Introduction

An infection is said to be nosocomial if it occurs during or following the treatment (diagnostic, therapeutic, palliative, preventive or educational) of a patient in a health care environment. Nosocomial infections pose a significant public health problem because of their frequency, severity and socio-economic cost, which represents a considerable burden for patients and the health system. According to Carling and Huang (2013), the patient’s environment, particularly treatment rooms and medical equipment, are key sources of multidrug-resistant pathogens that can be transmitted to other patients. This problem requires the development of more effective disinfectant agents to overcome the challenges of multidrug-resistant pathogens (Rolain et al. 2012). The World Health Organization (WHO 2019) recently published a report on antimicrobial resistance that classified this issue as a “public health problem and a serious threat affecting all countries”.

The use of natural antimicrobial compounds is important in various fields, particular in the control of human diseases (Choi et al. 2010). Thus, the search for safe, effective and readily available alternatives to current antimicrobial agents has become imperative. Plants have been used for the treatment of infectious illnesses since ancient times, even before any knowledge about microorganisms existed (Edris 2007).

Secondary bio-molecules extracted from aromatic and medicinal plants represent an excellent source of new molecules that can treat various infectious diseases, including nosocomial infections. Recent studies have focused on identifying new molecules that are effective against pathogenic bacteria that continue to develop resistance against antibiotics (Ez zoubi et al. 2020; Nieto 2017; Villalobos et al. 2016). Many of these studies have focused on different plants because of their biological activities, which are due to major and minor compounds in alcoholic extracts and essential oils. Indeed, some researchers have highlighted the role of phenolic extracts in biological processes, including anti-inflammatory (Muthuraman et al. 2011), antioxidant (Sarikurkcu et al. 2020) and antifungal (Tocci et al. 2018) activities. The antimicrobial activities of phenolic compounds are also well documented (Babar et al. 2020; dos Santos et al. 2017).

Lavandula stoechas, from the family of Lamiaceae, is one such plant with potential therapeutic effects. In Morocco, rheumatic diseases, polyarthritis, nephritis, cystitis and gastric infections have traditionally been treated with L. stoechas extracts (Ez zoubi et al. 2020; El-Hilaly et al. 2003). Several studies have reported that the aqueous and alcoholic extracts of L. stoechas are characterized by multiple biological activities. Extracts have been found to have antibacterial (Canlı et al. 2019), antifungal (Baptista et al. 2015), antioxidant (Celep et al. 2018; Gülçin et al. 2004), anti-hyperglycemic (Mustafa et al. 2019), anticonvulsant, antispasmodic, sedative (Gilani et al. 2000), and anti‐inflammatory properties (Kulabas et al. 2018; Algieri et al. 2016; Ez zoubi et al. 2016). These activities are related to the phenolic compounds identified in the extracts of L. stoechas. Phenolic acids and flavone glycosides (Fig. 1) are the most commonly reported phenolic compounds in L. stoechas extracts (Karabagias et al. 2019; Celep et al. 2018; Upson et al. 2000).

Fig. 1
figure 1

Chemical structures of phenolic compounds reported in Lavandula stoechas extracts. Phenolic acids (Caffeic acid; rosmarinic acid and chlorogenic acid) and flavone glycosides (apigenin 7-glucoside; Apigenin 7-O-glucuronide; luteolin 7-O-β-glucoside and luteolin 7-glucuronide)

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The objective of this study was to assess the phytochemical composition of L. stoechas plants native to central Morocco and to determine their total phenolic content. The antimicrobial effect of hydro-ethanolic, flavonoid and tannin extracts of this plant against pathogenic bacteria isolated from a hospital environment in Morocco was also determined for the first time.

