lower than initial fresh prepared emulsion.Formulation 2 with 0.5% CTSD revealed significant difference of viscosity after storage.The pH value is between 5.4 and 5.9.There is no significant difference between the different storage conditions of each formula.Furthermore, the prepared emulsion resistance to extrinsic factors was assessed by high-speed centrifugation.The four formulations showed stable appearance in various storage conditions.Taken together, overall consideration of different aspects formulas containing CTSD extracts have sufficient stability.

| DISCUSS ION
Plants have sophisticated protection systems to keep them alive in their environments, and are therefore a great source of pharmaceutical resources.5][26] Although development of plant-derived compounds for pharmaceutical use has been very active in the past few decades, still relatively very few studies have investigated the use of secondary metabolites infections, with a serial emergence of formidable epidemic strains. 32 aeruginosa is a Gram-negative bacterium, recognized as a lethal and resistant bacterium that can create critical infections.33,34 It is responsible for a wide range of healthcare-related infections in critically ill patients owing to its ability to easily acquire resistance to many classes of antibiotics.35 It is therefore important to develop novel antimicrobials or a new approach to prevent cases caused by these bacteria strains that cannot be controlled or destroyed by current antibiotics.Biofilms represent a system by which bacteria protect themselves from the host defense, disinfectants, and antibiotics.34,36 They play an important role in drug resistance, as biofilms limit drug diffusion and reduce substance delivery, leading to antibiotic failure. Biofimassociated drug resistance occurs via several mechanisms, such as lowering antibiotic penetration through the extracellular polymeric matrix, production of antibiotic-degrading enzymes that hydrolyze and inactivate antibiotics, and increased extracellular DNA production on the outer membrane, as well as induction of drug resistance through the stress response, quorum-sensing, and overexpression of efflux pump genes.37 Recently, various green nonlethal strategies for biofilm control have been improved.A promising alternative is to investigate naturally-occurring, plant-derived compounds that can block biofilm formation.
Treatment for biofilm-related infections is still very challenging, and thus new strategies to solve this problem need to be developed.
To achieve this purpose, new agents that can destroy biofilms would A recent study by Ye and coworkers 39 showed that saponins isolated from camellia seeds had significant inhibitory effects on biofilms of Escherichia coli and S. aureus in vitro and in vivo, bacteria that are likely protected by a biofilm.Another study also showed that tea saponins had antibacterial effects on E. coli and S. aureus, and the effect is associated with the ability to change membrane permeability and destroy the cell membrane structure of the pathogenic bacteria. 40though the MIC has been used as a standard to determine the susceptibility of specific pathogenic bacteria to specific antimicrobial agents, it does not necessarily mean that the agents will have the same effects in vivo, owing to bacteria being likely protected by a biofilm.In this study, we used the MIC to evaluate the susceptibility of planktonic S. aureus and P. aeruginosa cells, and showed that they were susceptible to CTSD.Biofilm antimicrobial susceptibility was evaluated, and the results showed that the MBECs against S. aureus and P. aeruginosa biofilms were two-and four-fold the MICs, respectively (Table 1).Additionally, CTSD showed a good antimicrobial activity against both Grampositive S. aureus and Gram-negative P. aeruginosa at a lower dose (0.5 × MIC) based on a leakage assay of potassium ions and materials that absorb at 260 nm.
Apart from the evaluation of CTSD's antimicrobial ability, we also explored its antioxidant and free radical scavenging activity.Plants have a complex defense system against diverse pests and pathogens in their environments, and are therefore a rich source for new drug development.The most appealing application area for plant extracts is inhibition of growth and antimicrobial and antibiofilm activities.Saponins are one of important groups of compounds that have antibiofilm activity against pathogenic bacteria.Our study demonstrated that saponins isolated from defatted camellia seed residue have good antibacterial effects on both S. aureus and P. aeruginosa.
For the conclusion, CTSD is a natural plant extract that can inhibit the formation of microbial biofilms, and also has antioxidant and free radical scavenging capabilities.In addition, it can also be used as an adjuvant compound to control infectious diseases, and can provide an alternative to related functional products in the future.

F
I G U R E 1 HPLC chromatogram and peak quantitation of CTSD.

