Chemical Composition, Antibacterial and Antifungal Activity of the Essential Oil from Cistus ladanifer L.

Cistus ladanifer L. is a plant widely used in folk medicine to treat various illnesses. This study aims to evaluate the effect of the plant flourishing time harvest on the chemical composition and the antimicrobial effect of its essential oil. Chemical analysis of the essential oil was carried out using gas chromatography-mass spectrometry (GC-MS). The antibacterial and antifungal proprieties were tested against four selected bacteria (Staphylococcus aureus, Salmonella Typhi, Escherichia coli, and Acinetobacter baumannii) and nine fungi (Yeasts (Candida tropicalis, Candida glabrata, Candida dubliniensis, Candida sp., Rhodotorula rubra, Cryptococcus neoformans) and molds (Penicillium sp. (P), Fusarium sp. (F), Aspergillus niger (A. niger)), respectively. The essential oil of C. ladanifer demonstrated a powerful antibacterial activity with an inhibition zone of 55 ± 0.22 mm for Staphylococcus aureus, 42 ± 0.11 mm for Escherichia coli, 35 ± 0.27 mm for Acinetobacter baumannii (Full resistant to antibiotics) and 30 ± 0.25 mm for Salmonella Typhi. It also inhibited all tested bacteria at 10 µL/mL. For the antifungal activity test, C. tropicalis and C. neoformans appeared to be the most sensitive strains to the essential oil with an inhibition zone of 13 mm, followed by R. rubra and Penicillium sp. (12 mm), then C. dubliniensis and C. glabrata (11 mm). The chemical analysis of the essential oil by GC-MS revealed that the major components of the essential oil were viridiflorol (17.64%), pinocarveol (11.02%), bornylacetate (9.38%), and ledol (8.85%). C. ladanifer exhibited a remarkable antimicrobial activity that could be more exploited to develop targeted natural remedies against specific diseases.


Introduction
Aromatic and medicinal plants (AMPs) have long been a part of man's everyday existence for a variety of purposes. AMPs play a crucial and fundamental role in traditional medicine and play a very significant role in drug discovery [1]. Natural substances account for around 25% of all medicines available for the treatment of illnesses (plants, animals, bacteria, and fungi) [2]. Cistus ladanifer L. is one of the medicinal plants, belonging to the Cistaceae family, the latter represented by seven genera (Cistus, Fumane, Halimium, Tuberaria, Helianthemum, Hudsonia and Lechea), the genus Cistus alone encompass 16 species particularly distributed in the Mediterranean region [3,4], this kind is widespread in Portugal, Spain, Italy, Algeria, and Morocco [5]. The most common species are C. ladanifer (Gum Cistus), C. monspeliensis (Montpellier Cistus), C. salviifolius (Sage Cistus), C. laurifolius

C. Ladanifer Moisture Content and Its Essential Oil Yield Percentage
The moisture content of C. ladanifer essential oil was 13.2%. The yield extraction of the essential oil was around 0.21 ± 0.01%. These results were quite high compared to those obtained by several other researchers. In France, Robles reported a yield of 0.119 ± 0.016% [27]. In Algeria, Bechlaghem reported a yield of 0.08% [28]. Zidane et al. from Morocco obtained a yield of 0.14% [29]. Grech et al., also from Morocco found a yield of 0.3 to 0.4% in the northern region [30]. Thus, we find that the C. ladanifer essential oil yield, although highly variable, remains relatively low regardless of the region and the time of harvest.

Mineral (Ash) and Organic Matter Content in the C. Ladanifer Essential Oil
The mineral content obtained was 3.7%, which was considered quite important. The organic content was around 1.35%. This variation can be explained by the mineral reserves of the soil, the efficiency of their root capture, and their movement towards the aerial organs of the C. ladanifer.

Refractive Index and Brix Index of the C. Ladanifer Essential Oil
The refractive index and Brix index are qualitative identification characteristics that may be used to evaluate the purity of essential oils [31]. Each substance has its specific refractive index. The purity of a product is determined by how near its refractive index is to the anticipated value. The refractive index of our studied essential oil is 1. 45. Mrabet et al. reported a refractive index of 1.49 for the essential oil of C. ladanifer var maculatus from northern Morocco [32]. The refractive index values of C. ladanifer essential oil extracted by hydrodistillation are comparable to those of standards, indicating that our extracts are of excellent purity confirmed also with the low Brix index (1.33) which is an indicator of the concentration (%) of all solids dissolved in the essential oil (Table 1).

