antimicrobial studies on creams obtained with clove oil ... · antimicrobial studies on creams...

Antimicrobial studies on creams obtained with clove oil and with waste aqueous phase remaining after the clove oil distillation

A. Wróblewska*,1, E. Makuch1, Ł. Kucharski2, A. Klimowicz2 and A. Markowska-Szczupak3 1 Institute of Organic Chemical Technology, Faculty of Chemical Technology and Engineering, West Pomeranian

University of Technology Szczecin, Pulaskiego 10, 70-322 Szczecin, Poland, [email protected] 2 Department of Cosmetic and Pharmaceutical Chemistry, Faculty of Health Sciences, Pomeranian Medical University in

Szczecin, PowstancowWielkopolskich72, PL-70111 Szczecin, Poland 3Institute of Inorganic Chemical Technology and Environmental Engineering, Faculty of Chemical Technology and

Engineering, West Pomeranian University of Technology Szczecin, Pulaskiego 10, 70-322 Szczecin, Poland

After distillation of dried cloves two phases are obtained: oil (clove oil) and aqueous. We also tested in our studies water remaining in the distillation flask after the cloves distillation and water which we used to wash the cloves earlier used in the distillation process (usually water phases are waste from the process of cloves distillation). All phases were analysed by GC/MS method and next their antioxidant capacity was tested. After these studies the clove oil and all aqueous phases were used for creams preparation. For obtained creams antioxidant capacity and antimicrobial tests were performed with Gram negative Escherichia coli K12 (ATCC 25922), Gram positive Staphylococcus epidermidis (ATCC 49461), yeast cause candidiasis Candida albicans (IIT&EE ZUT collection), and fungi cause fungal dermatitis Trichophyton rubrum (IIT&EE ZUT collection). Candida and fungi were isolated from patients with immunodeficiency. The presented studies are important because of the possible applications received creams in medicine for curing various skin diseases, among others: eczema, chronic wounds, and atopic dermatitis. Also prepared creams can be used in cosmetology as anti-age or soothing creams.

Keywords: creams with clove oil; antioxidant capacity; antimicrobial tests; eczema, atopic dermatitis

1. Introduction

In recent years, the increasing interest of producers and consumers in ecologically friendly and healthy safe cosmetics (without chemical ingredients) is noted [1-3]. Plants, in particular herbs, were the main source of natural ingredients used as skin care products, preservatives or pigments before the use of chemical substances. Essential oils isolated from many plant parts (e.g. fruits, flowers, leaves, buds, seeds etc.) are generally recognized as Safe (GRAS) substances and therefore could be used to prevent post-harvest growth of microorganisms [4]. Cloves are flower buds of tree in the family Myrtaceae - Syzygium aromaticum. They are commonly used as a spice in the cuisine of many countries (Asian, African, Middle East). They were traditionally used in medical application e.g. in Chinese medicine, due to their many health benefits. Cloves providing aid in digestion problem, fighting against cancers and possess anti-mutagenic properties, protecting the liver, boosting the immune system and having antimicrobial, antioxidant and anti-inflammatory properties. A clove taste and therapeutic properties is imparted by the chemical eugenol, which is a major constituent of the clove oil [5]. Although eugenol has been reported to inhibit the growth of molds, yeasts and gram positive and gram negative bacteria, to date, there has been relatively little or in some cases no reports, examining the eugenol containing creams against important bacterial and fungal pathogens. Eugenol (4-allyl-2-methoxyphenol) is a pale yellow colour liquid. It is a very interesting compound taking into account not only its applications in medicine (antiseptic agent in dental and medical practice) and cosmetics but also in organic syntheses and in polymerization processes (for example it is used as the antioxidant for plastics and rubber). Moreover, eugenol is widely used as a flavouring agent in backed foods, beverages, sweets and frozen products [6-9]. It belongs to biorenewable sources and it is cheap and easy available compound because it can be obtained from biomass (clove oil) by a simply distillation. It is also present in many other aromatic plants like basil, cinnamon and bay leaves. The aim of our work was to prepare not only creams containing clove oil (the organic phase obtained from the distillation of cloves) but also creams containing the aqueous phase obtained after the clove distillation, the aqueous phase remaining after cloves distillation in the distillation flask and the aqueous phase which was obtained after washing the cloves previously used for the distillation of clove oil. Usually water phases are waste from the process of cloves distillation and are not used in cream formula or in other formulations used in cosmetics and medicine. After checking the antioxidant capacity of these three aqueous layers we tried to use them in the cream formula. We checked for the obtained creams antioxidant activity and we also performed antimicrobial tests for these creams with: Gram negative Escherichia coli K12 (ATCC 25922), Gram positive Staphylococcus epidermidis (ATCC 49461), yeasts causing candidiasis - Candida albicans (IIT&EE ZUT collection), and fungi causing fungal dermatitis - Trichophyton rubrum (IIT&EE ZUT collection). For comparison also studies with commercially available eugenol were made. The presented studies are important because of the possible applications of the obtained creams in medicine for curing various skin diseases, among others: eczema, chronic wounds, and atopic dermatitis. Also prepared creams can be used in cosmetology as anti-age or soothing creams. Moreover, the ecological aspect of our research is very important. The

