The Biological Activity of Monarda didyma L. Essential Oil and Its Effect as a Diet Supplement in Mice and Broiler Chicken

The use of growth-promoting antibiotics in livestock faces increasing scrutiny and opposition due to concerns about the increased occurrence of antibiotic-resistant bacteria. Alternative solutions are being sought, and plants of Lamiaceae may provide an alternative to synthetic antibiotics in animal nutrition. In this study, we extracted essential oil from Monarda didyma, a member of the Lamiaceae family. We examined the chemical composition of the essential oil and then evaluated the antibacterial, antioxidant, and anti-inflammatory activities of M. didyma essential oil and its main compounds in vitro. We then evaluated the effectiveness of M. didyma essential oil in regard to growth performance, feed efficiency, and mortality in both mice and broilers. Carvacrol (49.03%) was the dominant compound in the essential oil extracts. M. didyma essential oil demonstrated antibacterial properties against Escherichia coli (MIC = 87 µg·mL−1), Staphylococcus aureus (MIC = 47 µg·mL−1), and Clostridium perfringens (MIC = 35 µg·mL−1). Supplementing the diet of mice with essential oil at a concentration of 0.1% significantly increased body weight (+5.4%) and feed efficiency (+18.85%). In broilers, M. didyma essential oil significantly improved body weight gain (2.64%). Our results suggest that adding M. didyma essential oil to the diet of broilers offers a potential substitute for antibiotic growth promoters.


Introduction
Antibiotic growth promoters (AGPs) have long been used in animal nutrition to reduce mortality and increase the feed conversion ratio and body weight gain, especially for broilers and swine [1][2][3][4][5]. The AGPs given for animal nutrition and agents used for treatment against bacterial infection in humans often belong to the same classes of antibiotics; this heightens the risk of bacteria developing resistance to antibiotics [6]. The enhanced resistance and the issues surrounding the excessive use of in-feed AGPs have led researchers to search out more natural alternatives. In Europe, AGPs have been prohibited for use in animal nutrition since 2006 [3].
In Canada, poultry production involves more than 2600 regulated chicken producers who have access to several AGPs approved as feed additives for poultry [6]. The use of AGPs in Canada is still permitted; however, since 2014, producers have been forced to remove those antibiotics considered as the most important for human health. This important restriction is part of a strategy involving the responsible use of antibiotics by producers and the search for new alternatives. A similar strategy for controlling antibiotic resistance is used in the United States, although there are some differences in regard to their classification of antibiotics compared to the Canadian and World Health Organization (WHO) classifications.
The drive for alternatives to standard AGPs involves finding compounds that ensure poultry health and decrease bacterial resistance while maintaining producer profitability. Public pressure and concerns about food and environmental safety have led to an active search for alternative approaches that could eliminate or decrease the use of antibiotics, such as the use of essential oil, probiotics, prebiotics, and acidifiers in diets. As natural and multi-active agents, plant extracts and plant-derived products offer an interesting potential as substitutes for synthetic antibiotics and inorganic chemicals. Essential oil has some advantages over the others, by (1) improving growth performance and nutrient digestibility through antioxidative and anti-inflammatory activity, and (2) improving intestinal and general health of the broiler via antibacterial and coccidiostatic effects [7][8][9][10]. Both the food industry and animal producers have increased their interest in the use of essential oils (EOs, volatile plant compounds). The positive effects of EO in the digestive tract of animals include the stabilizing of gut microbiota; this benefit could lead to further practices that improve intestinal health, digestion, and growth performance of livestock [6].
Some EOs have already been investigated in animal nutrition studies [11][12][13][14][15][16]. Plants from the Lamiaceae family are particularly promising [15][16][17][18][19]; for example, Denli et al. [20] added thyme essential oil to the diet of quail, resulting in significantly increased body weight gain and better feed efficiency relative to a control group. A member of the Lamiaceae, Monarda didyma L., has been used for millennia by indigenous people in North America to treat colic, flatulence, colds, and flu. This plant is also used in infusions for intestinal and stomach problems [21], and is particularly useful for treating digestive disorders [22].
At present, there are no studies on the bioactivity of M. didyma EO. Nonetheless, compounds such as thymol and carvacrol-often present in the EO of Monarda species [22]have marked antibacterial and anti-inflammatory activities [23][24][25]. Moreover, Ertas et al. [26] showed that thymol and carvacrol help stimulate digestion by affecting pathogenic microorganisms in the gut of broilers, thereby increasing their food conversion rate and weight. The protection and reinforcement of the intestinal microbiome in broilers is critical for maintaining the overall health and optimal absorption of nutrients in these animals [27].
In this study, steam distillation was used to extract M. didyma EO, and GC-MS and GC-FID were used to analyze the composition of the EO. The antibacterial, antioxidant, and anti-inflammatory activities of M. didyma EO in vitro was assessed and results compared with the positive control Thymus vulgaris [24]. The objective of this study was to substitute standard antibiotics with M. didyma essential oil, although the main compounds responsible for the biological activities were identified and the effects of M. didyma EO on the growth performance, feed intake, and feed efficiency of mice and broilers were then evaluated.
M. didyma EO also demonstrated anti-inflammatory activity. A concentration of 35 µg·mL −1 inhibited fifty percent (IC 50 ) of NO induced by LPS in RAW 264.7 macrophages. Carvacrol (IC 50 = 22.6 µg·mL −1 ) and p-cymene (IC 50 = 25.5 µg·mL −1 ) appeared to be responsible, in part, for this activity. Furthermore, a cell-based assay with an IC 50 of 4.6 µg·mL −1 highlighted the EO antioxidant activity (Table 3). Table 2. Antibacterial activity of M. didyma and T. vulgaris (as positive control) essential oils and the main compounds against selected bacterial strains.