Antibacterial Activity
In this study, the antibacterial activity was evaluated using two methods: the agar disk diffusion method and the dilution in a liquid environment. The aim of the tests is to highlight the inhibitory power of the essential oil vis-à-vis the tested bacteria after 24 h of incubation at an adequate temperature of 37 • C.

The Antibiotic Sensitivity Test
The antibiotic sensitivity profiles of the strains are developed according to the recommendations of the Committee on Antibiotic susceptibility of the French Society for Microbiology (CA-SFM) and presented in Table 3. From the table, it can be concluded that the strains of S. aureus and S. Typhi were sensitive to all antibiotics, while E. coli was only resistant to ticarcillin. At the same time, A. baumannii demonstrated complete resistance to all tested antibiotics. This may be due to the higher resistance of Gram-negative bacteria due to the complexity of their cell wall, containing a double membrane in opposition to the single glycoprotein/teichoic acid membrane of Gram-positive bacteria [35].  Table 4 and Figure 2 represent the results of the agar disk-diffusion method for C. ladanifer essential oil against the selected bacterial strains after 24 h at 37 • C. The agar disk diffusion method results indicated that the essential oil of C. ladanifer has a remarkable antibacterial activity compared to the concentration used (5 µL). From the analysis of the results obtained (Table 4), it was noted that the four microorganisms studied are sensitive except S. Typhi which demonstrated an inhibition zone of 30 ± 0.25 mm when the EO of C. ladanifer was used. The best inhibition diameter was against S. aureus (55 ± 0.22 mm) and E. coli (42 ± 0.11). It was also noted that contrary to the sensitivity test when A. baumannii demonstrated full resistance to the tested antibiotics, it demonstrated an inhibition diameter of 35 ± 0.27 when the EO was used, indicating a good and promising effect. Our results are superior to those found by Benayad et al. (S. aureus (28 mm), A. Baumannii (24 mm), and E. coli (18 mm)) (plant harvested in May) [11]. Same note for results obtained by Boukil et al. (S. aureus (14 mm), and E. coli (9 mm)) (plant harvested in August) [19]. As an outcome, harvesting the plant at the flowering stage is correlated to a potent antibacterial power.  (14 mm), and E. coli (9 mm)) (plant harvested in August) [19]. As an outcome, harvesting the plant at the flowering stage is correlated to a potent antibacterial power. The inhibition diameters were expressed in millimeters (mm): Mean ± SD.

Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentrations (MBC)
During this investigation, the determination of the MIC was evaluated by assessing the inhibitory power of the plant's essential oil at different concentrations against the selected bacteria (Table 5). In total accordance with the previous results obtained in the agar disk diffusion method and the observed inhibition zone, the results obtained indicated that the C. ladanifer essential oil MIC and MBC for all strains were 10 µL/mL. Regarding the MBC/MIC activity ratio, our results indicate that it was equal to 1 for all strains. This value allows us to affirm that the essential oil of C. ladanifer is bactericidal. This antimicrobial activity of this essential oil can only be explained by its chemical profile rich in 34.02% of oxygenated sesquiterpenes and oxygenated monoterpenes, and 33.14% of monoterpenes which are known as versatile anti-infective agents (antibacterial and antifungal) [36]. The structures of the functional groupings of the constituents of essential oils could play a crucial role in determining the antibacterial power of essential oils [37]. Nevertheless, minority compounds can interact directly, or in a synergistic or in an antagonistic way, to create a mixture with biological activity. Guinoiseau et al. [38], Rossi et al. [39], and Vieira et al. [40] demonstrated that essential oil from C. ladanifer has antimicrobial activity against Gram-positive and negative pathogens of clinical importance such as Staphylococcus aureus, E. coli, Streptococcus pneumonia, Pseudomonas aeruginosa, Enterobacter aerogenes, and Campylobacter jejuni.

