Preservative activity of lavender hydrosols in moisturizing body gels

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1 Letters in Applied Microbiology ISSN -8 ORIGINAL ARTICLE Preservative activity of lavender hydrosols in moisturizing body gels A. Kunicka-Styczynska,K. Smigielski, R. Prusinowska, K. Rajkowska,B.Kusmider and M. Sikora Institute of Fermentation Technology and Microbiology, Biotechnology and Food Sciences Department, Lodz University of Technology, Lodz, Poland Institute of General Food Chemistry, Biotechnology and Food Sciences Department, Lodz University of Technology, Lodz, Poland Significance and Impact of the Study: The presented research proved antimicrobial activity of hydrosols obtained from fresh or dried Lavandula angustifolia herbs or flowers in moisturizing body gel. The study shows the usefulness of lavender hydrosols as a natural, ecologically friendly component of cosmetics with potential preservative activity in formulations. Hydrosols are commonly regarded as waste in the production of essential oils. The use of lavender hydrosols in the cosmetic industry as a replacement for water phase in cosmetics may not only result in expenses reduction for chemical stabilizers and preservatives but also in substantial decrease in sewage disposal. Keywords antimicrobial activity, challenge test, cosmetic formulations, hydrosols, preservative. Correspondence Alina Kunicka-Styczynska, Institute of Fermentation Technology and Microbiology, Lodz University of Technology, 9-9 Lodz, Wolczanska /, Poland. alina.kunicka@p.lodz.pl /9: received May, revised 8 September and accepted October doi:./lam. Abstract The study was undertaken to verify the antimicrobial activity of Lavandula angustifolia hydrosols in moisturizing body gels. The inhibition efficacy of four lavender hydrosols (obtained from fresh or dry herbs or flowers) was tested against Staphylococcus aureus ATCC 8, Escherichia coli ATCC, Candida sp. ŁOCK 8 and Aspergillus niger ATCC in compliance with the standards of the European Pharmacopoeia Commission. Although the tested hydrosols did not express any remarkable antimicrobial action when tested via the macrodilution method, they show preservative activity in cosmetic preparations. Criterion A for fungi was fulfilled for the cosmetic formulation containing dried flower hydrosol (reduction of the inoculum by two logarithmic units within days with no increase up to the 8th day) and Criterion B for bacteria E. coli and Staph. aureus (reduction of the inoculum by three logarithmic units within days with no increase up to the 8th day). The fresh herb lavender hydrosol in the cosmetic formulation was regarded as the second one effectively satisfying Criterion B for bacteria, but its activity against fungi was below the acceptance value set out in the official regulations. Lavender hydrosols used as a replacement for water phase in cosmetics may contribute to maintaining microbiological stability of cosmetic formulations. Introduction Historically, plant material distillation was performed to obtain herbal waters called hydrosols or hydrolates; however, after the essential oils became the product of primary interest, they were considered undesirable by-products (Catty ). Hydrosols are much less investigated than essential oils, but they have become a subject of great interest in the last decade (Inouye et al. 8; Inouye et al. 9; Hussien et al. ). Due to the relatively high cost of essential oil production, it is economically beneficial for the producers to introduce to the market also the second product of oil distillation. The application of hydrosols as a raw material in the cosmetic industry results in both reducing expenses for sewage disposal and environmental burden as they are no longer discarded to the environment. The natural origin and aromatic properties of hydrosols can be additionally attractive to customers. Hydrosols contain droplets of oils and other plant metabolites usually absent in essential oil; Letters in Applied Microbiology, -- The Society for Applied Microbiology

