Assessment of Volatile Oil Composition, Phenolics and Antioxidant Activity of Bay (Laurus nobilis) Leaf and Usage in Cosmetic Applications

Received: May 31, 2017 Revised: June 16, 2017 Accepted: June 24 2017 Abstract: In this study, the components of the volatile oil obtained from Laurus nobilis leaves by steam distillation were determined using Agilent 6890 Gas Chromatography (GC) 5975 Mass Spectrometry (MS). The antioxidant activities of different extracts of L. nobilis leaves were determined by using DPPH (2,2-diphenyl-1picrylhydrazyl) free radical scavenging activity, β-carotene-linoleic acid bleaching assay and ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) cation radical decolorization assay. Determination of the total phenolic contents of L. nobilis leaf extracts were performed using the Folin-Ciocalteau procedure and total flavonoid contents were measured using a spectrophotometric assay. According to the GC/MS results, 1,8-cineole (46.16%), alpha-terpinyl acetate (10.62%), alpha-pinene (6.27%), terpinen-4-ol (5.07%) and sabinene (4.99%) were found to be the major compounds in volatile oil. The obtained volatile oil was used to make skin care lotion. Stability tests and organoleptic analyses of final product were performed after 1, 5, 30 and 90 days of production. The highest amounts of total flavonoid content were found to be 5.48 ± 0.65 and 8.60 ± 0.12 μg QEs/mg in ethyl acetate and ethanol extracts, respectively. The highest amounts of total phenolic compounds were found to be 54.42 ± 0.14 and 25.32 ± 0.10 μg PEs/mg in ethyl acetate and ethanol extracts, respectively. According to the results of ABTS, DPPH and β-carotene linoleic acid assays, ethyl acetate extract was found to be the most active extract (24.98±0.87 μg mL, 75.65±0.77 μg mL and 19.32±1.04 μg mL).