Antifungal Activity
The disc diffusion technique enabled us to demonstrate the antifungal activity of C. ladanifer essential oil against 10 different fungus strains (Figure 3). The antifungal effect of a volume of 5 µL, 10 µL, and 15 µL of C. ladanifer essential oil is presented in Table 6. The essential oil demonstrated a good inhibitory activity with a slight dose-dependent activity. Fluconazole used as a positive control presented the best inhibition zone diameters against all the selected strains. In relation to the essential oil activity, C. tropicalis and C. neoformans were the most sensitive with an inhibition zone of 13 mm, followed by R. rubra and Penicillium sp., which have the same inhibition diameter of 12 mm, then C. dubliniensis and C. glabrata with inhibition zones of 11 mm. No studies on the antifungal activity of C. ladanifer of our study area (Middle Atlas, Morocco) have been carried out, except for a few attempts that have been reported by Boukil et al. [16]. Plants 2021, 10, x FOR PEER REVIEW 8 of 15  Table 7 summarizes the values of the MIC and MFC of the essential oil determined in a liquid medium against the ten tested fungal strains. The essential oil of C. ladanifer showed an antifungal activity on all fungal strains tested with concentrations ranging between 16 and 64 µL/mL.    Table 7 summarizes the values of the MIC and MFC of the essential oil determined in a liquid medium against the ten tested fungal strains. The essential oil of C. ladanifer showed an antifungal activity on all fungal strains tested with concentrations ranging between 16 and 64 µL/mL. The results of the MFC were all compared to those of MIC except for Candida sp. strain. Those obtained results were interesting and in agreement with those obtained by Guinoiseau et al., Rossi et al., Vieira et al. which indicate that the essential oil from C. ladanifer has an antimicrobial activity on fungi (such as Aspergillus niger, Botrytis cinerea, Mucorracemosus, and Verticilliumalboatrum) [38][39][40].

Discussion
This study, among others, focused on revealing the potential bioactivity of the essential oil of C. ladanifer. The study's novelty was that plant material was collected during its time of flowering and in a different region (Middle Atlas), which is known for its semi-arid climate. The edaphic and climate parameters and others have influenced the variation of the composition revealed in the chromatographic analysis. The essential oil major components were viridiflorol (17.64%), trans-pinocarveol (11.02%), bornyl acetate (9.38%), and ledol (8.85%) indicating the domination of the oxygenated sesquiterpenes (34.02%), oxygenated monoterpenes (33.14%) in the overall composition.
The oxygenated sesquiterpenes and monoterpenes are a well-known group of compounds with antibacterial and antifungal proprieties [41,42] and their presence among the EO composition explains the majority of the outstanding outcomes obtained.
The strains chosen for this research are of great interest in the areas of clinical and public health. Their increasing resistance to conventional drugs has prompted further research into new, more effective options, particularly natural products [43,44]. S. aureus (Gram-positive) is a member of the indigenous human microflora and may be found asymptomatically in a variety of bodily locations. Diseases caused by transmission from these locations are both endemic and epidemic [45]. Infection with S. aureus is a leading cause of skin, soft tissue, respiratory, bone, joint, and endovascular diseases. Many strains of S. aureus are becoming resistant to current antibacterial treatments, posing a significant issue in medical microbiology [46].
S. Typhi, one of the representatives of the Salmonella family, is the direct causative organism of typhoid fever [47] (accompanied by weakness, headaches, mild vomiting, abdominal pain, and constipation). Symptoms may persist for weeks or months if not treated [48].
E. coli usually colonizes human babies' gastrointestinal tracts within a few hours after birth. E. coli and its human host often live in excellent health and mutual benefit for decades [48]. These commensal E. coli strains seldom cause illness unless the host is immunocompromised or the usual gastrointestinal barriers are broken, as in peritonitis. Diarrhea induced by E. coli infection is a growing issue in both the developing and developed worlds, with significant rates of death in newborn infants and animals [49]. Although most commensal representatives found in human and animal gut flora are non-pathogenic, certain strains are very dangerous.
The genus Acinetobacter, over the past 30 years, has experienced considerable taxonomic evolution. Its most prominent example, A. baumannii, has emerged as one of the most problematic infections for healthcare facilities worldwide [50]. A. baumannii strains resistant to all known antibiotics have now been discovered, indicating a sentinel occurrence that should be addressed by the worldwide health care community as soon as possible. It often attacks the most susceptible hospitalized patients, those who are severely sick and have compromised skin integrity and airway protection [51].
In terms of fungus, non-albicans candida species are increasingly being reported as both colonizers and pathogens causing nosocomial fungal bloodstream infections, accounting for nearly half of all non-superficial candida infections, with C. glabrata, C. tropicalis, and C. dubliniensis being the most common [52].
One other life-threatening fungi exploited in this study is C. neoformans which is responsible for cryptococcal meningitis, the most prevalent type of cryptococcosis, often chronic and deadly if left untreated [53]. This virulence is none less than those presented by other fungi such as Aspergillus niger, a fungus that causes the "black mold" on certain fruits and vegetables (contaminant of food) which its consumption (as it secretes ochratoxinsmycotoxins) causes nephrotoxicity and renal tumors [54]. The EO of C. ladanifer and through this study demonstrated a strong and real potential that could be better exploited to fight against the threats presented by all the microbial strains studied with slight differences in terms of efficacy.