2 Preservative activity of lavender hydrosols A. Kunicka-Styczynska et al. thus, they may share some of the biological activities of oils or even possess the specific ones (Catty ). Hydrosols as water solutions are extensively used in aromatherapy and thus can be easily applied topically without dilution. True lavender hydrosols can be directly applied on every type of skin, particularly the impure and oily. It exerts cooling, hydrating toning action similar to oil, and it alleviates mental fatigue and stress states. Lavandula angustifolia hydrolate exhibits also revitalizing and relaxing properties when consumed in the form of an additive to water or food (Rose 999; Catty ). However, literature data on antimicrobial activity of hydrosols from lavender are not extensive (Inouye et al. 9), some other hydrosols are reported to cease the development of micro-organisms. According to Tornuk et al. (), black cumin, sage, rosemary and thyme hydrosols are efficient even against pathogenic bacteria Escherichia coli O:H and Salmonella Typhimurium inoculated on fruit. It was also noted that hydrosols of thyme, oregano (Sagdic ; Hussien et al. ), basil, clove, cinnamon and cardamom (Hussien et al. ) pose antibacterial properties and can be a potential preservative in food. Fungicidal effect was noted for sater and pickling herb (Boyraz and Ozcan ). Clover, monarda and santolina hydrosols are able to inhibit both forms of the dimorphic Candida albicans growth in in vitro experiments (Inouye et al. 9). Taking into consideration the abovementioned proven antimicrobial activity of hydrosols, it seems to be interesting to check the preservative activity of lavender hydrosols in situ in cosmetics. In the formulations, they may act as both valuable biologically active components and potentially preservative agents. The Lavandula angustifolia hydrosols, replacing water, were introduced into moisturizing body gel. During the presented study, we applied hydrosols obtained from fresh and dried plants to verify the usefulness of the raw material. Results and discussion The tested lavender hydrosols differed in their chemical composition and contained from 99 mg (dried herb hydrosol) to 9 mg (dried flower hydrosol) of volatile compounds in ml hydrosol (Table ). The total number of identified compounds ranged from to 8 depending on the hydrosol and was the highest for the two hydrosols obtained from flowers. The main component of the volatile fraction of all hydrosols was linalool (9 %), and the next in concentrations were a-terpineol, borneol and geraniol ( 88%, 8 and 9 %). In the antimicrobial activity tests in vitro, no hydrosols showed any activity against the tested bacteria and fungi. In line with the findings of Moon et al. (), it could be demonstrated in this work that these Table Main components of volatile organic fraction of Lavandula angustifolia hydrosols in % Component Fresh herb hydrosol FHH Dry herb hydrosol DHH Fresh flower hydrosol FFH Dry flower hydrosol DFH Octan--one,8-cineole 9 nd,-cineole nd nd nd (z)-linalool oxide nd nd nd (e)-linalool oxide nd nd nd 8 Linalool oxide 9 Linalool 8 9 Oct--en--yl acetate nd nd Camphor 9 nd Borneol 8 8 Lavandulol nd nd nd Cryptone nd nd Terpinen--ol 9 a-terpineol 8 88 Verbenone Nerol 9 Geraniol 9 Caryophylene oxide Carotol nd nd d-cadinol 9 nd Total number of identified components 8 8 Volatile organic compounds (mg in ml hydrosol) nd: not detected. 8 Letters in Applied Microbiology, -- The Society for Applied Microbiology

3 A. Kunicka-Styczynska et al. Preservative activity of lavender hydrosols liquids are too highly diluted to produce an antimicrobial effect. However, some other plant hydrosols are not deprived of their ability to inhibit the growth of microorganisms. As good examples in this context serve thyme, oregano and savoury hydrosols, expressing considerable antibacterial activity in vitro (Sagdic ; Sagdic and Ozcan ). The activity of hydrosols against fungi varies from insignificant to even cidal (Boyraz and Ozcan ; Wojcik-Stopczynska et al. ). According to Hussien et al. (), Candida sp. yeast are resistant to hydrosols, similar to our findings. Another research demonstrates L. angustifolia hydrosol s strong inhibitory effect on filament formation by Candida albicans but moderate yeast growth inhibition (Inouye et al. 9). However, different chemical composition of hydrosols and varied methods of research do not allow making explicit references (Kalemba and