Study of Phenolic Compounds and Antimicrobial Activity of Lavandula angustifolia L. Flowers Macerates

GABRIELA STANCIU1, FLORIN AONOFRIESEI2, SIMONA LUPSOR1*, ANTOANELA POPESCU3, RODICA SIRBU3 1Ovidius University of Constanta, Department of Chemistry and Chemical Engineering, 124 Mamaia Blvd., 900527, Constanta, Romania 2Ovidius University of Constanta, Faculty of Natural Sciences and Agricultural Sciences,124 Mamaia Blvd., 900527, Department of Natural Sciences, Romania 3Ovidius University of Constanta, Department of Pharmaceutical Science, 124 Mamaia Blvd., 900527, Constanta, Romania

The genus Lavandula belongs to the 'Labiatae' family which geographically grows mostly in Europe and comprises many different species. The agricultural practices directly influence the secondary metabolites produced in the plants, and thus the quality of polyphenolic compounds [4][5][6]. Lavadula species biosynthesizes and accumulates its volatile compounds primary in specialized secretor y capitate and peltate oil glands located abundantly on the surface of the calyx and secondary on the leaves [1-2, [6][7]. Generally, the flower is an important part of plant which contains a great variety of natural antioxidants such as flavonoids, phenolic acids, anthocyanin and many other precious phenolic compounds [3].
Lavender is well known from ancient times for its medicinal uses as sedative, cholagogue and antiseptic [3]. Due to its content in essential oils, triterpenes, flavonoids and other polyphenolic compounds Lavandula species are widely used in food industry and cosmetics [4]. Lavandula angustifolia presents a wide range of useful therapeutically properties, such as: antioxidant [1][2], antifungal and bactericidal [6], cytotoxic [7], antiseptic, antiinflammatory, analgesic [7][8], and other useful properties for pharmaceutical industries, food industries and cosmetics industries. Antimicrobial activity of the essential oil extracted from lavender has been well documented over time [9][10]. Besides essential oils, other components of Lavandula may have substantial antimicrobial properties [11]. Therefore, we found interesting to determine the amount of polyphenolic compounds present in Lavandula sp. flowers from Dobrogea area and to test it against both Gram negative bacteria, Gram positive bacteria as well as against several Candida strains in order to estimate their antimicrobial effect. The total content of phenolic compounds from two different types of macerates was measured using the Folin-Ciocalteu method and the phenolic profile of studied macerates was established using HPLC-DAD method. The antimicrobial activity of lavender extract was established by difusimetric well method and minimal inhibitory activity (MIC).

Experimental part Plant materials
Lavandula angustifolia L flowers were collected in August and September 2017, from organic culture in Topraisar, Constanta County, Romania. The plant was dried at ambient temperature until constant weight was achieved.

Apparatus
The chromatographic determinations of phenolic compounds were performed with HPLC-DAD system Agilent 1200 with quaternary pump, DAD, autosampler.
Spectrometric measurements were carried out using a UV-VIS JASCO V550 scanning spectrophotometer.

Sample extract
Extraction 1 was achieved by maceration of 5 g of dried plant in 60 mL methanol at room temperature and protected from light during five days. The mixture was strongly shaken several times every day. A sample of 39 mL of macerate (M1) was separated by filtration and then analysed.
Extraction 2 was achieved by maceration of 5 g of dried plant product in 60 mL methanol: water 1:1 (V:V) mixture in similar conditions as extraction 1. After 5 days of maceration a sample of 33 mL of macerate (M2) was separated by filtration and immediately analysed.

Identification and quantification of phenolic compounds by HPLC-DAD
The resulted macerates solutions were analysed by HPLC.
Adapted USP30 HPLC method [12][13] was used for separation, identification and quantification of the phenolic compounds.
The retention times of standard solutions have been determined (table 2). Standard deviations of retentions time were obtained after statistical processing of the 6 injections (soft SPSS 10).

