Downy Lavender Oil: A Promising Source of Antimicrobial, Antiobesity, and Anti-Alzheimer's Disease Agents

Lavandula pubescens Decne (LP) is one of the three Lavandula species growing wildly in the Dead Sea Valley, Palestine. The products derived from the plant, including the essential oil (EO), have been used in Traditional Arabic Palestinian Herbal Medicine (TAPHM) for centuries as therapeutic agents. The EO is traditionally believed to have sedative, anti-inflammatory, antiseptic, antidepressive, antiamnesia, and antiobesity properties. This study was therefore aimed to assess the in vitro bioactivities associated with the LP EO. The EO was separated by hydrodistillation from the aerial parts of LP plants and analyzed for its antioxidant, antimicrobial, anticholinesterase, and antilipase activities. GC-MS was used for phytochemical analysis. The chemical analysis of the EO composition revealed 25 constituents, of which carvacrol (65.27%) was the most abundant. EO exhibited strong antioxidant (IC50 0.16–0.18 μL/mL), antiacetylcholinesterase (IC50 0.9 μL/mL), antibutyrylcholinesterase (IC50 6.82 μL/mL), and antilipase (IC50 1.08 μL/mL) effects. The EO also demonstrated high antibacterial activity with the highest susceptibility observed for Staphylococcus aureus with 95.7% inhibition. The EO was shown to exhibit strong inhibitory activity against Candida albicans (MIC 0.47 μL/mL). The EO was also shown to possess strong antidermatophyte activity against Microsporum canis, Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton floccosum (EC50 0.05–0.06 μL/mL). The high antioxidant, enzyme inhibitory, and antimicrobial potentials of the EO can, therefore, be correlated with its high content of monoterpenes, especially carvacrol, as shown by its comparable bioactivities indicators results. This study provided new insights into the composition and bioactivities of LP EO. Our finding revealed evidence that LP EO makes a valuable natural source of bioactive molecules showing substantial potential as antioxidant, neuroprotective, antihyperlipidemic, and antimicrobial agents. This study demonstrates, for the first time, that LP EO might be useful for further investigation aiming at integrative CAM and clinical applications in the management of dermatophytosis, Alzheimer's disease, and obesity.


Introduction
e genus Lavandula (Lamiaceae), lavender, is a typical aromatic evergreen understory chamaephyte that comprises about 32 species [1], some of them being utilized in complementary and alternative medicine for a long time, either dried or as essential oils (EOs). ree native Lavandula species are growing wild in Palestine (West Bank and Gaza for relieving anxiety and associated sleep disorders [10], depression, and headache [11]. e EO of Lavandula species is also used widely in pharmaceutical fragrance, food, and household cleaners [12][13][14].
e products derived from the Palestinian Downy lavender (L. pubescens) (Arabic, Khuzama), including EO, have been utilized for centuries in Traditional Arabic Palestinian Herbal Medicine (TAPHM) as CAM therapies [17]. e LP EO is traditionally believed to have sedative, anti-inflammatory, antiseptic, antidementia, and antiobesity properties and has therefore been utilized for the management of, but not limited to, indigestion, neurological disorders, dementia, obesity, and microbial skin infections [17].
is study was, therefore, aimed at defining the chemical composition of EO attained from above-ground parts of L. pubescens plants collected from wild populations in the Dead Sea Valley in Palestine, and assessing its potential in vitro antioxidant, antimicrobial, anticholinesterase, and antilipase effects and thus to verify its use as a complementary medicine for the treatment of AD, obesity, and microbial skin infections.

Plant Material and Essential Oil Extraction.
e aerial parts of fully bloomed Lavandula pubescens were collected from Palestine (Dead Sea Valley) in May 2017 and used for EO extraction. Plants were authenticated by the first author. e voucher specimen (Lavandula pubescens Decne, Voucher No. BERC-BX603) has been deposited at BERC Herbarium, Til, Nablus, Palestine. 250 gm of the fresh aboveground plant parts were subjected to hydrodistillation using a modified Clevenger apparatus until there was no significant increase in the amount of EO collected [18].