Antimicrobial research: Novel bioknowledge and educational programs (A. Méndez-Vilas, Ed.)

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use of waste water from the cloves distillation process is environmentally friendly and allows us to utilize very valuable layers, containing valuable biologically active compounds, in the cosmetic industry and in medicine.

2. Experimental

2.1 Hydrodistillation of clove oil and obtaining the phases for further investigations

The clove oil was isolated by the hydrodistillation method as follows: 20g of all cloves (Eugenia caryophyllata) from Prymat company were placed together with 200 cm3 of deionized water in the round bottom flask with the capacity of 500 cm3, equipped with a reflux condenser and dropping funnel. The duration of the process was calculated from the boiling point of the flask. During the distillation process, the deionized water was added to the flask so that it does not stop the boiling of its contents. In total, 200 cm3 of deionized water was added dropwise. After the 180 minute distillation process, the condensate (containing water and oil phase) collected in the receiver is separated in a separator, after a slow shaking of the contents. As a result two fractions are obtained: oil fraction (from light yellow to brown) named L1 and cloudy watery fraction named L2. Also two water fractions were used in our studies: the aqueous phase remaining after cloves distillation in the distillation flask (named L3) and the aqueous phase which was obtained after washing the cloves previously used for the distillation of clove oil (named L4).The residue in the distillation flask, which is cloves and dark brown liquid, is filtered on a glass funnel to separate the cloves from the liquid. In this way we obtained the aqueous phase remaining after cloves distillation in the distillation flask. The cloves used in the distillation process, which are separated on a glass funnel, are washed with 5 cm3 of distilled water. It is the aqueous phase which was obtained after washing the cloves previously used for the distillation of clove oil. For comparison, commercial eugenol (with 98% purity, from Sigma-Aldrich) was also tested by these methods (named L5). All phases were subjected to GC-MS studies. GC-MS analyses were performed on a Thermo Quest series apparatus with a mass detector VOYAGER and using a capillary column DB5 (30m × 0.25μm × 0.5μm). The following separation parameters were used during analyses: helium flow rate of 0.7ml /min, sample chamber temperature 260˚C, detector voltage 350v (volt), thermostat temperature increased according to the following program: isothermally 50˚C for 1 minute, temperature increase at 8˚C/min, isothermally 300˚C for 5 minutes and cooling to 50˚C, sample split factor in dispenser 20, volume of dispensed sample 0.1μl and ionic mass range 32-350 mz.

2.2 Determination of the antioxidant capacity of L1-L5 fractions

During the studies on the antioxidant capacity of L1-L5 fractions the following reagents were used: ethanol (a.g., Chempur) and DPPH (2,2-difenylo-1-pikrylohydrazyl, a.g., Sigma Aldrich). Measurements of absorbance of the samples were carried out on a Spectroquant Pharo 300 spectrophotometer at wavelength λ = 517 nm and after a 10 minute incubation of the test sample at room temperature. First calibration of the spectrophotometer was performed using ethanol as a solvent. Then a DPPH solution in ethanol was prepared by adding an appropriate quantity of DPPH reagent to get the absorbance of the resulting solution of about 1.00 ± 0.02 (wavelength λ = 517 nm). In a plastic tube, 450 μl of the test fraction and 650 μl of the DPPH solution were placed and then the tube was sealed with a stopper and placed in a dark place for 10 minutes incubation at room temperature. After this time, the measurement was made. Each sample was tested three times, and the average absorbance value for each of them was calculated.