Table 1 THE GRADIENT OF ELUTION SOLVENTS
The parameters of chromatographic process were: 1.5 mL/min the flow rate, 20 µL injection volume and 22 min analysis time.
Quantification of phenolic acid was performed using absorbance measurements at 310 nm and 35°C. The retention times and DAD spectra were compared to available authentic standards.  . 1).

Total phenolic content (TPC)
The total phenols were estimated according to the Folin-Ciocalteu method [14][15][16]. During the procedure in a 5 mL extractive solution were added 1 mL of Folin-Ciocalteureagent 1:2 (V:V) and 1 mL of 20% (w/v) Na 2 CO 3 aqueous solution; after 10 min the volume was brought to mark up to 50 mL with distilled water. After 30 min of incubation at 25 o C the absorbance was measured at 681 nm; the total phenols concentration was determined using the calibration curve plotted with gallic acid as standard. The calibration curve was linear in the range of 0.68 -4.76 mg /L GAE (R 2 =0.9973) where GAE is gallic acid equivalents. Total phenols content of lavender extract was expressed as mg of gallic acid equivalents per 100 g of dry weight (mg GAE/100g d.w.). All samples were performed in triplicate and the mean value was reported. Calibration curve For calibration curve the volumes of 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL and 7 mL of gallic acid standard solution were used. To plot the calibration curve the Folin-Ciocalteu reagent 1 : 2 was added in 50 mL calibrated flasks to different volumes of standard gallic acid solution, then 1 mL sodium carbonate solution 20%, was added and after 10 min the volume was made up to 50 mL with distilled water. Each sample was homogenized and let 30 min at room temperature for the colour stabilization and after that the absorbance was read at 681 nm. (fig. 2) For TPC analysis, volumes of 10 mL of each macerate were transferred into 25 mL volumetric flask and brought to the mark with the same solvent previously used for maceration operation. To measure the total phenols content, samples of 1 mL of previously diluted solutions were added in 25 mL calibrated flasks, then 1 mL Folin-Ciocalteu reagent 1:2 (V:V), 1 mL sodium carbonate solution 20% and the process was the same like those used for calibration. After inoculation by spreading, the wells (d=6 mm) were performed on solid media in aseptic conditions. Lavender macerates M1 and M2 were tested against seven bacterial strains and three Candida strains (table 3).

Estimation of Minimal Inhibitory Concentration (MIC)
The extracts were diluted (from 1:4 to 1:20) in sterile Muller Hinton Broth (bacterial strains) and Sabouraud Dextrose Broth (Candida strains). Test tubes containing culture medium and extracts were inoculated with 10 ìL of cultures grown for 24 h. To estimate the effect of solvent, similar dilution were made with methanol and methanol:

Results and discussions Phenolic compounds separation, identification and quantification
In table 4 are presented the values of phenolic compounds determined by HPLC-DAD method and compared with the available authentic standards.
By comparing to the available authentic standards used for determinations, four individual phenolic compounds were found in tested macerate M1 and six individual phenolic compounds in macerate M2. A high concentration of gallic acid was found in both analysed macerates, but in the hydro-alcoholic macerate M2 the concentration is much higher than the one obtained for alcoholic macerate M1 (table 4). It's well known that gallic acid has mutagenic and teratogenic effect [17][18][19][20][21].
Significant amounts of ellagic acid were found in both macerates. The difference of ellagic acid values concentration in the analysed macerates indicates a higher concentration in M2 (514.249 mg/100 g d.w.) than in M1 (499.487 mg/100 g d.w.). Ellagic acid has antiproliferative properties and antioxidant properties related with its ability to directly inhibit the DNA binding of certain carcinogens, including nitrosamines and polycyclic aromatic hydrocarbons [1][2]. Also, ellagic acid has a chemoprotective effect in cellular models by reducing oxidative stress [19][20][21].
Significant amounts of chlorogenic acid were determined for both macerates. Macerate M2 showed a higher concentration of chlorogenic acid than macerate  M1. It is known that chlorogenic acid slightly reduces blood pressure, but also it has anti-inflammatory action and is studied as a possible chemical sensitizer involved in respiratory allergy [3].
In the case of cinnamic acid it can be noticed that methanolic macerate of lavender M1 presents a higher concentrantion (353.763 mg/100g d.w.) than the hydroalcoholic macerate (M2).
p-Coumaric acid and caffeic acid were identified only in hydro-alcoholic macerate (M2) of Lavandula angustifolia L. flowers.
From the obtained results it is obviously that when methanol : water 1:1 (V:V) (M2) mixture was used as solvent, the extraction of phenolic compounds from Lavandula angustifolia L. flowers proved to be more efficient than the case when extraction of phenols was done with methanol p.a. (M1). The concentration of all phenolic compounds present in Lavandula angustifolia L. flowers extract proves that lavender is a precious source of antioxidants with valuable benefits for human health.