Introduction
Volatile oils are secondary plant metabolites, which are found in different parts of plants including flowers, roots, bark, leaves, seeds, peel, fruit and wood produced in cytoplasm and plastids of plant cells [1].These oils, also known as essential oil, etheric oil by people, can contain terpenic hydrocarbons and their oxygenated derivatives as well as organic acids, alcohols, phenols and ketones [2].The main components of volatile oils are usually mono and sesquiterpenes.In some cases their main derivatives are hydrocarbons (e.g.turpentine, formed by alpha and beta-pinene), while in others the main constituents are oxygenated (e.g.cloves formed by eugenol) [3,4].Some of the aromatic plants have a volatile oil formed mainly of aromatic compounds derived from allyl or isoallyl phenol.The plants containing those compounds, although they are less frequent than plants containing terpenes, only allow such compounds to be selective.
There has always been a great deal of interest in volatile oils throughout history.Although many of the intended uses of volatile oils have disappeared over time, it has generally been accepted that since the beginning of mankind, humans have obtained these oils from aromatic plants.Volatile oils have various applications for different purposes.Volatile oils are not only used for cooking in order to improve the taste and health of the food, but also for the manufacture of perfumes and cosmetic products [5].
The ancient Egyptians have used volatile oils in perfumery, medicine and even in the preparation of bodies and organs for mummification.In ancient Asia, Vedas coded intended uses of perfume and aromatics for therapeutic purposes.Indeed, throughout history, many civilizations have used volatile oils and fragrances for a variety of purposes, including religious rituals, perfumes and therapeutic against infectious diseases.During the Renaissance period, the use of volatile oils in perfumery and cosmetic products has been spread to the world [6].
Volatile oils can be obtained by water distillation, water and steam distillation, or steam distillation alone, which are the most commonly used methods.The part of the plant where the volatile oil is to be obtained may be fresh, partially or completely dried, but if the volatile oil is to be obtained from the flower part, the flowers should be fresh [7].
Different methods have been used to control and analyze essential oils [8].Currently, however, the identification of essential oil components is usually carried out with the aid of gas chromatography-mass spectrometry (GC/MS) equipped with flame ionization detector (FID) and mass spectrometer (MS) detectors, a capillary column and a split.Test conditions may vary depending on the column and the sample [9].
The main sources of volatile oils are medicinal and aromatic plants, which are widely used since ancient times in medicine, cosmetics and preserving and improving the flavor of foods.Especially in recent years, there are numerous artificial chemical-free productions, mainly cosmetics, due to the increased interest in natural products [10].As a natural ingredient, volatile oils are a growing market trend, being used in skin care cosmetics (e.g creams, lotions), balms, shampoos, soaps and perfumes [11].
Laurus nobilis (Lauraceae), one of the main sources of plant volatile oils, is an evergreen shrub that can grow up to 8 meters tall.It has dark green leaves about 8-14 cm long and 3-4 cm wide [12,13].
This plant, belonging to Lauracea family and unique to the southern Mediterranean region, is widely grown in Europe and USA as an ornamental plant.It is cultivated commercially for aromatic oil, found in its leaves, in Turkey, Algeria, Morocco, Portugal, Spain, Italy, France and Mexico.Turkey is the leader bay leaf exporter of the World.[14].
The volatile oils obtained from the leaves of L. nobilis still maintains the importance in both traditional and modern medicine with its pharmacological activities.Studies have shown that L. nobilis volatile oil has antioxidant [15], anticonvulsant [16], analgesic, antiinflammatory [13], antiviral [17], anticholinergic [18], antibacterial [19] and antifungal activities [20].L. nobilis, which is a powerful medicinal and aromatic plant with these pharmacological properties, has been reported in cosmetic uses.L. nobilis leaf volatile oil is used for the preparation of hair lotion due to its antidandruff activity and for the treatment of psoriasis [21].
The usage of volatile oils in the production of cosmetics and similar products can both increase the dermo-cosmetic effects of these products and marketing trend for the final product.A great number of usages of plant materials such as volatile oils in cosmetics products provide extra benefits to the skin more than ordinary products.[22].
In this study, the chemical composition of L. nobilis leaf volatile oil was examined using GC/MS.The antioxidant activities of different extracts of L. nobilis leaves were determined by using DPPH • (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity, β-carotenelinoleic acid bleaching assay and ABTS •+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) cation radical decolorization assay.Determination of the total phenolic contents of L. nobilis leaf extracts performed using the Folin-Ciocalteau procedure and total flavonoid contents were measured using a spectrophotometric assay.In addition, we prepared skin lotion using the volatile oil obtained with steam distillation.After preparation of lotion, we performed stability tests and organoleptic tests on the final product.

Plant Material and Isolation of Volatile Oil
Laurus nobilis leaves were collected in June 2016 from the trees in Fethiye region of Muğla (Turkey).Authentication of the plant was performed by Dr. Ergun KAYA from Department of Molecular Biology and Genetics, Faculty of Science, Muğla Sıtkı Koçman University, Muğla (Turkey).Collected leaves were dried under ambient temperature (25 o C), shadow and airy place.Dry leaves were separated from the vimen.
Air-dried leaves of L. nobilis subjected to steam distillation for 120 min to obtain its volatile oil.The resulting volatile oil was dried over anhydrous sodium sulphate, filtered and stored in a dark glass bottle at -21°C until analyze by Gas Chromatography-Mass Spectrometry (GC/MS).

Determination of Chemical Composition of Volatile Oil Using GC/MS
GC/MS analyses were carried out using an Agilent 6890N Gas Chromatograph equipped with a Multi Mode Inlet (MMI) (280 o C), a DB-1 capillary column (30m × 0.25mm; film thickness 0.25μm) and coupled with an Agilent 5975C MS Detector (MSD), operating in the electron impact (EI) mode at 70 eV.Transfer line temperature was set at 250 o C. The carrier gas was He (2.1 mL/min), and the oven temperature was held at 60 o C for 5 min, then increased up to 220 o C at a rate of 2 o C/min and held at this temperature for 10 min.The injected volume was 2 µL and the split ratio 40:1.
In this study, the identification of the compounds was based on the comparison of their retention times (RT) and mass spectra with those from the NIST 2008, Wiley 2008 and Flavor2 libraries.Relative percentages of compounds were calculated based on the peak areas from the MS data.