Material and methods

Plant material

Leaves, stems and flowers of L. stoechas were harvested during the period of April to May of 2016 at the commune Timezgana (34° 33′ 02.7″ N 4° 40′ 49.3″ W) in Taounate province (in the north central region of Morocco), at an altitude of approximately 800 m. The botanical identification was conducted by Professor Abdeslam Ennabili, National Institute of Medicinal and Aromatic Plants, Taounate, Morocco (NIMAP-Taounate, Morocco). Authenticated voucher specimens have been deposited in the Herbarium of NIMAP-Taounate, Morocco.

Hydro-ethanolic extract of L. stoechas by ultrasound-assisted extraction

The extraction method was performed using an Elma–Transsonic TI-H-15 ultrasound bath (frequency 35 kHz, nominal power 100 W). The powder of the dried plants (20 g) was placed with 150 ml of 80% ethanol in a beaker. Next, the suspension was immersed in water in the ultrasonic device and irradiated for 45 min (Ez zoubi et al. 2016). After ultrasonic extraction, the sample was filtered by Whatman paper and evaporated under vacuum at 40 °C on a rotary evaporator.

Extraction of flavonoid fraction

Following the method of Lee et al (1995), 100 g of L. stoechas powder was subjected to Soxhlet extraction in a mixture of distilled water and absolute ethanol (100 ml/100 ml V/V) for 1 h at a temperature of 60 °C. The extract was filtered and the ethanol was removed by evaporation at 40 °C. The aqueous phase was then extracted with 200 ml of n-butanol and acidified with 10% HCl to pH 3 and the butanol phase was evaporated. The resulting residue was extracted with 200 ml of distilled water/ethyl acetate (100 ml/100 ml V/V) for 1 h at room temperature. The pH of the organic phase was adjusted with NaHCO3 to pH 9. After standing for 15 min, the organic phase (flavonoids) was evaporated at 40 °C.

Extraction of tannin fraction

Two hundred grams of L. stoechas powder was extracted by maceration in a mixture of acetone/distilled water mixture (35 ml/15 ml V/V) for 72 h at room temperature. The solution was filtered and evaporated at 40 °C to remove the organic phase (acetone). The aqueous phase was washed with 15 ml of dichloromethane to remove the pigments and the lipids. After separation of the organic phase, the aqueous phase was extracted twice with 15 ml of ethyl acetate. The mixture of the two phases was evaporated to dryness at 40 °C (Vu et al. 2017).

Phytochemical screening

Tests were carried out L. stoechas powder in order to determine the chemical families (flavonoids, mucilages, tannins, gallic tannins, catechic tannins, sterols, terpenes and quinones) present in the plant (Aiyegoro and Okoh 2010). These are qualitative analyses based on staining and/or precipitation reactions.

Flavonoids

Two grams of the L. stoechas extract was dissolved in 2 ml of methanol and heated. A chip of magnesium metal was added to the mixture followed by the addition of a few drops of concentrated HCl. The occurrence of a red or orange coloration was indicative of the flavonoids.

Tannins

One gram of L. stoechas extract was dissolved in 10 ml of distilled water and filtered. A blue coloration resulting from the addition of ferric chloride reagent to the filtrate indicated the presence of tannins in the extract.

Catechic tannins

One ml of choloridric alcohol (5 ml of 95 °C alcohol, 5 ml of distilled water, 5 ml of concentrated HCl) was added to 5 ml of hydro-ethanolic extract of L. steochas and the whole is brought to boil for 15 min. In the presence of catechic tannins, there is formation of a red precipitate soluble in amyl alcohol.

Gallic tannins

Fifteen ml of Stiasny’s reagent (10 ml of 40% formalin, 15 ml of concentrated hydrochloric acid) was added to 30 ml of infused then heated in a water bath at 90 °C for 15 min. After filtration, the filtrate was saturated with sodium acetate, then 1 ml of a FeCl3 (1%) solution was added. Obtaining the precipitate shows the presence of gallic tannins.