2.3 Method of creams preparation for fractions L1-L5

The following components were used to prepare the studied creams: vaseline (Coel), beeswax (Distributor - Company "Make Yourself a Cream"), cholesterol (Coel), distilled water and fractions L1-L5.Creams were prepared in such a way that vaseline and beeswax were added to the 200 cm3 glass beakers and then placed in an oil bath (80°C) to dissolve the contents of these beakers. Then the appropriate quantities of ingredients were added to the plastic containers and the contents of the containers were mixed with a glass baguette to achieve a uniform consistency.

2.4 Antimicrobial tests

The antimicrobial tests were performed with the following microorganisms: gram negative bacteria Escherichia coli K12 (ATCC 25922), gram positive bacteria Staphylococcus epidermidis (ATCC 49461), yeast cause candidiasis Candida albicans (IIT&EE ZUT collection), and fungi cause fungal dermatitis Trichophyton rubrum (IIT&EE ZUT collection). Candida and fungi were isolated from patients (throat smear) with immunodeficiency disorders. The antimicrobial activity of the oli and cream was tested by disc diffusion method [10,11]. Culture plates were prepared with 15 mL of the three solid selective media: Plate Count Agar PCA (BIOCORP, Poland) for E. coli, Brain Heart Infusion Agar BHI (BTL, Poland) for Staphylococcus epidermidis and Sabouraud Glucose Agar SGA (BIOCORP, Poland) for T. rubrum and C. albicans. Each Petri dish were inoculated with 0,25 mL of each of tested microorganisms (appx. 106 CFU/mL). The sterile paper discs (Whatman No.1, diameter 5 mm) impregnated with each oil (10µL/disc)


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were placed at different locations on the surface of culture plates. Creams were placed directly on the plate (0.016-0.245g). The treated plates were incubated at 37°C for 48h (E. coli and Staphylococcus epidermidis) or at 30°C for 72h (T. rubrum and C. albicans). Then any zone of inhibition around the paper discs or cream were inspected. The sensitivity to the tested agents was classified by the diameter of the inhibition halos as: not sensitive (-) for diameters less than 5 mm; sensitive (+) for diameters 5–10 mm; very sensitive (++) for diameters larger than 10 mm and less than 15 and super sensitive (+++) for diameters larger than 15 mm. Three discs or cream inocula per plate were used, and each plate was set up in triplicate.

3. Results and discussion

Results of the studies on the composition of L1-L5 fractions (GC-MS method) and their antioxidant capacity are presented in Table 1. Table 1 Results from composition studies (GC-MS method) and antioxidant capacity for fraction L1-L5.

Fraction Composition of the fraction (main components) Mean DPPH radical scavenging rate for a given fraction

[average %]

L1 eugenol, ß-caryophyllene, α-caryophyllene, 4-allyl-2-methoxybenzoic aldehyde

92.41

L2 eugenol, ß-caryophyllene, α-caryophyllene 44.16 L3 eugenol, ß-caryophyllene,

4-allyl-2-methoxybenzoic aldehyde, ß-caryophyllene oxide

9.37

L4 eugenol, kopaen, ß-caryophyllene, α-caryophyllene, 4-allyl-2-methoxybenzoic aldehyde

86.45

L5 eugenol, ß-caryophyllene, α-caryophyllene, kadinen, 4- allyl-2-methoxybenzoic aldehyde, ß-caryophyllene oxide, isoeugenol

86.77

The results presented in Table 1 show that the highest antioxidant capacity was demonstrated by fraction L1. Slightly lower values showed fractions L4 (the aqueous phase which was obtained after washing the cloves previously used for the distillation of clove oil ) and L5 (commercial eugenol). Considerably lower antioxidant capacity presented fraction L2 (cloudy watery fraction). The lowest value of the antioxidant capacity was observed for fraction L3 (the aqueous phase remaining after cloves distillation in the distillation flask). For the next stage our studies (creams preparation) all fraction L1-L5 were taken. The compositions of the individual creams for fraction L1-L5 are shown in Table 2. Table 2 Compositions of tested creams and their names.