Preparation of Extracts
In the determination of antioxidant activities of L. nobilis leaf, four different extracts, hexane, ethyl acetate, ethanol and water, were used.The sample was extracted five times for 24 hours at room temperature with hexane, ethyl acetate, ethanol, water and then the combined extracts were filtered through Whatman No 4, separately.The combined extracts of hexane, ethyl acetate, and ethanol were evaporated (rotary evaporator Heidolph, Hei-VAP Precision) to dryness in vacuum, and the combined water extracts were lyophilized (Christ Freeze Dryer, Alpha 1-4 LD plus, Germany).The extracts were stored in deepfreeze until the time of study.The results were given as 50 % inhibition concentration (IC50).The sample concentration (µg.mL -1 ) inhibiting 50 % antioxidant activity (IC50) was calculated from the graph of activity percentage against sample concentration.

DPPH • Free Radical Scavenging Assay
The free radical scavenging activity of L. nobilis leaf extracts was determined using the method, DPPH • free radical protocol [23] with slight modifications.The extract solutions prepared in different concentrations (40 µL) and ethanolic solution (120 µL) containing DPPH • radicals (0.4 mM) were incubated in darkness at room temperature for 30 min.Absorbance was measured at 517 nm in SpectraMax 340 PC, Molecular Device (USA).The radical-scavenging activity (RSA) was calculated as a percentage of DPPH • decolorization using the following equation:

%RSA=[(ADPPH−AS)/ADPPH]×100
Where AS is the absorbance of the solution containing the sample and ADPPH is the absorbance of the DPPH • solution.

β-Carotene-Linoleic Acid Bleaching Assay
The total antioxidant activity was determined using β-carotene-linoleic acid test system based on the detection of inhibition of conjugated dien hydroperoxides due to oxidation of linoleic acid [24,25].β-Carotene (0.5 mg), dissolved in 1mL of chloroform, was mixed with linoleic acid (25 µL) and Tween 40 emulsifier (200 mg).Chloroform was evaporated under low pressure, 50mL of distilled water was added by vigorous shaking.Aliquots (1.60 µL) of this emulsion were added to 40 µL of the extract solutions at different concentrations.As soon as the emulsion was added to each tube, the zero time absorbance was initially measured at 470 nm, and then the absorbance measurements were done for every 30 min until 120 min.

ABTS •+ Cation Radical Decolorization Assay
The spectrophotometric analysis of ABTS •+ scavenging activity was determined according to the previously described method [26].The ABTS •+ (7 mM) in water and potassium persulfate (2.45 mM) reacted to give ABTS •+ , stored in the dark at room temperature for 12 h, and oxidation of ABTS •+ appeared immediately, however, the stability of absorbance was gained after 6 h.Then, the sample solution (40 µL) in ethanol at different concentrations were mixed with ABTS •+ solution (160 µL), giving the absorbance at 734 nm by using a 96-well microplate reader in 10 minute.The scavenging capability of ABTS •+ was calculated using the following equation: Where AS is the absorbance of remaining concentration of ABTS •+ in the presence of sample and AABTS •+ is the initial concentration of the ABTS •+ .

Determination of Total Phenolic Concentrations
The concentrations of total phenolic content in L. nobilis leaf were expressed as microgrammes of pyrocatechol equivalents (PEs), determined with Folin-Ciocalteu reagent (FCR) [27].The sample solution (1 mL) dissolved in methanol was added to distilled water (46 mL) and FCR (1 mL), and mixed thoroughly, 2% sodium carbonate (3 mL) were added to the mixture in 3 min and shaken intermittently for 2 h at room temperature.The absorbance was measured at 760 nm.The concentration of phenolic compounds was calculated according to the following equation that was obtained from standard pyrocatechol graph: Absorbance = 0.0073x -0.1665, r 2 = 0.9976

Determination of Total Flavonoid Concentrations
Measurements of total flavonoid concentration of the extracts were based on the previously reported method [24], and results were expressed as quercetin equivalents (QEs).An aliquot of the sample solution (1 mL) extracts in methanol was mixed with 10% aluminum nitrate (0.1 mL), 1 M potassium acetate (0.1 mL) and 80% methanol (3.8 mL) in test tubes, and then the absorbance was measured at 415 nm in 40 min, and stayed at room temperature.The concentrations of flavonoid compounds were calculated according to following equation that was obtained from the standard quercetin graph: Absorbance = 0.0082x + 0.0073, r 2 = 0.9998.