Kunicka ; Tajkarimi et al. ). In moisturizing body gel without hydrosols, only the viability of E. coli was affected after th day of a challenge test (Fig. ). The other tested micro-organisms were intact (Figs and ). The addition of lavender hydrosols changed the viability cells profile of E. coli substantially with the only exception of the hydrosol obtained from dry herbs. The most effective against these bacteria was the dry flower hydrosol (Fig. ). Staphylococcus aureus was more sensitive to the formulations with hydrosols comparing to the control gel with water. Although during the first week of the challenge test the cells viability was not deteriorated, the lethal effect was noted at the end of the experiment. The dry flower hydrosol expressed again the highest antibacterial activity. Neither Candida sp. nor Asp. niger were noticeably affected by three moisturizing body gels with lavender hydrosols (Fig. ). Surprisingly, both fungi were extremely sensitive to the formulations containing the dry lavender flower hydrosol, which resulted in population extinction after days with no recovery within 8 days of incubation period. The lavender hydrosols introduced to moisturizing body gels expressed not only antibacterial but also antifungal action. According to the criteria of the European Pharmacopoeia (E.P..) set for topical preparations, Criterion A for fungi was fulfilled for the cosmetic formulation containing the dried flower hydrosol. Criterion A for fungi means a reduction in the inoculum by two logarithmic units within days of a challenge test with no increase up to the 8th day. Although this moisturizing gel was characterized by excellent antifungal activity, its antibacterial action was weaker and for E. coli and Staph. aureus, Criterion B was fulfilled (a reduction in the inoculum by three logarithmic units within days with no increase up to the 8th day). The fresh herb lavender hydrosol in the cosmetic formulation was regarded as the second one effectively satisfying Criterion B for bacteria, but its activity against fungi was below the acceptance value set out in the official regulations. The effect of hydrosols on cosmetic microbiological stability has not yet been extensively researched, and no reference data are therefore available. Our findings for the dried flower hydrosol are comparable to the activity of some essential oils in cosmetics. The similar action against E. coli was observed for Eucalyptus globulus essential added in % to a hydrogel (Maccioni et al. ). Using the same formulation, none of the tested essential oils (% Laurus nobilis, % E. globulus and % Salvia officinalis) was active against Candida albicans, Viable cells in log (CFU ml ) E. coli Viable cells in log (CFU ml ) Staph. aureus Figure Growth inhibition of bacteria E. coli and Staphylococcus aureus in moisturizing gel formulations with lavender hydrosols, moisturizing gel (square), moisturizing gel with fresh herb hydrosol FHH (circle), moisturizing gel with fresh flower hydrosol FFH (triangle up), moisturizing gel with dry herb hydrosol DHH (triangle down) and moisturizing gel with dry flower hydrosol DFH (triangle left). Letters in Applied Microbiology, -- The Society for Applied Microbiology 9

4 Preservative activity of lavender hydrosols A. Kunicka-Styczynska et al. Candida sp. Asp. niger Viable cells in log (CFU ml ) Viable cells in log (CFU ml ) Figure Growth inhibition of yeast Candida sp. and mould Asp. niger in moisturizing gel formulations with lavender hydrosols, moisturizing gel (square), moisturizing gel with fresh herb hydrosol FHH (circle), moisturizing gel with fresh flower hydrosol FFH (triangle up), moisturizing gel with dry herb hydrosol DHH (triangle down) and moisturizing gel with dry flower hydrosol DFH (triangle left). expressing weaker antifungal effect that the dried flower lavender hydrosol tested by us. It should be noted that L. angustifolia essential oil at the concentration of % in aqueous cream (Muyima et al. ) and % in a body milk formulation (Kunicka-Styczynska et al. 9) produced a worse antifungal effect against Candida sp. and Asp. niger than that observed for the dried flower hydrosol in the body gel. Even though regarded as one of the most active Thymus vulgaris essential oil (Kalemba