Materials and Reagents
Ticarcillin (TIC), ceftazidim (CAZ), Meropenem 10 (MEM10), and Ticarcilline85 (TIM85), were purchased from Sigma Aldrich (St-Quentin Fallavier, France). All chemicals and solvents were of highly analytical grade and were used as received from the supplier without further purification.

Plant Material
C. ladanifer was harvested in April 2019 (time of flowering) in El Harcha forest, which is located in the province of Khemisset, circle of Oulmes, 150 km south-east of Rabat, it extends entirely over the rural commune of Teddas (Middle Atlas region with semi-arid climate). The plant material was then dried in the shade, at room temperature, and stored for various uses. Professor Mohammed Fennan, a botanical expert from Mohammed V University's scientific institute, identified this plant where a specimen of the voucher was set under collection number RTCBME10.

Moisture Content Determination of the Plant Material
The moisture content was calculated after drying three samples (5 g) of the plant material at 100 • C in an oven until a constant weight was obtained. The calculation formula was as follows: where %MC: Moisture content of the plant material, w1: Initial mass of plant material in g introduced into the oven at the time (t 1 ) and w2: Final mass of plant material in g removed from the oven at the time (t 2 ).

Essential Oil Extraction
Extraction of the essential oil was carried out by hydrodistillation using a Clevengertype apparatus (VWR, Pennsylvania, United States). The essential oils were recovered in small opaque bottles and stored at 4 • C until further use. The yield extraction is expressed as a percentage of the volume of essential oil (mL) about the mass of the vegetable material (g). The calculation formula is as follows: where YE: Efficiency of extraction of essential oil expressed as volume of essential oil per mass of vegetable material (V/m), w: Mass of vegetable material used for oil extraction, V: Volume of essential oil collected (in mL) and MC: Moisture content of the plant material.

Mineral (Ash) and Organic Matters Content of C. Ladanifer Essential Oil
The method used is that described by the "Association Française de Normalisation" (AFNOR) standard [55]. The raw ash is obtained after the destruction of the organic matter by incineration. In a desiccator, nickel crucibles were dried in the oven for 1 h and were deposited and tarnished. After cooling, each crucible was weighed at 0.1 mg. Approximately 3 g of sample was crushed at 1 mm and weighed in the crucibles at 0.1 mg. The solid crucibles are fed into the muffle furnace under the exhaust hood and their contents are burnt for 4 h at 550 • C. After this time, we let the temperature go down to 100 • C and the crucibles were taken out, cooled in a desiccator, and then weighed at 0.1 g. Total ash was calculated as follows: where %MW: Mineral matter (ash content) expressed as a percentage of the crude product, w1: Mass of crucible containing dry residue, in grams, w0: Empty crucible mass, in grams, and MT: Mass of the test sample, in grams. The organic matter content (OM) of the sample is the difference between the dry matter mass (DM) and the mineral matter mass expressed as a percentage: The refractive index of chemical compounds is important because it indicates characteristic physical properties. The refractive index of the essential oil was measured at 20 • C using a refractometer (ATAGO, Tokyo, Japan) [56].

Brix Degree
The Brix degree gives an idea of the concentration (%) of all solids dissolved in the essential oil (sugar, salts, protein, fatty acid, etc.). Indeed, a few drops of the essential oil was deposited on the refractometer tank (ATAGO). The light beam deflection through the sample (the essential oil) was varied and indicated by a colored delimitation of the degree Brix.