Preparation of Lotion
The lotion was prepared by following steps.The ingredients; oils, purified water and poly(acrylic acid sodium salt) as emulsifier polymer blend, were weighed separately.6 g of lilac-flavored hazelnut oil and 6 g of bay leaf volatile oil were added dropwise to 87 mL of purified water in a glass beaker at room temperature under magnetic stirring.
After the mixture was stirred with a magnetic stirrer for 10 min, the formulation is completed by addition of the homogenized emulsifier polymer blend.After lotion preparation was completely homogenized, it was poured gently (to avoid the presence of bubbles) into a container.

Determination of Viscosity
The rheology analysis of the lotion formulation containing L. nobilis leaf volatile oil was performed using Brookfield LVDV -I+ viscometer at 10 rpm, 25 o C. Rheological analyses were repeated 1, 5, 30 and 90 days after lotion preparation was completed.

Centrifugation Assay
The centrifuge test, which is often used for having preliminary information in the stability tests, was performed as described in [28].The lotion sample was centrifuged twice; each was 15 min at 3000 rpm and under ambient temperature.A 10 mL centrifuge tube was used for centrifugation and it was observed whether there was phase separation.

pH Measurements
pH change during storage is one of the indicator for chemical stability of cosmetic preparations.The pH of the skin care lotion was measured according to the method given in [29].The measurement was performed at room temperature with a Sartorius pH meter after 1/10 (v/v) dilution of the sample with purified water and filtration.Before the measurement, the pH meter was calibrated with the standard buffer solution (pH = 4, 7 and 10).

Organoleptic Tests of Cosmetic Preparation
Cosmetic preparation was evaluated for appearance, color, odor and spreadability.A visual evaluation was made by adding the sample to a glass container, placed over a white background, and compared it to the previous observations.Spreadability of cosmetic preparation was evaluated according to the expressions of user.All of the organoleptic analyzes were carried out in the same light, temperature and packaging conditions to avoid variations in appearance, color, odor and spreadability parameters.All tests were repeated on 1, 5, 30 and 90 days after the product was prepared.
Volatile oils have been used as antioxidants for the prevention of skin disorders such as skin cancer and wrinkles which caused by oxidative stress on skin surface [38].High amount of 1,8-cineol (46.16%) and terpinen-4-ol (5.07%) makes volatile oil a potent of cosmetic ingredient due to their high antioxidant activity [39].However, these compounds described as potent of antifungal agent by previous researches [40] and with this reason they can be considered as self-preserving ingredient for cosmetic products.
Considering the proven effects of L. nobilis leaf volatile oil and previous studies, the use of L. nobilis leaf essential oil in cosmetic formulations can result in high added value to the product and contribute to product functionality.

Antioxidant Activities
Antioxidant activities of extracts from L. nobilis leaves were investigated using three different methods (β-carotene-linoleic acid, ABTS •+ and DPPH • ).The antioxidant activities of L. nobilis leaf extracts were measured by using spectrophotometric methods after they were dissolved in appropriate solvents.When the total antioxidant activity values were examined, the 50% inhibition concentration (IC50) of ethyl acetate extract was found to be 19.32±1.04µg mL -1 in the β-carotene-linoleic acid assay.Compared to the other extracts and standards, ethyl acetate extract seems to be quite active.