GC-MS Analysis of Essential
Oil. Gas chromatographymass spectrometry (GC-MS) was performed to determine the EO composition by using the conditions reported by Ali-Shtayeh et al. [18]. Identification of the compounds was performed by comparing their relative retention indices (RI) with those of authentic compounds (e.g., carvacrol, terpinolene, ε-caryophyllene, and β-bisabolene) or by comparing their mass spectral fragmentation patterns with Wiley 7 MS library (Wiley, New York, NY, USA) and NIST98 (Gaithersburg, MD, USA) mass spectral database. e identified components along with their RI values and percentage composition are summarized in Table 1.

Enzymatic Inhibitory Activities.
e essential oils of L. pubescens and carvacrol were investigated for their enzyme inhibitory properties on acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and porcine pancreatic lipase (PPL) following previously reported spectrophotometric methods [21,22]. Neostigmine was used as a reference compound for AChE and BuChE enzymes, and orlistat was used for PPL enzyme. e effects of different doses of test compounds (LP essential oil, carvacrol and reference compounds) on the AChE, BuChE, and PPL activities were used to calculate the IC 50 values from dose-effect curves by linear regression.

Microbiological Assays.
Microorganisms used in this study are presented in Table 2.

Agar Disc Diffusion Assay.
is method was used to evaluate the antimicrobial activities of the EO and carvacrol against Candida albicans and bacterial strains as described by the Clinical and Laboratory Standards Institute (CLSI) [23]. e inhibition zone diameter for each sample was measured in mm and used to calculate the antibacterial and anticandidal activity index (AI) and % of inhibition (PI) at a concentration of 1 μL/disc using the following formulas [24]: AI � mean zone of inhibition of EO zone of inhibition obtained for standard antibiotic , All experiments were done in triplicate. Chloramphenicol and voriconazole were used as positive controls for bacteria and candida, respectively.

Broth Microdilution Assay.
e broth microdilution technique with some modifications was used to determine the minimum inhibitory concentration (MIC) values of the EO against bacteria and C. albicans strains [25][26][27]. Chloramphenicol (1 to 64 μg/mL) and voriconazole (0.019 to 1.25 μg/mL) were used as reference antibiotics for bacteria and Candida, respectively.

Determination of Antidermatophytic Activity:
Poisoned-Food Technique. Essential oils from L. pubescens and carvacrol were tested for their antidermatophyte activity against four dermatophytes species: Microsporum canis, Trichophyton mentagrophytes, Epidermophyton floccosum, and Trichophyton rubrum (Table 2) using the modified poisoned-food technique [28]. EO and carvacrol were tested at different concentrations (0.5-0.0039 mL/L). Mycelial growth inhibition % (PI) was calculated as follows: where DC is the average diameter of mycelial growth of the control, and DT is the average diameter of mycelial growth

GC-MS Analysis.
ere are no reports on the EO composition of L. pubescens growing wild in Palestine and only a few such reports are available worldwide [6,8,13,31]. Hydrodistillation of the L. pubescens leaves yielded 1.9 mL per 250 g fresh plant material.

Antioxidant Potential.
e antioxidant activity of EOs is a biological property of great interest because the oils that possess the ability of scavenging free radicals may play an important role in the prevention of some diseases that may result from oxidative stress damages caused by the free radicals, such as brain dysfunction, Alzheimer's disease, obesity, cancer, heart disease, and immune system decline [37][38][39]. e consumption of naturally occurring antioxidants that can be used to protect human beings from oxidative stress damages has therefore been increased [38]. is work reports the antioxidant activities of L. pubescens EO as assessed by ABTS and RP assays (Table 3). e antioxidant potential of LP EO was generally high with RP 50 and IC 50 of 0.16 and 0.18 μL/mL using RP and ABTS assays, respectively. Interestingly, carvacrol has shown comparable antioxidant activity (IC 50 � 0.03 μL/mL) relative to the potent antioxidant agent BHT using the ABTS assay and high antioxidant capacity (RP 50 � 0.07 μL/mL) comparable to the tested potent antioxidant agents (Trolox and BHT) ( Table 3). e antioxidant capacities of L. pubescens EO may be attributed to the high content of the oil's major phenolic constituents, especially carvacrol, which were confirmed as effective antioxidant compounds with potential health benefits [40]. Our results demonstrate that the EOs of L. pubescens and carvacrol have a significant strength to provide electrons to reactive oxygen species (ROS), converting them into more stable nonreactive species and ending the free ROS chain reaction.