Sterols and terpenes

Twenty ml of petroleum ether was added to 1 g of the L. stoechas powder. The mixture was left to macerate for 24 h, filtered and made up to 20 ml. After evaporating to dryness in a 10 ml capsule of extract, the residue was dissolved in 1 ml of acetic anhydride and then 1 ml of chloroform. 1–2 ml of H2SO4 was placed in the bottom of the tube using a pipette. At the contact zone of two liquids there is formation of a brownish-red or purple ring, the supernatant layer turning green or purple reveals the presence of sterols and tripetrpenes.

Mucilages

Five ml of absolute alcohol was added to 1 ml of the L. steochas extract, the formation of a precipitate, indicating the presence of mucilages.

Quinones

Ten ml of chloroform was added to 1 g of L. stoechas powder, the mixture was heated in a water bath for 3 min and the mixture was filtered. 10 ml of distilled water and 1 ml of concentrated hydrochloric acid were added to the mixture. The test tube was kept in the boiling water bath for 15 min. in a test tube of chloroform extract was added to 1 ml of diluted NH4OH. The red coloration indicates the presence of quinones.

Determination of total phenolic content

The total phenolic content (TPC) was determined using the Folin-Ciocalteu method, with slight modifications (Singleton et al. 1999). 100 μl of the hydro-ethanolic extract of the plant was mixed with 500 μl of the Folin–Ciocalteu reagent and 1.5 ml of 20% sodium carbonate (Na2CO3), and the volume was made up to 10 ml with H2O. The mixture was stirred and incubated in the dark and at room temperature for 2 h, and the absorbance was measured at 765 nm by a UV spectrophotometer (Selecta, USA). The results are expressed in mg gallic acid equivalent/g of dry plant matter (mg GAE/g) by referring to the calibration curve for gallic acid. TPC results are presented as mean ± standard deviation.

Antimicrobial activity

Bacterial strains

Six pathogenic bacterial strains were studied: one Gram-positive strain (Staphylococcus aureus and five Gram-negative bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumanii, Klebsiella pneumonia and Enterobacter cloacae). All of the pathogenic strains were isolated from different surfaces of a hospital in the city of Fez, and their identification was conducted at the Regional Laboratory for Epidemiology and Environmental Health in Fez using the biochemical gallery and the API gallery (Biomerieux®, France) methods. Three ATCC reference strains were also used in this study: Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213 and Pseudomonas aeruginosa ATCC27853.

Disc diffusion assay

The sensitivity of the different bacterial strains to hydro-ethanolic and phenolic extracts was assessed according to the method described by Alabri et al (2014). Muller-Hinton agar culture medium was used to assess the antibacterial effect of each extract (the hydro-ethanolic extract and the flavonoid and tannin fractions). The extracts were diluted in 10 ml of dimethyl sulfoxide (DMSO) at 5%, and 10 µl of each extract was impregnated in the filter paper discs (6 mm in diameter). Ampicillin and gentamicin were used as positive reference standards, and DMSO (5%) was used as a blind control. The inoculated Petri dishes were incubated at 37 °C for24 h and the inhibition zones were observed, including the diameter of the disc (6 mm).

The antibacterial activity is considered to be very good when the inhibition diameter exceeds 15 mm, moderate when the zone of inhibition is between 15 and 8 mm, and low if the zone of inhibition is less than 8 mm (Chen et al. 2016). The zone of inhibition results are presented as mean ± standard deviation.

Determination of minimum inhibitory concentration

The minimum inhibitory concentration (MIC) values were evaluated according to the method described by Amy (2016), with slight modifications. In test tubes containing 5 ml of Muller-Hinton broth, 0.5 ml of each bacterial inoculum was added to 1 ml of different concentrations of the hydro-ethanolic extracts, flavonoids and tannins studied (160, 80, 40, 20, 10, 5 and 2.5 mg/ml). The samples were incubated for 24 h at 37 °C. The MIC values correspond to the tubes that did not develop any bacterial growth.