Total Phenolic and Flavonoid Concentrations
Amounts of total phenolic and flavonoid contents in extracts of L. nobilis leaves are given in Table 3.The highest amount of total phenolic content with the amount of 54.42±0.14µg PEs/mg extract was determined in ethyl acetate extract of L. nobilis leaves and the minimum total phenolic content with the amount of 11.04±0.20 µg PEs/mg extract was measured in water extract of leaves.According to the results of total flavonoid content assessments of extracts, the highest total flavonoid content was found in ethanol (8.60±0.12µg QEs/mg extract) and the lowest flavonoid content was determined in the hexane extract (1.01±0.10µg QEs/mg extract) of L. nobilis leaves.
When we compared the results obtained, there is a good correlation between high antioxidant activity and high amount of phenolic compounds in extracts [45].In previous studies, some researchers have found such positive correlations between antioxidant activity and the amount of phenolic compounds of plants [46,47].The ethanol and ethyl acetate extracts obtained by the subsequent extraction appear to be rich in phenolic and flavonoid content in both extracts.Ethyl acetate extracts rich in phenolic compounds were found to have highest antioxidant activity in three different methods (βcarotene-linoleic acid, ABTS •+ and DPPH • ).
Based on the results, it can be said that phenolic compounds have an important effect on the antioxidant activities of extracts.In addition to the phenolic concentrations of extracts, flavonoid concentrations of L. nobilis leaf extract were investigated.The total amount of flavonoid contents in the ethyl acetate and ethanol extracts with high amounts of phenolic contents were found to be 5.48 ± 0.65 and 8.60 ± 0.12 μg QEs/mg extract, respectively.

Stability and Organoleptic Tests of Cosmetic Preparation
The results obtained from the stability tests and the organoleptic analyses of the cosmetic formulation made by the cold process following the analysis are given in Table 4.No phase separation was observed in both 15 min analyzes performed on the days 1, 5, 30 and 90 in centrifugation tests.The pH measurements made on the formulation showed that there was no significant change in the pH of the lotion even after 90 days.Only very small changes due to time have been identified.The viscosity of the product has changed to negligible level due to very small pH changes and it maintains product stabilization after 90 days.
The product was homogeneous and easy to spread when it was prepared.Lotion left a soft, non-greasy feel after it was subjected.There was no change in the product's spreadability and appearance after 90 days.

CONCLUSION
Nowadays, it is clear to see that the escape from artificial substances will further increase the importance of natural products.With this reason, investigation of composition and biological activities of natural substances is important.In this study, the volatile oil composition of L. nobilis leaves was accurately determined by comparative methods using GC/MS instrument.According to the our results, the amounts of 1,8-cineol, alpha-terpinyl acetate and alpha-pinene are similar to the previous studies made by different researchers.However, the amount of terpinen-4-ol in this study found to be higher than other studies.
The antioxidant potential of the extracts was determined using three complementary methods.The extracts investigated in this study have significant antioxidant activity.The highest antioxidant activity in all assays (DPPH, ABTS, β-carotene) was measured in ethyl acetate extract of L. nobilis leaf extract.
L. nobilis volatile oil has potency in cosmetic products due to its numerous antioxidant components.At present, the cosmetic formulation formed within the scope of the study has passed successfully the stability and organoleptic tests that were carried out.At the end of 90 days, there was no significant change in the pH and viscosity parameters of the formulation, and no change in organoleptic properties, such as product appearance, color, odor, spreadability were observed.Good stabilization of the formulation proves that the volatile oil has successfully performed its mission of acting as a preservative in the cosmetic formulation.
In the next step of the study, various dermo-cosmetic effects can be examined of the prepared formulation, and the obtained oil can be used on the formulations in different types of cosmetic products.

Table 1 .
GC/MS analysis of volatile oil composition of Laurus nobilis leafLaurus nobilis volatile oil.(Retention times of the components above the concentration of 1% are given).
a Comparison with standard compounds, b GC/MS analysis, c Literature comparison Figure 1.GC/MS chromatogram of

Table 2 .
Antioxidant activity of the extracts of L. nobilis by β-carotene-linoleic acid, ABTS •+ and DPPH • assays Number of studies have been done to evaluate the antioxidant properties of different extracts of L. nobilis leaves.Previous researchers have reported that ethanolic extract of L.

Table 3 .
Total phenolic and flavonoid concentrations of L. nobilis leaf extracts Results expressed as mean ± standard deviation.

Table 4 .
Results of stability tests and organoleptic analysis of formulation