Compounds
Antibacterial Activity MIC (µg·mL −1 ) >200 ND ND: Not determined; data are representative of three different experiments; mean ± standard deviation, n = 3; MIC is defined as the lowest concentration inhibiting 95% of bacterial growth. Table 3. Antioxidant and anti-inflammatory activity of M. didyma and T. vulgaris (as positive control) essential oils and the main compounds.
The antibacterial and antioxidant activities of other members of the Lamiaceae family have been observed, principally for T. vulgaris and Origanum vulgare [32][33][34][35][36]; however, the biological activities of M. didyma are less well known. Fraternale et al. [22] used a DPPH and lipid peroxidation test to demonstrate the elevated antioxidant activity of M. didyma EO. Numerous studies have highlighted the antibacterial and antioxidant activities of EOs that possess high levels of phenolic compounds such as thymol, carvacrol, and eugenol [9,24,25]. Thymol and carvacrol appear to be particularly effective against Gram-negative bacteria, in part because these compounds act on the outer membrane, such as by provoking the release of lipopolycaccharides, increasing the permeability of the cytoplasmic membrane, and depolarizing the cytoplasmic membrane. Furthermore, hydroxyl groups are highly reactive and form hydrogen bonds with active sites of target enzymes, inactivating them, and consequently create a dysfunction or rupture of the cell membrane [25]. Similar to our study, Guimarães et al. [25] also observed high and rapid thymol and carvacrol activity against E. coli. Antioxidant and anti-inflammatory effects of the EO can produce a positive effect in the gastrointestinal tract.
Given that M. didyma EO demonstrated in vitro activities, the effectiveness of different concentrations of EO in the diet of mice on body weight gain (BW), feed intake (FI), and feed efficiency (FE) was assessed. A significant difference between the treatment groups and the negative control group for BW (p < 0.001), FI (p < 0.001), and FE (p < 0.001) was observed (  No studies have yet tested EO diet supplements in mice; however, Denli et al. [20] observed that T. vulgaris EO caused BW gain and improved FE for quail. Platel et al. [37] concluded that adding various spices in food enhanced either enzyme activity related to digestion or increased the secretion of bile. Yang et al. [38] observed that EO supplementation during the growth period increased lipase, trypsin, and chymotrypsin activities significantly. EO also increased the fecal digestibility of dry matter and the digestibility of ether extract, fiber, fat, ash, and protein [18]. The improvement of BW and FE in our study of mice was attributed to these processes. When M. didyma EO was added to the diet of male broilers, during a growth period of 36 days, the treated broilers attained a BW of 2.65 kg with an average of 4.46 kg of FI by the animals. Control broilers with standard antibiotics weighed 2.58 kg after an average FI of 4.51 kg. Broilers fed with the EO-supplemented diet therefore showed a BW increase of 2.64% compared to control animals fed using standard antibiotics (Table 5). A significant difference was observed for BW between the control and M. didyma EO broilers in the first 10 days (268.5 g and 279 g, respectively). During the growth and finisher phases, broilers fed with the EO diet were significantly heavier than the control broilers with antibiotics and followed the same growth curve ( Figure S1). Table 5. Comparison of mean body weight gain (g), feed intake (g), and feed efficiency of the broiler treatments. Existing studies identify four different mechanisms that are important for EO actionsensorial, metabolic, antioxidant, and antibacterial activities [13]. Hashemipour et al. [39] demonstrated that supplementing a broiler diet with 100 mg/kg and 200 mg/kg of thymol and carvacrol, respectively, produced no effect on FI, but significantly increased BW gain and improved the feed conversion ratio. Antibiotics in animals improve the BW by 5-6% and the FE by 3-4%, with the most pronounced effects observed in young animals [6]. Khattak et al. [15], supplementing the diets of broilers with a natural blend of EO (including thyme and oregano), observed no significant difference in growth performance during the starter period. Although higher mortality rates could be expected in the EO test group due to the lack of antibiotic use, Khattak et al. [15] found no significant difference between the mortality of broilers having the EO-supplemented diet and that of the control broilers.