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I G U R E 2 (A) Cytotoxicity on CCD-966CK human normal skin fibroblast cells.Bars marked with "*" indicate statistically significant differences at p < 0.05, compared to medium control.(B) DPPH and ROS assay.The concentration units of CTSD and BHT are μg/mL, the concentration of H 2 O 2 treatment is 0.5 mM.The asterisk indicate significant difference, the treatment group in DPPH experiment was compared with the vehicle group; the comparison between treatment group and the H 2 O 2 group in the ROS assay.*p < 0.05, **p < 0.01, ***p < 0.001.

FF I G U R E 5
I G U R E 3 Time-and concentrationdependent effects of CTSD on (A) Staphylococcus aureus, (B) Pseudomonas aeruginosa.TA B L E 1 Susceptibility testing for planktonic bacteria and biofilm.as antimicrobial agents. 27-29Fast evolution of bacterial resistance has promoted the necessity of finding new antibacterial compounds from different natural resources as a new approach to overcome the problem.Thus, use of the potential antimicrobial activity of plants against resistant pathogens via different mechanisms of action is very promising.In this study, we showed that saponins from C. oleifera seeds have good antibacterial and antibiofilm properties against both S. aureus and P. aeruginosa.S. aureus is a Gram-positive bacterium that is infamous for its ability to resist many antibiotics.Infections caused by antibiotic-resistant S. aureus strains are becoming a serious issue and have attained epidemic levels worldwide. 30,31Successful treatment is still challenging, as this bacterium tends to cause healthcare-associated F I G U R E 4 Influence of various concentrations of CTSD on the intracellular potassium release kinetics of (A) Staphylococcus aureus, (B) Pseudomonas aeruginosa.Effects of CTSD on the release rates of 260-nm absorbing materials from Staphylococcus aureus and Pseudomonas aeruginosa.
be ideal, either for use alone or in combination with conventional antibiotics.Saponins are characterized by wide antimicrobial activities that occur through various mechanisms associated with the assorted structures of saponin molecules.Studies have indicated that the activities of plant-derived saponins in fighting infection are mediated by their detergent-like activity, which leads to damage to the bacterial cell membrane, causing augmentation of ion permeability, damage to vital intracellular components, or interruption of bacterial protein synthesis.38

TA B L E 2 6
Composition of cosmetic emulsion.Preservative efficacy of CTSD on (A) Staphylococcus aureus, (B) Pseudomonas aeruginosa.Oxidative stress resulted from ROS is one of the causes of skin aging.Moreover, ROS has been reported that associated with tumorigenesis and inflammation.It may be generated due to UV irradiation, air pollution, or microbial infection.Antioxidants can reduce reactive compounds by donating electrons to prevent them from suffering oxidative stress.In the cosmetics industry, there are also several products extracted from plants that have been proven to have antioxidant or free radical scavenging capabilities.CTSD was also found to have similar effects in this study.It can be seen that in addition to antimicrobial and biofilm formation, it should have considerable effectiveness in anti-inflammation or preventing skin aging.
. Huang J, Ahrends A, He J, Gui H, Xu J, Mortimer PE.An evaluation of the factors influencing seed oil production in Camellia reticulata L. plants.Industrial Crops and Products.2013;50:797-802.doi:10.1016/j.indcrop.2013.08.019 2. Yang C, Liu X, Chen Z, Lin Y, Wang S. Comparison of oil content and fatty acid profile of ten new Camellia oleifera cultivars.J Lipids.2016;2016:3982486.doi:10.1155/2016/39824863. Zeng W, Endo Y. Lipid characteristics of camellia seed oil.J Oleo Sci.Three storage conditions of the same formulation were compared.The "*" indicate statistical differences from initial (p < 0.05).Physical and chemical properties of each formulation under different storage conditions.
idation.Wen-Ling Shih: conceptualization, methodology, validation, formal analysis, investigation, original draft preparation, review and editing, visualization, supervision, project administration, and funding acquisition.All authors have read and agreed to the published version of the manuscript.R E FE R E N C E S12019;68(7):649-658. doi:10.5650/jos.ess18234Note: TA B L E 3