and Kunicka ), applied in cream formulations in % did not satisfy the required Pharmacopoeia preservation efficacy criteria against Asp. niger (Manou et al. 998), in contrast to the dried flower lavender hydrosol in the moisturizing body gel. The high preservative activity of two of the tested lavender hydrosols may be explained by interference among hydrosol constituents and the chemical components of the formulation. A similar phenomenon was noted for essential oils, where a different interference level was reported depending on the formulation type and oil concentration (Hammer et al. 999; Cox et al. ; Messager et al. ; Kunicka-Styczynska et al. ). Moreover, apart from micronized droplets of essential oil, hydrosols contain water soluble components (Hussien et al. ; Catty ) that may act in synergy with the constituents of cosmetic formulations contributing to the inhibitory activity. The tested hydrosols apparently differed in their chemical composition, but linalool was their main compound (Table ). Although the most active in the moisturizing body gel, the dry flower hydrosol, was characterized by the lowest linalool content, it was unique due to the presence of (z)-linalool oxide, (e)- linalool oxide and,-cineole. In contrast, the fresh herb lavender hydrosol with considerable antibacterial activity in cosmetic formulation contained the highest amount of linalool. Linalool is known for its antimicrobial activity, and according to the literature data, fungi are more resistant to linalool than bacteria (Kalemba and Kunicka ; Bakkali et al. 8). Interestingly, the fungicidal effect of linalool analogs differ (Bonikowski et al. 9) and the presence of two linalool oxides may accelerate the antifungal effect of the dried flower hydrosol in cosmetics. Lavender hydrosols seem to be promising cosmetic components and may contribute to maintaining their microbiological stability. However, the real usefulness of any particular hydrosol should be tested in specific cosmetic formulations due to the possibility of interactions between the hydrosol constituents and the cosmetic components. Materials and methods Micro-organisms Staphylococcus aureus ATCC 8, Escherichia coli ATCC, Candida sp. ŁOCK 8 and Aspergillus niger ATCC were used. The micro-organisms originated from ATCC and Collection of Pure Cultures of the Institute of Fermentation Technology and Microbiology, Lodz University of Technology, ŁOCK. The micro-organisms were activated through double passaging: bacteria on TSA medium (Trypticase Soy Agar) Oxoid ( C, 8 h), yeast and mould on SDA medium (Sabouraud Dextrose Agar) biomerieux (8 C, h yeast and days moulds). Letters in Applied Microbiology, -- The Society for Applied Microbiology

5 A. Kunicka-Styczynska et al. Preservative activity of lavender hydrosols Lavender hydrosols The plant material (Lavandula angustifolia L.) was originated from Poland (Wielkopolska), Herb Factory KAWON-HURT. Identification of plant material was performed at the Quality Control Department of KAWON-HURT, and voucher specimen // was preserved in KAWON-HURT, Gostyn, Poland. To prepare the dry plant material, the fresh herb or flowers were prepared in the air steam of C temperature for weeks. The hydrosols were obtained from both fresh and dried flowers and herbs by hydrodistillation method (Inouye et al. 8). Isolation of hydrosols was carried out in a hydrodistillation unit: the plant material was put into a two-necked round bottom flask ( ml) connected to an external source of steam. g of dried lavender flowers or g of fresh lavender flowers (equivalent to g of dried flowers), or g dried lavender herb or 8 g of fresh lavender herb (equivalent to g of dried herb) of Lavandula angustifolia were placed in the flask, and about ml of distilled water was added. Distillation was finished upon receipt of ml of hydrosol (three repetitions). The composition of the hydrosols was determined by GC-MS as described earlier (Smigielski et al. ). The total amount of volatile organic compounds in the hydrosols was estimated according to Smigielski et al. (). At the extraction step, ml of the hydrosol was salted with g NaCl and extracted five times with ml pentane. The extract was concentrated to a volume of about ml and transferred into a