Plant Material and Extraction of Essential Oils
M. didyma L. EO was extracted by steam distillation for three hours from freshly harvested aerial parts (refractive index = 1.4977; density = 0.939 g·mL −1 ; yield (%) = 0.49). The plant came from Saint-Fulgence, Québec and was harvested at the beginning of August (Voucher QFA0625784). Thymus vulgaris L. EO (refractive index = 1.4797; density = 0.923 g·mL −1 ) was purchased from Aliksir (Grondines, QC, Canada). The EO yield from extraction depended on the total amount of the raw material. The EO was then stored in the dark at a temperature of 4 • C until needed.

GC-FID and GC-MS Analysis
All chromatographic analyses were run on an Agilent 6890N GC (Agilent Technologies, Santa Clara, CA, USA) equipped with a non-polar DB-5 column and a polar SolGel-Wax column (30 m × 0.25 mm × 0.25 mm) as well as two flame ionization detectors (FIDs) (Agilent Technologies, Santa Clara, CA, USA). The oils were injected in an undiluted (0.1 µL injection volume, split 1:235) and undried state ( Figure S2). The temperature program began at 40 • C for 2 min, then rose 2 • C·min −1 up to 210 • C. The temperature was then held at 210 • C for 13 min. Samples were also injected into an Agilent 7890A GC (Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent 5975C InertXL EI/CI mass spectrometer (MS) equipped with a DB-5MS column using the same temperature program as above and a split of 1:1000. Compounds were identified from their retention indexes as calculated from even-numbered C7 to C36 alkane standards and from Wiley 6N, MS databases (NIST08) and standard injection when available (LASEVE, UQAC, Chicoutimi, QC, Canada). Quantification was derived from the FID response on the DB-5 column without any correction factor.