Fraction Composition of appropriate cream Name of cream

L1 vaseline (15.500g), cholesterol (0.750g),beeswax(0.500g),fraction L1 (0.500g) 1A (3 wt% of L1) L1 vaseline (15.5g),cholesterol (0.750g),beeswax (0.500g),fraction L1 (1.000g) 1B (6 wt% of L1) L1 vaseline (15.500g), cholesterol (0.750g),beeswax (0.500g),fraction L1 (1.500g) 1C (8 wt% of L1) L1 vaseline (15.500g),cholesterol (0.000g),beeswax (0.500g),fraction L1 (1.500g) 2 (9 wt% of L1) L1 vaseline (15.500g),cholesterol (0.000g),beeswax (0.000g),fraction L1 (1.500g) 8 (9 wt% of L1) L2 vaseline (15.5g),cholesterol (0.750g),beeswax (0.500g),fraction L2 (1.000g) 3B (6 wt% of L2) L2 vaseline (15.5g),cholesterol (0.750g),beeswax (0.500g),fraction L2 (1.500g) 3C (8 wt% of L2) L3 vaseline (15.5g),cholesterol (0.750g),beeswax (1.500g),fraction L3 (1.500g) 7 (8 wt% of L3) L4 vaseline (15.5g),cholesterol (0.750g),beeswax (1.500g),fraction L4 (1,500g) 6 (8 wt% of L4) L5 vaseline (15.5g), cholesterol (0.750g),beeswax (0.500g),commercial eugenol

(1.500g) 5 (8 wt% of L5)

Figure 1 presents the selected cream samples based on L1-L5 fractions.


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Fig. 1 Selected cream samples based on L1-L5 fractions: a) sample 1C, b) sample 2, c) sample 8, d) sample 3B, e) sample 7, f) sample 6 and g) sample 5

a) b)

c) d)

e) f)

g)


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The results of the inhibition trials for obtained cream, performed by the disk diffusion method are presented in Table 3. Table 3 The results of the antimicrobial tests (sensitivity and diameter of growth inhibition zone [mm]) of the obtained creams.

Name of cream

Fraction E. coli St. epidermidis C. albicans T. rubrum

1A L1 (++) 15.0 (+)10.0 (+++)20.0 (+++)35.0 1B L1 (+)10.0 (+)7.0 (+++)25.0 (+++)34.0 1C L1 (+)10.0 (+)10.0 (+++)23.3 (+++)26.3 2 L1 (+)10. 0 (++) 12.0 (+++)28.0 (+++)25.3 8 L1 (+)5.0 (+)5.0 (++) 14.0 (-)0.0 3B L2 (-)0.0 (-)0.0 (-)0.0 (-)0.0 3C L2 (-)0.0 (-)0.0 (-)0.0 (+)8.0 7 L3 (-)0. 0 (-)0.0 (-)0.0 (-)0.0 6 L4 (-)0.0 (-)0.0 (-)0.0 (-)0.0 5 L5 (-)0.0 (+)10.0 (+++)25.0 (+++)25.0