Antibacterial Activity.
Results for the in vitro antibacterial activity of L. pubescens EO and carvacrol are presented in Figures 1 and 2  e strong antibacterial activity of the EO may be ascribed to the presence of high % of oxygenated monoterpenes (73.26%) such as carvacrol (65.27%), which was found to destroy cell morphology and biofilm viability in typical biofilm construction by increasing the permeability and reducing polarization of the cytoplasmic membrane [41][42][43].
e antibacterial activity of carvacrol has been mainly attributed to its hydrophobicity and the free hydroxyl group in its structure [44]. With the appropriate hydrophobicity of carvacrol, the compound can be accumulated in the cell membrane, while its hydrogen-bonding and its proton-release abilities may induce conformational modification of the membrane resulting in cell death [45]. Our results can, therefore, explain the association of the use of the LP EO in TAPHM as an antiseptic, due to the antibacterial action of carvacrol which has been previously confirmed [46,47].

Anticandidal Activity.
Candidiasis is a mycotic infection caused by several species of Candida, which can endorse superficial and systemic opportunist diseases worldwide. e current treatment against candidiasis is based on synthetic antimycotic drugs. Most presently available anticandidal drugs have limitations that hamper their use, which  Figure 1) and MIC values of 0.47 and 0.24 μL/mL for EO and carvacrol, respectively (Figure 2). e strong anticandidal activity of EO can, therefore, be correlated with its high content of carvacrol owing to the anticandidal activity of carvacrol which has been previously confirmed [48].

Antidermatophytic
Activity. Aromatic plants EOs are known to be mycostatic or fungicidal and represent a potential source of new antimycotics [49]. In view of the increasing resistance to the classical antimycotics, the EOs and their active constituents may be beneficial in the management of mycoses, especially dermatophytosis [50]. In the present study, the L. pubescens EO showed strong activity against M. canis, T. rubrum, T. mentagrophytes, and E. floccosum as indicated by their PI, MIC, MFC, and EC 50 values (Figure 3). e EO of L. pubescens and carvacrol showed a dosedependent activity against the tested dermatophytes (Figure 4). Overall, as the dose of the EO or carvacrol increased, the inhibitory activity against the tested dermatophytes increased indicated by heightened mycelial growth inhibition. e radial mycelial growth of all tested isolates was completely inhibited by the EO and carvacrol at 0.5, 0.25, and 0.125 μL/mL concentration. However, at lower doses (0.004-0.063 μL/mL), the EO was still more active on the mycelial growth of T. mentagrophytes than other tested dermatophytes at 0.63 μL/mL, PI � 89.7% (Figure 3). e MIC and EC 50 values of the EO of L. pubescens on the tested dermatophytes were in the ranges of 0.08-0.16 μL/ mL and 0.05-0.06 μL/mL, respectively. However, EO showed a fungicidal effect on the four studied dermatophytes and the MFCs were in the range of 0.16-0.25 μL/mL. T. mentagrophytes were more susceptible to L. pubescens EO than the other tested fungi with MIC, MFC, and EC 50 values of 0.05, 0.08, and 0.16 μL/mL, respectively. e strong antifungal property could be attributed to the major component of the EOs, carvacrol, and the oxygenated monoterpene, which exhibited strong inhibitory activity against the tested dermatophytes ( Figure 3) with PI, MIC, Evidence-Based Complementary and Alternative Medicine EC 50 , and MFC values ranging from 76.7 to 100%, 0.063-0.125 μL/mL, 0.01-0.1 μL/mL, and 0.03-0.63 μL/mL, respectively. e monoterpene alcohols are water soluble and possess functional alcohol groups that explain their strong antidermatophyte activity [49].