volumetric flask. Subsequently, ml of % tetradecan C internal standard in pentane was added to a volume of ml. Calibration curves were prepared using essential oil of lavender (Lavandula angustifolia) and the internal standard of % tetradecan C in toluene (y = x). A Trace GC Ultra gas chromatograph coupled with a DSQ II mass spectrometer from Thermo Electron Corporation was used. An Rtx- ms capillary column was obtained from Restek (length m, internal diameter mm, film thickness mm). Signals were simultaneously collected from two detectors (FID, MS) using a flow divider (ms-column Flow Splitter) from SGE. The following parameters were used: programmed temperature C ( min)- C ( min), at a gradient of C min, injector temperature (splitless) 8 C, detector temperature (FID) C, carrier gas helium, carrier gas flow rate at a constant pressure of kpa and split :. The mass spectrometer parameters were as follows: ionization energy ev, ion source temperature C, and full scan mode in the mass range. TheNIST Library, Wiley 8th edition and Adams th edition were used. The main components of four lavender hydrosols: fresh herb hydrosol (FHH), dry herb hydrosol (DHH), fresh flower hydrosol (FFH) and dry flower hydrosol (DFH) are presented in Table. Antimicrobial activity of hydrosols in vitro Antimicrobial activity of the hydrosols was estimated by liquid macro dilution method. Trypticase soy broth (TSB, Oxoid) and sabouraud dextrose liquid broth (SDLB, bio- Merieux) were used for bacteria and fungi, respectively. The appropriate hydrosol was used to dissolve the media, which were further sterilized by membrane filtration method and inoculated up to a cell concentration of CFU ml. The pure medium without hydrosols acted as the positive control. The negative controls were the bacteria and fungi cultures without hydrosols and with addition of novobiocin ( lg ml ) and cycloheximide ( lg ml ), respectively. All the samples were incubated at temperatures optimal for the growth of individual micro-organisms for h for bacteria and yeast and days for fungi. Then, the viability of micro-organisms was controlled by a surface culture on TSA (bacteria) or SDA (fungi) media. Plates were incubated for days for bacteria and yeast and for days for mould at temperatures optimal for the growth of the particular micro-organisms, C for bacteria and 8 C for fungi. Moisturizing body gel Formula ingredients of cosmetics: extract of Aloe vera (Naturex s.c., Katowice, Poland) g, glycerol (Surchem Sp zo.o., Lodz, Poland) g, Carbomer Ultrez (Azelis Poland sp. z o.o, Poznan, Poland) g, SESAFLASH (glycerine, acrylates copolymer, VP/polycarbamyl polyglycol ester, hydrolysed sesame protein PG-propyl methylsilanediol) (Seppic, Warsaw, Poland) g, TEA (triethanolamine) (POCH S.A., Gliwice, Poland) 8 g and lavender hydrosol 9 g. Moisturizing body gel with water replacing the hydrosol acted as a reference sample. Challenge test Challenge tests of cosmetics were prepared by introducing ml suspension of bacteria or fungi after activation to ml moisturizing body gel. The suspensions of the tested bacteria and yeast cells were prepared in a physiological salt solution (NaCl 8 gl ), the ones of the mould in a physiological salt solution with the addition of gl polysorbate 8 R and later standarized to the density of about 8 CFU ml. Moisturizing body gels were mixed thoroughly and incubated in the dark at C. The number of viable cells in formulations was determined by the count plate method immediately after inoculation and at,, and 8 days. A sample of Letters in Applied Microbiology, -- The Society for Applied Microbiology

6 Preservative activity of lavender hydrosols A. Kunicka-Styczynska et al. ml moisturizing body gel was transferred to 9 ml suspending liquid (peptone K gl, NaCl 8 gl,ph ). The prepared suspensions were inoculated on TSA plates for bacteria and SDA plates for fungi, prior to being resuspended so as to obtain colonies on a plate. The plates were incubated at and 8 C for bacteria and fungi, respectively. The results were expressed as log CFU ml. Statistical analysis of the results Results were analysed using a -way ANOVA test at a confidence level of P <. Results of the population viability were presented as an arithmetic mean of three determinations