The best results were obtained for cream’s samples described as: 1A, 1B, 1C, 2 and 5 thus we decided to present figures only for these, the most active in the antimicrobial tests samples. Figures 2-4 illustrated representative pictures of the zone inhibition obtained for the cream 1A (Figure 2), for the cream 1B (Figure 3), for the cream 1C (Figure 4) and for the cream 5 (Figure 5). A zone of inhibition around the discs shows a bactericidal or bacteriostatic activity, while the absence of inhibition zone proved no effect of the tested creams against the microorganisms. The results of the disk diffusion method showed that the creams containing fraction L1 (creams 1A, 1B and 1C) had an inhibitory effect against all tested microorganism (Table 3) independent of content of L1 fraction. Thus the lowest concentration of this fraction should be selected as the most beneficial (3 wt%). It is important to note that when the commercial eugenol was used for cream preparation (cream 5), this cream showed worse antimicrobial properties than creams with fraction L1 (1A, 1B and 1C) and, moreover, this cream did not have the inhibitory effect against E. coli. The research showed that the most sensitive organism was Candida alabicans – opportunistic fungal pathogen that is responsible for candidiasis in human hosts and Trichophyton rubrum – deramtophytic fungus that causes skin, nail and hairs infections e.g. athlete's foot. Among tested bacteria Gram positive S. epidermidis and Gram negative E. coli showed lower susceptibility to creams 1A, 1B, 1C, 2, 8 and 5. It is not quite a surprising result due to the Gram positive bacteria are more sensitive to the antimicrobial properties of plant essential oils than Gram negative bacteria. An explanation for observed difference between these two groups of bacteria is various structure bacterial cell wall. The lack of an outer membrane, as well as the presence of a thick cell wall, in Gram positive bacteria, plays a crucial role in biocidal agent passage through the cell and then cell membrane. Moreover, active substances such as plant essential oils can disrupt the permeability of bacteria capsule that lead to loss of chemiosmosis control. According to Hanamanthagouda et al. [12] it is the most likely reasons for their lethal action. The mechanisms by which plant essential oils can inhibit fungi involve different models of action. The most frequently considered included: the changing structure of the lipid bilayer, loss its permeability, leakage of cell content (after disruption of outer membrane), and inhibition enzyme activity (after penetration of inside of cell). The factors influenced on the antifungal activity are type, composition and concentration of the oils, and also the type and concentration of the target fungi [13,14]. Although the cream 8 composition was similar to creams with the highest antimicrobial activity (1A-C, 2 and 5), the lack of beeswax in creams formula was crucial for its antifungal as well as antibacterial activity (Table 3). Moreover, the fractions L2-L4 from clove oil distillation were not good as antimicrobial agent in creams (creams 3B, 3C, 7 and 6). These fractions, despite of presence of eugenol and, in some cases highly antioxidant activity, have no inhibitory effect on the growth of the tested microorganisms. The cause may be the introduction with these fractions also water into creams - water is a good environment for the development of microorganisms. The obtained results may also indicate that not only eugenol shows antimicrobial activity but also other compounds contained in the analysed fractions, particularly in the L1 fraction. This, however, requires further research to determine which of these compounds in the presence of eugenol has shown such a high antimicrobial activity. The second explanation of a low antimicrobial activity fractions L2-L4 is lower content of eugenol in these fractions. It was showed by many authors that most of the essential oils, including eugenol, exhibited significant inhibitory activity with MIC (Minimal Inhibitory Concentration) or MCF (Minimum Fungicidal Concentration) with values of 0.01% and 0.10%, respectively, only when are used in pure form [4,11-13,15]. On the other hand, Sanla-Ead et al. [16] presented that eugenol at relatively low concentration - 50 µl/ml


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incorporated methyl cellulose films, revealed antimicrobial activity against Aeromonas hydrophila and Enterococcus faecalis.

Fig. 2 Zone of inhibition of the cream 1A obtained for: a) E. coli, b) Staphylococcus epidermidis, c) C. albicans and d) T. rubrum

Fig. 3 Zone of inhibition of the cream 1B for: a) E. coli, b) Staphylococcus epidermidis, c) C. albicans and d) T. rubrum

a)

a)

b)

c) d)

c)

b)

d)


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Fig. 4 Zone of inhibition of the cream 1C for: a) E. coli, b) Staphylococcus epidermidis, c) C. albicans and d) T. rubrum

Figure 5 presents the results of antimicrobial test obtained for the cream 5.

Fig. 5 Zone of inhibition of the 5 for: a) E. coli, b) Staphylococcus epidermidis, c) C. albicans and d) T. rubrum

4. Conclusions

In conclusion, we propose the following composition of creams: vaseline (15.500g), cholesterol (0.750g), beeswax (0.500g) and fraction L1 (0.500g) from distillation of clove oil for the treatment of mycoses (dermatoses), caused by C. albicans or T. rubrum. Fractions L2-L4 were not good fractions for the preparation of creams with high microbial activity. Probably the reason for this behavior of these creams was the presence of large amounts of water in them, which is a very good environment for the growing of microorganisms. Even the content of eugenol in these fractions did not affect the increase in their microbial activity, probably due to the low content of this compound. One way to solve this problem may be to use for eugenol extraction from water layers or to wash cloves after distillation ethanol as a solvent and re-preparing the creams. Perhaps ethanol extracts will exhibit greater antimicrobial activity - taking into account the antimicrobial properties of ethanol itself, this may be a good solution. The second direction of research on

a) b)

c) d)

a) b)

c) d)


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the proposed creams, which has to be developed in the nearest future, is to investigate the correlation between the composition of the layers, especially the content of extra compounds next to eugenol and the microbial effect of the creams. Perhaps some of these additional compounds in combination with eugenol will increase the antimicrobial properties. Taking into consideration possible future use of the tested creams, the continuation of these tests seems very purposeful.

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antimicrobial studies on creams obtained with clove oil ... · antimicrobial studies on creams...

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