Bacterial Strains
The in vitro antimicrobial activity of M. didyma EO was tested against Gram-negative Escherichia coli (ATCC 25922), Gram-positive Staphylococcus aureus (ATCC 25923), and Grampositive Clostridium perfringens provided by the Chicoutimi Hospital, Saguenay, Canada. Bacteria were grown in a humidified atmosphere at 37 • C.

Culture Methods
Bacteria were stored at −80 • C until use. For culturing the bacteria, all bacteria were placed in a nutrient broth base (Difco) for 16-18 h at 37 • C; C. perfringens was grown in an anaerobic vial. The cellular density of the inoculum was measured via optical density at 600 nm for E. coli [40], 660 nm for S. aureus [41], and 450 nm for C. perfringens using a Multiskan™ GO Spectrophotometer (Thermo Fisher Scientific). The inoculum was rediluted in the nutrient broth to obtain the required bacterial concentration.

Measurement of Anti-Inflammatory Activity
The inhibition of nitric oxide (NO) production by M. didyma EO and compounds was evaluated following Legault et al. [42]. Control L-NAME was used as a positive control. The murine macrophage RAW 264.7 cells were incubated with EO or dissolved compounds in DMSO, then stimulated using 100 ng·mL −1 LPS and incubated at 37 • C. After 24 h, the cellfree supernatant was collected and the NO concentration immediately determined using the Griess reaction. The absorbance was read at 540 nm using an automated Varioskan Ascent plate reader, and the presence of nitrite was quantified by comparing with a NaNO 2 standard curve, and IC 50 expressed 50% of NO inhibition.

Evaluation of Antioxidant Activity Using Cell-Based Assays
The antioxidant activity was evaluated using the DCFH-DA assay as described by Girard-Lalancette et al. [43]. Human skin fibroblasts WS1 were incubated for 1 h with a growing concentration of EO or dissolved compounds in DMSO. One hundred microliters of 200 µM tert-butylhydroperoxide was then added and fluorescence immediately measured, and again after 90 min. Measurements were made on an automated plate reader (Fluoroskan Ascent FL, Labsystems, Milford, MA, USA) using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Antioxidant activity was expressed as the concentration of extract inhibiting 50% (IC 50 ) of DCFH oxidation.

Evaluation of Antioxidant Activity Using ORAC
The method described by Ou et al. [44] with some modifications was followed. Briefly, the ORAC assay was carried out in black 384-well microplates (Nunc) on a Fluoroskan Ascent FL™ plate reader (Labsystems) equipped with an automated injector. Four concentrations of Trolox (the control standard) (1.56, 3.13, 6.25, and 12.5 µM) were used in quadruplicate, and a gradient of 16 concentrations of the samples (compounds dissolved in DMSO or pure EO) was prepared without replication. The experiment was conducted at 37.5 • C and in a pH 7.4 phosphate buffer with a blank sample run in parallel. The fluorimeter was programmed to record the fluorescence (λ ex.: 485 nm/em: 530 nm) of fluorescein every minute after the addition of 375 mM of 2,2 -azobis(2-amidinopropane) dihydrochloride (AAPH), for a total of 60 min. The final values were calculated using the net area under the curves of the sample concentrations for which a decrease of at least 95% of fluorescence was observed at 60 min. ORAC values were expressed in micromoles of Trolox equivalents (TE) per milligram (µmol TE·mL −1 ).