GC-SM Analysis
The chromatographic analysis was carried out on a HP 6890 series gas chromatograph (Hewlett Packard, Palo Alto, CA, USA), equipped with a DB-5 (5% phenylmethylsiloxane) capillary column (30 m × 0.25 mm × 0.25 µm film thickness), an FID detector set at 250 • C and lied by a mixture of H 2 /air gas. The injection mode is split; the carrier gas used is nitrogen with a flow rate of 1.7 mL/min. The column temperature was programmed with a rise of 4 • C/min from 50 • C to 200 • C for 5 min. The device was controlled by a HP Chemstation computer system managing the operation of the device and allowing to follow the evolution of chromatographic analyses. The GC-MS was coupled to a mass spectrometer (HP 5973 series). The fragmentation was carried out by electronic impact at 70 eV. The column used is a capillary type DB-5SM (30 m × 0.25 mm × 0.25 µm). The column temperature was programmed at a rate of 4 • C/min from 50 to 200 • C for 5 min. The carrier gas used is helium with a flow rate of 1.7 mL/min. The injection mode is split. The identification of the constituents of the essential oils studied was carried out both by the index identification method of Kovàts [57], and Adams [58], and from the mass spectral database. In this study, four bacterial strains and nine fungal strains were collected from the Mohammed V University Hospital in Rabat-Morocco (Table 8). Microbial strains were seeded in petri dishes containing nutrient agar, the incubation time and temperature were 24 h and 37 • C for bacteria, 48 h and 28 • C for yeast, 7 days and 28 • C for molds to obtain well-isolated colonies. Isolated colonies were collected from young cultures obtained using an inoculator. The colonies were placed in 5 mL of sterile physiological water with NaCl (0.9%) so that the solution contained about 10 g/mL at a density ranging from 0.08 to 0.1 to 625 nm [59]. The inoculum is adjusted either by adding culture if it is too weak or sterile physiological water if it is too strong. Seeding was done directly after the preparation of the inoculum. The antibiotics used for bacterial testing were ticarcillin (TIC), ceftazidime (CEF), vancomycin (VAN), tetracycline (TET), cefalexin (CEF); colistin (COL) and amikacin (AMI).

Diffusion Method on Agar Medium
This test was carried out on Petri dishes (90 mm) by the diffusion method in the agar media of Mueller-Hinton and Sabouraud by determining the diameter of the zone of inhibition [60,61]. Disks of 6 mm in diameter, filter paper, were cut and sterilized, then deposited delicately in the center and on the peripheries of the surface of a medium previously inoculated with the microbial inoculum (this inoculum was obtained in the form of a suspension in saline solution). Then, volumes of 5 µL of essential oil of C. ladanifer relating to the antibacterial tests and volumes of 5, 10 and 15 µL relating to the fungal tests were deposited on these discs. A sterile disc flooded with 6 µL of sterile physiological water was used as a negative control as well as another negative control disc impregnated with 2 µL of DMSO to check the growth of different bacterial and fungal strains. The inhibition diameters were measured around the disc after incubation in an oven, each test was repeated three times to obtain as precise an estimate as possible of the true experimental value sought.

Liquid Dilution Method
This method was used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal (MBC) and fungicidal (MFC) concentration of the essential oil of C. ladanifer L. [62,63]. An amount of 2 µL of DMSO was added to the 10 tubes. Hemolysis containing the culture medium (1 mL) for each bacterial strain. Each volume of essential oil (2, 4, 6, 10, 12, 14, 16, 18, 20 and 22 µL) was introduced into a hemolysis tube. Then, a volume of 6 µL of the bacterial suspension with a concentration of 105 CFU/mL was taken and then was deposited in each of the preceding tubes and then 2 µL of DMSO was added for each microbial strain. Two sets were made, one containing the culture medium (1 mL) plus a strain (bacterial or fungal) and the other containing the culture medium (1 mL) plus 2 µL of essential oil alone. After an incubation of 24 h at 37 • C for bacteria, 48 h at 28 • C for yeasts, and 7 days for molds, the MIC of the essential oil was determined. We then inoculated the surface of a Mueller Hinton agar cast in a Petri dish with 100 µL of the contents of the tubes having a concentration greater than or equal to the MIC to determine the CMB and CMF.

Conclusions
In this study, it was demonstrated that the essential oil of C. ladanifer is high in oxygenated sesquiterpenes, which mediated the activities seen. The results of the antimicrobial activity showed that this essential oil was able to inhibit the growth of a wide variety of bacterial and fungal strains. The observed activity was correlated to the harvest time of this plant done in May, which influenced the phytochemical profile of the EO in comparison to its harvest in other months as seen in other studies from the literature. The essential oil demonstrated an excellent antimicrobial activity and could serve as an alternative to some medicine and antibiotics. More studies should be done to determine the toxicity and the therapeutic margin for a more controlled and oriented application.