Determination of minimum bactericidal concentrations

The same concentration range carried out by the macrodilution technique in liquid media was used to determine the minimum bactericidal concentration (MBC) of the hydro-ethanolic, flavonoid and tannin fractions. Fifty microliters of each dilution were placed in the control tube, and each of the tubes of extracts were spread on tryptic soy agar Petri dishes. The Petri dishes were then placed in an oven for 24 h at a temperature of 37 °C. Petri dishes devoid of any bacterial growth were considered to represent the MBC.

Results

Phytochemical screening

The phytochemical screening of the L. stoechas powder revealed the presence of flavonoid, mucilage, tannin, catechic tannin, sterol and terpene compounds. Quinones and gallic tannin families were not identified (Table 1).

Table 1 Phytochemical families of Lavandula stoechas areal part
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Total phenolic content

Based on the gallic acid calibration curve shown in Fig. 2, the total phenolic compounds (TPC) in the hydro-ethanolic extract of L. stoechas revealed a value of 130.15 ± 3.72 mg of GAE/g.

Fig. 2
figure 2

The standard curve obtained using Gallic Acid for total phenolic content determination

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Antibacterial activities

Inhibition zones using disc diffusion method

The antibacterial activity of the hydro-ethanolic extract of L. stoechas and its phenolic fractions against microorganisms assessed using the disc diffusion method are summarized in Table 2. The hydro-ethanolic extract of L. stoechas was moderately active against E. coli ATCC, S. aureus ATCC and A. baumanii, with inhibition zones of 13.5 ± 2.4 mm, 14.2 ± 1.4 mm and 14.34 ± 1.6 mm, respectively. The flavonoid extract prepared from the L. stoechas powder demonstrated inhibition zones greater than 14.6 ± 2.2 mm in diameter, as shown in Table 2, with the greatest zones between 20.3 ± 1.5 mm and 22.5 ± 3.4 mm recorded against the hospital strains P. aeruginosa and S. aureus as well as the P. aeruginosa ATCC and S. aureus ATCC strains. Tannin extracts did not demonstrate antimicrobial activity against E. coli, K. pneumonia, S. aureus, E. cloacae or A. baumanii.

Table 2 The inhibition zones of hydro-ethanolic extract of L. stoechas and its phenolic extracts (flavonoids and tannins)
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Among the antibiotics (Table 2), ampicillin showed poor activity against all bacteria, with inhibition zones between 7.3 ± 0.4 mm and 10.7 ± 1.2 mm. Gentamicin had the widest coverage against all bacteria (between 14.5 ± 0.8 mm and 22 ± 3.2 mm).

The minimum inhibitory and bactericide concentrations (MIC and MBC)

The MIC and MBC assays of the hydro-ethanolic and phenolic fractions from Moroccan L. stoechas showed widely different activities against all microorganisms tested (Table 3). The flavonoid extracts had the highest effect against the bacteria, with MIC and MBC values varying between 10–40 mg/ml and 20–80 mg/ml, respectively. The results also revealed MIC values of the hydro-ethanolic extract of 80 mg/ml against all bacteria except A. baumanii and P. aeruginosa ATCC (MIC = 40 mg/ml).

Table 3 MIC and MBC values of hydro-ethanolic, flavonoids and tannins fractions of L. stoechas against pathogenic and reference strains
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The tannin fraction exhibited weak activity against all bacteria, with MIC and MBC values more than 160 mg/ml, except S. aureus and S. aureus ATCC strains, with MIC values at 160 mg/ml. This activity is likely due to the presence of flavonoid compounds in the hydro-ethanolic extracts and phenolic fractions from L. stoechas.

Discussion

The objective of this study was to determine the phytochemical screening of the hydro-ethanolic extract from L. stoechas and evaluate the antimicrobial activity of hydro-ethanolic and phenolic fractions (flavonoids and tannins) against bacteria of nosocomial interest.