Enzyme Inhibitory Activities of Essential Oil
3.6.1. Anticholinesterase Activity. Cholinesterase inhibitors (ChEIs) have recently become the most widely used drugs for the management of Alzheimer's disease (AD) [54]. ChEIs play a crucial role in the memory enhancement of AD patients through increasing ACh concentration in neural synaptic clefts and thus improving the brain cholinergic transmission and decreasing β-amyloid aggregation and neurotoxic fibrils formation [55][56][57]. However, synthetic AChEIs including galanthamine and tacrine have restrictions owing to the short half-life and adverse side effects such as digestive disorders, nausea, and dizziness [58,59]. Hence, it is necessary to explore new safe alternatives with superior characteristics to deal with AD.
Several plants and phytochemical compounds have revealed cholinesterase inhibitory capacity and therefore can be valuable in the management of neurological disturbances [21]. In this study, LP EO was investigated for its in vitro cholinesterases (AChE and BuChE) inhibitory activities. e EO and carvacrol have shown to possess high AChE (IC 50 � 0.9, and 1.43 μL/mL, respectively) and medium BuChE (IC 50 6.82, and 7.75 μL/mL, respectively) inhibitory activities (Table 4).
us, the high AChE inhibitory effect of the L. pubescens EO in the current study may be mainly associated with its major component, carvacrol, and with its high phenol content. Overall, the tested EO was shown to be more selective inhibitors for acetylcholinesterase than butyrylcholinesterase with a selectivity index (SI) of 7.58.
Our results demonstrate that LP EO could be a valued natural source of AChEIs, e.g., carvacrol, with effective inhibitory activities against the principal enzymes associated with AD and could signify a basis for developing a new treatment strategy for Alzheimer's using plant-derived AChEIs.
3.6.2. Pancreatic Lipase Inhibitory Activity. Pancreatic lipase, the principal enzyme associated with obesity, plays a key role in the efficient digestion of acylglycerols [60]. e hydrolysis of glycerides to glycerol and free fatty acids is performed by lipases. Taking into consideration that 50-70% of the total dietary fat hydrolysis is performed by pancreatic lipase, enzyme inhibition is one of the approaches used to treat obesity [60]. e mechanism involves inhibition of dietary triglyceride absorption, as this is the main source of excess calories [61]. Besides, pancreatic lipase inhibition does not alter any central mechanism, which makes it an ideal approach for obesity treatment [62]. e pancreatic lipase has been widely used for the determination of the potential efficacy of natural products as antiobesity agents [62].
In the present study, L. pubescens EO and carvacrol were assessed for their activity against pancreatic lipase. e EO exhibited high inhibitory activity against PPL with IC 50 of 1.08 μL/mL (Table 5). e high antiobesity activity of L. pubescens EO may be mainly ascribed to its high content of carvacrol which has been reported to inhibit visceral adipogenesis and adipocyte differentiation in animal cells and decrease body weight and plasma lipid levels [63,64]. However, carvacrol on its own cannot explain the high activity of EO, and therefore the totality of constituents of the EO may act synergistically to exert such high antiobesity activity. e higher pancreatic lipase inhibitory effects of L. pubescens EO may, therefore, be attributed to its high content of bioactive phenolic acids and flavonoids acting together in a synergistic style [22]. e current study has indicated the ability of the EO to exercise health benefit attributes by inhibiting the pancreatic lipase enzyme (responsible for digestion and absorption of triglycerides) and thus lead to the reduction of fat absorption.

Conclusions
e main constituent of L. pubescens EO was determined as carvacrol in wild plants. e results demonstrate that the plant is a valuable natural source for carvacrol-rich EO with promising potential antimicrobial, antiobesity, and anti-AD health effects ( Figure 5). Our results support the use of L. pubescens EO as a natural complementary treatment in TAPHM. is is the first report on the antidermatophytic, AChE inhibitory, and antiobesity effects of L. pubescens EO. In conclusion, our results might be useful for further investigation aiming at clinical applications of L. pubescens EO and carvacrol in the management of AD, obesity, and microbial skin infections including dermatophytosis, candidiasis, and others.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.   Figure 5: Beneficial health effects of Lavandula pubecsens essential oil and its main active constituent, carvacrol.
Evidence-Based Complementary and Alternative Medicine 7