Evaluation of Antibacterial Activity
The antibacterial activity of M. didyma EO and compounds was tested using the antibacterial hydrophobic assay as described by Côté et al. [45]. Briefly, after micro-organisms passed 16-18 h at 37 • C in a nutrient broth base (Difco), 20 µL methanol containing growing concentrations of EO and compounds (3.1 to 200 µg·mL −1 ) was transferred onto nutrient agar in 96-well plates. Bacterial strains having a concentration of 2.5 × 10 5 colony-forming units (CFU) were then added per mL of nutrient broth. Bacterial suspension without treatment was used as a negative control, and bacterial suspension plus solvent were tested in parallel to demonstrate the absence of solvent toxicity. The blank consisted of a culture medium only and was subtracted from all subsequent measurements of each well. The 96-well plates were then incubated at 37 • C for 5 h to foster bacterial growth. One hundred microliters of resazurin sodium salt solution with a concentration of 50 µg·mL −1 (Sigma R-2127, St-Louis, MO, USA) was then added to each well. Fluorescence was read on an automated Fluoroskan Ascent FLTM plate reader (Labsystems, Milford, MA, USA) after 2 h for S. aureus and 3 h for E. coli and C. perfringens. The MIC was determined as the lowest concentration resulting in 95% inhibition of bacterial growth.

Experimental Design in Mice
Mice were fed with a mouse diet specifically designed to support the growth and maintenance of animals (LabDiet 5015; 20% protein, 25% fat, 55% carbohydrate). The treatments were prepared by adding concentrations of M. didyma and T. vulgaris (used as the positive control) EO at 0.2% and 0.1%, to the original control diet. The food containing EO was prepared every week to ensure its freshness and quality. Our experiment was assessed for its ethical acceptability and was approved by the APC (Animal Protection Committee of UQAC, Université du Québec à Chicoutimi). Mice were housed in cages in an environmentally controlled room, having 12 h of darkness followed by a period of 12 h of light during the entire run of the experiment. Each cage was provided with a single feed and water system to provide ad libitum access. Fifty (50) mice (five-week-old Charles River males) were placed in cages; each cage contained five randomly selected mice (two replicates). Each cage was assigned to one of five dietary treatments (four EO treatments and one negative control), giving a total of ten (10) experimental cages. The study was completed after 80 days of treatment. BW was measured twice a week and average FI determined on a weekly basis. For each week, the FE per cage was calculated based on the average BW and FI per mouse.

Experimental Design for Broilers
Male broilers (n = 8216) were divided into four treatment groups: two control (n = 2054 each) and two treatment (n = 2054 each). The broilers were installed in an insulated room with facilities for control temperature, light, and humidity according to industry standards. Broilers were fed with the Nutrinor standard diet for poultry. The feeding program consisted of a starter (1-10 d), growth (11-20 d), and finisher (21-35 d) diet given to each broiler ad libitum. The treatment group received 0.5% M. didyma EO in the starter diet, 0.1% EO in the growth diet, and 0.05% EO in the finisher diet. The control group received standard antibiotics: Maxiban TM (0.5 kg/metric tons (MT)) and Tylosine (0.25 kg/MT). Broilers were weighed daily using an automatic balance installed in the hen house. Mortality was recorded daily and total FI was determined at the end of the experiment.

Statistical Analysis
Analysis of variance (ANOVA) was run on all our collected data using the statistical analysis system Sigma STAT. If appropriate, multiple comparison procedures were performed with the Holm-Sidak method. Statements of statistical significance are based on p < 0.05.

Conclusions
Our results confirmed the in vitro antibacterial, antioxidant, and anti-inflammatory activities of M. didyma EO. A diet supplemented with M. didyma EO significantly increased mouse BW and suggested good biological activity in broilers. EO has good potential as an alternative to synthetic antibiotics used in animal nutrition. Future research must explore M. didyma EO in vivo antibacterial activity against E. coli and C. perfringens. Nonetheless, the different dosages of M. didyma EO in the starter, growth, and finisher phases of the broiler diets remain to be tested to determine the optimal concentrations. Supplementary Materials: The following are available online, Figure S1: Body weight gain for M. didyma essential oil and antibiotics groups for day 0-36. Data represent de mean of the two groups, Figure S2: Chromatograms with GC-FID obtain for M. didyma essential oil. Table S1: Composition of Thymus vulgaris L. essential oil.