Total phenolic content (TPC)
The results of total phenolic contents from the analysed macerates of Lavandula sp., expressed as mg/100g dry weight, are presented in table 5. Also, the weight percentage (%) of total phenols was reported to the major phenolic compounds determined by HPLC-DAD.
The obtained values in case of TPC analysis indicate that Lavandula angustifolia L has a high amount of phenolic compounds comparable to other literature data [17][18][19][20][21].
The results for TPC indicates that Lavandula angustifolia L macerate M2, therefore when as solvent was used methanol: water 1:1 (V:V), is more efficient for phenolic compounds extraction than in case of macerate M1, when as solvent was used only methanol. The difference between the values of total phenolic compounds determined by both methods for both macerates is due to the presence of other phenolic acids that were not determined in the applied HPLC-DAD conditions.

Difusimetric evaluation of lavender extracts activity
The activity of both macerates was almost similar on average, ranging between 3.4-3.5 mm of inhibition zones. However, individual species demonstrated a distinct behaviour with more variable responses and inhibition zones, ranging from 2 mm (E. coli 2) to 6 mm (Streptococcus sp.) (table 6).   (table 7). Both diffusimetric test and MIC indicated a moderate antimicrobial activity of lavender macerates. In spite of this, the macerates might be useful in cosmetic formulations where they can act as bacteriostatic agents and as such they can prevent contamination and spreading of undesired microorganisms. Moreover, their activity might be increased in ointments and creams where other ingredients may act in a synergistic way and improve their antimicrobial potential.
The obtained data proved that hydro-methanolic and methanolic macerates of Lavandula sp. had a moderate  effect on the bacterial and Candida growth. Their antimicrobial activity was more or less in the same range as other plants products. Macerates of lavender might be used in different pharmaceutical formulations to control contamination and microbial spoilage due to their microbiostatic potential.

Conclusions
The extracts of Lavandula angustifolia L. flowers as hydro-methanolic and methanolic macerates have been analysed to find phenolic composition and to test their antimicrobial activity. Six individual phenolic compounds were identified and quantified by HPLC-DAD in the hydromethanolic macerate M2 of lavender, while in the methanolic macerate M1 only four phenolic compounds were found. The presence of high concentrations of ellagic acid in both macerates together with gallic acid and chlorogenic acid are remarkable for improving the benefits of using Lavandula sp. macerates also as chemoprotective agent.
The Lavandula sp. macerates present high concentrations of total phenolic contents, but also in this case it was noticed that hydro-methanolic macerate (M2) was much richer in phenolic compounds than the one when for maceration was performed only with methanol (M1). This indicates that the comprising solvent methanol: water 1:1 (V:V) mixture is more efficient for phenolic compounds extraction.
The antimicrobial activity of hydro-methanolic (M2) and methanolic (M1) macerates indicates that Lavandula sp. has a moderate effect on the bacterial and Candida growth. Comparing to other plants products, the antimicrobial activity of lavender macerates was more or less in the same range. Therefore, macerates of lavender might be used in different pharmaceutical commercial products with microbiostatic potential.
In general, the lavender use may represent an alternative source of antioxidants with anti-microbial, anti-cancer, anti-inflammatory and anti-diabetic effects.