The phytochemical screening of the hydro-ethanolic extract of the L. stoechas aerial part allowed the determination of several chemical families, including flavonoid, mucilage, tannin, catechic tannin, sterol and terpene compounds, which are secondary synthesized compounds. The presence of flavonoids, tannins and coumarins has been revealed in several species of the Lavandula family, such as Lavandula angustifolia (Yadikar et al. 2018).

Concerning the TPC, Bajalan et al. (2016) found that concentrations of methanolic extracts varied between 31.45 and 105.39 mg of GAE/100 g in populations of Iranian Lavandula, while, the TPC of lavender flowers growing in botanical gardens from Romania ranging between 74.98 and 89.88 mg of GAE/g for Lavandula angustifolia and Lavandula hybrid, respectively (Silva et al. 2012).The variations observed in the TPC can be explained by variation in several biotic and abiotic factors, including genetic variation, plant populations, distinct environmental and geographic origins (Ghasemi Pirbalouti et al. 2013). In addition, the harvesting period, gathering site, age of the plant, part of the plant used and extraction techniques influence the nature of the chemical composition and the rates of presence of the different constituents; this is also manifested in the phenolic compounds of plants (Moghaddam and Mehdizadeh 2015).

Few studies have addressed the subject of the antibacterial effects of L. stoechas extracts against bacteria isolated from hospital environments. This study, carried out for the first time in Morocco, evaluated the antibacterial power of phenolic extracts of L. stoechas against several bacterial strains isolated from a hospital environment. Indeed, Al-Niaame and Aziz (2013) evaluated the effect of aqueous and methanolic extracts of L. officinalis against Gram-positive and Gram-negative pathogenic multiresistant bacterial strains. The results of this study proved that the methanolic extract of the plant was effective against all bacteria with a variable inhibition zone between 15 and 20 mm, and MICs of 25 mg/ml. While the hydraulic extract did not inhibit the bacteria tested.

The results of our study have shown that the flavonoid extract fraction of L. stoechas is characterized by an antibacterial effect superior to the hydro-ethanolic and tannin extracts. The work of McClure (1975) has shown that the flavonoids have a key and major role in the self-protection of plants against bacterial, fungal and viral diseases. This is an important aspect in understanding the defense of plants. According to Lima et al (2019), phenolic compounds have an interesting antibacterial power against pathogenic bacteria. These compounds can act in several ways to destroy bacteria, in particular by inhibiting the manufacture of nucleic acids, the manufacture or function of the plasma membrane, and the enzymatic activity or the energetic metabolism of bacteria (Cushnie and Lamb 2005).

Several studies have shown that flavonoids act as enzymatic inhibitors or antioxidants. Hong et al (2006) and Verdrengh et al (2004) found that genistein, an isoflavone, inhibited the in vitro growth of S. aureus. Another study conducted by Wang et al (2010) showed that isoflavones inhibited nucleic acid synthesis in S. aureus. Mitani et al (2018) and Alves et al. (2013) showed that phenolic acids, namely coumaric, 2,4-dihydroxybenzoic and protocatechuic acids, displayed significant antimicrobial activity against both Gram-positive and Gram-negative bacteria.

Conclusion

This paper provides information on the TPC of L. stoechas hydro-ethanolic extracts and the antibacterial activity of hydro-ethanolic, flavonoid and tannin extracts of this plant. The TPC of this plant was 130.15 ± 3.72 mg of GAE/g. The flavonoid extract had the highest effect against bacteria, with inhibition zones between 20.3 ± 1.5 mm and 22.5 ± 3.4 mm recorded against P. aeruginosa and S. aureus isolated from the hospital environment, and for the P. aeruginosa ATCC and S. aureus ATCC strains. In addition, the MIC and MBC values of the flavonoid fractions ranged from 10 to 40 mg/ml and 20–80 mg/ml, respectively.

The use of these natural phenolic extracts, especially flavonoids, in antibacterial treatments could be a promising alternative to replace synthetic antibiotics and may lead to new research on natural products.