with standard deviation not exceeding logarithmic units. Results of the analysis of the hydrosol volatile organic compounds were presented as an arithmetic mean of five determinations with SD. Conflict of interest No conflict of interest declared. References Bakkali, F., Averbeck, S., Averbeck, D. and Idaomar, M. (8) Biological effects of essential oils a review. Food Chem Toxicol,. Bonikowski, R., Sikora, M., Kula, J. and Kunicka, A. (9) Thienyl analogues of acyclic monoterpene alcohols and their biological activity. J Sci Food Agric 89, Boyraz, N. and Ozcan, M. () Antifungal effect of some spice hydrosols. Fitoterapia,. Catty, S. () Hydrosols: The Next Aromatherapy. pp. 9,, 8. Rochester: Healing Art Press. Cox, S.D., Mann, C.M. and Markham, J.L. () Interactions between components of the essential oil of Melaleuca alternifolia. J Appl Microbiol 9, 9 9. European Pharmacopoeia Commission. (E.P..) () Efficacy of Microbial Preservation, Group of Experts no. CM (Microbial Contamination). Strasbourg: Council of Europe. Hammer, K.A., Carson, C.F. and Riley, T.V. (999) Influence of organic matter, cations and surfactants on the antimicrobial activity of Melaleuca alternifolia (tea tree) oil in vitro. J Appl Microbiol 8,. Hussien, J., Teshale, C. and Mohammed, J. () Assessment of the antimicrobial effect of some Ethiopian aromatic spice and herb hydrosols. Int J Pharmacol,. Inouye, S., Takahashi, M. and Abe, S. (9) Inhibitory activity of hydrosols, herbal teas and related essential oils against filament formation and the growth of Candida albicans. Jpn J Med Mycol,. Inouye, S., Takahashi, M. and Abe, S. (8) A comparative study on the composition of forty-four hydrosols and their essential oils. Int J Essent Oil Therapeutics, 89. Kalemba, D. and Kunicka, A. () Antibacterial and antifungal properties of essential oils. Curr Med Chem, Kunicka-Styczynska, A., Sikora, M. and Kalemba, D. (9) Antimicrobial activity of lavender, tea tree and lemon oils in cosmetic preservative systems. J Appl Microbiol, 9 9. Kunicka-Styczynska, A., Sikora, M. and Kalemba, D. () Lavender, tea tree and lemon oils as antimicrobials in washing liquids and soft body balms. Int J Cosmet Sci,. Maccioni, A.M., Anchisi, C., Sanna, A., Sardu, C. and Dessi, S. () Preservative systems containing essential oils in cosmetic products. Int J Cosmet Sci, 9. Manou, I., Bouillard, L., Devleeschouwer, M.J. and Barel, A.O. (998) Evaluation of the preservative properties of Thymus vulgaris essential oil in topically applied formulations under a challenge test. J Appl Microbiol 8, 8. Messager, S., Hammer, K.A., Carson, C.F. and Riley, T.V. () Assessment of the antibacterial activity of tea tree oil using the European EN and EN standard suspension tests. J Hosp Infect 9,. Moon, T., Wilkinson, J.M. and Cavanagh, H.M.A. () Antibacterial activity of essential oils, hydrosols and plant extracts from Australian grown Lavandula spp. Int J Aromatherapy, 9. Muyima, N.Y.O., Zulu, G., Bhengu, T. and Popplewell, D. () The potential application of some novel essential oils as natural cosmetic preservatives in an aqueous cream formulation. Flavour Fragr J, 8. Rose, J. (999) Essential Oils and Hydrosols. pp.. Berkeley, CA: Frog, Ltd. Sagdic, O. () Sensitivity of four pathogenic bacteria to Turkish thyme and oregano hydrosols. Food Sci Technol,. Sagdic, O. and Ozcan, M. () Antibacterial activity of Turkish spice hydrosols. Food Control,. Smigielski, K., Prusinowska, R., Krosowiak, K. and Sikora, M. () Comparison of qualitative and quantitative chemical composition of hydrolate and essential oils of lavender (Lavandula angustifolia). J Essent Oil Res, Tajkarimi, M.M., Ibrahim, S.A. and Cliver, D.O. () Antimicrobial herb and spice compounds in food. Food Control, Tornuk, F., Cankurt, H., Ozturk, I., Sagdic, O., Bayram, O. and Yetim, H. () Efficacy of various plant hydrosols as natural food sanitizers in reducing Escherichia coli O: H and Salmonella Typimurium on fresh cut carrot and apples. Int J Food Microbiol 8,. Wojcik-Stopczynska, B., Jakowienko, P. and Wysocka, G. () The estimation of antifungal activity of essential oil and hydrosol obtained from wrinkled-leaf mint (Mentha crispa L.). Herba Pol 8,. Letters in Applied Microbiology, -- The Society for Applied Microbiology